Advances in Carbohydrate Chemistry and Biochemistry, Volume 40

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Advances in Carbohydrate Chemistry and Biochemistry

Volume 40

This Page Intentionally Left Blank

Advances in Carbohydrate Chemistry and Biochemistry Editors R. S T U A R T TIPSON

DEREK HORTON Board of Acluisors H E N G r LINDBERG HANSPAULSEN NATHANSHARON ;LIAURICE STACEY ROY L. WHISTLER

LAURENS ANDERSON STEPHEN J. ANGYAL CLINTON E. BALLOU GUYG. S. DUTTON ALLANB. FOSTER

1982

ACADEMIC PRESS A Sub5idiary 01 tiarc our1 H i a c r lovdnovich Publirhrrr

Paris

San Diego

New York London San Francisco SSo Paulo Sydney

Tokyo

Toronto

COPYRIGHT @ 1982, BY ACADEMIC PRESS, I N C . ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY F O R M OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND R E 1 RIEVAL SYSTEM, W I T t l O U T PERMISSION IN WRITING F R O M T H E PUBLISHER.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

United Kirigdoni Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W I 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER:45 - 11 35 1 ISBN 0-12--007240-8 PRINTED I N T H E UNITED STATES OF AMERICA

82 83 84 85

9 8 7 6 5 4 3 2 1

CONTENTS C O N ~ R I B L J.I O . R. S PKElACt:

. . . . .

. .

. . . . . . . . . . . . . . . . .

. .

. . . . . . . . . . . . . . . . .

vii ix

T h e Synthesis of Sugars from Non-Carbohydrate Substrates AI I K S A N D F R

ZAMOJSKI

. A N N AH Z N A S I t K . A N D G R Z F C I O R Z

. . . . . . . . . . . . . . . . . . . Acetylrnrs m t l Alkc~lrcs . . . . . Sl.irtlrcses from Dc~rivativt~s of I>ili~dro-2H-pyrans Synthcsc~sfroin Derivatives of Fiirair . . . . . . . S>.iitheses from Vitryleiie Carl)onatca . . . . . . . \tiscellairc.ous Syiit1wsc.s . . . . . . . . . . . . . Totnl Syiitlrc~w\of Optictkll! . ..\(.ti\.( (:ai-l)olr?.dratcs

1. Introtlriction

f1Olll

III. I\. . \'.

\'I . \'I1 .

.

GRYNKltWltZ

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . .

1 3

.30

60 84 96 . 112

Chemistry. Metabolism. and Biological Functions of Sialic Acids I

25

the ainide group was hydroly~ed.The product was a mixture of the 1,4- and 1,5-lactones (135 and 136). Reduction of this mixture with diisobutylaluminum hydride (11IBAH) gave practically pure 137. This principle was further exploiteds22ain the synthe\is of DL-

1. EF3-Et,0 129

NH,-MeOH

2 . Me,CO-AlCl,

134

$' C02Me

o&COzMe

NMe,

135

139

138

t

-

NMe, 142a R = H 142b R = Ac

(52a) I. Dyong and H . Bendlin, Chefti Bet

,

112 (1979)717-726

136

26

A L E K S A N D E R ZA,MOJSKI et

(11

inegosamine (142). Epoxicle 129 reacted with dimethylamine at - lo”, to afford methyl 4,5-anhydro-2,3,6-trideoxy-3-(t~iinethylatnino)-~~lyxo- and -xylo-hexonates (138 and 139) in the ratio of 3.4 : 1. Alkaline hydrolysis of this mixture led to the amino acid 140, which, without isolation, was carefully treated with very dilute hydrochloric acid, to afford, in one step, the stereoisomeric mixture of‘ lactones (141). Reduction of the ribo isomer 141 (isolated by fractional recrystallization) with DIBAH under carefully controlled conditions led to the very labile 142a (“inaskecl” P-aminoaldehyde of the Mannich type), which was immediately acetylated to the 1,4-di-O-acetyl derivative

142b. (iii) Use of cis,&- and trans,trans-2,4-Hexadiene-1,6-diols.These diols were used a s substrates in an attempted synthesis of anhydrohe~itols~“; epoxitlation was, however, unsuccessful. When 1,6dibroino-truns,trc~1ls-2,4-hexadienewas treated with ni-chloroperoxybenzoic acid, 2,3:4,5-dianhydro-1,6-dibroino-1,6-dideoxygalactitol was obtained in 15% yield. An exchange reaction with silver methanesulfonate in acetonitrile afforded 2,3:4,5-dianhydro-1,6-di-O-mesylgalactitol. The synthesis of isomeric DL-allitol succeeded when the hydroxyl groups in cis,cis-2,4-hexadiene- 1,6-diol were protected with benzoyl or mesyl groups. Epoxidation gave the appropriate derivatives of 2,3:4,5-dianhydro-~~-allitol (16%), which were then reduced with lithium aluminum hydride to ~~-erytlzro-2,5-hexanediol. (iv) Conjugated Ketones.-There have been few reports on the utilization of conjugated ketones for the synthesis of carbohydrates. However, the acetoxyniethyl ketone 143 has been applied54for a successful synthesis of DL-glycero-2-tetrulose (145b). Although a terminal double bond conjugated with a carbonyl group usually has low reactivity to electrophilic addition, prolonged treatment of 143 with N-bromosuccinimide at room temperature afforded the desired 3-bromo ketone 144 in 46% yield. The conversion of 144 into the DL-& cero-Ztetrulose derivative 145a was readily effected with silver acetate in acetic acid. $H,OAc

:H,OAc

I

c=o I

-

I

c=o

I1 CH,

HCBr I CH,OAc

143

144

HC . ~.

YH,OR

I

-

I

c=o HCOR

I

CH,OR 145a R

= Ac 145b R = H

(53) G . Schneider, T. Horvlith, and P. Sohiir, Curbohytlr. Res., 56 (1977) 43-52. (54) T. Ando, S. Shioi, and h l . Nakagawa, Bull. Chern. S O C . J p r i . , 45 (1972) 2611-2615.

3-Pentadienone (divinyl ketotrt,) w a s epoxidize(1551)y nicaiis of h y drogen peroxide in alkaline solrition, to give ;i inistiire o f DL- and me.r.o-l,2 :4,S-~lianhydro-3-~~eiit~~ii01ies i n the ratio of 13 : 7. Reduction of the ketone group in the DL-dieposide with sodiuni Iiorohydride, followed b y alkaline hydrolysis i n tliiiietliyl sulfositle, was fitlly stereospecific, and afforded DL-aral)iiiitol. ‘rhe same r~.actioii-seclrieiiceperfoniied on the meso-diepoxitle led to 11 mixture of’ ribitol and sylitol. l-Chloro-1,4-hexadien-3-oiic (146) w;is used 1)y llatsiiiiioto ancl coworkers 558 for a stereos peci fi c s y n t 11e s i s of met h y I UL- daiiiio s am i n icle (150, methyl 3-ari~ino-~,3,Ci-trideox).-ol-DL-/!/.~~~-~iex~~~iyr~~1iosi Compound 146 was converted into x e t a l 147, which was cis-hyclroxylated with potassium pemiangan;ite, to yield the keto-diol 148. This compound was transformed i n t o tlic oxiine, which was reduced to the aniinodiol 149. Treatment of 149 with methiinolic hydrogen chloride yielded the methyl glycositle 150. Interestingly, no epinieric compound was formed during this synthesis.

146

150

147

149

148

g. Syntheses from Unsaturated 1Diok-A few syntheses of carliohydrates have been based oil xetylenic or alkciiic diols. In the first place, the commercially availiilile 2-liutyne- 1,4-diol, and tlie products of its partial hydrogenation, iianiel>,,cis- and tr-ciri.s-2-l~utetie-1,4-diol, have been exploited for tlie s),itthesis of four- and five-carlion sugars. Fraser and Raphael56 devised a synthesis of 2-deosy-~~-er-!ltlzr-opent o se (2) starting from 2- 11u t y n e- 1,4-dio I . M on ot ie n zoy 1at i on, follow e d b y react ion with ph ( ) s ph o rii s tri 1)rom i de , fu rii is h e cl 1-(lienzoyloxy)-4-bromo-e-l,utyne ( 151). Reaction of 151 with d i e t h y l sodio(55) P. Chauteiiips, C . R. Acucl. Sci.,S v r . C‘, 284 (1977) 807-808. ( 5 5 4 I. Iwataki, Y . Nakamura, K. Tak;iliashi, a n d T. Mataritnoto, H n I I . C / I [ , I TS oI .r . J p n . , 52 (1979) 2731-2732. (56) M. M. Fraser and R. A . Raphael, 1. C h v t t i . Soc. (1955) 4280-4283.

28

ALEKSANDER ZAMOJSKI et

rnaloiiate gave diethyl 5-(benzoyloxy)-3-pentyne-l,l-dicarboxylate (152), which was converted into diurethan 155 b y the sequence of reactions shown. Hydrolysis of the diurethan proved to be very difficult; only a small proportion of 2-deoxy-D~-erythro-pentose(2) could be obtained. H Et0,C:C 0, Et CH,Br

I

c

7%

FH2

-

111 C

H EtO,CyCO,Et

c

I CH,OBz

c

c Ill c

CH,OBz

CH,OH

152

153

CH(NHCO,Et), I

CH(NHCO,Et),

P

111

I

I

151

t I% H,SO,

-2

YH2 HCOH I HCOH

-

I

CH I

HC I1 HC

I

I

CH,OH

CH,OH

155

154

Another stereospecific synthesis of 2-deoxy-DL-eryt1zro-pentose was based'" on cis-l-(tetrahydropyran-2-yloxy)-2-penten-5-o1 (156) as the substrate. cis-Hydroxylation of 156, followed by oxidation of the unprotected, primary hydroxyl group to an aldehyde group according to Barton's procedure, yielded the desired sugar 2 in low yield. CH,OH

I

FH2 HC

II

HC I CH,OThp 156

-

CH,OH I CH, I HCOH I HCOH I CH,OThp 157

-

CH,OH

I

7%

HkO, I ,CMe,

HYO

CH,OThp 158 I

1. c o c 1 , . quinoline 2 . Me,SO. Et,N 3. H+

SUGARS FROM NON-(:.4HBOtIYI~RATE SIJRSTHATES

29

froiii 2-butyne- 1,4u ~ - ~ l r ~ c e r o - 2 - T e t r u l(14511) o s e was diol, which was partially hydrogeriated over Liiitllar catalyst, and the product acetylated to cis-1,4-tliact.toxy-i2-buterie (159b) o r , altematively, it was reduced with l i t h i u m aluminum hydride, and the procluct acetylated to the trans isoinchr 160b. Treatment of each alkeiie with N-broniosuccinimide furnishetl t h e stereoisomeric 1,4-diacetoxy-3I,r~~mo-2-butaiioIs,161 and 162. Oxidation of 161 a n d 162 with clrroniiuiii trioxitle yielded l , ~ ~ - d i - O - a c e t y l - 2 - l ~ r o u i o - 2 - d e o x y - ~ ~ - ~ / ~ ~ cero-Ztetrulose (149),which reacted with silver acetate to yield 1,3,4t r i ~ - a c e t y l - D L - ~ ~ y c e r o - 2 - t e t i - i(145a). i l o s ~ This w a s dcncetylated with barium hydroxide to the DL-tetrulose. CH,OAc

CH,OR I HC

. I

HOCH I HCni I C €I $1 Ac

I1

HC I

CH,OR

1590 R 159b R

= H

161

Ac

=

g

KOAr. EtOH (moist)

CH,OAc

CH,OR

I

CH

I

-

II HC I

HCOII

1

HCRr

CH,OR

1600 R

=

160b R

= Ac

H

KOAc , AcOH (dry)

DL-Threitol

I

CH,OAc 162

160c R = CH,Ph

Bromohydrins 161 and 162 were converted5i into peracetylated erythritol and DL-threitol on treatnient with potassium acetate in anhydrous acetic acid. An alternative route to erythritol m d DL-threitol involves5xreaction of cis- and tnl~is-1,4-diacetoxy-2-l~iiteiie (159 and 160) with hydrogen peroxide solution in tert-biitanol containing a catalytic arnount of osmium tetraoxide (the Milas reagent) o r with peroxy acids. The second reaction, on 159a and 160a, gave 2,3-aiihydro-erytliritol arid -DL-threitol, respectively, in excellent yield. 2-Aiiiino-2-deoxy-~~-erythritol ( 164b), a degradation product of (57) R. A. Raphael,]. Claem. Soc. (1952) 401-405. (58) R. A. Raphael,]. Cheni. Soc. (1939) 41-48.

30

ALEKSANDEH ZAMOJSKI et

(I/

sphingosine, was obtaineds9 f r o m tmn.s-1,4-cli(l~enzylox~)-2-bi1teiie (160c). Epoxidation arid ammonolysis of epoxide 163 led stereospecifically to raceiiiic 164a. (For resolution of 164, see Section VII.) Catalytic debenzylation of 164a gave 164b.

160c

-

CFI,OCH,Ph I ,CH HC I CH,OCH, PI1

CH,OR I

HCNH, I HCoH I CH,OR

1. NH, 2 . H2,Pd

1640 R 164b R

163

~

CH,Ph H

111. SYNTHESES FROM DERIVATIVES OF DIHYDRO-2EI-PYFtANS

The parent compounds of all pyranoses, the 2H- arid 4H-pyrans (165 and 166), and their derivatives, are impractical a s substrates for the synthesis of sugars. They a r e usuallv difficultly available and are rat her un st ah 1e .6o

165

166

167

168

In contrast, the dihydro derivatives of 165 and 166, namely 3,4-dihydro-2H-pyran (167) arid 5,Bdihydro-W-pyran (168), and their simple derivatives, have found wide application i n the \ynthesis of sugars of various types.

1. 3,4-Dihydro-W-pyran 3,4-Dihydro-W-pyran (167) is well known in organic chemistry as a reagent employed for the temporary protection of primary and secondary hydroxyl groups. Addition reactions to the double lmnd of 167 afford compounds that fill into the category of 3,4-dideoxypentoses. or cliloriiie62,63 leads to a 2.3-dildoReaction of 167 with (59) J. Kiss and F. Sirokinan, Helu. Chirn. Actci, 43 (1960) 334-338. (60) Only simple alkyl derivatives of W - p y r a n are known. For the synthesis and properties of N - p v r a n , see S . Masamune a n d N . T. Castellacci,J. A m . Ckenz. Soc., 84 (1962)2452-2453; J. Strating, J. H . Keijer, E. Molenaar and L. Brandsuma,Aiigew. Chem., 74 (1962) 465. (61) H. Paul, Bull. Soc. C h i m . Fr. (1934) 1397-1405. (62) H. Paul, C. R. Accid. Sci., 218 (1944) 122-124. (63) 0. Riobe, Ann. Chini. (Pnri.r.),4 (1949) 593-620.

geiiotetrahydropyraii (169, X = 131- or CI). 0 1 1 treatiiientW2J~’ of coinpound 169 (X = C1) with an dcoliol i i i the presence of t i base, the chlorine atom on C-2 is readily rc~iilacecl131. ;in alkosyl group, to give 170 (X = Cl). Compound 170 (X = Hr) inwy lie o l i t a i ~ i e c lb~y~bromina~(~~ tion of 167 in an alcohol; its coiivcrsion into 2-alkoxv-5,Ci-diliydro-2Elpyran is described in Section 111, 2. Alkylsdfenyl chlorides readily add to 167, affording 3-(dkj-lt l r i o ) - 2 - c l i I o r o t e t r ~ ~ l i y ~ l r o ~( 171), ~~~r~~iis which may be Lvith sodiuin methoside into ci.r,tr-crri.v-3(alkyltliio)tetrahydro-2-metlroxypyr~~iis (172) in good yields. Hydroxylation of 167 with the Milas reagent tetrahytll-op?iraii2,3-diol (173, R = H) along witli s o m e other products from which a “disaccharide” (174), of uiiknowii stereochemistry, was isolated.

e

OR

x X

QX

SR

X

169

170

OH

171 X = C1 172 X = OR

173

0

HO 174

OH

175

Oxidation of 167 with perosy acids i n the presence of alcohols led7] to tmn.s-2-alkoxytetrahydropyr;in-3-o1 (173) in 65-80% y i e l d . Further oxidation of 173 (with dimethyl sulfoside) g a ~ e ~ ’ -the ~ ’ 3keto compound 175 (see also, Kef. 75). (64) 0. Riobe, Bull. SOC.Chini. F I - . (1951) 829-832. (65) F. Sweet arid R. K. Brown, C U J /. J . C / I ( , ? J46 I . , (1968) 707-713. (66) G . F. Woods and H. Sanders,/. : \ I J J , C h e i t i . Soc., 68 (1946) 2483-2485. (67) H. U. Leiiiieux and B. Fraser-Heitl, C t r ~ iJ. . C h e m . , 43 (1965) 1458- 1473. (68) hl. J. Baldwin and R. K. Browir, C C I I.II.. C l i u n . , 45 (1967) 1195-1200. (69) 1 4 . J. Baldwin and R. K. Browii, ( , ’ o J L . ./. C ~ C J J46 I I (1968) ., 1093- 1099. (70) C . D. Hurd and C . 11. Kelso,J. , \ ) I I . C / I W J SI O. C . , 70 (1948) 1484- 1486. (71) F. Sweet and R. K. Brown, C U , I . /C. h c ~ t i i . ,44 (1966) 1571-1.576. (72) M.Anker, D. Desconrs, and 1-1. I’aclit.co, C:. H. Accid. Sci. Ser. C, 277 (1973) 215217. (73) D. Descours, D. Anker, H. Put.heco, hl. Chareire, and C . Carret, E u r . / . .!led. Chem. Chitn. Tlzer., 12 (1977) :31:3-316. (74) A. Saroli, D. Descours, D. Arikt.1, H . Pnclieco, and M . Charelre, /. Hrtcroc!yc/. C h w i . , 15 (1978) 765-768.

32

A L E K S A N D E R ZAMOJSKI e t (11

A substituted dihydropyran, 3,4-dihydro-2-(hydroxymethyl)-2Hpyran (176), was eniployed b y R. K. Brown and coworker^^"^'^ as the substrate in a total synthesis of hexoses (see Section 111,2). The first steps in this method consisted in ( 1 ) intramolecular addition of the hydroxyl group to the double bond, to afford the bicyclic compound 177, and (2) broniiiiation of 177 to the nionobronio derivative 178.

176

177 X = H 170 X = Br

2-Ethoxy-3,4-dihydro-6-niethyl-W-pyran (179) has been used78 as the substrate for the synthesis of inethyl 2,3,6-trideoxy-a-~~-erytlzrohex-2-enopyranoside (180). The synthesis consisted in hydroboration of the double bond, followed b y oxidation, bromination at C-2, and elimination of HBr, to give 180. This compound was, in turn, converted into methyl ~ ~ - m y c a i n i i i o s i d~e~, ~ - o~l e a n d r o s i dand e , ~ other ~ sugar glycosides (see Section III,2).

C H,

180

Br

(75) J. E. Roffand R. K. Brown, Cut1.J. Cheni., 51 (1973) 3354-3356. (76) F. Sweet and R. K. Brown, Curl. J . Cheiti., 46 (1968) 2289-2298. (77) T. P. Murray, C. S.Williains, ant1 R. K. Brown,]. Org. Chem., 36 (1971) 1311-1314. (78) S. Yasuda and T. Matsuinoto, Tetrcrliedron L e t f . (1969) 4397-4400. (79) S. Yasuda and T. Matsunioto, 'f'etrtrhedroti Lett. (1969) 4393-4396.

SUGARS FROM NON-(:ARHOHYIIRATE SUBSTRATES

33

Hydroboration was also einployedxOin the synthesis of methyl 2,3d i d e o x y - 4 - O - m e t h y ~ - a - D ~ - ~ ~ ~ ~ ~ ~ ~ , - o - l ~ e n(t182) o p y rfrom a n o ~3,4-diide hydro-2-methoxy-W-pyran (181). Catalytic denlcoholation of 182 nfforded 3,4-dihydro-3-methoxy-2H-pyran (183), which, on bromination in methanol, gaveX" methyl 2-bromo-2,3-dideoxy-4-O-methyl-cr-~~cr!jthro-pentopyranoside (184) a s the main product. 1 I3& 2 liLOz OH3 NaH. Me1

$OM.

6~ OMe

Me0

181

182

Br 183

184

Similar, first steps were ~ i s e d i~n "the ~ synthesi\ of 3,6-dideoxy-DLurubino- and -ribo-hexopyranoses ( 184c and 184d) from 3,4-dihyclro-6niethyl-2-(2-methylpropoxy)-2fl-pyran (184a). Hydroboratiori of the double bond in 184a, followed l)y acetylation and dealcoholation gave glycal 184b; reaction of 184b with perouybenzoic and acid in ether saturated with water afforded 184c and 184d.

184a

1841,

(80) R. M. Srivastava and R. K. B r o w t ~ Cntt. , J . Cliem., 48 (1970) 2334-2340. (80a) G. Berti, G. Catelani, S . Magi, and I-.. Monti, Gosz. Clzim. l t u l . , 110 (1980) 173178.

34

ALEKSANDER ZAMOJSKI et

(11.

A highly stereoselective synthesis of methyl 2,4-diamino-2,3,4,6tetradeoxy-a-DL-arubilto-hexopyranoside (187, methyl DL-kasugaminide) from 3,4-dihydro-6-methyl-2H-pyran-2-one (185) was performed.81,x2 Glycoside 187 was also synthesized in another way, using Me

185

b

O

H

f -

A

c

H

N

b

O

-

&(

I

HCNH, ( p

I HYOH

AcHN

AcHN

CO,H

+(ooj

AcHN

AcHN

NO

NO

186

CH"

LN

HO

I

187

OH

188

(81) Y. Suhara, F. Sasaki, K. Maeda, H. Umezawa, and M. Ohno,./. Am. Chem. Soc., 90 (1968) 6559-6560. (82) Y. Suhara, F. Sasaki, G. Koyama, K. Maeda, H. Umezawa, and M. Ohno,./. Am. Chem. Soc., 94 (1972)6501-6507.

S U G A R S FROM N O N - C A R B O H Y D R A T E S U B S T R A T E S

3Ei

179 a s the substrate. (For resoliitioii of 187 into its eiiantioiiiers, see Section VII and Ref. 323.) Intcrnic~diatecornpound 186 w a s treated with different alcohols; reaction with 1D- 1,2:S,6-(li-O-isopropyl idenechir-o-inositol, followed by rvcliiction, and removnl of tlie protecting groups, led to kasuganobios~~iirii~~ 188 in low yield. A more-efficient synthesis of the LL-enantiomer 188 wits perfonlied 1)y l>avid and his coworkers (see Section VII). The s>mtliesis of both of the stereoisoiiieric methyl 2 , 6 - b i s ( a c e t a 1 i i i t ~ o ) - ~ , : 3 , 4 , 6 - t e t r ~ ~ c ~ ~ ~ o x y - ~ - ~ ~ - l i e x o sides [acetylated methyl DL-l)iirpiirosaiiiinide C (189) and methyl DL-epi -purpurosaminide C (190)] from 3,4-dihydro-2-(hydroxyinethyl)-2H-pyran (176) was ;ic~coiii~lished~" in 10 steps.

2. 1. Ac,O NOCl 176

3 . MeOH

GoMe ooM steps

*

t

NOH

R2

189 R' 190 R'

= =

H. R2 = NHAc NHAc, R 2 = H

2. 5,6-I)ihydro-2€l-pyran S,6-Dihydro-UI-pyran (168) has not vet foiind any application in sugar synthesis. However, its 2-allioxy derivatives (such a s 193 and 196) are, as will be shown i n this Sr.ction, usefiil su1)strates in tlie total synthesis of pentoses, hexoses, ant1 m a n y other sugars. In Gact, a general method of carbohydratc synthesis is Iiased on derivatives of 2-alkoxy-5,6-dihydro-2H-pyr~i11. Iiitlividual stages of the synthesis are described in Sections 111, 2, a-(1.

a. Synthesis of 2-Alkoxy-5,6-dihydro-W-pyrans.- Four routes to 2alkoxy-5,6-diliydro-~-pyr~~ ha\~e i i s been descri1)ed in the literature. (i) From 2-Alkoxy-3-bromotetrahydropyrans (192).-Sul)strates 192 are obtained either by reactioir of I)romine with 3,4-~liliydro-~-p),raii (167) in an alcohol (see Section 111, l), or by direct bromination of 2-alkoxytetrahydropyraiis (191).Elimination of'the elements of hyclrogen bromide from 192 occurs readily on treatment with bases. This method was introduced b y Woods and Saiidersti6 and was iised b y Sweet and for the preparation of 6,8-dioxabicyclo[3.2.1]oct-3eiie (194), a key substrate in their synthesis of racemic hexoses. Also, (83) J. S. Brimacornlie, I . Da'Ahoul, and L. (:. N. Tucker, .I.C h e m . SOC.,Per-kit1 ?'r-UJl.\. I , (1974) 263-267.

A L E K S A N D E H ZAMOJSKI e t n l

36

Br

191

192

193

194

other 2-alkoxy-5,6-dihydro-W-pyrans have been ~ b t a i n e d " ~ in~this ~,~~ way. The mechanism of a-halogenation of acetals has been discussed.77

(ii) From 5-Substituted Acetals of 5-Hydroxy-2-pentyn-1-a1(195).Hydrogenation of the triple bond in 195 to a cis double-bond, followed by acid-catalyzed cyclization of the alkene, leads to 6-substituted 2-alkoxy-5,6-dihydro-2H-pyrans (196).This method was used by H. NewmanX6for the preparation of 2-ethoxy-5,6-dihydro-6-methyl2H-pyran (196, R' = Et, R = Me). R

195

196

(iii) By Diels- Alder Condensation of l-Alkoxy-1,3-butadienes with Compounds Containing an Activated Carbonyl Group.-This route has been very thoroughly studied. As dienophiles, f ~ r m a l d e h y d e , ~ ~ esters of glyoxylic (oxoethanoic) and iiiesoxalic (oxopropanedioic) a ~ i d s ,chloral,Xs ~ * ~ ~ ~and alloxan were Besides 197 (R = Me, Et, (84) F. Sweet and H. K. Brown, Cuti. J. C h e m . , 46 (1968) 2283-2288. (85) R. M .Srivastava and R. K. Brown, Cair. J . Cheni., 48 (1970) 2341-2344. (86) H. Newman,J. Org. Clzem., 29 (1964) 1461-1468. (87) D. G . Kubler,J. O r g . Chetri., 27 (1962) 1435. (88) A . Konowd, J . Jurczak, and A. Zarnojski, Rocz. Chern., 42 (1968) 2045-2059. (89) 0. A. Shavryyina and S. M. Makin, Z h . Org, Kkirn., 2 (1966) 1354-1357. (90) V. B. Mochalin, J. N . Porshnev, A . N. Vnlfson, and C . I. Saniokhvalov, Z h . Org, K l z i m . , 4 (1968) 16- 18.

SUGARS FROXI NON-(:41iHOHY I j R A T E SUBSTRATES R1

FP= O

+

A

OR

37

R’ R‘ I I C=CHR3

EtO-CHZC-

202 R‘, R’. R3 = H or CH, 197

193 R’ = R? H 198 R’ = C0,R2. R2 = H 199 R’ = R? = CO,R Rl ccl,, R 2 ~

201 R’.

R2 =

0,C -N,H C CO \ / OC -NH

MeOCONH-CH=CII-CH=CHR 203 R

=

M e or P h

Pr, or B U ) , alkyl-substituted ~~ l-alkoxy-1,3-butadienes (for example, 202) were also Condensation with truns-dienes occurs readily; sometimes, an exothermic effect is observed.y2 Overall yields are in the range of 70100%. Cyclo-additions are regioselective, and affordYLmixtures of c i s and trans adducts if R1 # R2. Usually, cis adducts are preponderantgz (see, however, Ref. 353). Equilibration of products with Lewis acids (ZnCl,, BF,, p-MeC,H,SO,II) occurs readily, and leads to mixtures containing > 90% of the trans isomers.“’-” cis-l-Alkoxy-1,3-butadienes polymerize under cyclo-addition conditions. However, with diethyl mesoxalate, a highly reactive dienophile, they can enters5 into cyclo-addition to afford 199 (14’ = Et). From 199, esters (199) have been obtained by way of d e c a r l ~ o a l k o x y l a t i ~ n . ~ ~ Attempts were made in order to obtain adducts 198 i n enantionieric form b y cyclo-addition of l-alkox~-1,3-butadienesto optically active esters of glyoxylic acids6; the enantiomeric purities of the adducts were, however, Optically active butyl 2-alkoxy-5,6-dihydroW-pyran-6-carboxylates (198, R’ = Bu) were obtained when the R group in 197 was a carbohydrate moiety. The diastereoisomers resulting from cyclo-addition were separated by chromatography (see Section VII and Ref. 350). Cyclo-addition has been extended to 4-substituted 1-(methoxycar(91) 0. A. Shavrygina, S. D. Jablorro\.ckayu, and S. M . Makin, Z h . Org. K h i n i . , 5 (1969) 775-778. (92) A. Zamojski, A . Konowax, and J . Jurczak, Rocz. Clzetri., 44 (1970) 1981- 1986. (93) S. D. Jablonovskaya, N . M . Shechtni;ur, N . D. Antonova, S. W. Bogatkw, S. hl. Makin, and N . S. Zefirov,Zh. Of-g.K h i v i . , 6 (1970) 871-877. (94) V. B. Mochalin, J. N . Porshriev, a r i d G. I. Samokhvalov, Z h . Ohshch. K h i m . , 39 (1969) 109- 112. (95) S. David, J . Eustache, and A. Ltibiiieati, /. Cheni. Soc., Perkin Trciiis. 1 (1979) 1795- 1798. (96) J. Jurczak and A. Zamojski, ?‘c,tr-ci/icxfr.cm, 28 (1972) 1.505- 1516.

ALEKSANDEH ZAMOJSKI et d.

38

bonylaniino)-1,3-butadieiie~ (203), which were successfully condenseds7 with formaldehyde or ethyl glyoxylate. Substituted 5,6-diliydro-Zf-thiopyrans 204 and 205 were obt a i ~ i e dby ~ ~condensation ,~~ of l-niethoxy-l,3-hutadieneor 1,4-diacetoxy-1,3-butadiene with methyl cyanodithiofoiiiiate; this reaction opened an approach to the total synthesis of sulfur-containing monosaccharides. R'

R'

I

I

OR?

R 2 0

204 R' = H. R2 = M e 205 R' = OAc. R' Ac

(iv) By Partial Reduction of DL-Parasorbic Acid (107).-Reduction of 107 with diisobutylaluniinuiii hydride leads to 5,6-dihydro-2-hydroxy-6-methyl-2H-pyran (207, R' = CH,, R2 = H), which can be acetalized with or tho ester^^^ to 196 (R = CH,, R1 = Me, Et, i-Pr).

k & -

H,O H'

P

Hf

OH

_____)

OR

CHO

207

206

b. Fundamental Physical and Chemical Properties of 2-Alkoxy-5,6dihydro-W-pyrans.-The conformation of the 2-alkoxy-5,s-dihydro2H-pyrans 199 and 200 was analyzed on the basis of their 'H-"'" (97) V. B. Mochalin and I. S. Varpakhovskaya, ZIz. Org. K h i m , 12 (1976) 2257-2258. (98) D. M. Vyas and G. W. IIay, Chem. C o m n m n . (1971) 1411-1412. (99) D. M. Vyas and G . W. Hay,]. Cheni. Soc., Perkin Trcriis. 1 (1975) 180-186. (100) 0. Achinatowicz, Jr., J. Jurczak, A. KonowaY,and A. Zamojski, Org. A4agn. Reson., 2 (1970) 55-62.

and lT-n.m.r."'lspectral data. Tlie e.i.1n.s. fragment;ition patteriis of 198 and 199 were also deterniined."'"1'''3 2-Alkoxy-5,6-dil1ydro-2H-p~~i~ans are readily li~~clrolyzed"X~X9 in di1u te , aqueous acids to t r(in s -a$- 11 11sat 11 rat e d alcle11 y de s ( 206). H y d rol ysis in a neutral medium leads to a i r eq~iilibri~im mixture of 5,6-tlihyd r o - ~ - p y r a n - 2 - 0 1(207) and tlic, corresponding cis-aldehyde 208. (For syntheses of stereoisomeric cteoxy-DL-aldoses from aldehyde 79, see Section 11.)The thermal isomerization of 193 h a s heen investigated."'.' The alkoxyl group in 193 ancl 198, being a part of the acetal and allylic systems, may be readily clisplaccd b y another OR, o r N€i1R2,group in an acid-catalyzedg2 or therin;i11"5(13O-2Oo") reaction i n the presence of an appropriate alcohol o r arnine. The acid-catalyzed reaction of213,214, and analogous coniporiiids with thiols leads"'" to l-thioglycosides (209), which are often accoinpanied by 3-S -alkyl-1,G-anhydro2,4-dideoxy-3-thio-D~-er!itli,-o -dd-1-enitols (210). The ester group in 198 was converted into a varic,ty of derived groups, thus fiirnisliing a series of substrates (211-217) which were further exploited i n carliohydrate synthesis. R'

R'

209

R' = CONH,, CH,NHAc

R2 :M e , C,H,3

RZS 210

211 R' CH,OH(Ref 139) 212 R' CH, ( R e f . 123) 213 R' = CONH,(Refs 94 and 121) ~

214

R'

215 R'

= CH,NH, ~

C l i (Ref. 121)

216 R' = COCHR*CO,R'(Ref 120) 217 R' COCH,R-'(Rel. 120) ~

The double bond in 193, 198, and 199 exhibit5 it5 normal properties; a variety of electrophilic. reagents may readily be ndded, for ex(101) 0.Achmatowicz, Jr., M . Chriiit.lewski, J. Jurczak, a n d L. Kozerski, H o c z . Cheni., 48 (1974) 481-490. (102) A. Zamojski, A. KonowaX, J . J i i w z i i k , and K. Jankowski, Hocz. ( : h e m . , 43 (1969) 1459- 1468. (103) J. Jurczak, A. Konowd, and A . Zamojski, Rocz. C/iem., 43 (1969) 2095-2101. (104) M. Lissac-Cahu arid G . Descotes, C . R. Acad. S c i . , 369 (1969) 1574-1576. (105) V. B. Mochalin, A. N . Koriiilo\., A . N . Vulfson, .ind I. S. Vai-pakhovska).a,K h i m Geterotsikl. Soedin., 2 (1975) 167- 170. (106) W. Priehe and A. Zainojski, 7'c,tr-~i/ic.tlr-ori,36 (1980)387-297; P o l . / . Chc.,ri., 54 (1980)731-739.

ALEKSANDEH ZAMOJSKI rt

40

(I/.

ample, hydrogen chloride,lo7 hypochlorous broinine,H~~loX-lln ethanesulfenyl and others."' The stereochemistry of the Base-catalyzed deproducts obtained has been deter~iiiiied.~~,'"~,~~'~.~~~ hydrobromination of 2-alkoxy-3,4-dibroinotetrahydropyrans has been studied.1119.111,11:3 Also, nucleophilic reagents, alcohols, and acetic acid may be added across the double boric1 under acid catalysis. Compounds 193 and 198 r e a ~ t l ' ~ Jwith l ~ methanol containing hydrogen chloride, to afford stereoisomeric tetrahydro-2,4-dimethoxypyrans (218 and 219, respectively). In this way, methyl a-DL-oleandroside (221) and methyl WDLcymaroside (222) were o b t a i ~ i e d from ~ ~ J methyl ~~ 4-0-benzyl-2,3,6-trideoxy-a-~~-erythro-hex-2-eiiopyranoside (220).

MeO'

220

1

210 R = n 219 R = CO,Me

1. MeOH, Hi 2. H,. Pd/C

Me0 221

222

Addition of acetic acid to 6-(acetoxymethyl)-5,6-dihydro-2-niethoxy2H-pyran (223) gave117.11xthree stereoisomeric 2,4-diacetoxy-6-(ace(107) hf. Cahu and G. Descotes, Bull. Soc. Cliini. FI-. (1968) 2975-2978. (108) R. M. Srivastava, F. Sweet, T. P. Murray, and R. K. Brown, ]. Org. Chem., 36 (1971) 3633-3636. (109) G. F. Woods and S. C. Teniin,]. Ani. Cherti. Soc., 72 (1950) 139-143. (110) M. Chmielewski and A. Zamojski, Roct. Cheni., 45 (1971) 1689-1700. (111) M. J. Baldwin and R. K. Brown, Can. J . Claetn., 47 (1969) 3553-3556. (112) hl. J. Baldwin and R. K. Brown, Coti. ]. Chem., 47 (1969)3099-3106. (113) R. M. Srivastava, F. Sweet, and R. K. Brown,]. Org. Chem., 37 (1972) 190-195. (114) F. Sweet and R. K. Brown, C a n . J . Cherti., 46 (1968) 1543-1548. (115) A. Zamojski, M . Chmielewski, and A. Konowd, Tetrahedron, 26 (1970) 183- 189. (116) S. Yasuda and T. Matsumoto, Tetrahedron, 29 (1973)4087-4092. (117) M. Chmielewski, J. Jurczak, and A. Zamojski, Rocz. Chem., 46 (1972) 627-632. (118) M . Chmielewski, J. Jurczak, and A. Zamojski, Pol. /. Chetn., 52 (1978) 743-749.

SUGARS FROM NON-('.\HHOI-IYI)RATE SLJHSTKATES

41

toxymethy1)tetrahydropyrans (224).Compounds 218,219, and 224 are derivatives of a little-known class of monosaccharides, 2,4-dideoxypentoses and -hexoses. CH,OAc

Ac

223

0' 224

c. Synthesis of 4-Deoxy Sugars.-The most direct approach to sugars from 2-alkoxy-5,6-dihytlro-2H-pyran and its derivatives involves introduction of suitallle substituents at the double bond, leading to alkyl 4-deoxyaldopyraiiosides.A survey of the syntheses perfonned will start with the hydroxylation reaction; next, epoxidation will be discussed; and finally, oxirane ring-opening reactions will be reviewed. (i) cis-Hydroxy1ation.-Introduction of two hydroxyl groups at the double bond of 193, 198, and some other derivatives occurs without difficulty with such reagents ;is dilute, aqueous potassium pemianganate,107.122 hydrogen peroxide in trr-t-butanol containing a catalytic amount of osmium tetraoxide (the Milas reagent),'2:j,124 arid osmiutn tetraoxide.12s,126 The yields of ci,r.-hydroxylated products are in the range of 25-70% with the first two reagents, and 80-100% with the last. Usually, both hydroxyl groups enter trcitis to the 2-alkoxyl group ofthe substrate (for example, 225 and 211, R = Me), yielding products 226 R'

R' I

225 R ' = H,

RZ = El

211 R' = CH,OH. RZ = M e

226 R' = H , RZ = Et 227 R' = CH,OH. R2 = M e

(119) J. Jurczak, A. Konowd, and A . Zainojrki, Hocz. Chcwi., 44 (1970) 1587-1590. (120) A . Konowax, K. Belniak, J. Jnrcmk, M . Clrnrielewski, 0. Achniatowicz, J r . . and A. Zamojski, Rocz. Chern., 50 (1976) 505-514. (121) K. Belniak and A. Zainojski, Rorz. Chein,, 51 (1977) 1545-1547. and A. N . Vulfbon,Zli. O r g . (122). V. €3. Mochalin, A. N . Komilov, I . S . V~irp~~khovskaya, Khim., 12 (1976) 58-63. (123) A. Banaszek and A. Zarnojski, H(Jc.z.C / w r t t , , 45 (1971) 391-403. (124) A. Konowd and A. Zamojski, U O C Z .( : h i t i , , 45 (1971) 859-868. (125) R. M. Srivastava and R. K. Browti, Ctrri. J. Cheni., 49 (1971) 1339-1342. (126) T. P. Murray, U . P. Singh, nnd I<. K. IIrown,Can.J. C h m i , , 49 (1971) 2132-2138.

ALEKSANDER ZAMOJSKI c t

42

(11

and 227, respectively having the p-erythro and a-lyxo configuratioii.1n6aThus, synthesis of methyl 4-deoxy-a-~~-l!/xo-hexopyranoside may be achieved in oiie step, starting from the readily available substrate 211. This m a y be contrasted with the synthesis of the II enantiomer of 227 from D-galactose, which r e q i ~ i i - e d ~ six ' ~ steps. cis-Hydroxylation of the dihydropyrans 204 and 209 was effec.ted106J2x exclusively b y use of crsrniuin tetraoxide, and afforded sulfur-containing sugars in good yield.

(ii) Epoxidation.- Alkyl2,3-anhydro-4-deoxy-~~-pentoand -hexopyranosides may be obtained from such unsaturated precursors as 193 and 198 (and many others) by epoxidation with peroxy acids,"".7", R-1~X8,90,1W,107,123,129-1.1X or with a mixture of 30% hydrogen peroxide and benzo- or aceto-nitrile.':'7,139,'"1 The other (less popular) method consists in base-catalyzed c1ehydrol.lalogenatioii of alkyl 3,4-dideoxy-3halogeno-DL-ttzreo-pento- (or -trrcihino- or -xyEo-hexo)-pyranosides.1"7, 130,137

Epoxidation of 2 - a l k o x y - 5 , 6 - d i l i y d r o - ~ - ~ ~ (193) y ~ ~ i i with peroxy acids leads to both possible epoxides, with the P-erythro stereoisomer preponderating.65

(126a) Cahu and D e ~ c o t e s ' ~described ' a v e r y sinlilar hydroxylation of225 and wrongly assigned the a-erythro configuration to the product (obtained in 30% yield). (127) M . &mi$ and J. Pacik, Collect. Czrcli. Claem. Cornrnun., 27 (1962) 94-104: 11. &my, J. Stanek, Jr., and J. Paciik, iM., 34 (1969) 1750-1765. (128) D. M. Vyas and G . W. Hay, Con. J. Chenc., 53 (1975) 1362-1366. (129) R. M . Srivastava and R . K. Brown, Can. J. Chem., 48 (1970) 830-837. (130) V. B. Mochalin, I. N. Porshnev, and G . I. Sainokhvalov, 211.Ohshch. Khini., 38 (1968) 85-90. (131) V. B. Mochalin, I. N. Porshnev, and G . I. Saniokhvalov, Z h . Ohshch. Khini., 38 (1968) 427-428. (132) V. B. Mochalin, I. N. Porshnev, G. I. Sainokhvalov, and M. C. Janotovski, Zh. Olishch. Khirrc,, 39 (1969) 116-119. (133) V. B. Mochalin, I. N. Porshnev, and G. I. Samokhvalov, Zli. Obsltcli. Klaim., 39 (1969) 681-684. (134) V. B. Mochalin, I. N. Porshnev, and G. I. Saniokhvalov, Zh. Ohslaclt. Khint., 39 (1969) 420-424. (135) V. B. Mochalin, I. N. I'orshnev, and G. I. Samokhvalov, 2 1 2 . Ohshch. Khirn., 39 (1969) 701-706. (136) A. K o n o w d A. Zaiiiojski, 51. Masojitlkova, and J. Kohoutova, Hocz. Cheni., 44 (1970) 1741-1750. (137) A. Banaszek and A. Zaniojski, Rocz. C h e n ~ .45 , (1971) 2089-2095. (138) A. Konowd, 0. Achmatowicz, Jr., and A. Zamojski, Rocz. C h e m . , 50 (1976) 879893. (139) M. Chmielewski and A. Zainojski, Rocz. Chertl., 46 (1972) 1767-1776. (140) J. Mieczkowski and A. Zamojski, Car-6ohyclr.Res., 55 (1977) 177-192.

193

R R

~

Me Me,C

228 (25 1 230 ( - 1 0 )

229 (75 1 231 ( - q O

)

The stereocheiiiical outcoirics of tapoxictatioii of 6-sul)stitutcd 2-idkoxy-5,6-dil?ydro-2H-pyrans witlr peroxy acids is ckpendeiit o i i the con fi gura ti oii and o 11 t h e type ( ) 1' s 111 s t i t 1ie i i t s i ii t h e s I i b s t ra t e ; fro 11i either isomer, t h e ci.9 o r t m t i , ~or , ( u s n a l l y ) Iioth. stereoisoiueric t'poxitle(s) a r e formed. I n t h e c;isc' of' tliv t r t / t L , $ isonicr, tlie a-l!/ro epoxide (232)preponderates over the tu-rilio coiiipouiid ( 2 3 3 ) .Froiii t h e ri.v iso-

mer, t h e p-riho epoxide (234) is iii;iiiily foi-iiie(I."~'~ These results m a y be inteipreted in terms of stcric, Iiiirclrance exerted 1)y t h e O R g r o i ~ p . ~ . ~ ~ I n contrast, epoxidation of 2 - a l k o x ) - 5 , 6 - d i h y d r o - ~ ~ - p y r a iwith i s ;i iiiixture of hydrogen peroxide and I)eiizonitrile leads mainly to products containing an oxirane ring a i i t l a i r OH group 011 t h e same side of t h e six-niembered ring. I t m a y Iw iissiiiiiecl that transient liycll-ogen-l,oii[ling between the peroxyiiiiiiiol~ciixt,icacid and t h e OR group (236) is responsible for this result.

236

The total yields from epoxitlatioii are in t h e I-ange of 3S-90% . The rate o f t h e reaction with pei-o\!' acids is rather low: one to seven days are usually necessary for coiiiplc'tioii of epositte form. t 1' 0 1 1 . Cl

(141) G . Bert], 7'011. SIereochenl.. 7 (1973) 9:3-251.

44

ALEKSANDEH ZAMOJSKI rf crl

The configuration of individual alkyl 2,3-anhydro-4-deoxyaldopyranosides was determined on the basis of their 'H-n.m.r. spectra,'", 1 4 2 ~ 1 2 Ror, even niore readily, their 1:3C-n.n~.r. data.144There is uncertainty regarding the configuration ofthe epoxides obtained b y Mochalin and coworker^.^^,^:^^^^^"^ These authors erroneously ascribed the cis configuration to the more stable isomer of 6-substituted 2-alkoxy-5,6dihydro-2H-pyrans. Therefore, the epoxides described must have a configuration different from that assigned. Although a correction of the configuration of the stereoisomeric 2-(benzyloxy)-5,6-dihydro-6methyl-W-pyran was later made,105a reconsideration of the epoxidation results has not yet been published. (iii) Ring-opening Reactions of Epoxides.- Stereoisoineric 4deoxy-DL-pento- or -hexo-pyranosides having truns-oriented substituents at C-2 and C-3 may be olltained from alkyl 2,3-anhydro4deoxyaldopyranosides b y oxirane ring-opening with nucleophilic reagents. Among the reagents einployed were ~ a t e r , ' " . ' " ~ .metha'~~ llol ,4x. 123,129.145 amiIle ,AH. 12:3.130.1:11,1:38- 110.14B- 148 am in on i a,14!' hydrochloric acid, I o i and lithium a1u in inum 11y dride .Ii5,26, 15" The results of epoxitle-ring opening in alkyl 2,3-anhydro-4-deoxyDL-aldopyranosides are dependent on the configuration of the substrate and the reaction conditions. The type of substitution at C-5 appears to be of minor importance. The results achieved may be sui~imarized a s follows.12"( ( 1 ) Epoxides ofthe 0-erytlzr-o (232, R1 = H) or aand 0-lyro (232 and 235, R' = CH3, CH,OH) configuration are opened i n acidic and basic media at C - 3 , to afford alkyl4-deoxy-~~-aldopyranosides (237) of the a-tlireo or a- and 0-urubino configuration. ( b ) aerythro Epoxides (233, R1 = H) in acidic and basic media are also opened at C-3, furnishing alkyl4-deoxy-~-DL-tlzreo-pentopyranosides (238). Alkyl 2,3-anhydro-4-deoxy-a- and -P-DL-riho-hexopyranosides (142) D. H. Buss, L. Hough, L. D. Hall, a i i t l J . F. Illaiiville, Tetr-cihedroii,21 (1965)6974. (143) F. Sweet and R. K. Brown, Ctrri. J . Chc.rri., 46 (1968) 1481- 1486. (144) M. Chmielewski, J. Mieczkowski, \Y. Prielx, A. Zaniojski, and H. .4damowicz, Tetmhedroii, 34 (1978) 3325-33:30. (145) F. Sweet arid R. K. Brown, C~iri. J . Cherii., 46 (1968) 1592- 1594. (146) A. Banaszek a n d A. Zainojski, C:cir-boh!ytlr. Re.)..,25 (1972) 453-463. (147) J. Mieczkowski a r i d A . 'Zxnojski, H i i l l . Actrd. P O / . Sci., Ser. Sci. (:him., 23 (1975) 581- 583. (148) D. Descours, D. Anker, J. Solland, J. Legheaud, H . Pacheco, and M. Chareire, Eur-. J . .!fed., Cheni., CJiiiii. Tlier., 14 (1979) 67-76. (149) L). Descours, D. Anker, and H. Pacheco, C . H . Accid. Sci. (1976) 691-693. (150) M. Chmielewski, A. Koiiowd, and .4. Zainojski, Cnrhoh!ydr. H e y . , 70 (1979) 275282.

react differently in acidic and I)iisic inedia. I n acids, opening occurs exclusively at C-3, leading to cottrpou~ids(239)having tlic scjlo configuration. In basic media, openitig takes place at Imth C-3 (sr~locoiiipounds, 239) a ~ i dC-2 (urabiiio coiirpounds, 240).

Nu

OH

MU

237

238

239

NU: OH. OR. o r NH,

I10

240

A single, rather understandable exception to these rules was foiiiid opening of 1,6:2,3-dianliydri~-4-deoxy-~-~~-]!l.ose (241)b y dimethylamine, which led151to lioth possible dimethylatiiiiiodeoxy derivatives 242 and 243. I t is evident that, here, nncleophilic in

attack at C-2 allows the f:,lvoretl diaxial opening of the epoxitle. The oxirane-ring opening reactioiis of alkyl 2,3-a1ihydro-4-deoxy-aldopyranosides discussed here closel]. resemble analogous reactions of alkyl 2,3-anhydro sugars.'S2These reactions provide simple syntheses of a variety of alkyl 4-deoxy-o~-s!ylo-and -cirtibiiio-hexopyr~in~~sicles, (151) K. Hanganayakiilu, U. P. Singh, T. P. hlrlrray, and R. K . Brown, C o t i . J .C h c t r i . , 52 ( 1974) 988 - 99 1 . (152) J. G. Buchanaii and H. Z. Sal)lv, i i i H. S. Thy;igaraj,iii (Ed.), Sc/Pcticr Oraciiiic Trunsfor,nutiotis, Vol. 2, \.l;ilc.y~IiiteIsciellce, N e w York, 1972, pp. 1-95,

ALEKSANDEH ZAMOJSKI et

46

(I/.

among them, methyl ~ ~ - d e s o s a i i i i i i i d e ~(244), ’ . ’ ~ ~ methyl DL-chalcoSide48,iz:3 (245), and ezoaininouroic acid methyl g l y c o ~ i d e ”(246), ~ inonosaccharides whose D forms are components of aiitil,iotic~.’”,~~~

OH

OH 246

244 R = N M e , 245 R - O M e

The majority of the total syntheses of desosarnine described in the literature have been based on the same reaction-sequence, namely, 2alkoxy-5,6-dihydro-6-methyl-W-pyran (247) was epoxidized and the epoxide (248) was then opened with aqueous diiiiethylainine to afford a mixture of the dimethylainino sugars 249. Interestingly, despite the simple reaction-pathway, all syntheses performed between 1962 and 1969 met with only limited success. Korte and coworkerstssand H. NewmanX6did not separate their mixtures of stereoisomeric alkyl2,3-anhydro-4,6-dideoxy-~~-hexopyranosides (248) in which the undesired a-l!4~0 stereoisomer was certainly the preponderant component. In consequence, raceniic alkyl desosaminide was obtained only as a minor product in admixture with alkyl 3,4,6-trideoxy-3-(dimethyla~~ino)-~~-~~rtibiiio-hexopyraiioside (250),

OOR - OOR 0 ArC0,H

Me,NH

0 247 R

=

M e or Et

248

OR

Me,N

OH 249

Me,N 250

(153) J. D. Dutcher, Aclc. CarhohtJtZr.Chetti., 18 (1963) 281-308. (154) K. Sakata, A. Sakurai, and S. Tamura, Tetruhedron Lett. (1974) 1533- 1536 (155) F. Korte, A. Bilow, and R. Heinz, Tetrc~hedroir,18 (1962) 657-666.

the niaiii product. Only Korte i t i i d were able to isolate a sniall quantity of a material whose i .r. spectrum w a s superposal~leon that of desosamine. In Mochaliii a i r t l coworkers’. synthesis, a mistake i n the configurational assigiiirieiit of 2 - e t h o s y - f j , 6 - d i l i ~ ~ l r o - ~ - 1 i i ~ ~ t l i ~ l 2H-pylan (cis instead of tr-cirm) caiised a series of unfortunate coilelusions. The ethyl 2 , ~ - a i i ~ ~ ~ ~ d r . o - 4 , 6 - d i d e o s ) ~ - D L - h e s o ~ ~ y r ~ ~ 1 i o s obtained was probably of the tw-/!/so configuration (not p-r-ibo iis s u g gested in the paper). The reaction with tlimetliylaniine led niost proba b l y to ethyl 3,4,6-trideox)~-3-(dilnethylanliiiiiii(i)-~-~~-~~r-tr~,irro-hesopyranoside (250, R = Et), instcacl of to ethyl 1)L-desosaiiriuitle. This conclusion is supported b y the 11i.p. repoitetl b y h l o c h d i i i aiid coworkers for the glycositle, naitiely, 48-49”, fairly close to the 1 i i . p . of Newman’s “cirubino” glycosidc, 12.5-45.5”. Ethyl D-desosanlinide is a syrup.X~

(iv) Other Syntheses.-Aiirotig iiiore-complex syntheses of sugars having a 4-deoxy grouping, the preparation of acetylated methyl 4deoxyneosaniinide C (251) m i d piirpurosainine B (252) should be

mentioned. Methyl 4-deoxy-~~-rieosaiilinide C, the D enantiomer of which is a component of lintirosiiis C, and C,, was prepared’stifrom 198 (R’ = CO,Bu, Rz = H, R = M e ) . Purpurosamine B (252),a coiiiponent of gentaniicin C,, lielongs to a new class of arnino sugars, the 2,6-tlianiiiio-2,3,4,Ci-tetradeox?altlo-

48

ALEKSANDER ZAMOJSKI et ul

pyranoses. A synthesis of 252 from 6-acetyl-5,6-dihydro-2-1nethoxyW-pyran is shown.lsfia

J

LINK,

y3 AcHNCH I

d. Alkyl 3,4-Dideoxy-~~-ald-3-enopyranosides.-Although derivatives of 2-alkoxy-5,6-dihydro-W-pyran can be readily converted into a variety of 4-deoxy sugars, their utilization for the synthesis of inonosaccharides having a substituent at C-4 of the pyranose ring is more complicated. Many attempts aimed at introduction of oxygen or halogen atoms b y means of “allylating” reagents, for example, N-bromosuccininiide, 1,3-dichlorohydantoin, selenium dioxide, lead tetraacetate, tert-butyl peroxybenzoate, and oxygen in the presence of cuprous chloride, were u n ~ u c c e ~ ~ f Allylic u l . ’ ~ broinination ~ succeeded for the acetylated, hydrolysis product of compound 198, namely, the trnns -a,P-unsaturated aldehyde 79. Reaction of acetylated 79 with N bromosuccinimide yielded157a mixture of stereoisomeric, allylic bromides (253)in high yield. Difficulties were encountered in attempted recyclization of 253,and therefore this route was not further exploited. C0,R’ COzR’

I

HC=O

RZO&OR’

Rzcv

(AcO),HC

OR2

253

254 R1 = Et, R2 = M e 255 R’ = Bu. R2 = AC

(156a) M. Chmielewski, A. Konowd, and A. Zamojski, Carbohydr. Res., 70 (1979)275-

282.

Another approach to derivatives of 2-alkoxy-5,6-dihydro-2H-pyrnn “oxygenated” at C-5 (that is, at C-4 of the future sugar molecule) was based on application of 1,4-climethoxy- or 1,4-diacetoxy-l,3-1,utadienes for Diels- Alder condelisations with alkyl glyoxylates. 1,4-Diinethoxy-l,3-\~utadieneis highly reactive and affords15Hcycloadduct 254 in high yield. Wider application of the diene i n sugar synthesis is seriously hampered by its difficult availability and low stability. traizs,trui~s-1,4-Diacetoxy-1,3-l~utadieiie is stable, a n d may be readily synthesized from cyclooctatetraeiie. Its condensation with hutyl glyoxylate leads to butyl 1,4-(li-(~-acetyl-2,3-dideoxy-a-DL-c.r!ltlz,-oand -tlzreo-hex-2-enopyranuroiiroii~~tes (255) in the ratio of 1 : 1, and 75% overall yield.ls9 This route certainly deserves further study. Most attention has been devotc>d to the conversion of eposides derived from 2-alkoxy-5,6-dihydro-2H-pyrans into unsaturated all ylic alcohols, that is, alkyl 3,4-dideoxy-u~-ald-3-eiiopyr~iiiosides (256). Ad-

-

O

O

R

2

-

QOR’

OH 256

ditioii reactions to the doulile Iioiid of 256 open a lxoad access to a variety of sugar structures. For the conversion of 2-alkoxy-3,4-epoxytetrahydro~~yrar~s into 256, three methods have been u s e d . (IL) The most direct approach was employed b y Singh and Brown’””. 161 for the conversion of 1,6:2,3-c~ianhydro-4-deoxy-p-~~-riho-~~~xopyranose (257) into 1,6-anhydro-3,4-dideoxy-P-~~-erythro-hex-3-eiiopyranose (258); it consisted in treatiiient of 257 with butyllithium at 0”. The direct isoiiierization of epoxides to allylic alcohols b y means of strong bases is well known in the literature.’“’ Biityllithium-promoted isomerization to the corresponding allylic alcohols also succeeded in the case of 1,6:3,4-dianhytlro-2-deoxy-~-~~-ri60-hexo~yrai~ose’~~ (259) and methyl 2,3-anhytlro-4-t~eoxy-~-~~-l!qxo-hexop).ranoside’~~ (260) (see also, Ref. 163). (157) M . Chmielewski, Pol. J . Chenr., S4 (1980)1913-1921. (158) 0. A. Shavrygina a n d S . M. Makiii, K l i i i t i . Farm. Z h , 3 (1969) 17-20. (159) R. R. Schmidt and R. Anyerlxinrr, Angeru. Chetn., 89 (1977) 822-823. (160) U . P. Singh and R. K. Brown, C ‘ u n . ] , Ckeni., 48 (1970) 1791-1792. (161) U . P. Singh and R. K. Brown, C‘(LII. J . Ckerii., 49 (1971) 3342-3347. (162) A. Rosowskp, in A. Weissberger (Ed.), Heterocyclic Compounds w i t h TlrrccJ-c Four-Menibered R i n g s , Part 1, Interscience, New Yo&, 1964, pp. 1-523. (163) K. Hanganyakulu a n d R. K. Hrowri,]. O r g . C h e m . , 39 (1974) 3941-3943.

~ t d

AL,EKSANDEH ZAMOJSKI e t (11.

50

BuLi ___t

82%

OH 258

257

H,C -

261

&;;4' Q OMe

@

OMe

260

262

For the conversion of 1,6:2,3-dianhydro-4-deoxy- and 1,6:3,4-dianhydro-2-deoxy-/?-~~-lyxo-hexopyranoses(261 and 262) lithium diethylamide was successfully eniployed.15' However, attempts at isomerization of methyl 2,3-anhydro4-deoxy- or 4 , 6 - d i d e o x y - ~ ~ - h e x opyranosides (263; R1 = CH,OH, CH3, R' = Me) with butyllithium failed, because of predominance of secondary reactions (for example, opening of the oxirane ring with B I I L ~ ) The . ' ~ ~yields of desired products were negligible or nil. ( b )Another approach to 256 is based on the following sequence of reactions: (i) opening of the oxirane ring in an alkyl 2,3-anhydro-4(263) with dimethylamine, (ii) oxidation of deoxy-~~-aldopyranoside the alkyl 3,4-dideoxy-3-(dimethylamino)-~~-aldopyrai~oside (264) obtained to the N-oxide (265), and (iii) pyrolytic elimination of N,N-dimethylhydroxylamine (the Cope degradation) to afford the alkyl 3,4dideoxy-~~-ald-3-enopyranoside (256). This method of synthesis was based on an observation made b y Celmer164 and P. H. Jones and Row(164) W. D. Celmer,]. Ant. Chein. Soc., 87 (1965) 1797-1799.

ooR2 0 0 SUGARS FROM NON-CARBOHYDRATE SUBSTRATES

Me2NH

-

OR2

-

OR2

[O]

OH

Me,N

OH

Me,NO

26 4

263

51

265

I

J

130-140'

2 56

ley,165who found that pyrolytic decomposition of glycosides of desosamine N-oxide led to derivatives of 3,4-dideoxy-~-erythro-hex-3-enopyranoside. Further investigations have shown that this degradation is general, and may be applied'"x~1~0,'~6~151~166 to any stereoisomeric alkyl 3,4-dideoxy-3-(dimethylarnino)-~~-aldopyraiioside (264). In Table I, the conditions for the Cope degradation, and the yields of methyl 3,4-unsaturated DL-pento- and -hexo-pyranosides, are collected. The pyrolysis of alkyl 3,4-dideoxy-3-(dimethylamino)aldopyranosideN-oxide (265)is reniarkably regioselective; to date, no prodTABLEI Cope Degradation of Methyl 3,4-Dideoxy-3-(dimethylamino)-~~-aldopyranoside N-Oxides

0

O

Me,NO

R

M

.

OH

Configuration

H CH,OH

a-threo

CH,OH CH,OH CH,OH CO,CMe,

a-arabino

CHE tN HAc

a-altro

ff-Xyl0

e

G

Reaction conditions 130", neat 130- 140"/0.2

O

M

OH

e

Yield References

Configuration

a-glycero a-ery thro

69 67

139 146

a-threo p-ery thro p-threo a-thrco

66 59 64 39

146 146 146 140

a-arabino

59

138

Torr, neat P-do

p-ara bino a-arabino

refluxing xylene soln. refluxing 1,4dioxane soh.

(165) P. H. Jones and E. K. Rowley,,/. Org. Chem., 33 (1968) 665-670. (166) V. B. Mochalin, Z. I. Smolina, and B. W. Unkovskii, Zh. Obshch. Khim., 41 (1971) 1863- 1866.

A1,EKSANDER ZAMOISKI e t (11

52

ucts derived from 2,3-elimination have been detected. The main drawback of the Cope degradation consists in deoxygenation of the N leading to the starting amino sugar. In most cases, the amino sugar can be re-oxidized arid retiiriied to the reaction mixture. ( c )The third route to 3,4-unsaturated sugars is based on Shaiyless and Laiier’s16H method of conversion of epoxides into allylic alcohols. This method consists of opening of the epoxide 263 with selenophenol, oxidation of the product (266) to the selenooxide (267), a i d

PhSeH

263

f

c

\

o

~

2

5O

-\\ri

R

*

-

256

EtOH A

OH

PhSe

O

OH

PhSeO 267

266

thermal deconiposition of 267 to the allylic alcohol 256. This reaction sequence resembles the Cope degradation of N-oxides. The advantage ofthe Sharpless-Lauer sequence lies in the milder reaction-conditions and the possibility of performing the synthesis in a one-flask operation. This method of‘ preparing 3,4-dideoxyhex-3-enopyranosides attached to sugar derivatives (for exaniple 268) was employed by David and coworkers (see Ref‘s. 355 and 356, Section VI).

CH,OH

CH,OH

boRQOR

0

269

+

CH,OH

QoR-

HO

270

CH,OH

OH

QoR 271

OH 272

The yields of stereoisomeric analogs of 268, compounds having the a-D-threo, p-L-threo, and a-L-thr-eo configuration, were 74, 66, and (167) J. ZLivada, M . Pdnkovd, and kl. Svohotla, Collect. Czech. C h e m . Comrnun., 38 (1973) 2102-2120. (168) K. H. Sharpless and R. F. I,auer,,/. ‘4111, Chcrri. Soc., 95 (1973) 2697-2699.

SUGARS FROM N O N C A R B O H Y D R A T E SUBSTRATES

5G

72%, respectively. However, application of tlie Sharpless-Lauer method to the a - r i h epoxitle 269 gave unsatisf:,ictory results; opening of the oxirane ring with PliSeH gave two regioisomeric plienyl selenides, 270 and 271, in similar yields. Oxidation and degradation of the 3-(phenylseleno) compound 271 gave the desired 3,4-unsaturated hexopyranoside 272 in - 11%)y i e l d , m d the re-closed, starting epoxide (for other examples, set: Scactiori VII). In conclusion, each of the tlirec synthetic approaches to 256 has its advantages and disadvantages. Rase-catalyzed isomerization of e p o x ides is certainly the simplest iiretliocl; however, it is not app1ical)le in

all cases. The use of a specific. I)ase, not prone to side reactions, would be tlie best approach to the synthesis. The second method, based o i l thcl Cope degradation, is fully applicable to all stereoisomeric dinlctliylainino sugars, and gives moderate to good yields of unsaturated prodricts. The only disadvantage is the rather lengthy procedure conirected with the necessity of isolation of

each inteiiiiediate product. The third method makes iisc ot‘tlie “one-flask” procediire, which is advantageous from the preparative point of view. However, opening of certain ste re o i someric e pc )x i de s ( 263) with s el e no ph e no 1 s u ffe rs from low regioselectivity, resulting in a low yield o f tlie final prodiict. occasioned b y The other disadvantage is tlie Iiasic. reaction-mecliui~~ the method used for the generutioii of selenophetiol, namely reduction of diphenyl diselenide with sodiuin borohydride it1 solution in anhydrous alcohol (see Ref. 356); some epoxides are sensitive to Ixisic media. However, David (see I k f . 356) did not observe s i d e re.1c.t’ions in h i s syntheses of 256. A simple access to benzyl 2 , : ~ , 4 , 6 ; - t e t l a t l e o x ~ - ~ ~ - 1 , ~ - ~ ~ ~ / ~ ~ ~ r ~ ~ - h

qMe PhCH,OH. ArSeBr

ArSe O O C H 2 P h H 273

2 74

AL.EKSANDEH ZAMOTSKI et rrl

54

opyranoside (276) coiisists169in a two-step synthesis from trunsd-hexenal (273),which reacts in the first step with (p-chloropheiiy1)selenyl bromide aiid benzyl alcohol. A mixture of selenium-containing products, 274 aiid 275, is formed. Under equilibrating conditions, the desired, six-membered product 275 is t h e main isomer (62% yield). Oxidation of 275 with 30% hydrogen peroxide gives 276 in 80% yield.

( i ) Addition Reactions to the Double Bond in Alkyl 3,4--Dideoxy~~-ald-3-enopyranosides.-Discussion of addition reactions of value in the total synthesis of nionosaccliaricles will be liiiiited to cis-hydroxylation and oxymercuratioii-clenierc~ir~~tioii. Epoxidations will lie discussed in Section 111,2cl(ii). cis-13ydroxylation of alkyl 3,4-dideoxy-~~-aldoliex-3-enopyranosides (256) by ineans of such reagents as osmium tetraoxide, the Milas and the Woodward reagents, or potassium perniangaiiate proceeds readily, and affords alkyl DLurubino- or -ribo-hexopyranosides, or analogous hex~pyranosides'~".'~' of the d o , altro, gulucto, or talo configuration. cis-Hydroxylation of 276 to beiizyl 2,6-dideoxy-a-~~-riboand -1yxo-hexopyranosides in the ratio of 2 : 1 and methyl 2 - d e o x y - ~ ~ erythro-pentopyranoside was obtained siiiiilarly from methyl 2,3,4-trideo~y-DL-peiit-3-enopl/ralloside.~~~~'~~

HO

R' = H, CH,OH, or CH, 277

R'

27-6:l

R'

2 78

R'

279

10-3:l

280

(169) S. Current a n d K. B. Sharpless, Tetrahedroll Lett. (1978) 5075-5078. (170) A. Banaszek, Bull. Actid. Pol. Sci., S e r . Sci. Chivt.,20 (1972) 925-933. (171) A. Banaszek, Bull. Acad. Pol. Sci., Ser. Sci. Chint., 23 (1975) 585-592. (172) V. B. Mochalin and A. N. Kornilov, Z h . O h h c h . Khini., 43 (1973) 218-219. (173) V. B. Mochalin a n d A. N . Kornilov, Zh. Obshcli. Khim., 44 (1974) 2334-2337. (174) M. Chmielewski and A. Zaniojski, B u l l . Acud. Pol. Sci., Ser. Sci. Chim., 20 (1972) 751-754.

S U G 4 R S F R O M NON-CARBOHYDRATE S U B S T H I T R S

55

0sin iuni te traox i de-promo t e c I rcsact i o n s are s te ri call y control 1e d ; that is, in all instances, the prc~tlomiiiantformation of products haviiig trans-oriented substituents at C-2 and C-3, and C-2 arid C4, is observed, and, consequently, 277 and 279 preponderate over 278 and 280. However, for 2-0-acet~l-l,Ci-~ti~1iydro-3,4-dideOxy-p-1)~-el-~~~~~ hex-3-enopyranose (281), reiiction with osmium tetraoxide leadsi75to 2-O-acety~-1,6-anhydro-~-~~-dlositlc (282; 88%) and -galactoside (283; 8%).Obviously, the l,Ci-aiih!.dro bridge creates grc,ater steric

OAc

HO

281

OAc 282

OAc

283

11:l

hindrance than the 2-0-acetyl groiip, and this coiiclusioii finds support in other results of cis-hyclroxylation of the sanie skeletoii, described by Singh and Brown.ii5 Woodward hydroxylation of 256 leads mainly to sterically disfavored products having cis-oriented substituents at c - 2 and c-3, and C-2 and C-4. In fact, in this case also, the attack of I' occurs froiii the less-hindered side. The iodoniuin ion (284)is then opened b y the acetate anion, to afford iodo acetate 285 which, in the following substitution reaction with silver acetate, gives the more-hindered, cis-hydroxylation product 286.

Q

OMe

*&[Q

OMe

L S L Ar

Q

OMe

0 Ac

OAc

0 Ac

285

284

/

J

AgOAc

Qe

Ac 0

OAc

AcO 2 86

(175) U. P. Singh and R. K. Brown, Coti. J . (;/w?ti., 49 (1971) 1179-1186.

56

ALEKSANDER ZAMOJSKI e t d .

Application of the oxyiiiercuration-deinercuratioll reaction176to provides17i easy access alkyl 3,4-dideoxy-c~-~~-hex-3-e1iopyranosides to alkyl 3-deoxyhexopyranosides (for example, 288). Interestingly, both stereoisomeric fornls of the alkene are apparently attacked b y mercuric acetate from the same side. It has been assumed17i that the transient, mercurinium ion 287 is stabilized by bonding to the l-methoxyl group.

288

287

R' =

cn,. cn,on

R2 = H or OAc R3 = OAc or H

(ii) Epoxidation of Alkyl 3,4-Dideoxy-~~-ald-3-enopyranosides and Oxirane-ring-opening Reactions.- Formation of alkyl 3,4-anhydroDL-aldopyranosides (289) from unsaturated precursors (256) b y means of the epoxidating reagents coinmonly used presents no difficulty. The yields are in the range of 6 O - Y W o . From each, stereoisomeric, compound 256, both epoxides possible are usually formed.

256

or

n,o,. RCN ArC03H

-

ooR2

0

OH

289

The steric course of epoxidation is g ~ v e r n e d ' ~essentially ~ * ' ~ ~ b y two factors: (1) the presence of a free, allylic hydroxyl group at C-2, which promotes the formation of an oxirane ring from the same side as that occupied by the hydroxyl group, and (2) steric hindrance exerted b y allylic substitueiits of other types (CH,OH, CH3, OR, or OCOR) and the alkoxyl group at C-1. Thus, the stereochemical outcome of epoxi(176) H. C . Brown and P. Ceoghegan, Jr.,]. A m . Chein. Soc., 89 (1967) 1522-1524. (177) A. Banaszek, Bull. Accid. Pol. Sci., Ser. Sci. Chim., 22 (1974) 1045-1051. (178) A. Banaszek, Bull. A c c d . Pol. Sci., Ser. Sci. Chim., 20 (1972) 935-943. (179) M . Chinielewski and A. Zamojski, Roct. Ckem., 46 (1972) 2039-2050.

SUGARS FROM NON-CAHHC)IIYDRATE SUBSTHATES

57

-0

n7 - C lC, H,CO,H

Q

O

M OR

R = H R = Ac R = PhCH,

1 OR R = H R = Ac R = PhCH,

e

* Overall yield (%)

( d o M e 0

+

Q

O OR M

e

OR

70 64 93

Overall yield (8)

6

64 13 16

51

75 53 56

5 17 45

77

'0

80 70

81

Scheme 1

dation remains within the bouiidaries established earlier for cyclic alkenes.l4I The examples s h ~ w ninl ~Scheme ~ l demonstrate clearly the interplay of both factors. The next stage of sugar synthesis consists in opening of the oxirane ring in 289 with nucleophilic reagents, primarily with water,12"'4""ss~ 160,161,180-lH2 or amines .166,183,18-1 The direction of oxirane ring-opening in the stereoisomeric compounds 289 depends on several filetors; for example, fkivored axial attack of the nucleophile, anchiineric. assistance of neighboring groups, and "conformational control" of the approaching nucleophile. The exaniples shown in Scheme 2 provide support for the importance of each factor. There are, however, exceptions to these rules (see, for example, Ref. 184).Therefore, the results of oxirane ring-opening in 289 cannot always be foreseen. Epoxide-opening reactions of d k y l 3,4-anhydro-~~-aldopyranosides enabled stereoselective preparation of a wide variety ofmonosac(180) A . Banaszek, Bull. Acud. Pol. Sci.,S a r . Scz. Chin!., 22 (1974) 79-89. (181) M . Chniielewski and A. Zaniojski, B u l l . Accitf. P o l . Sci., Ser. Sci. Chiin., 20 1972) 755- 757. (182) M.Chrnielewski and A. Zanwjski, Roc;. (:hem., 46 (1972) 2223-2231. (183) V. B. Mochalin, Z. I. Smoliira, ;cnd B. W. Unkovskii, 211.Org. Khim.,7 1971) 1502- 1505. (184) A. Banaszek, Bull. Actid. Pol. Sci., S e r S c i . Chini., 23 (1975) 633-6.36.

AL.EKSANDER ZAMOJSKI e t

58

-

(I/.

1,6- Anhydro-p-o~-glucose

Axial attack of the nucleophile Methyl u 2.6-Di-O-acetyl-nDL-

&

mannopyranoside

CH,OAc

OMe

L<'

0

Neighboring-group participation

0- CH

\ H3

+

O

O

O

M

. )

Methyl 2.6-Di-O-acetyl-aoL-idopyranoside

doMe

-0

0eL+

0

OH

Two 1,3-diaxial interactions

-.--

Methyl CY-OL-L~XOpyranoside

OH

Conformational control

No 1,3-diaxial interactions

Scheme 2

charides, including racemic ethyl 4-arnin04-deoxy-cr-lyxoside,~~~ methyl a-lyxoside,lx2a-xyloside,lx2a-guloside,'SQ a-idoside,lxncr-mannoside,'*" 1,6-anhydro-~-gluco~e,~~".~~~ cr-curamicoside,1x4 a-kanosaminide,lH4and many others. If the epoxide ring-opening does not proceed stereoselectively, two products are formed that can usually be readily separated by chromatography. It must be stressed that the

chemistry of epoxides disciissc,tl h e r e is, in flict, an extension of that already known from reactions of eiiaiitiomeric alkyl 3,4-a1ihvdro;iltlop y ran o s i de s .

e . Other Syntheses-Two c,xamples of syntheses involving :3,4-unsaturated sugar precursors elesclrve closer attentioil. The first example is the synthesis of DL-mycarosetX5 and its 3-epimer, which started from the lactone of 5 - h y d r o x y - 3 - 1 1 i c ~ t l i ~ I l ~ e x - 3acid - e i ~ ~(290). ~i~ Partial reduction of 290 with lithium iiliiniinrim hydricle gave 5,6-dih~dro-6-

hydroxy-2,4-dimethyl-2H-pyrall (291, ~,3,4,6-tetradeoxy-3-C-nlethylDL-hexopyranose), which was converted with mi orthoester into the 6methoxy (292) or 6-ethoxy (293) derivative. ci.E-Hydrox).latioii of 293 with iodine- silver acetate (thc Woodward reagent) occurred with partial hydrolysis of the acetal grouping, and led to DL-mycarose (65) in 6%' yield. A better yield (129%)of this sugar could lie achieved when the 6-acetoxy derivative (294) wiis used for ci.v-hvdroxylation. Epoxidatioii of 292 with peroxybeiizoic acid gave epoxicle 295, which, on

290

2 9 2 R = Me 293 R = Et 294 R = A c

291

PhC0,H

292

-

boMc -0 HC10,

HO

OR

0

H,

I

c

H3 c 295

293

R

= Me R = H

~

I

HO 65

(185) F. Korte, U . Claussen, aiid K. C;ohring. Tetrcihedrort. 18 (1862) 1257- 1264

ALEKSANDEK ZAMOJSKI

60

f>t(11.

hydrolysis with M yerchloric acid, yielded a mixture of products from which methyl 3-epi-DL-mycarosite was isolated; acid hydrolysis thereof yielded 3-epi-DL-mycarose. 5-C-Methyl hoinologs of both of these sugars were1xsobtained in the same way. The second example demonstrates the applicability of the general approach to the synthesis of a higher sugar. Methyl 7-deoxy-cu-~~-lincosaminide (299)was preparedI3* from 5,6-dihydro-2-methoxy-6-propanoyl-2H-pyran (296) in a few steps, involving synthesis of the 6acetamido derivative (297), which was then converted into the 3,4-unsaturated sugar 298. cis-Hydroxylation of 298 afforded two stereoisomeric methyl 6-acetainido-6,7,8-trideoxy-a-octopyranosides, of the DL-gl~cero-DL-gnl~icto (299) and DL-glycero-DL-uIlo (300) configuration, respectively. Et I

Et

Et I

AcHNFH

OH

297

296

298

po,. Et

Et

oso,

I

I

AcHNCH

A~HNCH

OH

HO

t

300

OH 299

IV. SYNTHESESFROM DERIVATIVES OF FUFUN The formation of furan derivatives in acid-catalyzed dehydrations of carbohydrate substrates is a well known reaction, first reported b y DGbereiner'XGin 1832. Ainong the plethora of compounds formed, 2-furaldehyde is the main product obtained from all of the pentoses, whereas 5-(hydroxymethyl)-2-furaldehydeis the major product (186) J. W. Dobereiner, Ann., 3 (1832) 141-146

SUGARS FROM NON-CARBOHYDRATE SUBSTRATES

61

formed during acidic degradation of hexoses.lx7Hence, reverse transformation would be a plausible route for the synthesis of monosaccharides. Indeed, it has been found that simple derivatives of furan may be converted into racemic monosaccharides. Most of the syntheses thus far completed involve 2,s-addition to the furan nucleus: (i) on treatment with oxidizing reagents, and (ii) in Diels- Alder or related reactions. There are also a few syntheses starting with addition across the 2,3-double bond of a furan derivative; they are described at the end of this Section.

1. Transformations of 2,5-Dihydrofurans In 1947, Clauson-Kaas reportedlRX the reaction of furans 301 with alcohols in the presence of bromine and a weak base, leading to 2,5dialkoxy-2,s-dihydrofurans. In the following years, these compounds have found wide, synthetic application, mainly as a source of 1,4-dicarbonyl substrates for the preparation of heterocycles; for example, in the Robinson- Schopf methodlRgof tropinone synthesis. From the parent compound (301), the resulting cis- and trans-2,5-dihydro-2,5dimethoxyfuran [302 and 303 (R = H)] have been separated, and their hydroxylation with potassium permanganate described.lgOIn both cases, only one 3,4-cis-diol resulted, and, as neither compound could be resolved into its optical antipodes, no configurational assignment has been made. [The cis compound 302 could yield two meso-diols on treatment with permanganate, whereas the DL pair would result from cis-hydroxylation of the trans isomer 303 (R = H).]

301

302

303

The 3,4-diols thus obtained are closely related to tetroses, but no direct correlation with natural compounds has been made, apparently because of the unusual stability of the acetal ring towards acid hydroly~is.'A ~ 'number of studies have been devoted to alternative function(187) M. S. Feather and J. F. Harris, Ado. Carbohydr. Chem. Biochem., 28 (1973) 161224. (188) N. Clauson-Kaas, K. Dun. Vidmsk. Selsk. Mat.-Fys. Medd., 24 (1947) No. 6; Cheni. Abstr., 42 (1948) 193Of. (189) N. Elniing, Ado. Org. Chem., 2 (1960) 67- 115. (190) J. T. Nielsen, N . Elming, and N. Clauson-Kaas,Acta Chem. S c a d , 12 (1958) 6367. (191) K. Zeile and A. Heusner, Chem. Bet.., 87 (1954) 439-443.

62

ALEKSANIIER ZAMOJSKI et

(11.

alization of the 3,4-doubk bond in 2,5-dihydro-2,5-dii~~ethoxyfurans. For example, the addition of alcohoIs,lYL,lS:l lmnnine,1S4a i d hypohalous acids1Y5p1s8 has been described. The application of the aforementioned fiiran derivatives a s sulistrates in the total synthesis of monosaccharides was first explored b y Srogl, Janda, and coworkers. The first report in the series described an atteinpted synthesis of furanosides through addition of bromine to the double bond in 2,5-dihydro-2,5-dimethoxy-2-(1~ietlioxyc~~rl~oiiyl)f~ir~ii~ (304), followed by replacement of the bromine atonis b y acyloxy groups. As eliinination of hydrogen bromide occurred (instead of the nucleophilic siilistitution expected), the authors turned their attention to direct hydroxylation. Oxidation with potassium permiinganate of the following dihydrofiirans (304-307; c i . ~plus frtins mixtures not separated) gaveL".'low yields (10%)of the corresponding cis-diols (308-311) having unspecified, relative configura t'1011. M e z y y z M e

M e Z G O M R' ?

HO

R' = H,

R2 = C0,Me 305 R' = H, R2 = M e 306 R' = Me, R2 :C0,Me 307 R' = H. R2 = CH'OCOPh 304

OH

308 309 310 311

Only much later was the hydroxylation of pure trans-307 and cis307 perfonned, and the corresponding cis-diols were obtained in 2226% yield. The compound obtained from truns-307 was converted199 into erythro-pentopyrarios-4-ulosehydrate b y successive alkaline debenzoylation, and hydrolysis of the acetal in the presence of Dowex W-50 resin. An attempt at direct trua.r.-hydroxylation of trcins-307 b y Woodward's or Prkvost's method failed, as have attempts at its epoxidatioii with peroxy acids.")(' Nevertheless, a number of 3,4-epoxides A. Stoll, A. Lindenniann, and E. lucker, Helc. C/ii?n.ilctci, 36 (1953) 1500-IS05 N . Clauson-Kaas, J. T. Nielsen, and E. Boss,Actri Chem. S c u d . , 9 (1955) 111115. J. Sroyl and F. Liska, Collect. C z e c h . Clierri. Conitnuti., 29 (1964) 1277-1281. A. Stoll, B. Becker, and E. Jucker, Helo. Chini. Actu, 35 (1952) 1263- 1269. J. C. Sheehaii and B. M. Bloom,J. A m . Chem. Soc., 74 (1952) 3825-3828. D. Ginsburg, B u l l . Rcs. COZLIK. I.sJ-., 2 (1952) 268-269. J. Kelvxle and P. Karrer, Helt;. Chinl. Acto, 37 (1954) 484-494. J. Srogl, M. Janda, I. Stibor, arid J. Kucera, Collect. Czech. Cheni. C o v i ~ n u n .38 , (1973) 455-458. N. K. Kochetkov, L. I. Kudriashov, N. V. Molodtzov, and N. 11. Khoniutova, Z h . Oh.rhch. Khinz., 31 (1961) 3909-3916.

(312-316) hnve been obtnined, ,Jtliough i n pool !ield\ (1 1- 1 G 9 ) . 11) the addition of hypochlorou\ ,ic.id ot tcrt-but) 1 111 pochloritc~,followed b y dkaline dehydrochloi in'itioii ."L1

Only for the parent conipouiid (312) in this series has detailed analysis of the reaction mixture Iwen performed; the isoniers were sepa-

rated, and configuration was assigned to them on the basis of their 'H-n.1n.r. spectra arid the iiieiisiireiiieiit of their dipole iiiomeiit.LO* Synthesis of soiiie 3,4-aiiiiii~alcohols b y opening of tlie epoxide rings with ammoiii a was a1so descri 1)ed .202 F ui-the rm ore, the a11p 1ica t ion of 3-substituted furans to the syiithesis of branchetl-chain sugars was examined. It has been demonstrated that 3-(acetoxyinethyl)-2-inethylfuran and 3-fonnyl-2-methylf11i-an readily undergo electrolytic 2,5~ n e t h o x y l a t i o n . ~ "Similarly, ~~~"~ 2,5-tliinethoxy derivatives of 3-(1hydroxyethyl)furan, 3-(l-acetoxyethyl)furaii, and 3-acetylfuran [in the last case, 3-(l,l-diiiiethoxyc~tliyl)-2,5-dihytlro-2,5-dimethoxyf~ir~~ri was formed in 2 1.5%yield] were ol>tained,and their ci.~-hyclroxylation was studied.205In the case of crystal line tetrahydro-3,4-dihydroxy-3(l-hydroxyethyl)-2,5-dimethoxvtiir~in,tlie relative configuration 317 was assigned.205

HO

317 I< 318 I?

= =

OH CH(OH)CH, A(.

(201) J. Srogl and J. Pavlikovi, Collect. C z , e d , Chcin. Cotntn~iti., 33 (1968) 1954- 1957. (202) L. N. Kralinina, H. I. Kruglikow, ; i i i d V . I. Hogornoleva, Kliini. CkterotsiX-/. Soedin. (1970) 229-301; Chern. Ahstt-.,73 (1970) 55,89fjj, 76 (1972) 140,589. (203) M. Jatida and P. Novak, CollcJct.( : z c c / i . (:hem. Conit,iuti., 29 (1964) 1731-1734. i . , ( 1966) (204) X I . Valenta, M .Janda, a n d A. Kl;irck, ( : o / l w t .Czech. C h c , r r i . C o r ~ i t n t ~ t:31 2410-2415. (20s) J. Srogl, M. Janda, and I. Stilloi, C o 1 l c ~ c . t .Czech. C / i e r t i . C o n z m t r t i . , -38 (197.3) 3666- 3674.

AL.EKSANDER ZAMOJSKI el

64

(I/

On the basis of the nuclear Overhauser effect, the cis arrangement of the methoxyl group on C-2 and in the side chain was established. The trans relationship of H-4 and H-5 was deduced from their coupling constant (j4,53.7 Hz). The post-hydroxylatioi1 mixture was shown to contain two other isomers of 317, as well as some of 318. The compounds were reported unsuitable for further transformation involving hydrolytic cleavage, as they polymerized readily in acidic media.205 This difficulty has been overcome in the following synthesis of racemic streptose tetramethyl acetal. 3-Formyl-2-1nethylfurari was converted in 75% yield into derivative 319 by electrolytic rnethoxylation. A corresponding mixture of cis-diols (320) was treated with Dowex W-50 ion-exchange resin in methanol for four days, to afford 5-deoxy-3-C-(dimethoxyinethyl)-~~erythro-4-pentulose dimethyl acetal (321). Reduction of this compound with lithium aluminum hydride or sodium borohydride gave a mixture of 5-deoxy-3-C-(dimethoxymethyl)pentoses, which was separated by column chromatography on silica gel, to givezo6DL-streptose tetramethyl acetal (322) and the isomeric 5-deoxy-3-C-(dimethoxymethyl)-DL-ribose diniethyl acetal 323 (lyxo: ribo = 13:7). Detailed, combined gas- liquid chromatographic-ii~ass spectrometric analysis of the compounds related to streptose (in the form of their trimethylsilyl derivatives) has heen H MeOCOMe

I

HCOH Me:Qtie

I (MeO),CH-&OH

MeQ : e:t CH(OMc),

HO HO

319

c=o I c H3

CH(OMe), 321

320

321

-

H MeOCOMe I HCOH

(MeO),CH-&OH

H MeOCOMe

I

HCOH

HOCH

(MeO),CH-&OH I HCOH

CH,

CH3

I

I

322

t

I

323

a. Unsaturated Pyranosu1oses.-A new approach to the utilization of 2,5-dihydro-2,5-cli1nethoxyfuralls in monosaccharide synthesis was (206) J. Srogl, M . Janda, and I. Stibor, Collect. Chevz. Commtrn., 39 (1974) 185-191. (207) I. Stibor, J. Srogl, and M. Janda,J. Clzromatogr., 91 (1974) 767-773.

SUGARS FROM N O S - ( :AHHOHYDRA'IE SUHSTR.4TES

65

elalmrated by Achtiiatowicz a i i d coworkers. The key step of this synthesis involves transformation of a fury1 alcohol derivative (324) into an ald-2-enos-4-ulose (325) 1)). tileatis of hydrolytic cleavage of the furatiosyl acetal ring, presumd)ly tlirough an interniediate dicarlionyl compound.20x

o(--oH 324

325

This conversion of readily availalile %fury1 alcohols into unsaturated pyranosuloses proved a very effective route to racemic nionosaccharides, through stepwise, selective fiirictioiializatioii of the e n w e grouping in 325. The shortest s)mthesis of a natural compound b y following this scheme involves pallacliiim-cataly~e~~ hydrogenation of the aldosulose (325, R = M e ) ohtained from 1-(2-fiiryl)etlianol,resulting209in cinerulose A, the sugar component of the antibiotic cinerubin. Although the configuration of the starting 2,5-dihyclro-2,5-diinethoxyfuran is not relevant for prodiicts having pyraiioid structui-es (325-327), a series of cis - t r c i ~ i isomers .~ of 324 have been separated, and their configuration has bee11assigned by comparison of their H-5 chemical-shift values in the l H - i i . ~ i i . r spectra.21" . The hemiacetal hydroxyl groiip of the aldopyranosuloses 325 was usually protected in the form of the methyl glycoside b y treatment with methyl orthoformate in the presence of a Lewis-acid catalyst.20ox Higher yields of the corresponding methyl glycosides could lie obtained b y treatment of a glycop!.raiiosulose with methyl iodide in the presence of silver oxide."' Oiic-stcp transfomiation of the dih\drofurati derivatives 324 into the niethyl glycosides (326, 327) b y treatment with methanol and formic o r trifluoroacetic acid has been reported.212 (208) 0. Achmatowicz, Jr., P. Bukou,\ki, 13. Szechner, Z . Zwirrzcllowska. and A. Zamojski, Tetrahedron, 27 (1971) 197,'3-1996. (209) 0. Achmatowicz, Jr., and B. Szechner. H i r l l . Accitl. Pol. Sci., Ser. Sci. Chit?&.,19 (1971) 309-311. (210) 0. Achinatowicz, Jr., P. Bukowhki, G . (:rynkiewicz, B. Szechner, A. Zaiiwjski, and 2. Zwierzchowska, Roc;:. C ~ I V I16 J I (1972) ., 879-888. (211) R. Lalibeite, G . Medawar, and Y,I,efel)vre,J. Med. Clwnl., 16 (1973) 1084- 1089. (212) P. D. Weeks, D. E. Kuhla, R. P. .4llinghain, H. A. Watson, Jr., a n d H. Wlotlecki, Curbohydr. R e s . , 56 (1977) 1YS- 109.

66

ALEKSANDER ZAMOJSKI et

(I/

The reaction of 2,s-dihydrofiiryl derivatives with hydrogen chloride i n inethanol leads to a rather complex mixture of acyclic products. The presence has been revealed of significant proportions of stereoisodiinethyl acetals and tetrameric tetraliydro-2,4,5-trirnethox~~furfural hydro-2,3,3,5,6-pentamethoxyfurans among the products of treatment of ~-acetoxy-2,S-dihydro-5-nitro-2-fural~lehyde diacetate with inethanolic hydrogen chloride s o l i i t i o i ~ . Glycopyranosiduloses ~~:~ hiring a more complex aglycon group m a y lie obtained by exchange of the hemiacetal ester group, catalyzed b y stannic chloride.214Alternatively, some pentenosiduloses, including disaccharides, were obtained b y treatment of glycosulose 325 ( R = H) with an appropriate alcohol in the presence of the cliethyl azodicarboxylate-triphenylphosphinemercuric 11 rom i de reagent . The reduction of niethyl ald-2-enopyranosid-4-uloses (anomers separated b y column chromatography) with complex, metal hydrides has been studied in detail, and found to proceed with a high degree of stereoselectivity due to stereoelectronic control of the approach of the hydride ion.21fiThus, the reduction of methyl 2,3-dideoxy-DL-pent-2enopyranosid-4-ulose (326, R = H) with sodium borohydride in aqueous oxolaiie gave a-DL-ghjcero (328, R = H) and P-DL-gltjcero (329, R = H) glycosides in the ratio of 45: 4 (additionally, 2% of the saturated a-glycoside was found in the mixture). Similarly, reduction of methyl 2,3,6-trideoxy-a-~~-hex-2-erlopyranosid-4-iilose (326, R = Me) with lithium aluminum hydride in diethyl ether afforded2172,3unsaturated glycosides having the a-DL-er!jthro (328, R = Me) and aD L - ~ ~ W(329, O R = M e ) configuration in the ratio of 9: 1. However, prolonged contact of the substrate with an excess of the hydride may lead to formation of the glycal b y way of attack of hydride ion at C-3, apparently concerted with fission of the glycosidic bond.21X Reduction (327, R = of methyl 2,3,6-trideoxy-P-~~-hex-2-enopyranosid-4-ulose Me) afforded217the corresponding P-DL-erythro (330, R = Me) and pDL-threo (331, R = Me) unsaturated alcohols in the ratio of 1: 1. This result reflects the conformational mobility of the p-ald-2-enosid4-

(213) B. B. Greene and K. C. Lewis, Auct. J . Chem., 31 (1978) 627-638. (214) C. Crynkiewicz, B. Barszczak, and A. Zamojski, Synt/ze.c.i.v (1979) 364-365. (215) G. Grynkiewicz and A. Zarnojski, Syrith. Commun., 8 (1978) 491-496. (216) 0. Achmatowicz, Jr., a n d P. Bukowski, Rocz. C k e m . , 47 (1973) 99-114. (217) 0. Achmatowicz, Jr., and B. Szechner, Rocz. Chent., 49 (1975) 1715- 1724. (218) 0. Achmatowicz, Jr., and B. Szechner, Tetrahedron Lett. (1972) 1205- 1208.

SUGARS FROM NON-(:ARBOHYDRATE SUBSTRATES

67

uloses as compared to the a anoniers (see Scheme 3), which probably exist almost exclusively in the 'IS, conformation.*19,220

oVo),,OMe

-

~

O

w

,

0

Scheme 3

2,3-Unsaturated methyl glycosides (328-331), readily separable by column chromatography, were subjected to hydroxylation of the double bond, yielding racemic pyranosides. The efficiency and selectivity of the transformations involved are illustrated by the following examples. On treatment with the Milas reagent, methyl 2,3-dideoxy-aDL-glycero-pent-2-enopyranoside (328, R = H) gave inethyl CX-DL-~YXopyranoside (332, R = H) as a single product in 45% yield. cis-Hydroxylation of the corresponding P - D L -ycero-pent-2-enopyranoside ~~ (330, R = H) with the same reagent afforded a mixture of two compounds (42%), in the ratio of 3: 2, which was resolved221into methyl P-DL-ribopyranoside (335, R = H) and methyl P-DL-lyxopyranoside

(336, R = H). A preparation of methyl pentopyranosides having the arabino and the xy20 configuration was achieved by epoxidation, followed by opening of the oxirane ring. Both methyl a- and P-DL-pent-8-enopyranoside (328 and 330, R = H) underwent epoxidation on treatment with m-chloroperoxybenzoic acid, affording mixtures of 2,3-anhydro compounds in the ratios of 9 : 1 and 10 : 1, respectively. With the aid of the 'H-n.m.r. spectra of the corresponding .l-acetates, it was shown that products having the oxirane ring cis to the hydroxyl group [that is, a-rib0 (337) and p r i b o (341),R = HI preponderated in each case; the minor products were assigned the structure of methyl 2,3-anhydro-a-

(219) 0. Achmatowicz, Jr., and M. H. Burzyliska, Pol. J . Chem., 53 (1979) 265-276. (220) 0. Achmatowicz, Jr., P. Gluzinski, and B. Szechner, Bull. Acad. Pol. Sci., Ser. Sci. Chim., 23 (1975) 911-916. (221) 0. Achmatowicz, Jr., and P. Bukowski, Can. /. Chem., 53 (1975) 2524-2529.

2

X

5

i

) -0

1

a "'

-

SUGARS F R O M N O N - C A R B O I H Y D R A T E S U B S T R A T E S

69

ilnd -P-DL-lyxopyr~inoside(338, aird 342, H = H ) . During alkaline h>.drolysis of methyl ~ , 3 - a n l i ~ ~ d r o - a - ~ ~ - r i l ~ o ~ ~ prcadoniinant y r ~ ~ t i o s i at~l~~, tack of the reagent occurretl at C-3; the resulting mixtiire o f inethyl pentopyranosides, obtained iI I 7 3 4 , yield, cont;iinetl 4 parts of t h e ND L - X ~ I ~and O 1 part of the N - l > L - ( i r u h i t i o glycoside. Under sinrilar conditions, illethyl 2 , 3 - a n l i y ~ l r o - ~ - ~ ~ - l y x o p y r ~ ~ r i o s i d e = H) underwent oxinme-ring migratioii, b u t its 4-(I-acctyl derivative could lie cleaved I)y treatiiient with 80%' acetic acid, to afford methyl cr-DL-2irabinopyraiioside (60%))as the o n l y isolablc product."' An a1te rnati ve met hod for fu I i ct i o t I a1 i zut ion of'ii pen te 11 o s i d 1I 1o s e (326, R = H) was applied i n a highl!. selective synthesis of DL-ribose derivatives. Direct hydroxylation of the pentenosidrilose with the ostniuni tetraoxide-potassium chlornte reagent, follo~vedIiy protection of the 2,3-diol grouping with an isopropylidene group, and reduction of the 4-ketone group, resulted in the formation of methyl 2,3-O-isopropylidene-P-DL-ribopyranoside. When 2,3-dideox?.-~~-pent-2-enopyranosyl-4-dose benzoate was used a s the substrate, the saine sequence of reactions, supplemented b y alkaline hydrol>sis, afforded 2,3-0-isopropylideiie-DL-rilofLi~~nos~,.~~~ Aiialogo~dy,a total synthesis ot' the methyl glycosides of' all of the raceinic 6-deoxyhexoses froin 1-(2-fiiiyl)ethanol was completed. Methyl 2,3,6-trideoxy-a-DL-~r.!/flir.o (328, R = Me), p-DL-or.!/thro (330, R = Me), and a-DL-threo (329, R = hle)-hex-2-eiiopyr~~iioside4 were hydroxylated with the Milas reagent, to afford :i moderate yield (3643%) of the respective meth!.l 6;-tleoxyhexop),r~iiioside.The selectivity of the hydroxylation is apparently govel-ned b y the disposition of the substituents on C-1 and C:-4. 'Thus, the cu-er!/tliro (328, R = Me) glycoside yielded methyl 6-deoxy-a-DL-manliiio~yr~~tioside (rhamnoside) exclusively, and the /3-otyt/t t-o (330, R = Me) compound gave only methyl 6-deoxy-P-DL-allopyr~~ii(~side. On the other hand, hydroxylation of the a-threo (329, R = M e ) glycoside resulted in a mixture of two compounds; these were separated b y coliinin chromatography, and identified"' a s methyl 6-deoxy-n-DL-talopyranoside (19%)and ide methyl 6 - d e o x y - a - ~ ~ - g u l o p y r ~ i 1 1 o s(24%). All of the stereoisomeric methyl 2,3-aiihydro-6-tleoxy-~~-hexopyranosides have been prepared b y epoxidation of 2,3-unsaturated pyranosides (328-331, R = M e ) with hydrogen peroxicle-benzonitrile. The 'H-n.1n.r. data of their 4-0-acetyl derivatives were reported, and discussed in connection with configurational and conformational assignments.22" The rc.gio- and stereo-selectivity of the

(338, R

(222) 0. Achmatowicz, Jr., and G . (;r> nkiewicz, C ~ i r h h ~ dh rs .. , 54 (1977) 193- 198. (223) 0. Achmatowicz, Jr., and B. Srech~iel-, Cctrbohydr H w , SO (1976) 23-33.

70

ALEKSANDER ZAMOJSKI et

(I/

hydrolysis of the oxirane ring in methyl 2,3-anhydro-6-deoxyhexopyranosides having the a-DL-?r~(~ui~o, a-DL-clllo, C X - D L - ~ U and ~ Oa-DL, tnlo configurations have been exainined. The muiino epoxide underwent selective opening ( b y attack of hydroxide anion at C-3) in an alkaline inediuin, to give methyl cu-DL-altropyranoside, but, on acid hydrolysis, it was shown to give an approximately equal yield of both the latter and methyl a-uL-glucopyn~noside.The same pair of coinpounds was obtained by hydrolysis of the 2,3-(111o epoxide (337, R = Me). The a-gulo epoxide was hydrolyzed in an acidic medium to give inethyl 6-deoxy-a-~~-galactopyranoside a s the sole product (61%), whereas rearrangement to the 3,4-anhydro compound was observed to occur under basic conditions. Selective opening at C-3 took place during alkaline hydrolysis of the tcilo epoxide (340, R = Me), which gave methyl 6-deoxy-a-~~-idopyranoside in 63% y i e l d z z 4A simple syiithesis of noviose (5,5-diC-methyl40-methyl-~-lyxose),the sugar component of the antibiotic novobiocin, in the form of the racemic methyl glycosides, b y traiisfoimatioiis parallel to those mentioned for methyl DL-lyxoside, has been described. 2-(2-Furyl)-2-propanol was the starting material, and the target molecule was reached in six steps.225 Synthesis of hexopyranosides b y the method outlined required 1-(2furyl)-l,2-dihydroxyethane as the substrate. This compound was shown to be readily available b y thermal, or acid-catalyzed, condensation of furan with glyoxylic esters, followed b y reduction with lithium aluminum hydride of the furylglycolate thus ol)tained.226It was further converted into the dihydropyranone intermediate (325, R = CH,OH) in the usual manner b y way of the 2,5-dihydro-2,5-dimethoxy derivative. The corresponding methyl glycosides (326 and 327) were separated, and reduced, to afford the 2,3-u11saturatedpyranosides (328-331, R = CH,OH). Treated with hydrogen peroxide and a catalytic amount of osmium tetraoxide, methyl 4,6-di-O-acetyl-a-~~erythro-hex-2-enopyranoside afforded an 80% yield of crystalline methyl a-DL-mannopyranoside 4,6-diacetate. Epoxidation of the double bond in the same substrate with the hydrogen peroxide-acetonitrile reagent gave two products which, after acetylation, were separated by column chromatography, and assigned, on the basis of theJ,,2 and],, values, the Configurations a-niunno (338, R = CH,OAc) and aullo (337, R = CH20Ac) in the ratio of 3:2. The mutino epoxide was further hydrolyzed to methyl 4,6-di-O-ace(224) 0. Achmatowicz, Jr.. arid B. Szechner, Rocz. Chem., 50 (1976) 729-736. (225) 0. Achmatowicz, Jr., G. Crynkiewicz, and B. Szechner, Tett-uhedr-on, 32 (1976) 1051- 1054. (226) 0. Achmatowicz, Jr., and A. Zamojski, Rocz. Chcin., 42 (1968) 453-459.

SUGARS FROM NON-CAHBOHYDKATE SUBSTHATES

tyl-cu-DL-altropyranoside.c.z.r.-H y d r o x y latioil of the corresponding

71

p-

er-ythro diacetate (330, 4-OAc,, 1% = CH,OAc) with osmium tetraoxide in pyridine, followed by ;icvtylwtion and c ~ i r o i n ~ i t o ~ r u j ,purificaIii~ ti on, afforded a single p r o d 1I ct , i tl ell t i fied a s 111e t h y 1 2,3,4,6-t e t ra-O acetyl-p-DL-allopyranoside, i i i 70“r )ield.22i Starting from the monoberizyl ether of 2,5-di(I~ydroxymethyl)furan, methyl glycositles of the following hexuloses have I ~ c o1,taiiied n by

way of iiiterinediate n~eth)rl~ , 4 - ( ~ i d e o x y - D L - h e x - ~ 3 - e i i o ~ ~ ~ r ~ ~ i ~ o s i ~ ~ diuloses: a-uL-sorbose, a-Dr.-tqatose, p-DL-fructose, and a-DL-psicose. Analogously, 1 , 2 - d i h ~ d r o s y - 2 - ( 5 - l ~ y d r o x y i i i e t h ~ l f i 1 r ~ l ) ehas th~~1~e been transformed in to me tli 1.1 U - I I L - I~I co ~ -hep tu 1o s i cle .22x The selectivity of the reduction of methyl cu-DL-ald-2-enopyranosid4-ulose 326 and, consequently, t h e low avai1al)ility of the 2,3-unsaturated pyranosides of the cu-thrm (329) configuration, required the inversion of the configuration of C-4 (C-S in the glpculopyr~tnosides)for completion of several syntheses. The benzoic acid-tliethyl iizodicarboxylate-triphenylphosphine reagent was reported to effect the esterification specifically, with inversion of the configiiration; the yields were significantly higher t h m those olitained i n the two-step, sirlfonic ester, displacement procecliircB.229 The scheme of synthe outliiied may readily be adapted for the preparation of modified sugars. For example, a Michael addition of some active-niethylene substrates to inethyl 2,3-dideox)-DL-pent-21,2-trrr 11s adducts reenopyranosid-4-ulose was f o i i ~ i d ~to : ~ produce ” lated to pyranosides branclietl at C-2. The saint‘ methyl pentenosidulose (326, R = H) has also served as the su1)strate in t h e synthesis o f a deoxyinonosaccharide analog coiitaiiiing a pliosphorirs atom in the five-membered ring. The forination of the carl,on-phosphorus Iiond was achieved2:31.2::” by additioii of methyl phenylphosphoi-lite to the [ I tolylsulfonylhydrazone of the satiirated ketone obtained b y the hydrogenation of326 (R = H); see Section VI. The synthesis of biologicall>,iinportant, arninodeoxy derivatives of (227) 0. Achinatowicz, Jr., R. Rielski, and P. Rukowski, H o c z C h c w t . , 50 (1976) 15351543. (228) M. H. Burzyliska, Ph. D. Thesih, Institute of Organic. C h i n i s t i - y , Polish Academy of Sciences, Warsaw, 1976. (229) G. Grynkiewicz and M. H. Rrirqliska, ?’etrcihetlr-oti, 32 (1976) 2109-21 11. (230) G . Grynkiewicz, 0. Achinutowicz, J r . , and €3. Rantoli, Roc;. O’/wt!i., 51 (1977) 1663- 1674. (231) M. Yamashita, Y. Nakatsuk,is.i, hl. Yoshikane, H . Yoshida, T. Ogata, and S. Inokawa, Corhoh!ydr. Res., 59 (1977) ~ 1 2 - ~ 1 4 . (232) M ,Yamashita, M. Yoshikane, ‘I. O g a t a , and S. Inokawu, ?’ofruhcdrori, 35 (1979) 741-743.

72

ALEKSANDER ZAMOJSKI et a / .

monosaccharides has been approached in three different ways: (1) splitting of the oxirane ring with ammonia served as a k e y step in the synthesis of methyl N-acetyl-2,4,6-tri-O-acetyl-a-~-kanosainiiiide,~~~ (2) introduction of a required substituent at the stage of furyl alcohol was applied in the synthesis of methyl 6-deoxy-6-C-nitro- and 6-acetamido-2,3-4-tri-O-acetyl-6-deoxy-a-~~-n~aiinopyr~~nosides,'~'~ and ( 3 ) exchange of the all ylic hydroxyl group of hexenopyranosides for a phthalimido group, pronioted b y the diethyl azodicarboxylate- triphenylphosphine reagent. The result of the last reaction was found to depend on the structure of the substrate. Thus, for 2,3-unsaturated pyranosides, an exchange involving inversion of the configuration of C-4 occurred. In the case of 3,4-unsaturated substrates, regio- and stereo-isomers resulting from nonspecific, allylic rearrangement were identified.z35As a rule, stepwise functionalization of ald-2-enopyranosid-4-uloses (326 and 327) does not involve the separation of complex mixtures. Moreover, there is no possibility of isomerizatioii at any stage of the synthesis. It has been demonstrated that transformation of optically active furyl alcohols into monosaccharide derivatives proceeds without loss of optical purity (see Section VII). Furfuryl alcohol is oxidized directly to 2,3-dideoxy-DL-pent-2-enopyranosid-4-dose (325, R = H) b y treatment with m-chloroperoxybenzoic acid.2"6A variety of substituted furfiiryl alcohols have thus been converted into over 60 enediulose derivatives (345) in connection with studies of their antimicrobial activity.211It was later found that pyridiniuni chlorochromate m a y be applied in this reaction, instead of a peroxy R

34 5

b. Enedia1one.s.- Several workers have reported that oxidation of furaldehyde with bromirie-water2"x~2"1 or chlorinez42yields an acyclic (233) 0. Achmatowicz, Jr., and R. Bielski, Rocz. Cheni., 51 (1977) 1389-1394. (234) 0. Achmatowicz, Jr., a n d G. Grynkiewicz, Rocz. Chem., 50 (1976) 719-728. (235) A. Banaszek, B. Szechner, J . Mieczkowski, a i d A. Zaniojski, Rocz. Chem., 50 (1976) 105- 119. (236) Y. Lefebvre, Tetrahedrori Lett. (1972) 133- 136. (237) G. Piancatelli, .4. Scettri, and hi. D. Auria, Tetrahedron Lett. (1977) 2199-2200. (238) E. E. Hughes and S. F. Acree,]. Res. N o t ! . B u r . Stand., 24 (1940) 175-180. (239) N . Clauson-Kaas and J . Fakstoip, Acto Chenz. Scund., 1 (1947) 415-421.

S UG ARS F R O M

N C ) N 4 :AH H O HTUH ATE S LIB STKA7'I.: S

73

derivative, namely, 2-penteii-l,5-dii~l-4-0ne[eneclialoiie, isolated a s the bis(phenylhydrazone)]. The slwtliesis of raceinic peiitoses, elaborated by BognAr and Herczegh,24:4.244 in which the carlmnyl group of 2-furaldehyde becomes C-1 of the alclose, makes use of this trimsformation. First, it was necessary to protect the aldehyde group against both acidic (oxidation) and basic (1)orohydride reduction) conditions. and The hydrolysis of several 2-fiiraldehyde acetuls w a s 2-(2-furyl)-4,4,5,5-tetr~~methyl-1,3-dioxolai~e (346), stable at pH 3 at room temperature, was fiiially chosen a s the substrate. Oxidation of the cyclic acetal 346 with Iiromi ne water-tcr-t-l)utyl alcohol-phosphate buffer gave the unstahlc eiieclialone derivative 347, characterized as its nionophenylhytllazoii~.Reduction of 347 with sodium borohydride gave DL-2-( 1,4-dili~droxy-ci.~-2-l)iite1lyl)-4,4,~5,~5-tetr~~methyl-l,3-dioxolane (348), and r,poxidation of compound 348 with iiionoperoxysuccinic acid afforded DL-t~~r.eo-2-(2,3-epoxy-1,4-dihy-

d r o x y b u t y l ) - 4 , 4 , 5 , 5 - t ~ t r a m e t l ~ ~ l - l , ~(349), 3 - d ea 3,4-anhydroarabinose derivative, which, o i l hydrolysis with warm 0.5 M sulfuric acid, gave DL-xylose. Hydroq~lationof the clilmizoate of 348 with hydrogen peroxide-osmium tetraoxide gave a mixture of 350 and 351, debenzoylation of which, followetl b y acid hvdrolysis, gave a mixture of DL-ribose and DL-ara1)iiiOse.2i' HO 0-CMe, '0-CMe,

I

HCT&{H

0-CMe,

0

346

347

CH,OH 348

MezC-

CMe,

I

I

C

I I

HOCH HC 10 ' HC'

I

CH,OH 349

Me,C-CMe,

I

O,H,O

I

c I

HCORz

I

HC'OH I H COH

I

CH,OBz 350

Me,C-CMe,

I

O,H

C' I DzOCH

I

0

I

HCOH I HCOH I CH,OBz 351

(240) M. Szaklics-Pinter and L. hl;iror, A c f n Cliirn. Accitl. Sci. H ~ r t ~ g56 . , (1968) 87-91. (241) M.Szaklics-Pinter and L. %faro\,.4clciC h i i r i . Accici. Sci. H i t r i g . , 56 (1968) 199-213. (242) J. B. Petersen, J. Lei, N. CILIIISOII-K~~~IS, arid I<. Norris, K . I l c i r I , Vidcntk. S e l . c k . M a t . - F y s . Aledd., 36 (1967) N o . 5; ('/ierri. A h t r - . , 69 (1968) 59,06:h. (243) R. Bognlir and P. Herczegh, ,lfcig. Kc7rrr. F d ! l . , 83 (1977) 298-304, Clrc.r,t. L i / l . s t r . , 87 (1977) 1 8 4 , 8 1 4 ~ . (244) R. Bogniir and P. Herczegh, C(ir/~(h!/cfr-.R e s . , 52 (1976) 11- 16.

74

ALE:KSANDEH ZAMOJSKI rt

(I/.

Similarly, 5-methyl-2-fiiraldehyde was converted into 3,4,6-trideoxy-~~-hex-3-eiiopyranosides. 4,4,5,5-Tetrainethy1-2-(5-methyl-2furyl)-1,3-dioxola1iewas oxidizecl with I,romine-water, and the unsaturated dioxolaiie resulting was immediately reduced with sodium borohydride, to give a mixture of' D L - ~ -1,4-dihydroxy-cis-pentenyl)( 4,4,5,5-tetrai:iethyl-l,3-~1ioxolane. Methanolysis gave the known inethyl 3,4-6-trideoxy-a-~~-threoand -er!ithro-hex-3-enopyr~~iiosides, identified by 'H-n.in.r. spectroscopy.245

2. Bicyclic Precursors Considerable effort has been directed towards the synthesis of' monosaccharide derivatives, and, in particular, C-glycosyl compounds, from Diels-Alder adducts of furan. Just and his group investigated the reaction of furan with a nuinljer of dieiiophiles, and described inany interesting tl-ansfonnations of the adducts obtained from

352 X = CO,Me, Y = NO, 353 X = NO,. Y = C 0 2 M e

354

356

355

357

Me2C<&

H20H CH,OH

358

NaQ

HO 359

(245) R . Bognir and P. Herczegh, Carbolrydr. R e s . , 54 (1977) 292-294

HO 360

inethyl 2-nitroacrylate and diiiietliyl acetyleiir~clicar1,oxylate The reaction of methyl 2-nitroacryl;tte with furan at r o o m temperatiire gave ii quantitative yield of the regioisoiiiers 352 and 353, which were then hydroxylated, to give the pair of (Jvo-ci.vdiols354 a i i d 355, separable 1)y recrystallization. Treatinviit of'the isopropylidene acctal o1)tainetl from diol 354 with diazabicyc~lo[ij.~.O]rinclec-S-c~~i~~ (IIRU) gave a high yield of the alkene 356, wliic~liW;IS convertecl into the ozoiiicle 357. Hediiction of the ozonide, 01. of the product (358) of its reaction with climethyl sulfide, with sotliiitii 1)oroliydride g a v e a uiixtrire of'tlie epiineric triols 359, which, after cleavage with periodate, afforded'-"' 2,5arihydro3,4-0-isopropylidcirc~-1>1.-allose (360) i n 1S% yie!d, based 011 the inethyl 2-nitroacrylate r i s c d . T h e same allose derivative \vets 01)tained from the Diels-Alder titltliict (361) of fiiran with ditiiethyl ;icetylenedicarboxylate. Selecti\ze Iiytlroxylation of 361, follonred 1)y acetonation, gave dinic~tliyl 5,6-C)-isopr~~pylidei~e-7-0sal,icyclo[2.2.l]hept-2-ene-cxo-5,6-tliol-2,:3-dicarl~ox~l~ite (362), from which a quantitative yield of the \tal)le, ciystalliiie ozonicle 363 w a s 011tained. The ozonide was retlricetl with lithiiini aluniinum hydride to tetraol 364, which was repoi-tcd to give 360 o n periodate Careful reinvestigation of' these reactions rcvea1ed"l' that reduction of ozonide 363 with lithiinn ;tlriiriiiium hydridc produces trio1 359, in addition to the tetraol 364. To account for the loss of a skeletal carlion atom during the reduction, the thernid rearrangement of ozonitle 363 was studied. On heating i:i various solvents, compound 363 gave mainly the stable oxalate 365, a n d , presumably, the mixed anhydride 366 (not isolated), from which thtl keto ester 367 was obtained on hydrolysis. Hydrogenation of owlate 365 in the presence of a palladium catalyst, and subsequent riwthanolysis, gave two products, namely, methyl (methyl 2 , 3 - 0 - i s o p r o ~ ~ ~ ~ l i d e 1 ~ e - ~ - ~ ~ - t a l o f u l . a r ~ o s i d ) u r o n a (368; 6770) and the isomeric (1110 compound (369; 22%)).Configurational assignments were basctl mi the transforniation of the major rednction-product (368) into thc corresponding mcthyl 2,3-O-isopropyliclenehexofuranoside, which proved not to he identical with an authentic sample of' methyl 2 , 3 - O - i s o p r o p y I i d e i i e - ~ - ~ - ~ t l l o f i i r ~ ~ i i ~ ~ s i d e ; however, the two compountls gave' the saine aldehyde upon periodate cleavage.24xTogether with ii tiill accorint of the synthesis of the anhydroallose derivative 360, sevvral new derivatives thereof' have heen (246) G . Just and A. Martel, Tetrrihdi-ori I , e t t . (1973)1517- 1520. (247) G. Just and K. Groziiiger, T ~ t i ~ i l ~ t / Lett. i - ~ ~ (1974) i i 4165-4168. (248) G. Just and K. Grozinger, Ctrri.J. C / t ~ t t t .53 , (1975) 2701-2706. (249) G. Just, A. .Ifartel, K. Grozinyc>r,a i i d X I . Hanijjeesingh, Carl. / . L ' h e i ~ i , 5, 3 (1975) 131-137.

ALEKSANDEH Z A M O J S K I rt

76

(11.

Me2C<& 0

C0,Me

b,Me

362

361

C0,Me I C=O

Me0,C I

Me0,C

0

o=c OH 0,

/o CMe,

0, 366 R 367 R

365

/o

364

CMe, = ~

CI1,CO

n

365

I

Hkwe +

MeO,?

Me0,C

H o c w

Treatment of lactol 360 with semicarlxizide or thiosemicarbazide gave the corresponding derivatives 370 (one geometric isomer in each case), whereas a mixture of the s!yt1- and ciiiti-oximes was obtained 011 reaction with hydroxylamine hydrochloride. Compound 360 reacted tol igive o s ~ lthe i o ra$~ne with e t h o x y c a r l ~ o n y l m e t l i y l e ~ ~ e t r i p l ~ e ~ ~ ~ l ~ ~ unsaturated, trczris ester :371 in good yield, but failed to react with nonstabilized Wittig reagents. The reaction of e,S-anhydroallose 360 with

ethyl triphenylphosphoraiiyli(l~.iiel,yruvatedid not give the expected, cy,P-unsaturated, keto ester; i i i s t t d , the intranioleciilar, Michael-udtlition product was obtained."" 'I'licl unstalile aldehyde 373 w a s prepared24gfrom 360 by way of osuzolidine 372. Syntheses oftlie following C -nucleoside analogs, 1) ased o n clerivatives of 360, have I)een described: acrylic ester 371 reiicte[l with a n excess of ethereal diuzomethane to give the isomeric pyrwolines 374, which, b y successive t rea time n t with b roin ine and iiic t h i 11o I i c am iiio 11ia, afforded cry s t a1 1i ne ~ - ( c a r b o x a n ~ i d o ) ~ - ~ - D L - r i ~ ~ o t ~ ~ r ; i ~ (375); i o s ~ ~ seinicar~~azone ~pyrazo~e 370 was cyclized to 2-aiiiiiio-~-~-DL-ribofuranosy~-1,.3,4-oxac~iazo~e (376) by treatment with I c d tetrxacetate, and lactol 360 WHS coilveited,25'by w a y of the corresponding allolactone 377, into 3-aniino-5-

P-ribofuranosyl-l,2,4-triazolc (378).

//' 360

1

\

H,N-CO,

co2t':l

NH

I

\o / o

CMe,

370

371

372

377

I

I

I

I

t ROCH

podH0

R30cG9w R'O

374 R' 376

OR'

=

OEt

R2 = >CMc,

373

378

RJ = (Me,V)MvlSi

375 R'

=

NH,

R2 = R3 =

H

(250) G. Just, M. Hainjeesingh, a i d T. Liah, C : U I L . 1. C h c w . , 54 (1976) 2940-2917. (251) G. Just and M. Hanijeesingh, '/'(,I,-CI/IC.C(J-OII I,ett. (1975)985-988.

ALEKSANDER ZAMOJSKI ct

78

(I/

Raceniic analogs of showdomycin have been synthesized from adducts (352 and 353) of inethyl 2-nitroacrylate with furan. Synthesis of ~ ~ - 2 - e ~ ~ i - s h o w d o i i i yinvolved ciii treatiilent of the adducts with nz chloroperoxybenzoic acid, followed b y nitrous acid elimination, to give tlie alkenic epoxide 379. Opening of the oxirane ring, and a sub-

C0,Me

379

sequent ozonolysis-reduction sequence gave, after appropriate protection of the liydroxyl groups of tlie ara1,iiiofuraiiose portion, and oxidation, the key interniediate, namely, the keto ester 380. Compound 380 was then converted into 2-epi-sllowdomycin b y treatment with ( c a r b a i ~ i o y l m e t h y l e n e ) t r i p h e n y l p h o s p eand , also into DL-2-e)iipyrazofuriii A in a reaction with ethyl hydrazinoacetate hydrochloride, followed tiy cyclization under tlie influence of sodium methoxi&'S2

The same, general scheme was applied for the synthesis of DL-2deoxyshowdoiiiyciii. The reaction of the em-nitro adduct 352 with diborane, followed b y oxidation, resulted in the formation of an isomeric mixture of alcohols that could not be separated (neither, after eliinination of nitrous acid, could the corresponding, unsaturated alcohols or acetates). The isomeric alcohols olitained b y hydroboration of adduct 353 were separated as the corresponding acetates, but they could not be assigned configuration at this stage of the synthesis. Recognition of regioisoniers became possible after eliniination of the nitrous acid molecule, due to differences in the 'H-n.m.r. spectra, and the desired, unsaturated acetate 381 was transformed, by way of the ozonide, into the keto ester 382. The reaction of 382 with (carbanioylmethy1ene)triphenylphosphorane, followed b y deprotection of the hydroxyl groups, completed the nine-step synthesis of ~ ~ - 2 - d e o x y s h o w d o m y c i n . ~ ~ ~ CO,Me

o w

Ac()&

Me I MesCSiO- CH, I

Me

RO

381

380 R = SiMe,CMe,

(252) M .Lim, Ph. D. Thesis, hlcCill University, Montreal, 1976. (253) G . Just and M. Lim, C a n . ,I. Clzern., 55 (1977) 2993-2997.

C0,Me

vo I

Ac 0

382

It has been found that 1,.3-di(etlioxycarbonyl)allenereacts readily with such heterocyclic dieneh a s fiiran, pyrrole, and pyrone derivatives. The fiiran adduct 383, o1)tained in 87% yield, was hydroxylated, and after acetonation, was c l c ~ i i ~ e dwith ~ ~ . 'ozone, to afford inteiiiiediate 384. An approach to the coii\,ersion of 384 into C-glycosyl compounds b y hydride-promoted scissioii of the C-5- C-6 bond was disclosed.255 C0,Et

Me2C/@

&-

C0,Et

'0

HC-C0,Et

0

383

384

The known adduct (385) of furan and vinylene carlxmate, previously used for the synthesis of some cyclitols,2S6,2Si h a s been transformed into DL-ribose derivatives. After hydroxylation of 385 and subsequent formation of the isopropylidene derivative, the carlmnate group was removed by treatnient with barium hydroxide, and the resulting diol was cleaved by oxiclation with pennanganate. Dicar1,oxylic acid 386 gave, upon treatment with acetic anhydride, cyclic anhydride 387. The reaction of 387 with azidotriiiiethylsilane produced

385

R02CwNHC02Me

R02CvNC0

0,

387

10

CMe,

388

0,

9

CMe,

389

(254) A. P. Kozikowski, W. C. Floyd, .ind 51. P. Kuniak,]. Chent. Soc. Chrttt. C o m m t r n . (1977) 582-583. (255) A. P. Kozikowski and W. C. Floyd, ?'c.trcr/wdron Lett. (1978) 19-22. (256) Y. K. Yurev and N . S. Zefirov, Zh.O / m / i c / t ,Khini., 31 (1961) 685-686. (257) C. R. Kowarski and S. Sarel,]. O r g . C h e w . , 38 (1973) 117-119.

80

ALEKSANDER ZAMOJSKI et

(I/.

isocyanate 388, and, on treatment with methanol, 388 afforded carbamate 389. The effective resolution of a monoester obtained from anhydride 387 with (R)-l-(2-naphthyl)ethylamine has been achieved.258 A new approach to the general, stereocontrolled synthesis, from furan, of C-nucleosides in which the ribose skeleton is constructed b y use of a polybromoketone-iron carbonyl reaction has been described by N ~ y o r i . The ' ~ ~ starting material, bicyclic ketone 390, was prepared in a cyclocoupling reaction of 1,1,3,3-tetrabroino-2-propaiioiie and furan, promoted with diiron nonacarbonyl, followed by reduction with a zinc-copper couple. The hydroxylation of390 with a catalytic amount of osmium tetraoxide, followed by reaction with acetone, gave a single acetal, namely, 391, in 65% yield. Subsequent Baeyer-Villiger oxidation with trifluoroperoxyacetic acid afforded lactone 392, from which the dimethylaminomethylenelactone 393, a common intermediate for the synthesis of natural, pyrimidine C-nucleosides and their analogs, was Alternatively, lactone 392 was condensed with 2-fiiraldehyde, and the aldol adduct was dehydrated to give 394. Treatment of 394 with methanolic sodium methoxide afforded the methyl ester 395, which, after (trialkylsilyl)ation, was transformed by ozonolysis into the unstable keto ester 396. Compound 396 was converted into showdomycin, of a,a'-dibroas well as into some 6 - a z a p s e ~ d o u r i d i n e s A . ~ number ~~ moketones react262with furan, to give substituted analogs of the bicyclic ketone 390. Appropriately substituted substrates have been converted,2fi3by way of Baeyer-Villiger oxidation and treatment of the resulting lactone with tert-butoxybis(dimethylarnino)methane, into pseudouridines 397 modified at C -5.

3. 2,3-Dihydrofurans A novel synthesis of branched-chain monosaccharides was based on the finding that 3-furoic acid readily undergoes Birch reduction, affording 2,3-dihydro-3-furoic acid. Treatment of its methyl ester 398 with methanol and acid gave,2mi n quantitative yield, methyl tetrahydro-5-methoxy-3-furoate 399 as a mixture of the isomers. Compounds (258) R. R. Schmidt and A. lieberknecht, Arigeiu. C h e m . , 90 (1978) 821-822. (259) R. Noyori, Acc. Cheni. Res., 12 (1979) 61-66. (260) R. Noyori, T. Sato, and Y. Hayakawa,J.A m . Chem. Soc., 100 (1978) 2561-2563. (261) T. Sato, R. Ito, Y. Hayakawa, and R. Noyori, Tetrahedron L e t t . (1978) 1829-1932. (262) R. Noyori, S. hlakino, T. Okita, and Y. Hayakawa,J. Org. Chem., 40 (1975) 806807. (263) T. Sato, M. Watanabe, and R. Noyori, Tetrahedron Lett. (1978) 4403-4406. (264) T. Kinoshita, K. Miyai-lo, and T. Miwa, Bull. Chem. SOC. Jpn.,48 (1975) 18651867.

SUGARS FROM NON-(:AHHOHYLIRATE SLJHSTK.4TES

81

390 391

C0,M.x

Me

I

393 X = CHNM,,, 394 x = C H - 0 - u r y l

C0,Me

396

0

397

399 sewed as i he substrate in ;i synthesis of raceniic apiose. Bronnination of 399, followed by deliytllol~romination,resulted in fonnatioii of the 2,5-dihydr:)furan derivative 400 which, b y successive hytlroxylation, isoprop!rlidenation, a n c l reduction, gave methyl 3-C-(hydroxy-

398

399

400 R = H 402 R = M e

4 01

ALEKSANDER ZAMOJSKI et

82

(I/

m e t h y ~ ) - 2 , 3 - O - i s o p r o p y l i ~ e i i e - ~ - D L - e r y t h o s i d (401). e This compound was identified b y comparing its physical constants with those of a sample of 1,2:3,3'-di-O-isopropyIidene-[3-C-(hydroxy~ n e t h y ~ ) - a - D - e r y t h r o f u r ~ t r i o sprepared e ] ~ ~ ' ~ from 1,2-0-isopropylidenea-D-erythrofuranose. DL-Dihydrostreptose and its r-ibo isomer were similarly obtained. Birch reduction of 2-methyl-3-furoic acid, followed by addition of methanol, bromination, and dehydrobrominatioii, gave 402 a s a mixture of the isomers. Hydroxylation of 402 with osmium tetraoxide-sodiuin chlorate, and subsequent treatment with acetone-sulfuric acid afforded three isomeric acetals (403-405). The structures of these compounds were assigned on the basis of their 'H-n.m.r. spectra. In addition, the relationship between 403 and 404 was established h y hydrolysis and reglycosidation. The methyl esters 403-405 were quantitatively reduced to the corresponding alcohols. The mixture of alcohols obtained from 403 arid 404 was converted into crystalline 5-deoxy-3-C-(hydroxymethyl)-l,2-O-isopropyliclene-c~-u~-ribofuranose (406),which was compared directly with a sample prepared from D-xylose. Methyl 5-deoxy-3-C-(hydroxyrnethyl)-2,3-0-isopropylidenep-DL-Iyxofuranoside (407),obtained by reduction of 405 with lithium aluminum hydride, was hydrolyzed with dilute hydrochloric acid, to give a,P-DL-dihydrostreptose.~lj~;

0,

/o

0,

CMe,

403

/o

0,

CMe,

/o

CMe,

405

404

H3 c

HO

0-CMe, 406

407

Another synthesis of' streptose derivatives has been completed in the authors' laboratory. Among a number of' oxetanes obtained from (265) T. Kinoshita and T. Miwa, Curbohydr.Re.s., 28 (1973) 175- 179. (266) T. Kinoshita and T. Miwa, B u l l . Clieni. Soc. J l ~ n . ,51 (1978) 225-228

furan a i d carb m y 1 compounds l)y a Paterno-Biichi reaction, and used as inteiiiiediates in t h e prcxpmitioii of 3-sul,stituted fiirana,"" 6iiiethyl-2,7-dicxal~icyclo[3.2.O]licpt-3-eiie (408) s e e m e d i i i i appropriate substrate for t h e synthesis of 3-deoxystreptosr. Iiideetl, hydroxylation of 408 v.ith potassium ~~eriiiaiigariate, followed b y reaction of the resulting i iol with acetoil(,, ga\ c' two isopropyl i d e n e acetals, itlentified as 409 k.nd 410. The con figtiration of' the niaiii product (410), assigned on t:ie basis of '€I-ii.m.l.-sl,ectral duta, was confinntrd b y conversion i n to 3,5-dideox>.-I ,2-O-i so~~rog)..liclene-3-C -1nethyl-P-1>~arabinofurano:,e 412 ( b y way ot'thc clihydro deri\.ative 411, and R a n e y nickel reduction of t h e corrc)spontliiig iodo coinpound), earliei- ohtained from tk e natural inoiiosaccli~iri(1~~. The tr-ci ri.s-h>~dros)latiollof 408 b y epoxichtion with ni-cl ~ l o r o ~ ~ e r o s ~ l ~ e acid i i z o wi ca s also exiiiiiined."jx

CH, 409

408

H

3

C

!

T

@-CMe, 410 X 411 X 412 X

= = =

CIIO CH,OH CH,

The use of t c trahydrofuryl tlerivatives for synthesis of racemic sugar derivatives ha:; also b e e n reported. I t was found that iodine tris(trifluoroacetate) oxidizes tetrahytlrofiiryl trifluoroacetate, to yield a mixture of four d i ;is t e re oiiie ric 3-(1e o x spe 11tofu raiio s e s (413- 4 16) in t h e ratios of 25 : 15 : 4 : 6. The maiii procluct, 3-deoxy-tlireo-pentofiir~~iiose tris(trifli1oroacl:tate) (413), readily crystallized out o f t h e reaction mixture .269 (267) A. Zamojski ;ind T. Koiluk,J. O j g . C/ivrri., 42 (1977) 1088-1090. (268) T. Koiluk, Ph.D. Thesis, Institute of Organic Cheinistry, Polish .4cadem). of Sciences, Wars;.w, 1978. (269) J. Buddrus and H. Herzog, C / i f l r t i . Uer.., 112 (1979) 1260-1266.

ALEKSANDEH ZAhlOJSKI r t

84

413

414

(I/

415

416

x = COCF,

V. SYNTHESES FROM VINYLENE CARBONATE 1,2-Ethenecliyl carbonate (1,3-dioxol-2-one; viiiylene carbonate,

417) is a readily available,270versatile synthon having pronounced dieiiophilic p r o p e i - t i e ~ . ~ Diels~ " - ~ ~Alder ~ adducts of 417 with 1,4-diacetoxy-l,3-butadiene and furan were selectively converted into cylito] ,256.257 2 7 5 and also served as precursors of DL-ribose derivativeP8 (see Section IV, 2). Another possibility of applying 417 as an equivalent of a 1,e-dihydroxyethane unit has been demonstrated in a synthesis of raceinic apiose. Photochemical cycloaddition of 417 to 1,3-diacetoxy-2-propanone (418) gave the oxetaiie derivative 419, which, on alkaline hydrolysis, afforded DL-apiose (420) in 23% yield.'

417

410

419

419

-

I

HOH,C--C-CH,OH HO 420

A fundamentally different approach to the total synthesis of monosaccharide derivatives from 417, based on the telomerization principle, was elaborated by Kuiiieda and coworker^.^^^,^^^ These authors (270) M . S. Newinan and H. W. Addor,J. ,4m.Cliem. SOC., 75 (1953) 1263-1264; 77 (1955)3789-3793. (271) H. Kwart and W. G. Vosburgh,]. A m . Cherti. Soc., 76 (1954) 5400-5403. (272) J. B. Lambert antl A. G. Holconib,J. A m . Cliena. Soc., 93 (1971) 3952-3956. (273) M. Z . Haq,]. Org. Clwni., 37 (1972) 3015-3019. (274) J. Daub, U . Erhardt, antl V. Trantz, C h e m . Bcr., 109 (1976) 2197-2207; J. Daub and V. Trantz, Teti-nliedroii Lett. (1970) 3265-3268. (275) R. Criegee and P. Becher, C h e m . Bcr., 90 (1957) 2516-2521. (276) T. Tamura, T. Kunieda, and T. Takizawa, Tetruhetli-on Lett. (1972) 2219-2222. (277) T. Kunieda and T. Takizawa, Yuki C h e i Kogcikir Kyokai S h i , 33 (1975)560-571; Cheni. Alwtr-., 83 (1975) 177,527,.

found that carbonate 417 is capable of undergoing ready reaction with polylialogenoriiethanes, in thc. presence of berizoyl peroxide or azobisisobiitanonitrile, under a nitrogen atmosphere, to give teloiners of the type of421. A compreheiisi\.e rc3view on various synthetic applications of these coiiipoiiiids h a s ; ~ p p ~ e d . ~ ~ ~

417

421 R = CC1,. C R r , , CH,Br. i ) r CHBr, X H . C1. 111‘ R r t2 = 1. 2 ur 3 2

The lower telomers (ti = 1-:3) \\‘ere extracted froni tlie mixture o f products with c~ichlorometliaiie,a r i d separated b y coliiinii chroniatography. The distribution of tlie teloiiiers was f o i ~ i i d to ~ ’ depend ~ on the kind of polylialogenoniethane i i s e t l , ;is well as on the ratio of the reagents. For exainple, when four eclriivdents o f bromotrichloroiiietliarie reacted with 1 mole of 417, c‘onipound 429 could I x isolated in 92% yield, whereas reaction with five rnolar proportions of c a r l ~ o ntetrachloride produced a mixtiirc containing 20.4%’ of 4-cliloro-5-trichloromethyl)-1,3-dioxol-2-one (422, I I = l), 1870of telomers 422 ( 1 1 = 2), and 6% of the corresponding coiiipounds containing three tlioxolane units (422,n = 3 ) .These :ire' tlie highest yields reported for I I = 2 and rt = 3 telomers. As a rille, teloiners 421 contain a teriiiiiial c d i o n atom bearing halogen and o x y g e i i siilistituents, in aii arrangement equivalent to an aldehyde groiip. Alternatively, the R group coiitainiiig two or three halogen atonis could be converted2ixinto C-1 of an aldose. The synthetic utility of’ t h e 1,3-dioxol-2-oiie-li~~Iogeiiomethaiie telorners as intermediates i n the synthesis of racemic inoiios~ccharides is further enhanced I)!. tht, high stereoselectivity of their formation. Carlion tetrachloride, chlorotoriri, clibromoiiiethniie, trilironioniethan e , tr il)roiii oc h 1orome t hail cl, 11 I 1 d t c‘ t rab rornoiiie t hane we r e e xai 11i n e (1 as te 1ogen s , and 11 ro mom e t 11 ;LI i e s we re foii lid zxO to foI 1ow reaction pathways inore complicated tlian those for chlorinated compounds. Thus, chlorofoiiii reacts exclusively b y hydrogeii transfer, giving rise to 423 and a small proportion of higher telomers whereas, for dibromo(278)T. Kunieda and T.Takizawa, f f ( . / o - o c , ! / c . l c 8 . , ,(1977)661-694. (279)T.Tamura, T.Kunieda, and T. ‘l‘akiz;iwa,/.Org. C l i c ~ i i i . ,39 (1974)38-44. (280) K. Hosatla, T.Kunieda,and ‘I. ‘l’cikiu\v;i,C / i ( > i t i . P l i c i i ~ i i i .B i i / / . , 24 (1.976) 2.9272933.

ALEKSANDER ZAMOJSKI e t ol.

86

methane, abstraction of a hydrogen or a bromine atom is equally proliable; this results in formation of two series of telomers (424, 425, and their t i = 2 analogs). Tribroinoethane likewise reacts by bromine- or b y hydrogen-transfer, to give 425 and 426. Both triliroino- and tetra-

H

CHBr,

H

H

A

425 n = l ( n = 2 )

424n=l(n=2)

417

+ 426 n

426

+

429 n

=

1

(427 f 428)

=

1

Scheme 5

oToBroKo 0

0

427, 428

lironio-nietliane gave,’x0 adtlitionally, “two-fold addition” products (427, 428) arising from 1,3-real.1.~iirgeiireiitof the intermediate radical. The same isomeric mixture ot‘ 427 and 428 WQS ol,tained2x0i n high yield from the reaction of417 wit11 426. Only one stereoisomer has Iwen ol)tained for each constitutionally different 421 (n = 1).Out of eight stereoisomers possible for I I = 2, only two sterically different tc.loinc.1-s could be isolated front reactions of 417 with polyhaloniethaiies. Examination of the 422 ( 1 1 = 3 ) fraction of the teloniers resulted i i r isolation of foitr isomeric compounds oiit of the 32 possible.27xThe liiglr selectivity o1)served in these reactions is explained by the itrfitteircc of steric effects and a strong tendency for trcins-addition cliiriiig nitlical telomerization.2x’ tr-(irisStereocheniistry of the l o w c ~ tctloniers w a s sii1)htantiatetl b y the small coupling-constants (I 2.0 IHz) of tlie vicinal protons of the c x h n a t e rings, consistently for the 421 ( 1 1 = 1-3) compounc!s. Additionally, it has been shown that the difference in spatial arrangement of the stereoisomers cont:iining two dioxolane units is litnitetl to the mutual dispositon of the carlioirate rings, ;is the two isomers give the saiiie acyclic pheny1liydr;izoite on reaction with (2,4-ditiitropheiiyl)liydrazine, or the siitiit’ monocyclic enol phosphate on treatment with trialkyl phospliitP2 (see Schenie 6). I t was therefore a s R

R

I? = CC1,.

X R’

1

R

CHRr,

C1, B r

M e , Et

Scheme 6

sumecl that telomers obtained in it free-radical process have all-trtins geonietry, the carbonate rings Ileing joined in ‘‘syt~” (for example, 440 or 441) or “anti” (442 or 443) manner. Ionic addition of methanol or ethanethiol to 417 has also I)reti reported. The telomers obtained in this way, containing two dioxolmie units, were fouiidzX:’to consist mainly of the “cis -syn” isonier, leading to DL-erythrose on hydrolysis. (281) C. Walling, Free Radiccil.s iir S o l r r t i o i i , Wile);, N e w York, 1966, p. 201. (282) N . Mitsuo, T. Kunieda, arid T. ‘Takizawa, C/ieni. P / w i . r r t . / 3 1 i ! / . , 25 (1977) 231-238. (283) T. Kunieda, Y. Abe, S. Sanri, m i d T. Takizawa, Iletc.i-oc!/c/e.c,12 ( 1979) 18:3.

ALEKSANDER ZAMOJSKI et

88

(11.

It has been foundzlc4that the trichloromethyl group in the 422 type of teloniers may b e selectively converted into a dichloromethyl group by reductive photolysis. Alternatively, tri- and di-halogenomethyl groups may be selectively dehalogenated under veiy mild conditions by treatment with nickel t e t r a c a r b ~ n y l .On ~~~ irradiation with a highpressure, mercury-vapor lamp, the readily available 422 gave the dichloromethyl compound 430, which was converted2X6(although in poor yield) into DL-glyceraldehyde by consecutive treatment with so-

E>o-

/-> CHC1,

0

c1

c1

422

422

430

DL-

CHO I

HCOH

CH,OH I

Glyceraldehyde

-c13c7--foH .Yo

-

HO-C-CC1, H 431

430

-

C1,HC

HoH

O HO

H

0 CHC1,

432

dium borohydride and aqueous silver nitrate. Compound 422 is hyd r o l y ~ e d ”on ~ dissolving in water, giving a high yield of 3,3,3-trichloro-3-deoxy-~~-glyceraldehyde, which dimerizes selectively to the five-membered-ring cornpound 431, characterized as the corre(284) N . Mitsuo, T. Kunieda, and T. Takizawa,]. Ot-g. Chem., 38 (1973) 2255-2257. (285) T. Kunieda, T. Tamura, and T. Takizawa,]. Cheiri. SOC.Cheni. Cotnmuri. (1972) 885-886; Cheiti. P h a n n . Bull., 25 (1977) 1749-1755. (286) H . Takahata, T. Kunietla, and T. Takizawa, Cheni. PlzcrrJti. B u l l . , 23 (1975) 30173026. (287) T. Matsuura, T. Kuniecla, a n d T. Takizawa, Chem. P l a c i n ~ z .B u l l . , 25 (1977) 239245.

SUGARS FROM NOK-(:BRROHYURATE SUUS'I'HAI'ES

89

sponding diacetate. Under siiniltrr cond.itions, the dichloroniethyl compound 430 affords the six-iiieinl,ered-ring dirner 432. On the other hand, treatment of 422 with rriethaiiol gives the dimethyl acetal 433 in high yield. The isomeric compountls 427, 428 also readily undergo methanolysis, and afford2xncyclic acetals 4.34 in high y i e l d . Small proportions (5-9%) of the sulxtitiition products 435 were also isolated. Methylation of 434 gave crystalline 436, compounds that were, i n turn, hydrolyzed to 437. Analysis of 'H-n.m.r. spectra of the cyclic

GOH

Me0 C~,C-CH-CH(OCH,), I

Me O'C0,

OH

OC0,Me

Rr 433

434

-

M o*M e,oe

Ko". 'To 0

0

427,428

435

Me0

MeOC02QOMe

H

B~

r

b

O

Br

OCO,M(?

436

M

e

OH

437

heiniacetals 436a, 4361, allowcd the assignment of configuration to the teloiiiers 427 and 428. Photoreduction of the g e m -dibronio group, and nucleophilic displacement of the secondary I,romine atom in "twofold addition" products, have also Iieen d e s c r i l d . 2 x ' 1

Br Br 0

42 7

43th

ALEKSANIIEH ZAMOJSKI e t ol

90

Me?

,o

I

OMe

Br 436 b

0

Br

428

Owing to a sufficient reactivity of the secondary halogen atoms towards nucleophiles, the telomers 421 ( n = 1)have also been transformed286into tetroses. Reaction of430 with sodium cyanide, induced b y a phase-transfer catalyst, afforded truris-438 and cis-439 nitriles in

DL-Threose HO

CN

CH,OH

438

CHCI,

-

o~-Erythrose

HO 439

equal amounts. After separation, these compounds were converted into DL-threose and DL-erythrose, respectively, by the sequence of reactions involving esterification, hydride reduction, and hydrolysis of the dichloromethyl group in the presence of silver nitrate. The telomers (424,425, n = 2 ) obtained from 417 and dibromomethane were directly correlated with natural monosaccharide derivative^.^^^-^^^ Compounds 440 and 442 were selectively reduced with nickel tetracarboiiyl to 441 and 443, which, after mild hydrolysis, afforded 5bromo-5-deoxy-DL-lyxose (444) and 5-broino-5-deoxy-~~-xylose (445), respectively. Authentic specimens of these derivatives were prepared, for comparison, from D-mannose and D-xylose by way of 446 and 447.

91 Br

440 R = CHBr, 441 R = CH,Br

444

446

442 R = CHBr, 443 R = CH,Br

445

447

Telomers containing a trichloroniethyl group (422, n = 2) were converted"x6 into raceinic aldopciitoses in bwo steps. After separation, compounds 448 and 450 were rediiced photochemically to the corresponding dichloromethyl derivatives 449 and 451, which were hydrolyzed with aqueous silver nitrate to DL-arabinose (in 56% yield) and DL-xylose (54%), respectively. Analogously, the sanie pentoses were obtained from chloroforin-derived teloiriers (423, n = 2). A variety of

?

cc4

cc13

, OMe

H0

- H HO O T c H 0

c1 452

448 R = C C 1 3 449 R = CHC1,

R

J

-

-

o

__f

o

~

-

Me0

0 0 c1 450 R = CC1, 451 R = CHC1,

454

c

c

l

~

/ j C C 1 3

HOC

Me0 455 453

92

ALEKSANDER ZAMOJSKI e t

(I/

pentose derivatives coiitaining a terminal trichloromethyl group have been o b t a i n e P 7 from the aforeiiientioned telomers. Reaction of 448 and 450 with methanol afforded the corresponding diiiiethyl acetals (452 and 453) in quantitative yield. Removal of the remaining carbonate ring b y the action of sodium borohydride or triniethylaniine, followed by treatment with a cation-exchange resin, gave S,S,5-trichloro5-deoxy-DL-lyxose (454) and S,S,S-trichloro-S-deoxy-DL-xylose (455) in 83 and 96% overall yield, respectively. Direct hydrolysis of teloiiiers 448 and 450 gives less satisf2ictory results, owing to side rextioiis. (Trichloromethyl)alditols, on the other hand, were obtained2X7 directly from the telomers, in high yield, b y reduction with sodium borohydride. On treatment with (2,4-dinitrophenyl)hydrazine, 448 and 450 afforded279the same phenylosazone derivative 456, whereas their reaction with trialkyl phosphites ledzx2 to the same eiiol phosphate 457. Mild, acid hydrolysis of 448 and 450 yieldedzH7 the cyclic dirners 458, which could be further hydrolyzed to the single, dimeric pentos-%dose derivative 459. 448,450

J// H OH

OH

0

NNHAr I1

I I CI,C-C-C-C-CH I I II HO H N N H A r 456

458

I

t

0

457

[T13C-i2-CH2-C-CH0 459

0 I1

I,

Telomers 422 ( n = 2) and other 4-halogeno-1,3-dioxolan-2-ones were shown2x8to react readily with amnionia or primary aliphatic amines, with formation of 4-hydroxy-2-oxazolidones (460). The latter, for which the trans arrangement of the hydrogen atoms of the oxazolidone ring was deduced froin 'H-n.m.r. data, readily underwent replacement of the hyclroxyl group by a phenyl group on reaction with (288) T. Matsuura, T. Kunietla, and T. Takizawa, Cheni. Phurm. B u l l . , 25 (1977) 12251229.

lieiizene, to give 461, or dehytlration under the influence of trifluoroacetic acid, with formation of'46.2. I t has Ixen pointed oiit'xx that analogous traiisfonnations of422 ( t i = 3 ) teloiners would provide a source of C-nucleoside analogs.

, , 7 H

Cl,C,

O K 0 0

CI,C

I'tl

OK 0 "

-

O 0

f

O 0

f

462

461

Synthesis of aldohesoses fl-on1 422 ( t i =: 2) telomers w w achieved b y way of cyanide in t ermedi at t, s . () I I treat in ent with pot :is s iiim c.yauide, each of the dichloroinethyl coinpoilids 449 antl 451 gave"'; d. pair of ep i 111 e ri c i i it ri 1e s ( 463-466) that \v e re ccmve rte d , i n in ode rate y i e 1d , into DL-galactose, DL-altrose, UL-iclose, and D L - ~ ~ I I C O bS y~ coiiventional procedures involving esterification, reduction, antl final h\.tlrolysis of the dichloromethyl group. CH,OH

R

I

nL-

Galactose

I

463 R CN. R' - H 464 R = H ri' = C N ~

YH,OH

Altrose

R

CHO DL-Idose

DL

CHO 465 E? 466 r i

CHO

CN. R' H H. R L = CN ~

nL-Glucose

94

A L E K S A N D E R ZAMOJSKI

tlt a /

Teloiners containing three carbonate units,2xsobtained from carbon tetrachloride and 417, were convertedzs0into heptoses, and, b y further reduction, into the corresponding heptitols. The procedure involved photoreduction of individual telomers 467-470 to the corresponding dichloromethyl compounds 471 -474, which were reduced with sodium borohydride, and the products hydrolyzed with the aid of aqueoils silver nitrate. The four heptoses obtained in this way were, without purification, reduced to alditols, which were identified2s0 ( b y gas-liquid chromatographic comparison with authentic specimens) as DL-gh4ce ro- LD-gdncto-, DL-glycero -DL-gductu-, DL-gl tlcero-DL-ido-, and uL-gll/cero-LD-itlo-heptitols. These results, combined with 'Hn.1ii.r. data for the starting telomers, and the conclusion derived from their reactions with trialkyl phosphites, provided unequivocal proof of the all-trans geometry of the adduct 422 ( i t = 3), and allowed assignment to them of the configurations 467-470. In an alternative route, the most readily available telomer, namely, 468, and its dichloroinethyl analog 472, were treated with methanol in R

c1 467 R = CC1, 471 R = CHCI,

468 472

I

469 4 73

470 474

I

i R

R

,OMe HO 475 R = CC1, 476 R = CHCI,

CHO HO

477 R = CCI, 478 R = CHCL, 479 R = CH,

(289) Y. Nii, T. Kunieda, and T. Takizawa, TetruAedron Lett. (1976) 2323-2326. (290) Y. Nii, T. Kunieda, and T. Takizawa, Chem. Phurm. Bull., 26 (1978) 1999-2006.

the presence of acid, to give tl tc corresponding diniethyl acetals 475 and 476, which were converted'"' into the raceinic 7 - d e o r y - ~ ~ ~ / c c ~ r o gulo-heptoses possessing a teimiin,il trichloromethyl (477), dichloromethyl (478), or, after reduction ~ . i t l an i organotin hytlritle, methyl (479) group. Treatment of the dicl~loromc~tltI\.1 deriviitive 472 with sodium cyanide g ~ an almost e ~quantitative ~ ~ yicxld of the epiineric iiitriles (480, 481). The mixture coitld I r o t l x t wpm-ated b y chroniatogfiiphic methods, but differences i i i rtl;wti\.ity of the stereoisomers towards niethanolic hydrogen chloritlc. allowcd isolation of only one ester, namely, 482; the t r a m cyanitk~rtntlt.r\vent read)- conversion into the conipoiiiid gave a rather complicated methyl ester, whereas the c i . ~ mixture of products, with ainiclt, 483 I)eing identified a s the major coniponent. Reduction of the vster group in 482, followed hydrolysis of the dichloroinethyl group, affortlcd D L - t h r ~ ~ - I ) L - i d ~ - o c t ochar:icse, terized as the heptaacetate, and itleiitifkc1 iis flir-co-itlo-octitol by cornpari s on with an authe 11tic' s iiiii 1)1e .

o>:zL CHCI,

CHO

HO

HO-

0'0'0-

R

HO HO

CH,OH

480 R = CN 482 R = CO,Me

CONH, HO -

HO 481

483

The approach of Kuiiiedii and Takizawa is unique, in that elements of the carbon skeleton of the iiiotiosaccliaride molecule fonn an acyclic frame up to the very final stage o f the s8ynthesis,arid y e t a high degree of selectivity is achieved, I)ecaiise of the all-truns geometry of the starting telomers. On the other h a n d , this situation limits the range of sugars synthesizable by this method. Only half of the aldohexoses,

96

ALEKSANDER ZAMOJSKI e t (11

namely, the gluco, gulucto, (11 tro, and ido, have been synthesized from telomers 463-466. Among the aldopentoses, only ribose could riot be obtained directly from the telomers.

VI. MISCELLANEOUSSYNTHESES The total syntheses of sugars described in Sections 11, 111, and IV demonstrate the general utility of starting materials employed as precursors of various sugars of desired structure. This Section deals with starting compounds that are not generally applicable. Some of these substrates have been used in syntheses directed at one particular sugar. However, most of them provide valuable routes to selected sugars of great importance, among them, amino or thio analogs in which 0-5 in the pyramid ring is replaced b y nitrogen or sulfur.

1. Pyridine Derivatives Some pyridine derivatives have been found by Natsume and Wadaz9*to be suitable starting-materials for the synthesis of isomeric 5-amino-5-deoxypyranoses. Assuming that the substituted l-acyldihydroxypyridiiies are stable291against oxidation agents, polyhydroxy functions could be introduced into the corijugated double-bonds. For the preparation of the antibiotic nojiriinycin (5-amino-5-deoxy-D-glucopyranose), nicotinonitrile was usedzY2 as the starting material. It was transformed, in a photochemical reaction with methanol, into the corresponding derivative (484) of 1,2-dihydropyridine possessing the desired skeleton of nojirimycin. Owing to differences in the reactivity of each double bond of 484 (because of the influence of the 3-cyaiio group), functionalization could be performed stepwise, b y use of the usual cis- or truns-hydroxylation reagents, and then N-broniosuccinimide in methanol, as illustrated in Scheme 7. An attempt to replace the bromine atom in 487 and 488 b y an acetoxyl group by treatment with tetra~utylaminoniuminiacetate”:’ failed, and, instead, elimination of bromohydrin acetate took place, leading to the unsaturated compound 489, whose cyanohydrin acetate grouping was transformed into an equatorial hydroxyl group b y reduction with sodium borohydride. The reaction was performed on the deacetylated compound, to ascertain if a ketone was fornied as an intermediate. The niethylnojiriinyciii obtained according to Scheme 7 was characterized as its N-benzoyl derivative 493. (291) M. Natsurne a n d M. Wada,Ahstr. PO^. S y i i i ) ~ Prog. . S!ynth. Recict., 1st (1974) 135. (292) M. Natsuine and M. Wada, Chern. Phcirin. H d I . , 23 (1975) 2567-2572. (293) M. Sakai, Tetrcihetlroii Lett. (1973) 347-350.

SUGARS FROM NON-(:ARHC)HYDRATE SUBSTRATES

97

QIMe

BzOCH,

ArO CN

CN

AcO 487

486

CN

489 C N

4aa

i

,CO,Me

OAc 494

CN

OR 491 R = R ’ = AC 492 R = Ac, R’ = Bz 493 I< = H. R ‘ = BZ

490

S( h e m e 7

By an analogous reaction sequence, 5-amino-5-cleoxyxylopyrnnose derivative 494 was prepared’!’l using 3-cyano-1,2-dihydro-N-(methoxycarbony1)pyridine as startiiig imiterial. Based on 484, synthesis of the stereoisomeric methyl 5-benzaniido, 24 (1976) 2651-2656. (294) M . Natsume and M. Wada, Clivr,r P h ( i ~ - mBull.,

98

AL,EKSANDEH ZAMOJSKI e t

(I/.

5-deoxyidopyranoside 501 was also achieved.295The k e y intermediate, 496, was obtained stereospecifically (7370yield) on heating 484 with N-bromosucciniinide in acetic acid. It was proved that the reaction proceeds through the 3,4-unsaturated 5-bromo derivative 495, which reacted with acetic acid to give 496. In order to obtain the epoxide 498, the acetyl group in 496 was selectively hydrolyzed with perchloric acid, and the resulting h o m o derivative 497 was treated with silver oxide, giviug 498. Coinpound 498 underwent acetylation, furnishing 499, the labile 1-0-acetyl group in which was replaced by a methoxyl group to give 500. Subsequent, stereoselective cis-hydroxylation, and conversion of the cyanohydrin acetate grouping as already described, led to compound 501, having the ido configuration.

CN

CN 498

1. Ac,O-AcONa 2. MeOH- p T s O H

CN

CN 499 R = Ac 500 R = M e

495

no 501

(295) M. Natsurne and M. W7ada, Chern. Phunn. Bull., 24 (1976) 2657-2660.

Application of dihydropyritline derivatives to the synthesis of 5amino sugars proved to lie ver)’ sutczssfiil when, in a photo-oxidation reaction, the singlet-oxygen, 1,4-adduct 503 was obtained.’s6 Due to its high reactivity, this new coiiipound, having the functionality of N metlioxycarbonylamine perosicle, offers the possibility of stereoselective introduction of various nricleophiles into tlie tetrahydropyridine ring. On the basis of careful stritlies, it was estnl,lished2s(ithat, for successful ring-opening of the e,ido-epoxitle, the simultaneous presence of an acid, a reducing agent, and a nucleophile is necessary. Thiol compounds were found to be itleal reagents possessing all of these requiremeiits in one molecule. Thus, “one-flask” photo-oxidation reaction of 502 with an excess oflirnzeiiethiol in dichloromethane at ii low temperature, followed b y treatment with p-tolaeriesulfonic acid, resulted in the formation of a 7 5 : 1 mixture of 504 and 505. On cis-hy-

I

I

C0,Me

CO,Mr

502

503

i(;J

,CO,Me

R,oQ

* HO

Ac 0

OR‘ 506

504 ,CO,Me

505

OM e

___)

-~

R’O

<

C0,Me

,CO,Me

AcO

OAc 508

C0,Me

507

,CO,Me

509

R = Ph R‘= H,Ac

(296) M. Natsume, Y. Sekine, and H. Soyagiml, <:hem.P h u r m . Bull., 26 (1958)21882 197.

ALEKSANDER ZAMOJSKI et (11

100

droxylation with osmiiiin tetraoxide in pyridine, these compounds yielded, stereospecifically, 506 and 507, respectively. Nucleophilic replacement of the phenylthio group by a inethoxyl group, facilated by the vicinal nitrogen atom, was effected with N-bromosuccinimide in methanol in the presence of silver nitrate, thus leading to methyl 2,3,4-triO-acetyl-5-ainino-5-deoxy-5-N - (niethoxycarbony 1)-ribopyranoside (508) and methyl 2,3,4-tri-O-acetyl-5-amino-5-deoxy-5-N-(111ethoxycarbony1)-lyxopyranoside (509) in high yield. Further extension of this approach the l,4-singlet-oxygen adduct (510) of the 3-cyano-1,2-dihydropyridine derivative 484, which was transformed into methyl 5-amino-5-N-benzoyl-kkoxy-DLmcN

BzOH,C

0

- eM: BzOCH, >(

Ac 0

RO

I BZ

CN

510

e

Bz OCH,

AcO

511

OAc 513

BzOCH,

CN

HO

512

514

H zo

OMe

HO

HOCH, @Bz

515

OMe

HOCH, oBz

OMe

HO HO

OH 516

HO

HO 517

OH 518

Scheme 8

(297) M. Nataume, M . Wada, a n d M. Ogawa,Chcni. Phcirni. Bull., 26 (1978) 3364-3372.

allopyranoside (516), 5-ami I 10-5-h7 - I ) enzo y l - 5 - d e o x y - D L - a l t r o ~~~, r ~n o side (517), and 5 - a n i i i i o - T i - h 7 - l ~ e i ~ ~ o ~ ~ l - 4 , 5 - ~ l i ~ l e o ~ ~ - r ~ ~ ~ ~ - h e x o p side (518) in the respective ratios o f 2 :2 : 1, b y a three-step procedure. The most important, oxygeii-ring-openiiig stage for 510 was completed b y the use of dimeth,,l sulfide in methaiiol as the reducing agent, in the presence of p-tolrienesulfoiiic acid. Compound 511, thus obtained in 69% yield, was separated from its byproduct 512 (970)by recrystallization of its acetyl derivative. Successful, stereoselective cis-hydroxylation to 513 (72%’),followed by conversion of‘ the cyanohydrin acetate into a hydroxyl groiip, as illustrated i n Scheme 8, allowed isolation of sugars 516, 517, ant1 518 i n 70% yield.

2. Esters of 3,4-Thiolanediol 1-Oxides The discovery that replaceinent i n sugars of the ring-oxygen atom h y a sulfur atom markedly affects their biological properties, making some of them active against ti~niors,”~ prompted elaboration of synthetic methods for the preparation of thio sugars containing the sulfur atom in the ring from non-cal-l,oh!.drate~rlj~jh,,[lrateprecursors also. McConnick and McElhinney realized the synthesis of various derivatives of 4th iofu ranos e s 299 and 5-thio pe n to p y raiio se s ,30‘1 and s onie of their branched-chain analogs. It wab found that the anomeric center of all of these compounds is reactive towards nucleophiles, leading to glycosides,”] l-thioglycosides?”’ aiid iiucleosides.“00.:’02 The key step in the synthesis consists in a Pumnierel- rearraiigei~~ent”~ of the appropriate, cyclic hydroxy sulfoxides on heating in acetic anhydride or benzene. The yield and stereochemical course of this reaction are strongly dependent on the type of‘group protecting the hydroxyl group of the hydroxy sulfoxide, as well as on the configuration, and do not depend on the configuration of the oxide function (cis or t r u m in relation to the sul,stituents). In general, the rearrangement of the sulfoxide cis-diesters 519 and 520 leads to cis-triesters 522 i n 40-60% yield, independent of the configuration of the S-oxide function (cis, 520, or trans, 519). Obviously, a certain proportion of the truns isomer is also formed. The proportion of trci 11.9 isomer 524 may lie unpredictably M . Bobek, A. Bloch, R. Parthasaratliy, and 11. L. Whistler,]. M a d . Cham. 18 (1975) 784- 787. J . E. McCorniick and R. S. McElhinriey]. Chenr. Soc., Chern. Corrrnri~ir..(1969) 171-172;J. Clzem. Soc., Pcrkirr ’I’rum. 1 (1976)2533-2540. J . E. McCormick and R. S. hlcEltrinney,./. Cheni. Re.r. S (1979) 52-5:3. J . E. McCorniick and R. S. McElhinney,]. Chem. Soc. Perkirr T w i t , % I. 1.1978)6470. J . E. McCormick and R. S. M(~Elhiiiney,./. Cherrr. S O C . Perkill l ’ r ~ ~ r 1r , ~(1978) . 500-505.

ALEKSANDEH ZAMOJSKI e t u l

102

large in the case of carbonate 519d, 520d; for the phenylboronic esters 519e, 520e, the trcins isomer 525 is the exclusive product of the reaction. The rearrangement of the trans sulfoxides 521a-c is evidently less stereoselective, although their reactivity is of the same order. For example, from the di-(1-benzoyl derivative 521c, both possible a#acetate dibenzoates (523) were obtained in the ratio of 1:1, and the mixture could be not separated by chromatography. It is noteworthy that the cis sulfoxide bis(methanesu1fonates) 519-520f are more reactive in the Pummerer rearrangement than the analogous carboxylates 519,52O(a-c), probably giving all-cis products (not separable b y chromatography). In contrast, their tnins isomers 521f were isolated unchanged under analogous conditions; more-vigorous reaction-conditions led to the rearranged products in 8-15% yield. The stereochemical directing-properties of the phenylboronic group were of great importance for preparation of glycosides, l-thioglycosides, and glycosylamines. Thus, the c ~ n d e n s a t i o n of ~ "525 ~ with the appropriate alcohol in the presence of a catalytic amount ofp-toluenesulfonic acid stereoselectively afforded glycosides 527 in high

c) 0

RO

OR

519

0

a R=H b R=Ac C

Q RO

OR

520

Q

R=COPh

d RR = co ef RR RR

= S0,Me BPh

OR

521

Ac

Q O

RO

OR

OR 522

523

X 524 X = CO 525 X = BPh

0\,9 I Ph 526 527 526 529

X X X X

=Br =OR = SR = purinyl o r pyrimidinyl R = alkyl o r a r y l

OBz 530 X = 6-chloropurin-9-yl

SUGARS FROM N O N ( ' A R H O H k 1lH.4IE SCBSTR4TES

103

yield. Displacement by thiols was, however, much less stereoselective, arid both isomers of 528 were formed in almut equal proportions. The condensation could also I ) e achieved b y the use of the stable, crystalline bromide 526, readily avai1al)le hy a Pummerer rearrangement of519 and 520 with trifluoroacetic anhydride and hytlrogen bromide in acetic acid. The broinide 526 was found to be less reactive towards alcohols than the acetate 525. The usefulness of 1)oronates 525 and 526 was demonstrated by satisfictory preparation:'"' of the corresponding 4-thiotetrofuranosyl p-nucleosides 529 of purines arid pyrimidines. The last condensation was catalized b y tirr(1V) chloride. Minor proportions (20%) of the (Y anomer were also forined. All of these results led to the conclusion that the stereoselectivity of glycosylation using the cis-phenylboronic esters decreases in the order 0 > N > S. The same glycosylation reactions performed on the \)enzoyl derivatives 522c and 523c showed that the trcins isomer 523c is again less reactive (14% yield), and the condensation conipletely lucked stereoselectivity, the isomer ratio l)eing 1: 1. Preparation of branched-chain 5-thio-~~-pentopyranose derivatives 532-535 included an intrainolecular, aldol reaction of diacetonyl sul-

531

1. PhB(OH),-C,H,N 2. H,O,-AcOH 3. Ac,O-C,H,

- WoAc 0

Ph 532

531

2. 1. Ac,O-NaOAc H,O,-AcOH

*

M e Ac 0

O

o

A

c

AcOQOAc

53 4

Me 535

AL,EKSANDER ZAMOfSKI e t al.

104

fide, leading to a cyclic hydroxy sulfide, its reduction with sodium borohydride to a mixture of cis and trciiis diols (531),and Punimerer rearrangement of their sulfoxides to give derivatives of l-O-acetyl-3deoxy4-C-methyl-5-thio-~~-erythro-pentopyranose (532), its 2-Cmethyl isomer 533, and their tlireo analogs (534 and 535, respectively). Interestingly, only the boroiiate of the 4-C-methyl derivative 532 condensed with alcohols and purines in the presence of p-toluenesulfonic acid to give the corresponding glycosides and glycosylamines; no glycoside was obtained from the 24-methyl-5-thiopyranoses 533 and 535;the last syntheses were reported only in preliminary form.

3. Ethyl Ethoxyfluoroacetate and Related Compounds In a series of papers, Kent and coworkers reported on the synthesis of the racemic 2-fluorotetritols and 2-fluoropentitols, and related carbohydrates from simple, lion-carliohydrate precursors containing a fluorine atom in the carbon chain. Thus, synthesis of 2-deoxy-2-fluoroDL-erythritol (537)and its threo isomer (538)was accomplished"":' by vigorous reduction of ethyl ethoxalylfluoroacetate (536)with lithium C0,Et

CH,OH

co I

HCF I CO,Et 536

CH,OH

I

I

*

HCOH I HCF

I

CH,OH 537

I

+

HOCH I HCF I CH,OH

-

538

1

CHO HCF I CH,OH 539

aluminum hydride. Isomer 537 was separated from the reaction mixture b y crystallization, mid its structure was deduced from X-ray data. Reduction of 536 with potassium borohydride in methanol led to a mixture of alditols 537 and 538, together with diiiiethyl DL-2-fluoro-3hydroxysuccinate, the product of siniultaneoiis transesterification. Two alditols containing fluorine atoms, namely, 2,4-difluoro-1,3-butanediols, were preparedso0"b y controlled reduction of ethyl 2,4-difluoro-3-oxobutanoate with potassium liorohydride. Higher honiologs having a iioiiterminal fluorine atom were synthesized3n5 b y Claisen condensation of ethyl fluoroacetate with methyl 2,3-0-isopropylidene-~~-glycerate, giving a mixture of the isomeric 2-deoxy-2-fluoro-4,5-O-isopropy~ideiie-~~-3-pentu~osonates (540).On (303) IV. F. Taylor and P. W. Kent,]. Chein. SOC. (1956) 2150-2154. (304) J . E. C . Barnett and P. W. Kent,/. Cheni. Soc. (1963) 2743-2747. (305) P. W. Kent a n d J. E. G . Barnett,]. Cheni. Soc. (1964) 2497-2500.

SUGARS FROM NON-(:AHBC)€IYDRATE SUBSTRATES C0,Et I HCF

-

I

co

I HCO,

I

H,CO' 540

CMe,

105

CH,OH I HCF I HCOH I HCOH

I

H,COH 541

reduction with potassium hydride i i i ethanol, these afforded the corresponding mixture of fluoropentitols. After removal of the isopropylidene group, the erythro and thr-cw isomers were separated b y chromatography. The structure of 2-cieoxy-2-fluoro-DL-ribitol (541) was established on the basis of X-ray data. In the course of studies on the reactivity306of fluoro carbohydrates, the 3,4-isopropylidene acetals of 2-deoxy-2-fluorotetritols were obtained. These react with the Purdie reagents under inild conditions, to give the corresponding O-methyl derivatives. More-vigorous reaction-conditions cause exprilsion of the fluorine atom, leading to 3,4-O-isopropylidene-1,2-di~)-itietliyl-~~-tetritols. Mild oxidation of 2-deoxy-2-fluoro-3,4-O-isopro~~ylidene-~~-erpthritol with Iiarium perinanganate gave methyl 2 - d e o x y - 2 - f l u o r o - ~ ~ - e r y t l i r o n ~readily ~te, convertible into the correspondiiig amicle by treatment with aminonia in methanol. Periodate oxidation of both tetritols 537 and 538 afforded 2-deoxy-2-fluoro-~~-glyceraldehyde (539). 4. Nitro Alcohols

A simple, interesting method of synthesis of aminopolydeoxy7 " ~approach is sugars has been elaborated b y a Jupanese g r o ~ p . " ~ ~The based on versatile, nitrogeii-containing staiting-materi~tls, namely, nitro alcohols, Thus, 2-nitroethanol, l-nitro-2-propano1, and l-nitro-2butanol react with acrylaldehyde in the presence of diethylamineformic acid (1:1.75 mol) with spontaneous cyclization, to give anomeric mixtures of 4-nitro-~~-pento-(542), -hexo-(543), and -1ieptopyraiioses (544) in reasonable yields. The steric course of the cyclization was found to be depentlc.nt on the kind of nitro alcohol used; 1nitro-2-propanol furnishes a- and p-anomeric, enythr-o isoiners (4 :3), whereas l-nitro-2-butanol affords iiii aiioineric mixture of both the er!lth-o and the threo isomers. Glycosidation of nitro sugars 542-544 with methanol catalyzed by hydrochloric acid gave an cr,p-anomeric mixture of the corresponding methyl glycosides 542-547, separable (306) R . Cherry and P. W. KelIt,j. C/IC,III. Soc. (1962) 2507-2509. (307) S. Zen, E. Kaji, and H. Kohno, ( ~ 7 / t ~ ~L~r~t tt .. (1974) 1029-1030. (308) E. Kaji, H . Kohno, and S. Z e i i , / j t r / l . C / z m t . Soc. J ~ I I .SO , (1977) 928-932

ALEKSANDEH ZAMOJSKI et

106

ti/

RL

542 R = R ' = H 543 R = H. R'

Me 544 R = H, R' = Et 545 R = M e , R' H 546 R = R ' = M e 547 R = M e . R L - Et -

548 R 549 R

= =

I1 Me

550 R

=

Et

551

~

b y chromatography. I n one instance, when 542 was iiiethylated b y use of methyl iodide-silver oxide, the cy anoiiier was formed a s the sole product. Reductioil of the nitro group of 545-547 in the presence of Raney nickel catalyst respectively afforded the corresponding 4-aniinopento-, -hexo-, a n d -1iepto-pyraiiosides 548-550. Methyl 4-ainino2,3,4,6-tetradeoxy-a- and -P-DL-eI-Ytlzro-hexopyranoside (549), characterized a s the N-benzoyl derivative, was identical in its 'Hn.1n.r.-spectral data with the analogous derivative of the natural, antibiotic sugar tolyposamine. On the other hand, reductive demethylation of 549 with formaldehyde-Raney nickel (under 3.5 kg/cm2 pressure of hydrogen) was effected, to yield another antibiotic sugar, methyl DL-forosaminide (551). A similar approach, involving condensation of 4-nitro-2-butanol with sodium glyoxylate"Ogin the presence of sodium hydrogencarbonate offers a route to 3-amino-3,4,6-trideoxy-~~-hexopyranoses. Two lactones, 552 arid 553, were formed during the reaction (overall yield, 42%) in the ratio of 3 : 1;these lactones rnay be considered to be potential precursors of a variety of isomeric 3-amino sugars. The synthesis reportecPYwas aimed at the preparation of desosainine (113). There-

ooto.. NO, HO

R'

Q

R3

552 R ' = R3 = H, RZ = NO,, R ' = OH 553 RZ = R4 = H , R' = NO2, R3 = OH

e

OH 554

555

(309) T. Kinoshita, Y. Kawashiina, K. Hayashi, a n d T. MiwaJ. C h e m . SOC.,Cheni.Commun. (1979) 766-767.

fore, lactone 552 was first coii\-c.rteclinto the 2-0-tetrali?-tllop!.raii-2-);1 derivative (to improve its so1iil)ilit)~i n toliiene), and the product reduced with diisobutylaluriiiiiiiiii hlrdride at - 70". Glycosidation w a s performed with iiiethanolic Ii!.tlrogen chloride, to give ;I mistiire of the methyl a- and P-glycosides (554) i n the ratio of 12 : 7. Further steps involved an equilibration reaction at C-3 of 554, with sodium hydrogeiicarbonate i n aqueous methaiiol at 50" (retro-aldol reaction),:"" iuid isolation of the product having tlic. desired, s!/lo configiiration (555) b y preparative t.1.c. Catalytic hyclrogenation of the nitro group over Adanis' catalyst in methanol, tollou~edb y Escli\Yeiler-Clarke N,N-dimethylation, gave raceinic methyl desosamiiiide (244). 0-CMe,

Me&-0

Me&-0

0-CMe,

K? - - k2 Me

OMe

0

55 7

556

e

Robw

+

AcObpGOMe

o,

0,

AcO

CMe, 558 R = Ac 560 R

=

,

H

CH,OR

C0,Me

561 R = H 562 R = Ac

(310) J. Kovii, K. Capek, and H. H . H,wr,

OAc 559

563

C:citi.

J . Chem., 49 (1971) 3960-3970

ALEKSANDER ZAMOTSKI e t crl

108

5. Inositols It has been found that suitably protected inositols readily undergo an oxygen-insertion reaction,"'IJl2 leading to a seven-membered heiniacetal which may be utilized for sugar synthesis. Therefore, DL-1,2:3,4-di-0-isopropylidei7e-5-~-1net~ly~-e~~~-inosito~ (556) was oxidized with the Pfitzner- Moffatt reagent313 to the corresponding epi-inosose derivative, which, in a Bayer-Villiger reaction with peroxybenzoic acid, afforded the hemiacetal lactone 557 in 8070yield. Acid-catalyzed rearrangement of 557, followed by acetylation, gave a mixture of methyl (methyl 2,3,5-tri-O-acetyl-~-DL-allof~iranosid)~iro1iate (559) and methyl (methyl 5-0-acetyl-2,3-0-isopropylidene-~-~~al1ofuraiiosid)uronate (558). The latter compound was reduced with lithium aluminum hydride to the corresponding derivative of ~ ~ - a l l o furanoside 561. On the other hand, on heating with 50% aqueous acid, compound 558 afforded, after acetylation, methyl 1,2,3,4-tetra-O-acetyl-P-DL-allopyranuronate (563).

-- K2J 0

564

CH,R

565

566

CH,OH

H2cv C0,Me I

I

HCOH

I

HCO, H(pMe,

I

y

0,

560 R = OH 569 R = OMS 570 R = H

/o

2

CH,OH

CMe,

571

567

/

J

CHO I HCO, HAO/CMe2

I 7H2 CH,OH 572

(311) E. F. Pmtt and J. F. V;inDeCastle,J. Org. Clicm., 26 (1961)2973-2975.

By a similar approach, an efficieiit synthesis:".' of methyl 5-deoxy2 , 3 - O - i s o p r o p y l i d e n e - ~ - ~ ~ - r - i b o - h e s o f o s i d(570) e and 4-deoxy2,3-0-isopropylidene-DL-er!/t~ir-o-peiitose (572) was xconiplished. Reductive, oxirane-ringcleavage with lithium aluminum hydride afforded the alcohol 565, which was rc.giospecifically converted into the hemilactone 566 b y oxidation and II Bayer-Villiger reaction. The regiospecifity of the latter reactioti w7iis proved l)y reduction of 566 to 2-deoxy-3,4-0 -isopropylidene-uL-ri!~o-hexitol(571), with concomitant elimination of one isopropylidene groiip. Compound 571 underwent oxidative cleavage with periodate, to give the corresponding 4-deoxyDL-ery thro-pentose derivative 572, which was reduced to 2-deoxy-DLerythro-pentitol. On the other hand, lactone 566 reacted with metlianol (acid-catalyzed), to yield methyl (methyl 5-deoxy-2,3-0-isopropylidene-p-DL-ribo-hexofranosi~~)iiroriate (567) which, on reduction of the methoxycarbonyl group with lithium aluminum hydride, afforded methyl 5-deoxy-2,3-O-isopropylide11e-~-~~-riho-hexofi1r~~noside (568). Mesylation of the 6-hydroxyl group, to give 569, and subsequent reduction of 569, produced the 5,e-dideoxy analog 570.

6. Miscellaneous Substrates lias been shown:1'5that alkanes and a. Alkanes and Ethers.-It ethers are able to react with iodine tris(trifluoroacetate), thus introducing vicinal hydroxyl groups into the carbon skeleton. Therefore, the title conipounds may be considered to be potential precursors of sugars. Such an approach to sugar synthesis is exemplified 11). the preparation of a cis,truns mixture of 3,4-dideosy- 1,2-di-O-(trifluoroacety1)-DL-pentopyranoses (573), formed by the action of iodine tris(triflu0roacetate) on tetrahy(1ropvran. Both pentoses were isolated in 42% yield, and were accoiiipnicd b y 574 (14%).

$OCOCF, 573

574

b. Methyl 2,3-Dideoxy-(1S)-~~-pentopyranosid-4-ulose. -There is one r e p o r P concerning the total synthesis of a racernic sugar analog (312) H. Fukami, H . 3 . Koh, T. S;ik;it;t, ant1 hl. Nakajima, 7 e t r u h e d r 0 1 ~L c f f . (1967) 4771-4776. ~ . 87 (1965)5661-5670; 5670-5678. (313) K. E. Pfitzner and J. G . Moffatt,/. C h e r ~Soc., (314) H. Fukarni, €I.-S. Koh, T. Sakata, ant1 M . Nakajima, T~ti-uhetlr-onLett. (1968) 1701- 1704. (315) J. Buddrus and H. Plettenberg, Aiigeic. C h e m . , 88 (1976) 478-479.

ALEKSANDEH ZAhlOTSKI et (11.

110

'ao

possessing a phosphorus atom instead of the ring-oxygen atom. The in a few stages, hv transformation of synthesis was T s H N N G o M e F L O

G

O

M

R(MeO)P TsN--N H H

e

575

576

577 R = O M e 578 R = P h

582 R = H 583 R = Ac

581

579 R = O M e 580 R = Ph

the title derivative of an aldosulose (see, also, Section IV). Thus, 575 reacted with p-toluenesulfonylhydrazide to give 576 in high yield. On respective treatment of 576 with dimethyl phosphite, or with methyl phenylphosphonite, adduct 577, or 578, was formed. Reductive removal of the p-toluenesulfonylhydrazide group was effected b y means of sodium borohydride in oxolane, furnishing 579 or 580. Compound 580 was reduced with sodium dihydrobis(2-methoxyethoxy)aluminate at o", to give, after acid hydrolysis, 2,3,4-trideoxy-4-C-(phenylphosphiiiyl)-DL-~lycero-pentofuranose(582) and its 1,s-diacetate (583).

c . Dihydrofuran Derivative.-A dihydrofuran derivative was used for the first time by Jar>; and coworkers"16for the synthesis of S,6-dideoxy-DL-ribo-hexito1 (586). The preparation of 586 was acconi-

H~OH H A 0 4

-

1

HCO

H~OR HCOR I

-

1

HCOR

584

585

586 R = H 587 R = Ac 588 R R = C H P h

(316) R. Luke$ M . Moll, A. Zobicovii, and J. Jary, Collect. Czech. Chem. Comrnun., 27 (1962) 500-503.

SUGARS FROM NOn’-(:4RnOHYDRATE SUBSTRATES

111

plished b y cis-hydroxylatioii of’the doul)le bond in %ethyl-5-oxo-4,5dihydrofuran (584), followed l)y redtiction of the lactone ring i n 585 with lithium aluminum hydriclc,. Coinpound 586 was characterized a s the tetraa-acetyl aiid 1,2:3,4-di-O-l,eiizylide1ie derivatives (587 aiid 588, respectively).

d. 3-Benzoyloxy-2,4-pentanedione.-The title compound served as the starting material for a five-step ~ynthesis:~” of 4-deoxy-~L-dallnosainine (598). As shown in Scheme 9, compound 589 was converted into the corresponding oxazole 590 b y heating with aniiiioniiiiii acetate in acetic acid. The reaction had to be performed under carefiilly controlled conditions in order to avoid diazole-ring formation. Photocatalyzed bromiiiatioii of 590 afforded S91, which was transformed into the cyano derivative 592, which, on treatment with hydrogen chloride in aqueous acetic acid, gave 593. Compound 593 was found to be very labile under the conditions used, and underwent decarboxylation, thus reverting to the oxazole 590. Therefore, the cyaiio derivative 592 was first converted into the methyl ester, and this was treated with hydrochloric acid in glacial acetic acid, to afford crystalline 593 in high yield. Compound 593 reacted with thionyl chloride, to give the k e y intermediate (595) desired. A three-stage transforniatiori of 595, iiivolving hydrogenation to the satiirated lactone 596, reduction of 596 0

Me

0

0

595

590 R = H

C6H5

591 592 593 594

589

H = Br R = CN H = CO,H R = C0,Me

H,- Pt 10,

WH e!-

1 . reduction 2 . HBr-AcOH

0

597 R =

-

596

598 R = H . H F 3 r Srhrme 9

(317) H.-K. Hung, H.-Y. Lam, W. Nic.iirczrii-a, M.-C. Wang, and C.-XI. Wong, C o ~ i/.. Chem., 56 (1978) 638-644.

ALEKSANDER ZAMOTSKl et (11.

112

to the hemiacetal 597, and removal of the N-protecting group led to the daunosamine analog 598 in moderate yield.

e. Dihydro-oxazine Derivatives.-The earliest work on the synthesis of 5-amino-5-deoxyhexoses concerned3I8the total synthesis of two 5-amino-5,6-dideoxy-~~-hexonic acids. The approach consisted in a proper functionalization of 3,6-dihydro-cis-6-methoxy-3-methyl-1,2-

602

599

Me0,C HO..,bg

CO,H

CO,H

HCOH

HCOH I HCOH

I I

HCOH

I

600

oxazine hydrochloride (599), obtained by a Diels- Alder condensation of 1-chloro-1-nitrosocyclohexanewith methyl sorbate (75% yield). A cis-hydroxylation step with osmium tetraoxide in pyridine was fully stereoselective, leading, after hydrogenolysis over platinum, followed by hydrolysis with concentrated hydrochloric acid, to 5-aniino-5,6-dideoxy-DL-allonic acid (601). Epoxidation of 599 was devoid of stereoselectivity, as evidenced by the formation of a 1 : 1mixture of epoxides 602 and 603. Acid-catalyzed, oxirane-ring-scission performed on this mixture led, however, after hydrogenolysis and hydrolysis, to 5amino-5,6-dideoxy-D~-gulonic acid (604)as the sole product, thus confirming that the isomeric epoxides underwent ring-opening at different positions; hence, the two diols possess the same configuration.

VII. TOTALSYNTHESESOF OPTICALLYACTIVE CARBOHYDRATES In previous Sections of this Chapter, total syntheses of sugars have been described which led to pure diastereoisomers in raceinic fonn. (318) B. Belleau and Y.-K. Au Young,]. A m . Cheni. SOC.,85 (1963) 64-71.

SUGARS F R O M NON-(:AHBOIIE’I)RATE

SUBSTHATES

113

One of the most serious objections that is often raised against the totally synthetic approach to sugars is tlie lack of optical activity of the final products. Attempts at overcontiiig this difficulty were undertaken quite early. Essentially three w a y s of obtaining opticall>,active products have been explored: (I) resolution of tlie racemate of the final product, or, of one of the inteniiediates, (2)chemical conversion of an optically active, non-carbohydnite precursor into a sugar, the c h i d center (or centers) of the sulxtrate Leirig preserved in the final product, and ( 3 ) stereo-differentiating synthesis. All of these approaches have turned out to be quite successful in obtaining enantiomeric sugars, although, at present, tlie configuration of the enantiomer resulting from resolution of a rac.eiitiite or from ;i stereodiffereritiating synthesis cannot be foreseen with disolute certainty. Although some of the syntheses described next might have been discussed in previous Sections, they are collected here in order to provide better insight into the results achieved thus t‘u.

1. Resolution of Racemates There are reports in the literature describing the resolution of substrates, a s well as of products. 2-(2-Furyl)glycolic acid (605), a substrate for the synthesis of hexoses (see Section IV) was r e ~ o l \ d ”into ’ ~ both enantiomeric forms by recrystallization of its B(R)-nienthyl ester. From the R eiiantiomer of 605, methyl ~-D-glucopyranoside”2” and methyl cu-D-mannopyraiiosidezz7were obtained by a sequence of reactions discussed in Section IV. No raceinization occurred at a n y stage of the synthesis.

605

Another substrate for the synthesis of hexoses, tl-aiis-5,6-dihydl-o-fi(hydroxymethyl)-2-methoxy-W-pyran (21I), was obtained i n h t h optically active fomis by resolution of its 6-cainphanyl ester.:’21From the 1evorotatoi-y enantiomer, methyl 2,3,6-tri-O-acetyl-4-deoxy-a-D-.r!/lohexopyranoside (606) was synthesized, thus confirming its (2S):(6S) configuration, corresponding to the D configurational series. (319) 0. Achmatowicz, Jr., a n d P. Bilkowski, B t t l l . Accid. P o / . Sci., Scr-. Sci. Chim., 19 (1971) 305-308. (320) 0. Achmatowicz, Jr., and R. Biel\ki, C u r / m h y d r , Re.,., 55 (1977) 165-176. (321) A. Konowd, J . Jurczak, and A. Zciiiiojski, Tetrahedron, 32 (1976) 2957-2959.

114

ALEKSANDER ZAMOJSKI et

(I/.

CH,OAc

OAc 606

3(H)-Hydroxy-4-peiiteIloic”l.:”” (R-94) and 3(R)-hydmxy-3(R)methyl-4-hexenoic (R-99,R = acids were obtained froin the racemic acids b y recrystallizatioii of their quinine salts. Compound H-94 as the sulistrate for the synthesis of 2-deoxy-~-erytliro-pentose as in 94 -+ 98. The enantiomeric acid H-99 was employeddfifor the preparation of D-everniicose (see 99 -+ 105). Racemic methyl mycaroside:” (63) (see 5%65) was resolved b y means of its 4-( +)-isobor~ieol-lO-sulfonicester, to yield L-mycarose (65) after hydrolysis. Methyl D-kasugaminide (187) was obtained”’“ b y resolution of the racemate of the mono-N-acetylated product with Lthrearic acid. The anhydride 387, serving for the synthesis of ribose derivatives, was converted with isopropyl alcohol into ester acid 607, which was with (H)-l-iiaphthylethylainiiie, or with bnicine, into the enantiorners. 2-Amino-2-deoxy-~-erythrito~ (164b) was obtained“’ b y resolution of the raceinate by means of L-glutamic acid. cis-4,5-Epoxy-2-hexenoic acid (128)was resolved b y means of the salt with optically active phenylethylamine. With the dextrorotatory ainine, compound 4 ( R ) , 5(R)-128was o l ~ t a i n e ~ This ~ . ” acid ~ ~ ~served ~~~ for the synthesis of D-forosamine (608) b y opening of the oxirane ring with diniethylaniiiie (the 4-dimethylaniino derivative was the major product), hydrogenation of the double bond, lactonization, and pzirtial

Me,NO

O

H

608 607

(322) G . Nakaminaiiii, M . Nakagawa, S. Shioi, Y. Sugiyama, S . Isemura, and M .Shibuya, Tetrcihedroii L e t t . (1967)3983-3987. (323) S. Yasuda, T. Ogasawara, S. Kawatxita, I. Iwataki, and T. Matsuinoto, Tetrahedron Lett. (1969) 3969-3972. (324) I. Dyong, R. Knollinaiin, arid N. Jersch, Angcw. Chem., 88 (1976) 301-302. (325) I. Dyong, R. K n o l l m a n n , N. Jersch, aiid H. Luftmann, Chem. Rer., 111 (1978) 559-565.

reduction of the lactone. LJse of lrvorotatory plieiiyletli~~latiiiiie idlowed the separation of 4(S),5(S )-128, from Lvliich N-ac~~t),l-L-acosamine (137) was 2. Chiral Precursors

For the synthesis of opticall). active sugars, a number of iiatural compounds has heen siiccessfrilly einployed. The rather obvious prerequisite of such syntheses is the retention of configuration of chiral centers during all operations that a r e involved i n conversiori o f the substrate into the desired sugar. Soiiie readily available, natiiral products as, for the example, tartaric and aiiiino acids have particularly often been used for that pui-pose. a. L-Threaric [2(R),3(R)-Tartaric] Acid.-Although, L-threaric acid is, indeed, a sugar derivative, nrairy chemists who have eniployed it in sugar syntheses have tended to reg;trcl it a s a noiic~~rboli~di-ate precursor. E. Fischer was the first t o recognize the potential value of this acid for the synthesis of tetrosths. However, atteiiipts:{2tj.:’2i at “asynirnetrizatioii” of tartaric acid by selective reduction of only one carl)osyl group apparently met no siiccvss. O n l y much later did Liicas and Bauiiigxteii:328obtain di-0-act.tyItartaric aiihydricle (609). Treatinelit of 609 with methanol yielded tuoiioniethyl diO-acetyltartrate (610), which reacted with thionyl chloride to afford the acid chloride (611). Roseiimund reduction of 611 gave methyl 2,3-di-O-acetyl-~threouronate (612). Reduction of the aldehyde group in 612 yielded L-threonic acid (10). Further rtduetion of 10 afforded ~-threitol,isolated as its di-0-benzylidene tleriv,‘1 t’1ve.

9

A cHCOAc O X a o

609

cox

‘

I HCOAc

AcOCH I C0,Me

-

610 X = OH 611 X = C1

CHO

’

I

HCOAc

AcOCH

I

-

10

C0,Me 612

Acid chloride 611 was u s e c P a s the substrate in the synthesis of^apiose (53)(see Scheme 10). Further workB:{”.3:{* extended the u s e of (326) E. Fischer, B e r . , 22 (1889)2204-2205. (327) E. Fischer, B e r . , 23 (1890) 930-038. (328) H. J. Lucas and W. Baumgarteii,,\. Ani. Chem. Soc., 63 (1941) 1653-1657. (329) F. Weygand iiiid R. Schmiecheit, C h e r t t . Bet-., 92 (19.59)535-540. (330) H. J. Bestniaiiii and R. Schniiechen, (J/tetri. Ber., 94 (1961) 751-757. (331) C . Nakaminami, H. Edo, and M . Nakagawa,Bull. Cheni.Soc.Jpii.. 46 (1973)266269.

116

611

A L E K S A N D E R Z A h l O J S K I ~t o /

CHZN,

CHN,

I c=o I HCOAc I

CH,OAc

CH,OAc

I c=o

I HCOAC

Ac OH

-.

I

C-CH, A,

I

CHZN,

HCOAc I AcOCH I C0,Me

I AcOCH I C0,Me

AcOCH I C0,Me

I

613

53 L-Apiose

CO,H I HCOH

HzO, Fe(OAc),

1. KOH, MeOH 2 . Amberlite IR-120 resin

CH,OH

-

I

HOCH I HOH,C-C-CH,OH I OH

I I

HOC-CH,OH HCOH

I

HOCH I CO,H

S c h e m e 10

diazoketone 613 for t h e synthesis of several pentose derivatives; for example, methyl 2,3-di~-acety1-5-deoxy-~-threo-4-pentulosonate (614), dimethyl 2,3-di-0-acety1-4-deosy-~-tlreo-pentarate(615), arid C0,Me I HCOAc

C02Me I HCOAc

I

I

AcOCH I

Ac OC H I

y*

c=o I c H3

CO,Me

614

615

S c h e m e 10 CH,OH HCO, +CMe, OC H

LiAlH,

CH,OH

I HFO\

c-CMe, OCH

CH,OTs

-

CHO

I I

I

c H3

CH3

616

I 1 . H - 0 . Ht 2 . [H] 3 . PhCOCl

I I

HN-CH *CM% HCO,

617

CH-C0,Et

II

CH2

OCH

CH

*

NH3

I

HFO\

(OdH CMe, I

SUGARS FROM NON-CARBOHYDRATE SUBSTRATES

117

others. Reduction of ~-threono-l,.l-lactone( l l a ) afforded331L-threose. 2,3-O-Isopropylidene-4-O-tosyl-~-threitol (616) was converted332in 7 steps into 3-benzamido-2,3,6-trideoxy-~-xyZo-hexose (617).

b. Amino Acids.-From L-alanine, both anomers of methyl 2,3,6trideoxy-~-glycero-hex-2-enopyranosid-4-ulose (326 and 327) were obtained. From the a anomer 326, methyl a-L-amicetoside (618), methyl a-L-mycaminoside (619),and methyl a-L-oleandroside (L-221)

CO,H H,N-C-H

I

-

I

2. I

CH,

I CH3

4 OMe 327

Me0

Me,N 618

CH3

326

619

221

were prepared,333 and 3-acetamido-2,3-dideoxy-~-gZycero-tetrose (621) was obtained334from methyl N-(trifluoroacety1)-L-aspartic chloride (620). The synthesis of racemic 621 from ethyl 2-alkyl-2-(formamido)malonate was also described.334 L-Threonine was deaminated to 2(S),3(R)-dihydroxybutanoicacid (622).Esterification of 622, and reaction of the ester with acetone furnished 623, which was reduced to 4-deoxy-2,3-0-isopropylidene-~threitol (624).This substrate was further employed332for the synthesis (332) G. Fronza, C. Fuganti, P. Grasselli, and G. Marinoni, Tetrahedron Lett. (1979) 3883-3886. (333) K. Koga, S. Yamada, M. Yoh, and T. Mizoguchi, Carbolaydr. Re.9. (1974)c9-cll. (334) S. David and A. Veyrieres, Curbohydr. Res., 13 (1970) 203-209.

ALEKSANIIEK ZAMOJSKI rt (11.

118 C0,Me I F,CCOHNCH I FH. COCl

C0,Me I H,NCH

-1. H,,

I

1. LiAlH,

YH, CH(OMe),

2 . Ac,O

Pd/BzSO,

2. MeOH, HC1

L

CH,OH I AcHNCH 1 7H2 CH ( OMe),

620

H,O. H'

CH,OH

I

AcHNCH

I

P

O

H

=

?HZ CHO

AcNH

621 C02H I H,NCH 1 HCOH I CH3

1

C0,Me

-

OCH ,el ihc,+( HCO

I

622

CH3

623

624 OCH,Ph

I

I

oo-H2 \

H,C

I

7H2

-

?H% CO,H

OCH,Ph H2CI

OMe

*

CHONa

625

OCH,Ph HzCe I

OCH2Ph H2Co I

OMe

Yo>

Br

626

OCH,Ph

OCH,Ph

I

I

I

AcO

OAc

627

2.7: 1

I

c H3

OCH,Ph

CO,H I H,NCH

CH,OH I OCH -+,cMe, HCO

I

CO,H I HOCH HCOH I CH3

628 S c h e m e 11

-

OMe

SUGARS FRO bl N0N -< : A HR( )I 1Y I > H A T E S U H STRATE S

119

of the D-enantiomer of 617. L-(;liitaii>icacid was iised’”’53:””’ for an ingenious synthesis of derivatives of D-ribose (627) and D-lyxose (628). The steps of the synthesis art. c ~ i d e n from t Scheme 11. Methyl 5 - 0 - l ~ e n z y l - 2 , 3 - d i d e ~ ~ ~ y ~ ~ e 1 i t - 2 - e n o f i 1(626), r ~ ~ i r ~air~ sinicle termediate in this synthesis, W I S epoxiclized:’:3iwith m-chloroperoxybenzoic acid, furnishing methyl ~ , ~ - a l l h y d r o - ~ ~ - ~ > - ~ , e n z ? . . ~ - D - p e n t o furanosides of the P - r i h (629) and a-lyxo (631) configur at’1011s. Reaction of 626 with calciiini hypoclilorite followed hy potassium hydroxide treatment gave:’:37 the reni;tining two stereoisomeric, 2,3-anhydro compounds having the w t - i l i o (630) and p-l!yxo (632) coiifigiirations. Reductioii of the epoxit1t.s with lithiunr aluniiniim hydride gave 3-deoxy compounds from 629 and 631, and 2-deoxy compounds fi-om 630 and 632. Also, other D-peiitose derivatives, for instance 633 and 634, were b y three-iiieiiiljered-riiigopening of the p-riho and a-Zyxo epoxides with appropriate reagents. Lactone 625 was cI Sed:l:%8 for the synthesis of thc I)r~~iichecl-cliain sugar derivative 635.

629 R’ = OMe, RZ = H 630 R’ = H . R2 = OMe

631 R’ = H , R 2 = OMe

632 R1 = OMe. R2 = H

PhCH,OCH,

W OAc

634

PhCH,OCH, I HOCH I (YH2)2

MeMgI

Me

A~NH

633

625

O

MeqMc

~

FHZN, Ac OC H I +

-

I/ (p.)2

MeCMe

W

I

OH

AH

635

(335) K. Koga, M .Taniguchi, and S . Ymiiada, Z’rtrciliedrori Lett. (1971) 263-266. (336) M. Taniguchi, K. Koga, and S . Y~iiiirda,TcJtrcihecfroii,30 (1974) 3547-3552. (337) M.Taniguchi, K. Koga, a i d S . Yiuiiida, Clieni. P ~ u ~ ~B uJ lLl . ,. 22 (1974)2318-2323. (338) W. A. Szarek, D. M. Vyas, airtl I,. Cheii, Ccirhohydr. Res., 53 (1977) C I - C ~ .

120

A L E K S A N D E R ZAMOJSKI et

(I/

c. Other Natural Products.- Jary and coworkers investigated the conversion of (S)-parasorbic acid [S-107, the lactone of 5(S)-hydroxy2-hexenoic acid] into sugar derivatives. cis-Hydroxylation afforded339 4,6-dideoxy-~-ribo-hexonolactone(109) as the sole product. Ruff degradation of 109 yielded 3,5-dideoxy-~-erythro-pentose(636).Epoxidation of (S)-107 gave”O a single 2,3-anhydro compound [(S)-108], having the ~ 4 xconfiguration. 1 Opening of the epoxide with ainmonia or dimethylatnine yielded”“’the corresponding amides of 3-amino3,4,6-trideoxy-~-xyZo-hexonic acid (112). Amide 112 ( R = H) was readily hydrolyzed by heating in water, and, after formation of the hydrochloride of the amino group, cyclization to the 1,9lactone (637) could be effected. Partial reduction of the lactone grouping, followed b y methylation of the amino group, gave342L-desosamine (113), the eiiantiotner of the natural amino sugar. CHO HOCH Il

F

G

O

H

HO:H

(5)-107

H P 637

OH I

H 108

H I

I R,N 113 K

=

OH I

/

H

H. Me

D-Glyceraldehyde (usually in the form of its 2,3-isopropylidene acetal 638) has been employed several times for the synthesis of various sugars. Although such syntheses are, in the strict sense, beyond the scope of this article, those in which reactions other than the aldol type of reaction were used for chain extension are described. (339) R. LukeS, J. Jar,’, and J . Neiriec, Collect. Czecli. Cliem. Cotnmun., 27 (1962) 785741. (340) J . Jar?; and K. Kefurt, Collect. Czech. C h e m . Conitnun., 31 (1966) 1803-1812. (341) K. Kefurt, Z. KefurtovB, and J. Jar,’, Collect. Czech. Chetn. Conitnun., 37 (1972) 1035- 1043. (342) K. Kefurt, K. &pek, J . &pkovii, Z. Kefurtov6, and J. Jar,’, Collect. Czech. Chenl. Cornmun., 37 (1972) 2985-2993.

H ~ u g h " ~described :' a synthesis of 2-deoxy-D-er-ythr-o-pentose that was based on the reaction of' 638 with allylmajinesiiim hromide, ci.yhydroxylation of the product, and cleavage of the 1,2-diol system in 639 with sodium metaperiodate. Although a mixture of two diastereoiners should have been formed, 2-deoxy-D-erytliro-pentose was the main product of the reactions, a result consistent with expectations based on Cram's rule of 1.2-induction. CH, It T'H

H,C=CH-CH,MgBr

638

('HZOH I CHOH

639

Ishido and c o w o r k e r ~ ~ ~ foniid ~ ~ : ~ -that I ' ~ N-P).ruvylideneglycinatoaquocopper(I1) (640) reacts with 638 (or with its L enantiomer), furnishing 2-arnino-2-deoxy-~-or -L-pentonic acid (641). 0

0

+ 0

640

638 --H,O-Cu--N

Me,

I

O

C-

C-

H

H

I

CH,

1. NH, 2 . H,O

CO,H

I

CHNH,

I

CHOH I HCOH

H,COH 641

D-Erythrose, D-threose, and some of their derivatives were obtained from the diastereoisomeric products resulting from the reaction of638 (343) L. Hough, Cheni. Znd. ( L o T ~ o (1951) ~ I ) 406-407. (344) T. Ichikawa, S. Maeda, T. Okamoto, Y. Xi-aki, and Y. Ishido, B d l . ChcJiii.Soc.Jptl., 44 (1971) 2779-2786. (345) S. Ohdan, T. Okamoto, S. Mat&, 'I. Ichikawa, Y. Araki, and Y. Ishido, H n ( l . Chem. SOC.Ipti., 46 (1973) 981-985. (346) T. Ichikawa, T. Okamoto, S. Maetla. S. Ohdaii, Y. Araki, a r i d Y . Ishido, Tetrtrhedron Lett. (1971) 79-80.

ALEKSANUER ZAMOJSKI et

122

d

with ethyiiyln~agI~esi~iin h r o n ~ i d e : or ~ ~a' vinylmagnesium halide.34i*348 Ozoiiolytic cleavage of the multiple bonds in 642-645, followed b y hydrolysis, led to tetroses in good yields. 12

1;.

1. 0,

H'

I

I HCO,

HCO, I ,CMe, H,CO

H,(&O/CMe'

t

644

642

HC=CMgBr

R

638

-

H,C=CHMgBr

-

n. AC

=

cH

+ 638

CH

Ill

C

I

H2

ROCH I

-

It

CH

I ROCH

f-

HCO, I ,CMe, H,CO 643

645

110

f)-Threose

The reaction of 1,3,2-dioxaphospholene (646) with aldehydes leading to ketodiols was applied by David and coworker^"^ to the synthe-

He OMe

OH

HO

647

I

I

HO

OH 649

(347) D. Horton, J . B. Hughes, and J. K. Thomson,J.Org. Chem., 33 (1968) 728-734. (348) D. J. Walton, Curl. J . Cheni., 45 (1967) 2921-2925. (349) S. David, M.-C. Lepine, G . Aranda, arid G . Vass,j. Chem. Soc., Clzern. Cornmun. (1976) 747-748.

sis of a branched-chain sugar. 'Thus, 646 reacted with 638 to fiirnish the clioxaphospholane 647 as the main product. Hydrolysis of 647, followed b y methaiiolysis, gave m e t h y l l-deoxy-3-C-n~ethyl-~-~-r.il,ohexo-pyranosid- and -fiiraiiosic!-2-i1lose (648 arid 649) i n 53% yield. The most remarkable feature of this synthesis is the formation of a single eiiantionier having three c h i d centers from a snlxtrate containing a single, chiral atom.

3. Stereo-differentiating Synthesis a. Cycloaddition to Sugar lJ-Butadienyl Ethers .-David and his coworkers developed conveiiieiit tilethods for the preparation of a 1,3-l1utadienyloxy grouping attached to various sugar derivatives. Compounds of this type readily undergo cycloaddition with alkyl glyoxylates or mesoxalates, furnishing esters of 2-alkoxy-5,6-dihydro2H-pyran-6-mono- or -6,6-di-carl)oxylic acid. Suitable, chemical rnodifications of the dihydropyran ring, essentially along the lines discussed in Section 111, led to various disaccharides. This method has special and original features that justify a separate discussion of the results achieved. ROH

f

IIOMe,C-C~C-C~C-CMe,OH 651

650

fI

R?

/C=C \ H CGCH

' +

RO, ,CECH /C=C,

H

652

i

H 653

1

Ha, Lindlar catalyst

Ro\ /c=C H

P \

CH=CHz

654

RO,

,CH=CH,

,c=c\ n~ 655

R =

0-bMe, Scheme 1 2

OCH,Ph

124

A L E K S A N U E H Z A M O J S K I et

(I/.

Synthesis of 1,3-butadienyl ethers is different from that employed when R is a simple alkyl group. David and coworker^^^^,^^^ elaborated two approaches to dieiiyl ethers. I n the first method, a sugar derivative (650) having an “isolated” hydroxyl g r 0 ~ reacts p ~ ~ under ~ ~basic ~ catalysis with 2,7-dimethyl-3,5-octadiyne-2,7-diol (651) to yield two enynyl ethers (652 and 653). Partial hydrogenation of 652 and 653 gives t r a m - and ci.s-1,3-biitadie1iylethers (654 and 655); see Scheme 12. The second synthesis of 654 and 655 makes iise950,351 of the Wittig reaction. The (methy1thio)inethyl ether 656 is converted into the chloromethyl ether 657, which reacts with triphenylphosphine to yield a crystalline phosphonium salt (658). Reaction of 658 with phenyllithiuin gives a phosphorme, treatment of which with acrylaldehyde leads to ethers 654 and 655 in 50%yield. Pure truns-diene 654 was obtained352 in a “reversed” way consisting in preparation of a sugar ether acrylaldehyde (660) b y replacement of the p-tolylsulfonyl group in 659, followed by reaction of 660 with methylenetriphenylphosphorane.

-

RONa

+

ClCH,SMe

- C12

ROCH,SMe

ROCH,CI

656

+

Ph,P

RO-PPh,Cl-

657

658

1 PhLi 2 . CH,=CH-CHO

RONa

+

TsO,

,f[

- /c=c

/c=c \CHO

H

659

Ro,

H

P \

CHO

Ph3P=CH,

-

655

I

i 654

660

Application of the Wittig reaction permitted the preparation of dienes functionalized at C-4 of the butadiene system. Thus, by replacing acrylaldehyde with 3-(benzyloxy)acrylaldehyde in the reaction ~ ”obtain with the phosphorarie obtained from 658, it was p o ~ s i b l e “ to 1,4-dialkoxy-1,3-butadienes as a 2 : 3 mixture of the trans,traizs (661) and truizs,cis (662) isomers. By equilibration of the transient, Wittig betaine with a strong base, the percentage of 661 could be from 40 to 78%. Reaction of 660 with chloromethylenetriphenylphos(350) S. D a v i d , J. E u s t a c h e , a n d A. L u b i n e a u , C . H . Acatl. Sci., 276 (1973) 1465- 1467. (351) S. D a v i d , J . E u s t a c h e , a n d A. L u b i n e a u , ] . Chem. Soc., Perkiii Trcins. 1 (1974) 2274-2278. (351a) R. S. Tipson,Ado. Cnrbohydr. C h i n . , 8 (1953) 107-215; s e e p. 166. (352) S. D a v i d a n d J. Eustache,]. Cheni. Soc., Perkin Truris. I (1979) 2521-2525. (353) S. D a v i d a n d J . Eustache,]. Chem. Soc., Perkifi T ra m . 1 (1979) 2230-2234.

phorane 1ed”j’ to l-alkoxy-4-cliloro- 1,3-l,utadienes of the tr-ciris,tr-ci ti,y (663), and trtirzs,cis (664) configuration i n the ratio of 1: 1. H

RO

\

/c=C

H

/

\

C=C

H’

H

H

/

‘€3’

661 R’ = OCH,Ph R L = ~1

663

“0,

\

/c=C

/C=C H H

R’ /

H

662 R’ = OCH,Ph 664 R’ = C1

R , as i n Scheme 1 2

These dienes, especially the reactive

t i - u i ~ s , t t - ~ iisomers, ru

are v d u -

able substrates for the preparLition of cycloadducts already fiinctionalized at C 4 of the future sugar ritiit. The second important featiirc of 1)avitl’s approach to disiiccharides consists in generation of optical itcti\.ity in cyclic precursors. Cycloadditioii between sugar 1,3-biitaclieiiyl ethers and esters of glyo-iy-lic acid is, in fact, an example of a Iliastereof~~ce-cliffe~eritiatitig reactioii, and it leads to all four possible addric.ts, tlamely, the a- mid p-D,and aaiid 0-L,in unequal proportio~ra.”~” It was fouiid:15-k that endo d d i t i o n (leading to p-D and p-L coniporiiitls) is not necessarily operative, but, rather, attack of the dienophile on o r i t . f k e of the cisoid diene (leading, for instance, to a-L and p-u conipounds) is favored. Cycloaddition of esters ofgl).os)~lic acid occiirs only with the f r c i t i s 1,3-butadiene system (unless t h e very reactive, 1,4-dii~lkoxy-l,3-1~iitadienes are employed, see Ref. 353). It was found, however, that a more-reactive dienophile, clieth) 1 niesoxalate, also reactsYswith c i s 1,3-butadienyl ethers, although t h e reaction is much slower. Decktrboa1kox y lat ion accord in g to t hc K r qich o 111e t hod 1 e a d s to 111on oca rl )ONylic products. Reaction of diethyl mesosalate with ii iiiistiire ofchlol-o.. dieiies 663 and 664 (the secoirtl 1)eiiig inert in loaddi ti on) ga\re,’”products in which the substitlietits tit C- 1 and C-4 were cis-oriented, that is, of the a - aiid ~ a-L coiifigriratioii (665 anti 666), in the ratio of 69: 19. Condensation of the mixtiirt. o f clieties 661 m c l 662 with 2 ( R ) menthyl glyoxylate (until only t h e t r c i r t s , t r - u r i , y dieiie had entered into reaction) gave:’”:’a mixture of‘three products (667-669) in 63% overall yield. This inixtore was separated into its components. Reduction of R’ to a hydroxymethyl group i i i 667 i t l i d 668, und c.i.y-liydroxylatioiiof the double Iiond, gave disacchal-ides 670 m d 671, in which the newly created sugar units were identified a s D- and L-gulose, respecti\rely. .)

(354) S. Ilavicl, A. Lul,irieau, a r i d A . T l r i e f f q , Tctrrihadrori, 34 (1978) 299-304

ALEKSANDEH ZAMOJSKI et

126

(I!

The third adduct (669) was an ether; after similar functionalization of the dihydropyran ring to the L-gulo system, 3-0-(benzyl 4-deoxy-p-~gulopyranosid-4- y1)- 1,2:<5,6d i - 0 - isopropylidene-a-D- glucofuranose (672) was obtained. Cleavage of the ether 1)ond between the siigar units was possible355" b y alkaline p-eliinin,d t'1011.

668 (24';)

669 ( 1 9 ' 0

R . as in Scheme 1 2 R'. CO,C,,H,,

1. LiAlH,* 667

2.

oso, HO

OH

670

CH,OH

668

1. LiAIH, 2. oso,

671

0-CMe, 672

Functionalization of the dihydropyran ring to a hexose system allowed the preparation of a remarkable variety of di- and tri-saccharides; for example, hexopyranosyl-(l+3)-~-glucoses of the a - ~ -

SUGARS FROM NON-( \KHOHYDRATE SUBSTHATE5

127

ci/lot a-D-dtro, a-L-ultro, P-L-uIli-o, and p-D-gnltrcto co~lfiguratioiiS,””~ :mi 40-P-~-galactopyranosyl-u-glucose, ~naltose,”~‘ 2-acetaniido-2deoxy-a-D-galactopyranosyl-, -Lu-D-talopyranosyl-,and -a-L-fucopyrano~yl-(l~3)-D-gl~icoSeS~~~ 4~)-(~~-a-~-fucopyrariosyl-~-~-altro~~y osyl)-D-glucoSe, 4 ~ - ( 2 ~ ) - a - I . . - f U ~ O p y r a n O S y ~ - ~ - D - ~ l l t r ( ~ p y r a l l o S y ~ ) - D glucose,357and ~~-a-L-f~icopyrariosy~-~~-(~-acetamido-~-deoxy-agalactopyranosy1)-u-galactose(the tc.rinina1 trisaccharide in the bloodgroup A, antigenic detenin~tnt).:”.!‘ David’s approach was particiilarly cffective in a new synthesis of (- )-kasuganohiosainine””” ( ~ ~ 1 8 8addition ); of 2(R)-nienthyl glyoxylate to a butadienyl ether o f a protected inositol gave it cvcloadduct (673) which was functionalizetl t o the kasriganiine system (see Scheme 13).The total yield of the final product (LL-188)was 1.370, that is, - 100 times as much its in the previous synthesis.x2

1. H,. Pd’C 2. 20‘; AcOH

LL-

188

I I Me,C-0 Scheme 13

It was also possible to synthesize:”” a protected disaccharide (673c) containing N,N’-diacetylpui7)urosalriiirie C as the nonreducing unit. S. David, A . Lubineau, and J.-M. Vat+lt.,/. Chem. Soc., Clberii. Cornrriun (1975) 701-702. S. David, A. Lubineau, and J.-hl. VatCle, 1. Cheni.S o c . , Perkit1 Trciri6. 1 (1976) 183 1 - 1837. A. Thieffry, Doctoral Thesis, Uiiiversiti. tie Paris-Slid, 1977. S. David, A. Lul~inean,and J . - h l . \’ati.le, t o be published. S. David, A. Lul~ineau,and J.-hl. \7ati.le,,/.Clieiri. Soc., Chern. Cutnrrmri. (1972) 535- 537. S. David and A. Lubineau, N o t i t ,/. C / t i r ) t , , 1 (1977) 375-376. S. David and A. Lubineau,/. O r g . Ch~ni., 44 (1979) 4986-4988. ,

ALEKSANDER ZAMOJSKI et crl.

128

The CX-D-Z~XO epoxide [232, R = (3-deoxy-1,2:5,6-di-O-isopropylidenea-D-glucofuranose-3-y1), R’ = CH,OH] was converted into a 3,4-unsaturated ~-threo-2,6-diol(268),which was dimesylated and the diester treated with sodium azide. Two diazides (673a and 673b) were obtained and separated; the latter was hydrogenated in the presence of platinum catalyst and the product acetylated, to furnish 673c.

b. Other Syntheses. -Precursors of disaccharides (for example, 674) have been obtained as 1: 1 mixtures of the DD and DL diastereoisomers by condensation of 2,3-dideoxypent-2-enopyrmos-4-ulose (325, R = H) or its l-benzoate with monosaccharide derivatives having “isolated” hydroxyl g r o ~ p s . ” ~ , ” ~ ~ The unsaturated aldehydes 675 undergo363v:3w an enantiof‘xe-differentiating reaction when treated with fermenting bakers’ yeast, furnishing optically active diols 676.

p a

Ph,P

+

Et0,C-N=N-CO,Et, HgBr, (R = H)

/Q7 O-CH,

or SnC1, (R = Ac) R = H . Ar

Me,C-0

O-CMe, 674

b a k e r s ’ yeast,

R Y c H o R’

addition of “C,-unit“

675

R = Ph, P h R ’ = H, M e

: * R

R’ 676

w

(362) G. Grynkiewicz, Carbohydr. Res., 80 (1980) 53-62. (363) C . Fuganti and P. GrasselliJ. Cliern. Soc., Chern. Coinnitin. (1978)299-300. (364) C. Fuganti, P. Grasselli, and G. Marinoni, Tetrahedron Lett. (1979) 1161-1164.

1 H,. Pd 676 (R

=

P h , R'

=

H)

-" F 'O

2 . 0,. HCO,H 3. lactonization 4 . (Me,C H C H2) ,A1I1

OH

677

676 (R = P h . R'

=

Me)

Ho

1. A r C 0 , H

3 . H,, Pd

HO 678

2 (Me,CHCH,),AlH

t

L-Mycarose 65

Schenic 14

These diols have been employed for syntheses of L-amicetose (677), L-olivomycose (678), and L-mycarose (65) according to Scheme 13. ACKNOWLEDGMENTS T h e authors are grateful to Drs. R . Hognbl-, S. David, I. Dyong, G . Just, T. Kunieda, V. B. Mochalin, I. Stibor, and T. Takizawa for preprints of their relevant papers.

This Page Intentionally Left Blank

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 40

CHEMISTRY, METABOLISM, AND BIOLOGICAL FUNCTIONS OF SIALIC ACIDS BY ROLAND SCHAUER Biochemisches Institut, Christian-Albrechts-Unioersitiit,Kiel, West Germuny

I. Introduction

...

11. Occurrence of Sia

..........................

1. Acid Hydrolysis of Glycosidic Bonds

3. Ion-exchange Chromatography 4. Chromatography on Cellulose IV. Analysis of Sialic Acids . . 1. Colorimetric Methods .

....................... .................

4. Gas-Liquid Chromatography 5. Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Nuclear Magnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . . . . .

152

165 167

V. Biosynthesis of Sialic Acids, and

. . . . . . . . . . . . . . . . . 181

Linkages, and Further Degradation VII. Biological Significance of Sialic Acids

............

2. Influence of Sialic Acids on Macromolecular Structure . . . . . . . . . . . . . . 218 3. Anti-Recognition Effect of Sialic Acids . . 4. Sialic Acid as a Component of Receptors 232 VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-007240-8

132

ROLAND SCHAUER

I. INTRODUCTION Interest in the sialic acids has rapidly increased in recent years, especially since their involvement in the regulation of a great variety of biological phenomena was recognized. Based on such observations, which will be described in the last Section of this article, sialic acids play a strong, protective role in living cells and organisms. This remarkable function of sialic acids appears to be mainly due to their peripheral position in glycoconjugates and, correspondingly, to their frequent, external location in cell membranes. The name “sialic acids” was created in 1957 by Blix, Gottschalk, and Klenk’ and comprises all N - and O-acyl derivatives of neuraminic acid (Neu) isolated from natural materials.’” These compounds are also called “acylneuraminic acids.” The term “neuraminic acid” was first used by Klenk2 for an acidic compound isolated after methanolysis of submandibular-gland glycoprotein and later identified3 as neuraminic acid methyl P-glycoside (Neu-P-Me).N-Acetylneuraminic acid (Neu5Ac) was first isolated b y Gottschalk4 after the action of influenza viruses on ovomucoid or urinary mucins, and by Klenk and coworkers5 after acid hydrolysis of mucous substances. Shortly thereafter, N-glycolylneuraminic acid (Neu5Gc) was isolated from porcine, submandibular-gland glycoprotein,6 and several O-acetylated sialic acids were obtained from the corresponding bovine and equine m~cins.~ Elucidation of the structure of Neu or its N,O-acyl derivatives proved to be a time-consuming process. The first hints as to the fundamental structure were given b y an alkaline-degradation product, namely, pyrrole-2-carboxylic and the first synthesis of Neu5Ac was achieved Dy Gottschalkg b y an aldol reaction of 2-acetamido-2deoxy-D-glucose ( D - G ~ c N Aand ~ ) pyruvic acid, or of D-G~cNAcand oxalacetate.’”It was tentatively concluded from these experiments that D-G~cNA may ~ contribute to the structure of the Neu molecule. How-

(1) G. Blix, A . Gottschalk, and E. Klenk, Nature, 179 (1957) 1088. ( l a ) F. Zilliken and M. W. Whitehouse, Ado. Corbohyclr. Chein., 13 (1958) 237-263. (2) E. Klenk, Z. Ph!ysiol. Chem., 268 (1941) 50-58. (3) E. Klenk, Z. Ph!piol. Chein., 273 (1942) 76-86. (4) A. Gottschalk, Nature, 167 (1951) 845-847. (5) E. Klenk, H. Faillard, and H. Lempfrid, Z. Physiol. Cheiri., 301 (1955) 235-246. (6) E. Klenk and G. Uhlenbruck, Z. Physiol. Chem., 307 (1957) 266-271. (7) G. Blix and E. Lindbel-g,Acta Cheni. S c a d . , 14 (1960) 1809-1814. (8) E. Klenk and H. Faillard, 2. Physiol. Chein., 298 (1954) 230-238. (9) A. Gottschalk, Nature, 176 (1955) 881-882. (10)J. W. Cornforth, M. E. Firth, and A. Gottschalk, Biochem. I . , 68 (1958) 57-61.

ever, as was shown b y Coinl) m i d Roseinan," Neu5Ac is cleaved to pyruvate and 2-acetarnido-2-d~~ox).-D-iiiannose (D- h!lanNAc)b,, the action of aldolase acylneurarninate pyruvate-lyase (EC 4.1.3.31, and thus, the configuration of the substituents at C-5-C-8 of Neu corresponds to that of D - M ~ I I N A'The ~ . successfd synthesis starting from D-G~~N may A ~be explained 1)) the epimerization of u-GlcNAc to DManNAc under the alkaline conditions used.'2 After detenniiiation of the l)-gl!/wroconfiguration at C-4 of N e u by Kuhn and B a ~ c h a n g , the ' ~ stertwclreniistry of Neu was largely known. The conformation of NeuSAc was elucidated b y Lutz and c o ~ o r k e r s ' ~ b y studying its methyl ester derivative b y nuclear magnetic resonance (n.1n.r.) spectroscopy; it p r o v v d to have the T,(L) c~iifoiinatioii'~" as shown in formula 1. Furthenriore, the studics14 showed that the anoineric hydroxyl group at C-2 of free Ncu5Ac is mainly axial. Thus, the systematic name of the (levorotatory) NeuSAc is S-acetanrido-3,5-dideoxy-D-glycero - D - g u ~ u c t o - ~ - i ilopyranoson~~ 1-onic acid; alternatively, for naming its conforniatioir, its configuration m a y he regarded as D%rythro-Laruhiizo. In aqueoiis solution, as reported in Section IV,6, a sinall proportion ( 6 - 7%) of free NeuEiAc has the a-D configurat i ~ n . ~ " ' In ~ natural compounds, sialic acids are a-glycosiclically linked, with the exception of their CMP glycosyl esters.IX

6

?H

1

(11) D. G . Coinh and S. Roseman,,/. ' l r t i . (:hc.vr. S o c . , 80 (1958) 497-499.

(12) R. Kuhn and R. Brossmer,Atiti. ( : h c J t t r . , 616 (1958)221-225. (13) R. Kuhn and R. Baschang, C h e t t t . Bet-., 95 (1962)2384-2385. (14) P. Lutz, W . Lochinger, and G. Taigel, Chctn. Ber., 101 (1968) 1089-1094. (14a) It should be noted that the s t c ~ ~ - c ~ o c h ~of~the m i sC-6 t ~ ~sul>stituentdictate5 a\\ignment of the ( L ) designator to tlw coiiforinatioii~ilsvml)ol; see k;ur. / . ~ i o c h e i i ~ . , 111 (1980) 295-298. H . Friebolin, R. Brossmer, C;. Kt.ilic.11, 11. Ziegier, a d ?*l. Siipp, Z. Ph!/siol.Clbem., 361 (1980) 697-702. J. Haverkamp, L. Uorlantl, J . F. C;. Vlirgentliart, J . \lontreiiil, and H. Schauer, A h t i - . Znt. S y i n p . Carbo/z(/t/t-. ( , ' / t ( , t t t . , WI, Lotldorl (1978)D-7. J. Haverkamp, H. van Hallwek, 1,. I h r l a i i d , J. F. G . Vlicgenthart, H. Pfeil, arid H. Schauer, Eur. J. Biochem., 122 (1982)305-311. J. Haverkamp, T. Spoorrnaker, I ,. I)cirland, J. F. G . Vliegerithart, anti H. Schaiier, I. Aria. ChertL. Soc., 101 (1979)4851-4853.

134

ROLAND SCHAUEH

Great progress in our knowledge about sialic acids was made with the aid of gas-liquid chromatography (g.l.c.), mass spectrometry ( n i . ~ . )and , n.1n.r. spectroscopy. Structural analysis of the inany different N - and 0-acylated Neu derivatives occurring in Nature will be described in Sections IV,5 and 6. Emphasis will be given to the metabolism (see Sections V and VII) of sialic acids, which is connected with the biological roles of' these compounds. The sialic acid abbreviations used in the text are listed in Table I. They are drawn up on the basis of a Round Table discussion at the Fifth International Symposium on Glycoconjugates which took place at Kiel in 1979. 11. O C C U R R E N C E O F SIALIC ACIDS As excellent reviews on the occurrence of sialic acids in Nature only a short outline of the distribution of sialic acids will be given here in addition to recent findings. Neu is a relatively old "invention" in evolution, dating back 600 million years to the Precambrian period (see later). The occurrence of sialic acids in plants has never unequivocally been established, although some positive reports e x i ~ t .The ' ~ ~chromogeiis ~~ found in the dialyzates from some plant materials, leading to reddish colors in the periodic acid-thiobarbituric acid assay,2' were shown not to be due to the presence of sialic acids, as they did not lead to chroniophores characteristic of sialic acids in the orcinol assay.23The thiobarbituric acid chroinophore from plants is believed to be derived from 3-deoxy-2-glyculosonic acids not containing N-acyl group^.'^ Our studies have revealed the absence of sialic acids in mucous secretions from the European and African sundew species Drosera rotuiidifolia, D. ccipensis, and D. h i n n t ~ . ~ ~ Sialic acids are also absent from most bacteria. Known and firmly established exceptions are Escherichia coli, Neisseria meningitidis, ~"~~~; all of them are hosted by and Salmonella ~ t r a i n s ' ~ . remarkably, mammals, or are even pathogenic. Sialic acids are rare in viruses; they (19) S . 3 . Ng and J. A. Dain, in A. Rosenberg and C.-L. Schengnind (Eds.),Biological Roles of Sirilic Acid, Plenum, New York, 1976, pp. 59-102. (20) L. Warren, Comp. Riochem. Physiol., 10 (1963) 153-179. (21) A. Gottschalk, The Cheniistrtl antl Biolog!l of Sialic Acids aiztl Related Substances, University Press, Cambridge, 1960. (22) L. Warren,]. Biol. Cheni., 234 (1959) 1971-1975. (23) P. Bohm, S. Dauber, and L. Baurneister, Klin. Wocherischr., 32 (1954) 289-292. (24) K. Rost antl R. Schauer, Phytochemistry, 16 (1977) 1365-13fi8. (25) B. Kqlzierska, Eur. ]. Biochem., 91 (1978) 545-552.

TABLEI

Names, Abbreviations, and Indication of Nature and Position of the N - and 0-Substituents of Natural Sialic Acids" Substituent on

Name N-Acetylneuraminic acid

N-Acetyl-4-O-acetylneuraminicacid N-Acetyl-7O-acetylneuraminic acid N-Acetyl-84-acetylneuraminic acid R7-Acetyl-94-acetylneuraminic acid S-Acet~l-4.9-di4-acetyIneuramlnicacid N-Acetyl-7,9-diO-acetylneuranrlnlc acid .V-Acetyl-8,9-di~-acetylneuraminicacid N-Acetyl-7,8,y-tri~~)-acetylneuraminicacid N-Acetyl-9O-L-lact)iIneiiraminic acid N-Acetyl-44-acetyl-90-lact).lneuraniinic acid N-Acetyl-8-0-methylneuraminicacid N-Acetyl-94-phosphononeuraminicacid h

N-Glycolylrieuraiiiiiiic acid 40-Acetyl-h7-glycolylneu~iii~inic acid 7O-Acetyl-iC'-glycol~l1ieuraminicacid 9~)-Acetyl-~-glycolylneiirainiiiic acid 7,9-DiC)-acetvl-.~-glycolylneuraniinic acid 8,Y-DiO-acetyl-~-glyc~~lyl1ieuraiiiinic acid 7,8,9-TriO-acetyl-iC'-glvcolylneuramiic acid N-Glycoly I-8-O-methylnerlraininicc acid N-Glycolyl-RO-sulfoneiiraniinic acid "

Abbreviation Neu5Ac Neu4,5Ac, Neu5,7Ac, Neu5,8Ac, Neu5,9Aq

Neu4.S,9.4c3 N e u5,7,9Ac3 Neu5,8,9Ac3 Neu5,7,8,1-)Ac4 Neu5AcSLac N e u4 ,5Ac29Lac Neii5Ad3X21e Neu5Ac9P Neii2m5Ac NeuSGc

h'ei14AcijCc. Neu7Ac5Gc Neu9AcSGc Neu7,9Ac25Gc Neu8,9Ac25Gc Neu7,8,9Ac35Gc Neu5Gc8Me NeuSGc8S

0-4 (R)

N-5 (R' 1

0-7 (R2)

H

acetyl acetyl acetyl acetyl acetyl acetyl acety 1 acetyl acet>1 acetyl acetyl acetyl acetyl acetyl glycolyl glycolyl glycolyl glycolyl glycol yl glycol yl glycol ?.I glycolyl gl ycolyl

H H

acetyl

H H H acetyl

H H H H acetyl

H H H I-I acetyl H H H H H H

H

Compare formula 1. The occurrence of the individual sialic acids is described in the text.

0-8

(R" H H H

acetyl

H H H

acetyl

H H H

acet> I

H acet\.l

H H H

acety-l acet>1

H H

acetyl ,icet>1 ;1cet>I acetyl L-lactyl I>-lactyl

H phosphate

H acetyl

H H

acetyl

H H ti H H H H

acetyl

I1

H H H H

methyl

H H H H

acetyl

0-9 (RJ)

acetyl acetyl methyl

sulfate

'' See text (p. 146).

H H €1 H acetyl acetvl acetyl :tcet).l

H H

136

ROLAND SCHAUER

have been described as components of glycoproteins from, for example, Sindbis, Rous sarcoma, and vesicular stomatitis v i r u ~ e s .In ' ~ contrast to those in bacteria, viral sialic acids seem to be synthesized by host enzymes. Furthennore, occurrence of sialic acid has been demonstrated in and both NeuSAc and NeuSGc, in the molar ratio of 10: 1, have been isolated from Trypaltosomu c r u ~ i . ' ~ Sialic acids do not seem to occur in lower animals belonging to the Protostomia. This has been established by a wide variety of investigations made, and reviewed, by Warrenz0 and other research workers. However, exceptions have been reported in the literature: sialic acids were found in the primitive, turbellarian species Polychoerus curmelensis and the trematode Fascioloides inagnu . 2 0 Because whole animals were analyzed, no conclusion could be reached as to whether this sialic acid was synthesized in the animals, or was derived from their food. Segler and coworkers28gave support to the latter explanation, as they could not find sialic acids in the European turbellarian species Euplanaria gonocephula, even in the nerve-cell-rich, front portions of the animals. No sialic acids were found in the jelly fish, the sea anemone (Anemonia sulcutn), and the earthwonn.2YFurthermore, the absence of Neu in insects, reported by Warren,20was confirmed by investigation of the total larval and adult forms, as well as the eyes and ganglia of some fly species fed a diet free from sialic acids.2g Conflicting reports exist in the literature as to the occurrence of sialic acids in crustaceans and molluscs. Whereas, in some species (or by various investigators), sialic acids were found in small amounts, in other species and other laboratories no sialic acids could be determined.1g*,20*2* Thus, in a detailed investigation, Rahinann and coworkers'* could find no sialic acid in, for example, Helix pomatia. In contrast, sialic acid was found by the same investigators,28 and by Warren,20in the digestive gland of the decapod crustacean Uca tangeri, the lobster Homurus americanus, and the crayfish Astucus leptodactylus. The latter animal was investigated in more detail, but no sialic acids were found in individual organs apart from the intestine.'* It was unable to synthesize sialic acid from radioactive ManNAc as the precursor.2RThis observation, as well as the fact that the sialic acid found in these animals was not glycosidically bound, points to the probability that it is derived from foodstuffs or bacteria in the digestive organ.2XIn fact, feeding of the animals with chicken brain contain-

-

(26) M. E. A. Pereira, M. A. Loures, F. Villalta, and A. F. B. Andrade,J. E x p . Med., 152 (1980) 1375-1392. (27) R. Schauer, M. E. A. Pereira, G. Reuter and H. Muhlpfordt, unpublished results. (28) K. Segler, H . Rahmann, and H. Rosner, Biochem. S y s t . E d . , 6 (1978) 87-93. (29) R. Schauer and M. Wember, unpublished results.

SL:\I.IC ACIDS

1.37

ing bound, radioactive NeuiSAc. resiiltetl in free radioactive Neil5Ac in the digestive gland.2RThis finding of an exogenous origiii of mollusc sialic acids is in agreement w'ith the findings and considerations of Warren,'" who observed sialic acids mainly in a free state, antl exclusively in the digestive gland of' the, squid Loligo lircdii. I n contrast to protostornes, which, on the basis of the examples already given, appear to be unal)le to synthesize sialic acids, the tleute ro s tome s have acquired t h i s 1) i o s y n t he t ic route . The E ch inotlennata are the first group of animals i n the evolutionary ladder to contain sialic acids as glycosidic compoiients of indigenous molecules having a physiological function. These aliiinals, occurring since Precambrian times, are considered to be "inventors" of the Neu molecule. Warren described the occurrence of sialic acids, in high concentrations, in all five classes of Echinodemxita, and detemiined their nature antl concentration in different tissues and reproductive cells from various starfish and sea-urchin species, a s was reviewed i i i Ref. 20. Remarkably, apart from Neu5Ac, large relative atiiounts of sialic acids having N-glycolyl, O-acetyl, O-methyl, and O-sulfo groups were detected in these animals, as will be described later. In hemichordates and cephalocliordates (acranians), sialic acids, mainly "2115Gc, were also found."' Unexpectedly, in urochordates (tunicates),which are higher i n the phylogenetic tree than Echinoderniata and, together with the acraniutis, are considered to be the ancestors ofthe vertebrates, the occiirreiice of sialic acid is doubtful. In contrast to some reports in the literatiire,:"' Warren,2"as well as Rosner and Rahniann,"' could not find Neil i n several tuiiicate species, including Plzullusiu rnam~uillutu.The latter authors considered that the absence ofthis compound from tunicatrs is due to a secondary, sessile niotle of life b y which a possible, foriner acquisition of the sialic acid-coding genome was lost or inactivatt,tl, thus leading to a lack of sialic acidcontaining glycoconjugates. Sialic acids generally occur i i i vertebrates, as is suiiiinarized in Refs. 19-21. Neu5Ac was isolated i n our laboratory from the secreted mucus of the cyclostome M y x i t i c ) glrttino.sa,29a discovery in contrast to an earlier report by Wessler and Werner."* The sialic acid content of the dried mucus is 0.5%. The greatest variety of N- and O-acyl derivatives of Neu was found in niaininals, as will be reported later. Sialic acids usually occur iii very low concentrations as free molecules in biological materials, i n tissrtes having an active sialic acid rne(30) L. Svennerholm,Biochini. B k q ) h ! ~Actn, ~ . 24 (1957) 604-61 1. (31) H. Riisner and H. Rahniann, i ~ i t p ~ ~ l ~ l iresults. ~lied (32) E. Wessler and I. Wenier, A c f ( i C : h m i , Scc~,td., 11 (1057) 1240-1247.

138

ROLAND SCHAUER

tabolism (for example, 50 pM concentration in bovine, submandibular gland33),in normal human urine and serum34(1-3 p M ) , and in human saliva3” (25 pM). Higher concentrations of free, but exogenous, sialic acids have been found in the digestive gland of some lobsters and squids, as already described,2Hin fish eggs (for example, those of the rainbow trout Salriao guirdrieri~3s) with a maximum before hatching,36 and in body fluids in some human diseases. Several years ago, a mentally deficient boy was discovered who excretes over 10 g of Neu5Ac in the urine per day3’; this corresponds to an average concentration of sialic acid ofy445 nlM, a value > 10,000 times that in normal urine. Much higher concentrations of sialic acid were also fouiid in the serum and saliva of this patient, compared to those for healthy individu a l ~A. possible ~~ reason for this metabolic disorder, called “sialuria,” is discussed in Section V. Salla disease, found in Finland, is also accompanied b y an increased rate of secretion of free sialic acid in the urine (51-109 mg/day, corresponding to an 10-fold increase).3RAnother patient, suffering from a disorder most probably different from Salla disease, and excreting daily a few hundred mg of free sialic acids in the urine was discovered in Elevated amounts of sialic acid-containing oligosaccharides are found in the urine from sialidosis or mucolipidosis patier~ts.~” Sialic acids occur in a-glycosidic linkage as components ofoligosaccharides, polysaccharides, and g l y c o ~ o n j u g a t e s . ’ ~ - As * ~ - binding ~~

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(33) A. P. Corfield, C. Ferreira do Amaral, M. Wember, and R. Schauer, Eur. J. Biochem., 68 (1976) 597-610. (34) J. Haverkamp, R. Schauer, M. Wember, J.-P. Farriaux, J. P. Kamerling, C. Versluis, and J. F. G. Vliegenthart, Z. Physiol. Cheni., 357 (1976) 1699-1705. (35) L. Warren, Biochirn. Bicrphys. Actn, 44 (1960) 347-351. (36) H. Rahniann and H. Breer, Wilheltn Roux Arch. Entwicklungsmech. Org., 180 (1976) 253-256. (37) J. Montreuil, G. Biserte, G. Strecker, G. Spik, G. Fontaine, and J.-P. Farriaux, Clin. Chitn. Acta, 21 (1968) 61-69. (38) M. Renlund, M. A. Chester, A. Lnndblad, P. Aula, K. 0. Raivio, S. Autio, and S.-L. Koskela, Eur. J. Biochetrt., 101 (1979) 245-250. (39) J. P. Kamerling, J. F. G. Vliegenthart, R. Schauer, and F. van Hoof, unpublished re sillts . (40) G. Strecker, M. C. Peers, J.-C. Michalski, B. Foumet, G . Spik, J. Montreuil, J.-P. Farriaux, P. Maroteaux, and P. Durand, Eur. J . Biochem., 75 (1977) 391-403. (41) M. I. Horowitz and W. Pigman, The Gl!ycoconjugntes, Academic Press, New York, (a) Vol. I, 1977; (I)) Vol. 11, 1978. (42) A. Gottschalk, Gl!ycoproteins,Their Cotnposition, Structure o t i t l Fzinctioti, Elsevier, Amsterdam, 2nd. edn., (a) Part A; (I)) Part B, 1972. (43) C. C. Sweeley and B. Siddiqui, in Ref. 41(a),pp. 459-540. (44) S. DeLuca, L. S. Lohniander, B. Nilsson, V. C . Hascall, and A. I. Caplan,J. B i d . Chem., 255 (1980) 6077-6083.

partners, sialic acids, galactose (Gal), ;2-~icetainiclo-2-tleoxy-l~-g~~lactose (GalNAc), and GlcNAc h v e thus far heeu detected. Natural P-glycosidic linkages of sialic acids are known to occur only in the CMP glycosyl esters, the anomeric configuratiou of which was unequivocally established by n.ii1.r. stiiclic.s.'xWhere sialic acids are linked to each other, they usually form a-(2+8) I)onds, aiicl repetition of this binding type leads to the oligosialyl groups found i n the oligosaccharide chains of some g1ycol)roteins' ' and gaiigliosides46 (for reviews, see Refs. 19 and 43), or tlic hoiiiopolysaccharidc colominic acid produced by E . coli \Vhc,rc>asoligo- and poly-sialyl chains are mostly composed of Neu5Ac,, i i i the cat-erythrocyte ganglioside Neu5Gc-GD3 (abbreviations for gaiigliositles are a s recoininended b y Sve1~1ierlrolii1~~"), disaccharid(1s of NeuSGc have been and, in a glycoproteiri of rainbow-trout eggs, more than 15 Neu5Gc resiclues have been described a s p o l y ~ i i e r i z e d .Interestingly, ~~ tiisialyl groups made up of two diffc1-c~irtsialic acids have heen detected in gangliosides. In GD, from mb1)it thymus, a-Ner15Gc-(2~8)-a-Ne~iSAc groups have been f o u i ~ c l mrd, , ~ ~ i i r a trisialoganglioside from inouse 11 rain, a -N e u5 ,9Ac2-(2-8 )-a-N e I 15Ac re s idue s ,3'' S i a1ic aci d s hav i i i g a(2+9)-glycosidic bonds have Iwen found to occur i n capsular polysaccharides of bacteriaSs1 In the numerous types of c o i i i p l e x carbohydrates occurring in Nature (and reviewed, for exalrrplc, i n Refs. 19, 41, and 42), sialic acids are most frequently linked to Gal b y a-(2+3) or ~ ~ ( 2 4linkages. 6) Often they are also bound to G d N A c residues, niailily at 0-6. Whereas, frequently, sialic acids are hound to GalNAc in glycoproteins, such linkages have been discovered in a novel gangliosicle from human erythrocytes having ;ti1 a-Neu5Ac-(2+3)-GalNAc linkage."2 Evidence for binding of sialic acids to 0-6 of GlcNAc came from earlier studies of inilk oligosaccliaritles conducted in Kiihn's and Mon(45) S. Inorie and M . Iwasaki, Bioc,/i(,ttr.Bioj)h!/,v.Rrs. C o t t i r t i u t i . , 93 (1980) 162-165. (46) J. Haverkainp, J. P. Kainerliirg, iiiid J . F. C.Vliegeiithxt, F E B S Lctt., 73 (1977) 215-219. (47) F. Orskov, I. Brskov, A. Sutton, H . Schneerson,Mi. Lin, W. Egan, C . E . Hoff; and J. B. Hobhins,]. E x p . Met/., 149 (1!17R) (569-685. (47a) L. Svennerholm,]. Neuroclic~ttr, 1 0 (1963) 613-623. (48) S. Hamanaka, S. Handa, J. Inoiit,, 4 . Hawgawa, and T. Yainakawa, ]. H i o c h w t i . ( T o k l o ) ,86 (1979) 695-698. (49) M. Iwamori and Y . Nagai,/. Biol. (.'hc,m, 253 (1978) 8328-8331. (50) H. Ghidoni, S. Sonnino, G . Tctt.iiriaiiti, N. Bauinann, <;. Reuter, and R. Schauer, ]. R i d . Chetn., 255 (1980) 6990-6 (51) H. J. Jennings, A. K. Bhattachujw, 1). R. Bundle, C . P. Kenny, A. Martill, ;ind I. C. P. Smith,]. Infect. Dis., 136 (1977) s 78-s 83. (52) K. Watanabe mid S.-I. Hakomori. R i o c . / ~ ~ ~ i t r i s t18 r ! /(1979) , 5502-5504,

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treuil’s laboratories, and reviewed in Ref. 53. a-NeuSAc-(2+4)-P-Gal linkages have been found to occur in hunian, and bovine, plasma fib r ~ n e c t i i i , and ~ ~ ~ a-NeuSAc-(2+4)-P-GlcNAc groups, in oligosaccharides from rat sublingual glycoprotein.””I’ Sialic acids are usually bound at tenninal positions of oligosaccharide chains. In gangliosicles, a variety of glycoproteins and oligosaccharides, they can also be linked in side positions of the oligosaccharide chain, thus forming a branch. As binding partners of such sidepositioned sialic acid residues, GlcNAc (in the aforementioned, milk oligosaccharides) and, frequently, Gal or GalNAc, have been f o ~ n d . ~ ~ , ~ ~ Sialic acid residues located within the oligosaccharide chain have been reported to occur in a sialoglycolipid from the hepatopancreas of the starfish Putiriu pectiitiferu, which differs considerably from the sialic acid oligomeric or polymeric structures already described. In this unusual glycolipid, containing L-arabinose in a tenninal position, Nei15Ac connectss6 two Gal residues, fonning the sequence p-Gal(1+4)-a-NeuSAc-(2-+3)-p-Gal-. From Asterinu pectinifera, Sugita isolated three sialoglycolipids having similar structures, wherein Neu5Ac was replaced“’ b y NeuTjGc or NeuSGc8Me. In one of these compounds (“ganglioside 3 ” ) , a terminal Gal residue is P-glycosidically bound to 0-8 ofthe NeuSGc residue situated within the carbohydrate chain. Sialic acids are structural constituents both of insoluble and soluble components of tissues and cells. Accordingly, they are found in the glycoconjugates of membranous structures in cells, including the nucleuS.19.41.5H Golgi membranes involved in the biosynthesis of the different types of sialic acids and their glycosidic linkages are especially rich in sialic acids. Highly sialylated glycoproteins and gangliosides have been isolated that contribute to the remarkable, and functionally very important, N e u concentration on the surface of cell^^^+^^ (see Sec(53) A. Koljata, in Ref. 41(a), pp, 423-440. (53a) A. Kobata, Cell Struct. Ftrtrct., 4 (1979) 169-181. (531,) A. Slomianv and B. L. Sloiiiiany,]. Aiol. Chetn., 253 (1978) 7301-7306. (54) B. L. Sloniiany, V. L. N. Xliirty, antl A. Sloiuiany,J. B i d . Chetu., 255 (1980) 97199723. ( 5 5 ) A. P. Corfield, J.C.Michalski, and R. Schaiier, in G. lettainanti, P. Durand, and S. DiDonato (Eds.),Sicilitlnscs c i t i t l Sicdidoses, Pwvpectioes i n Inherited M ~ t u b o l i c Disccisea, Vol. 4, Edi Eriiies, hlilan, 1981, pp. 3-70. (56) G. P. Smirnova antl N.K. Kochetkov, Biochirn. Hiophys.Actn, 618 (1980) 486-495. (57) M. Sugita,]. Biochcrti. ( 7 o k y o ) , 86 (1979) 289-300. 765-772. (58) L. Warren, i n Ref. 19, pp. 103-121. (59) 41.C. Click antl H . Flowers, in Ref. 41(lj), pp. 337-384. (60) hl. Saito and T. Osawu, C u r h h / c / r .Re.s., 78 (1980)341-348. (61) H. W. Jeanloz and J. F. Codington, i n Ref’. 19, pp. 201-238.

tioii M I ) . The most highly sialylated, cell-surface glycoproteins are known to occur in the jelly coat of s e a urchins. They contain u p to 70% of sialic acids.6' Secreted glywproteins occurring in serum, urine, and, especially, products froni tniicous glands also frequently contain a considerable proportion of sialic acid. Thus, the contents of sialic acid of, for example, human a,-:tc,id glycoprotein,"3 c a l f f e t ~ i nedible ,~~ birds'-nest substancefi5or siil)iiiaiitiil)ular-glaii~lniucins from several ~ n a i n m a l s , ~are ' j ~in ~ ~the range of 9-36%. Neuratninic acid (~-aiiiino-~,5-dideoxy-D-fil!lcero-D-ga~ac~o-~-noiiulosonic acid) does not itself occiir i n Nature, because, in slightly acid or alkaline solution, its free aiiiino group immediately cyclizes with the glycosidic hydroxyl group to fonn an internal Schiff-base, 4-hydroxy-5-(~-arabino-tetritol-l-yl)-l-~~yrro~ine-2-c~~r~oxy~ic acid,6x which, under acidic conditioiis, is rapidly degraded to reddish and brownish, increasingly insoliible substances, earlier called "humic acids." These reactions do tiot occtir if either the aniiiio group of Neu is blocked, or a glycosidic linkage h a s heen formed. The natural derivatives of Neu are stabilized b y acylation of the amino group with an acetyl or glycolyl group. I n the laboratory, the Neu ttiolecule may readily be converted into its rather stable methyl p-glycoside.6H At present, 23 derivatives of nonglycosidically bound Neu have been identified in biological illaterials and their structures elucidated; they are listed in Table I. NertSAc is the most common Neu derivative, occurring exclusively, o r together with other sialic acids, in biological materials (for reviews, see Refs. 21 and 70). Tissues containing exclusively, or a high proportion oi; Neu5Ac (in addition to O-acetylatecl NeuSAc in some cases, I)ut without Neu5Gc) are, for example, those from man, several monkey species, sheep, whale, several fish species,",70 European dogs,'l the frog Rnna e s c u l e i ~ t aand , ~ ~ the hagfish Myxine glntiizosu.zYAs NeuSAc is a precursor of the other sialic acids (see Section V,2), it occurs, at least in traces, in such materials in (62) J. P. Karnerlirig, R. Schauer, J. F.C;. Vliegenthait, and K. Hotta, Z . Physiol. ( ; / w i n . , 361 (1980) 1511-1516. (63) R. W. Jeanloz, in Ref. 42(a), pp. 56.5-61 1. (64) E. R. B. Graham, in Ref'. 42(a), pi). 717-731. (65) 11. H. Kathan and I. D. Weeks. ,4rc/f,Hioc/ion. B i o p h / . s . , 134 (1969) 572-576. (66) A. Gottschalk and A. S. Bhargaw, i l l Ref. 42(b), pp. 810-829. (67) W. Pigman, in Ref. 41(a), pp. 137-152. (68) W. Gielen, Z. Physiol. C h e m . , 348 (1967) 329-333. (69) F. Wirtz-Peitz, R. Schauer, and k l . F'aillard,Z. Ph!/siol Chern., 350 (1969) 111-115. (70) J. A. Cabezas, Reu. E s p . Fisiol.. 29 (1973) 307-322. (71) S. Yasue, S. Handa, S. Mipagaw;i, J . Inoue, A. Hasegawa, and T. Yaniakawa,]. Biochem. (Tok!yo),83 (1978) 1101-1 107. (72) R. Schauer, J . Haverkamp, and K. Ehrlich, Z . P/i!/siol. Cheiii., 361 (1980) 641-648.

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which other sialic acids prevail, for example, Neu5Gc in horse or pig erythrocyte-membranes,21or in the egg glycoproteins from some seaurchin species.62 The best sources for isolation of Neu5Ac in larger quantities are edible bird’s-nest coloininic acid from the culture filtrate ofE. coli K-235 strain73after acid hydrolysis (see later), or the urine of sialuria patients excreting large quantities of Neu5Ac in the free For the preparation of Neu5Ac from other sources containing glycosidically bound, 0-acetylated sialic acids or Neu5Gc, methanolysis of the sialic acids may be performed (see Section III,l), followed by N-acetylation, and cleavage of the methyl groups of the methyl P-glycosides Materials readily obtainable for such purposes are the submandibular glands from pig or cow, which contain glycoproteins rich in sialic a ~ i d s . ” f i , ~ ~ , ~ ~ Neu5Gc also frequently occurs in oligosaccharides and glycoconjugates from both vertebrates and deuterostomian invertebrates, as is reviewed in Refs. 19-21 and 70. Polymerization of Neu5Gc in trout eggs has already been m e n t i ~ n e d Biological .~~ materials containing almost pure Neu5Gc are rare; examples of a high proportion (>go%) of Neu5Gc in the sialic acid fraction are the submandibular-gland glycoproteins from pig,66,67,70*74 erythrocyte membranes from cow,75horse,2L and fetal-calf skin,76horse-erythrocyte gangliosidz,77,7seggs of the rainbow several sea-urchin species,62 and the glycoproteins of the sea cucumber Holothuria forskuli.79 In the sialic acid fraction from mouse lymphocytes, 75% of Neu5Gc has been found.R0 Although there have been some reports on the detection of Neu5Gc in human materials, careful investigation excluded the presence of this sialic acid in normal, as well as in malignant, tissues of man.29In this connection, the observation that glycosidically bound Neu5Gc is antigenically active in man, leading to several pathological states, for (73) G. T. Barry and W. F. Goebel, Nature, 179 (1957) 206. (74) H.-P. Buscher, J. Casals-Stenzel, and R. Schauer, Eur. J. Biocheni., 50 (1974) 71 82. (75) E. Klenk and G. Uhlenbnick, Z. Physiol. Chern., 311 (1958) 227-233. (76) R. Bourillon and R. Got, Biochirn. B i o p h y s . Actu, 58 (1962) 63-73. (77) R. Schauer, R. W. Veh, M. Sander, A. P. Corfield, and H. Wiegandt, in L. Svennerholm, P. Mandel, H. Dreyfus, and P.-F. Urban (Eds.), Structure arid Function of Gangliosides, Plenum, New York, 1980, pp. 283-294. (78) R. Maget-Dana and J . C . Michalski, L i l ~ i d s ,15 (1980) 682-685. (79) J. P. Kamerling, J. F. G. Vliegenthart, K. Schmid, and R. Schauer, unpublished r e sults. (80) S. H. E. Kaufmann, M. Respondek, B. Wos, R. Schauer, and H. Hahn, in R. Schauer, P. Boer, E. Bucldecke, M. F. Kramer, J. F. G . Vliegenthart and H. Wiegandt (Eds.), Glycoconjugates, Georg Thierne, Stuttgart, 1979, 166-167.

example, serum-sickness tliscase, is of great interest. Thus, only Neu5Gc-containing gangliosidcs l ( ~ a dto Hanganutziu- Deicher antibodies, but notx' the corresponding compounds containing NeuSAc. Another interesting observatioii, made b y Yaiiiakawa a n d coworkers," is the occurrence of NeiiSGc in the erythrocyte hematoside of several Asian, including Japanese, species of dog, whereas the same compound from European dogs contains NeuSAc exclusively. I t was found that NeuSGc hematoside appears in some breeds of Japanese dogs by autosomal, dominant inheritance, whereas the gene for NeuSAc hematoside is recessive. Thus, in European dogs, the genotype is hoinozygous with regard to the nature ofsialic acids in evthrocytes. The occurrence of NeriSGc i n canine-erythrocyte glycolipids has been found to be related to a blood group i n the Japanese dogs.71 More and more evidence is accumulating for widespread occurrence of 0-acetylated sialic acids, from Echinodemiata to honiinides.1g-21,70 0-Acetyl groups have I ~ e e nfound at all of the positions possible in the Neu molecule, that is at 04, 7, 8, and 9. However, in no sialic acid molecule isolatcbd from natural sources have all these oxygen atoms been found to be esterified. The highest degree of 0-acetylation observed is in N-acetyl-7,8,Y-tri~~-acetylneurairiii~ic acid ( Neu5,7,8,9Ac4) and N-glycolyl-7,8,9-tri~-acetylneuraminicacid (Neu7,8,9Ac35Gc),found in small cluantities in bovine, submanclibular-gland glycoprotein.82In addition to these natural, tri4l-acetylated sialic acids having established structures, dia-acetylated sialic acids are also rare; however, they have been detected in several biological materials in quantities that can more readily be handled than those of the tria-acetylated species. Both Neu5,7,9Ac3 and the corresponding N-glycolyl derivative (Neu7,9Ac2SGc)have been obtained in relative yields of 5- 10%from bovine, submandibular gland^.^^,^^ Mono4l-acetylated sialic acids occur more frequently, and sometimes in large quantities, for example, in submandibular gla11ds.'~.~~ 0-Acetyl groups most frequently occur in the side chain of both NeuSAc and NeuSGc, and usually on 0-9. For instance, 2.5% of the sialic acids from bovine, sul)iiiaiidibular-gland g l y c o p r ~ t e i n ~is~ , ~ ~ NeuS,9Ac2, and 5% is NeuSAc5Gc. NeuS,9Ac2 has also been detected ~ of various in different tissues, sera, and saliva o f ~ n a nin, ~gangliosides

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(81) J. M . Merrick, K. Zadarlik arid F. Milgrom, Int. Arch. Allerg!/ A p p l . Zt?Lniutiol., 57 (1978) 477-480. (82) R. Pfeil, G. Reuter, J. P. Kamerliiig, J. F. G . Vliegenthait, and R. Schauer, nnpublished results.

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vertebrate^,^^*^^-^^ and in mouse,X5rat,x6rabbit,x6and Rhesus monkeyx7 erythrocytes. It has been estimated that, on the surface of BALB/c mouse erythrocytes, almost all of the sialic acid residues are O-acetyI a t e ~ lIn . ~ rabbit ~ erythrocytes, Neu9Ac5Gc also occurs.H6Neu5,9Ac2 has been isolated from human-tonsil B lymphocytes; 50% of the sialic acids from these cells are 0-acetylated. T-Lymphocytes contain only traces of 0-acetylated sialic acidsGxx Acetylation at 0-9 seems to be a phylogenetically old acquisition, as Neu9Ac5Gc occurs in the sea-urchin species Pseudocentrotus depres,sus (Okayama).'j2Acetyl groups at 0-9 of Neu have, with the aid of 13C-n.m.r. spe~troscopy,~' also been found in E . coli K-1 colominic acid. The occurrence ofsialic acids having an acetyl group on 0 - 7 has seldom been detected. Neu5,7Ac2 was isolated in large quantities from bovine, subinandibular-gland g l y c o p r o t e i ~ iThe . ~ ~ relative yield of this compound from bovine, submandibular-gland glycoprotein is strikingly variable, 0 to 25% occurring in the sialic acid fraction isolated from this material. A possible explanation for this phenomenon is migration of the acetyl group from 0-7 to 0 - 9 of the Neu side-chain under the conditions of purification of the sialic acid. This assumption is supported b y the observation, made by using thin-layer chromatography (t.l.c.), g.1.c.-m.s., and n.m.r. spectroscopy, that Neu5,7Ac2 is convertedYointo Neu5,9Ac2 at pH values lying between 7.2 and 8.0 in the course of 24 h at room temperature. Furthermore, the 74l-acetyl group of Neu5,7,9Ac3 migrates to 0-8, yielding Neu5,8,9Ac3 under similar c o n d i t i ~ n s . ~ ~ The occurrence of 7-0-acetylated sialic acid in colominic acid has also been reported.47Although the substance has not yet been obtained in pure forni, there is some evidence for the existence of

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(83) J. Haverkamp, R. W. Veh, M . Sander, R. Schauer, J. P. Kainerling, and J. F. G. Vliegenthart, 2. Physiol. Chem., 358 (1977) 1609-1612. (84) I. Ishizuka, M. Kloppenburg, and H. Wiegandt, Biochim. Bioph!/s.Acta, 210 (1970) 299-305. (85) G. Reuter, J. F. G . Vliegenthart, M . Wernber, R. Schauer, and R. J. Howard, B i o chent. B i o p h y s . Res. Commun., 94 (1980) 567-572. (86) R. Pfeil, J. P. Kamerling, J. M. Kiister, and R. Schauer, 2 . Physiol. Chem., 361 (1980) 314-315. (87) R. Schauer, R. J. Howard, and G. Reuter, Z . Physiol. Chem., 363 (1982) in press. (88) J. P. Kainerling, J. Makovitzky, R. Schauer, J. F.G. Vliegenthart, and M . Wemher, Abstr. F E B S Meet., 13th,Jen.rsa/e1n,1980, p. 180. (89) R. Schauer and H. Faillard, 2 . Ph!/sio/. Chem., 349 (1968) 961-968. (90) R. Pfeil, R. Schauer, J . P. Kainerling, L. Dorland, and J. F. G. Vliegenthart, unpublished results.

S1.4LIC ACIDS

145

Neu7AcSGc in sialic acid mixtures from bovine, suliniandibular glands, based on analysis b y g.l.c.-m.s.HL Evidence for the natural occurrence of 80-acetyl groups in sialic acids was obtained from g.l.c.-in.s.x' and n.1n.r. spectroscopy." In the mass spectra of sialic acid mixtures from bovine, suhmandiliular gland, mass fragments were obtained that were interpretedx2as Neu5,8Ac2. In contrast to this compound, the O-acetylated sialic acids Neu5,8,94c3 and Neu8,9Ac25Gc could he partly purified (by the isolation procedure described in Section 111) to an extent enabling clear interpretation of the spectra obtaincd 1)y g.l.c.--Iii.s.H2The occurrence of Neu7,8,9Ac35Gc and Neu5,7,8,9Ac4 i n hovine, s u l ~ n i a n d i ~ ~ u l a r - g l a r ~ ~ gl ycoprotein has already been mentioned.8' Hints as to the existence ot' 80-acetylated sialic acids in humaiicolon mucus have been obtainctl from histochemical studies based on periodate oxidation.Y1However, isolation of colon sialic acids after acid hydrolysis revealed the presence of Neu5,9Ac2 and Neu5,7,9Ac3; no evidence for an acetyl group at 0-8 was obtainedY2by g.1.c.-m.s. As will be discussed in Sections IV,2 and 8, structural analysis using periodate oxidation of sialic acids O-acetylated in the side chain may prove d e c e p t i ~ e . ~ ~ The occurrence of 4Q-acetyl groups in sialic acids has been finnly established in three animal species, namely, the horse, the donkey, and the Australian monotrenie Echidna (Tachyglossz~saczdc(ifus). Relatively large quantities of Neii4,5Ac2 occur in submandibulargland glycoproteins of the horse, together with smaller amounts of Neu4Ac5Gc, Neu4,5,9Ac3, N ~ U ~ , ~ A C , ~ Gand C ,Neu4,SAc29Lac.Ys ~~.~~,"~ 4-O-Acetylated sialic acids have a l s o been found i n other horse tissues, as well as in horse serum (a,-acid glycoprotein) and in horseerythrocyte membranes. In tlw sialic acid fraction from these eiythrocytes, Neu4AcSGc prepontlcrates,'" and it has been shown to be a coniponent of the ganglioside GM, purified to h ~ r n o g e n e i t y . ~ ~ Neu4,5Ac2 has been found in donkey serum.Bfi The sialyl-lactose from the milk of Tachyglossus ucu1ccitu.y is a unique substance containing

(91) C . F. A. Culling and P. E . Reid,,/ ,\lici-osc., 119 (1980)415-425. (92) C. M. Rogers, A. P. Corfield, G . Rt,rtter, M . 1. Filipe, K. B. Cooke, and H. Schauer, in Ref. 80, pp. 652-653. (93) J. Haverkamp, R. Schauer, M. W t , i n l w r , J . P. Kanierling, and J. F. G . Vliegeiithart, Z. Physiol. ChcJtn.,356 (1975) 1575- 1583. (94) J. P. Kamerling, J. F. G. Vliegeiithart, C . Versluis, and H. Schauer, Curhohydr. Rcs., 41 (1975) 7-17. (95) G. Reuter, R. Pfeil, J. P. Kamei-liiig,J . F. G . Vliegenthart, and H. Schurier, Bi(JChir?l. B i O p h ! / S . Actu, 630 (1980) 306-310.

146

ROLAND SCHAUER

Neu4,5Ac2 as the only sialic acid, as was shown b y using sialidase and colorimetric tests,96and b y SOO-MHz, 'H-n.m.r. studies.Ii The hydroxyl groups of some natural sialic acids have been found to be substituted by groups other than acetyl. Thus, L-lactyl groups have been detected at 0 - 9 ofNeu5Ac isolated from bovine, submaiidibulargland glycoprotein,9i and human tissues and ~ a l i v a , "and ~ at the same oxygen atom of Neu4,5Ac2 isolated from equine submandibular glands.s5 The occurrence of phosphoric ester groups at 0 - 9 is long established, as Neu5AcSP was recognized as the condensation product of enolpyruvate phosphate (PEP) and ManNAc 6-phosphate in the biosynthetic pathway of sialic acids (see Section V,l). There is some evidence for the occiirrence ofa glycolyl group at 0 - 4 of Neu5Ac in serum and subiiiaiidibular-gland glycoproteins from the horse, based on biosynthetic studies with radioactive precursors,"j and chemical and t.1.c. analysis of the sialic acid ester group^.'^ Sulfuric ester and methyl ether groups have been identified at 0-8 of sialic acids from some sea-urchin and starfish species. Neu5Ac8Me occurs in the starfish Distolasterias r ~ i p o nThe . ~ ~corresponding N-glycolyl derivative has been isolated from the starfish Asterias forbesiSg and Asterina pectinifera.5i In the latter animal, this unique sialic acid has been found to be a component of a novel ganglioside, and to constitute an internal, sialic acid residue. Neu5Gc8S has been detected in the sea-urchin Echinocardiuin cordaturn. loo The structures of these extraordinary sialic acids were established by inass spectrometry. A synthetic neuraminic acid derivative having a methyl ether group at 0 - 4 (Neu5Ac4Me) was synthesized by Beau and coworkers101*102 by using an oxymercuration-demercuration reaction.Io3 The metabolic behavior of this compound will be described in Sections V and VI. An unsaturated Neu5Ac relative, namely, 5-acetamido-2,6-anhydro3,5-dideoxy-D-g~ycero-D-gakicto-non-2-enonic acid (Neu2en5A~'O"~; (96) M. Messer, Biochein. J., 139 (1974) 415-420. (97) R. Schauer, J. Haverkamp, M. Wember, J. F. G . Vliegenthart, and J. P. Kamerling, Eur. J . Biochern., 62 (1976) 237-242. (98) N. K. Kochetkov, 0. S. Chizhov, V. I. Kadentsev, G. P. Smirnova, and I. G . Zhukova, Carholaydr. Res., 27 (1973) 5-10. (99) L. Warren, Biochim. Biophys. Actu, 83 (1964) 129-132. (100) N. K. Kochetkov, G. P. Smimova, and N . V. Chekareva, Biochin~.Bioph!ys. Acta, 424 (1976) 274-283. (101) J.-M Beau and P. Sinav, Carbohytfr. Res., 65 (1978) 1-10, (102) J.-M. Beau, P. Sinay, J . P. Kamerling, aiitl J. F. C . Vliegenthart, Carbohydr. Res., 67 (1978) 65-77. (103) J.-M. Bean, R. Schaiier, J. Haverkamp, L. Dorland, J. F. G. Vliegenthart, and P. Sinay, Carboh!/clr. Res., 82 (1980) 125- 129. (1034 Although this is a convenient abbreviation, it is technically incorrect.

147

Ac OH

2

2), which was synthesized b y Meindl and T~ippy"'"for inhibition of viral sialidases, was subseqiientl y discovered in sera, saliva, and iirine of lnan.:{A. 105 It can exist only i i i the free form, a s it lacks an anoineric 2-liydroxyl group. In the frec sittlic acid fraction froin serum aiitl urine of normal individual s , the F rc' n c ti s i a1 uria patient .IoTro r the Salla disease patients,:'X."9the propor-tioii ot' N e u 2 e n 5 k varies I)etween 1 and 3%. The cmrresponding \.alric\ was 17% i n the uriiic froin the Belgian sialuria patient."9 Altlwugh the French sialuria patient e x creted a few 100 m g ofNeu2en5Ac per (lay, the correspondiirg quantity from a healthy individual is:14110 higher than 5 0 - 100 p g . Kt~inarkalily, the proportion ofNeu2en5Ac i n the fraction o f f r t ~ sidic : acids i n saliva varies between 1 and 8070 wlrvn iiieasured in different persons, representing a maximum conceiitration of 20 FUJI Neu2eniJAc in this fluid.:32It is not known whethet these differences i n saliva art, due to variations in the metabolic state of the salivary glands or to other (for example, genetic) influences. This phenomenon needs fiiitht,r evaluation on a larger scale for recognition of its biological or pathophysiological significance. The nietalmlic origin of Neu2en5Ac will be discussed in Section V. ,:j4

111. ISOLATION

AND

PURIFICATION OF SIALIC ACIDS

The acylneuraininic acids ciiii he released from their glycosidic linkages either tiy dilute (aqucwus or niethanolic) acids or sialidases. Special care must be taken in the isolation of the rather labile, O-acylated sialic acids, which arc pnrtially non-susceptil,le to the action of sialidases. Furthermore, in gaiigliosides, non-0-acetylated sialic acid residues occur, and these are a l s o inore or less resistant towards these enzyrn e s. 1. Acid Hydrolysis of Glycosidic Bonds Almost total release of sialic. acicls from glycoproteins or oligosaccharides can be achieved106~l"i b y heating the material in 0.05 A 1 sul(104) P. Meindl and H. Tuppy, M o i i c i f s l t . C / w i t t . , 100 (1969) 1295-1306. (105) J. P. Kamerling, J . F. G. Vlicgt~iithait,H . Schauer. G. Strcckcr, m t l J . Ilontreuil, Eur. J . Biochriti., 56 (1975) 253-258. (106) R. Schauer, A. P. Corfield, 11. lf't.iiil)c~r,mid D. l h i o n , Z. Ph!I.,io/. L ' / w t t i . , 356 (1975) 1727-1732. (107) R. Schauer, .2let/zotls E t t z y i t t o / . . 30C ( 1078) 64-89.

ROLAND SCHAUEH

148

furic or 0.1 M hydrochloric acid for SO inin at 80". For gangliosides, the heating time should be extended to 60 or 70 inin. A greater resistance of the glycosidic bonds towards acid hydrolysis has also been reported for 0-acetylated sialic acids as compared to the unacetylated con1pounds.108During acid hydrolysis, however, 10% of the sialic acids are decomposed, and this must be taken into consideration when calculating the original sialic acid content of the substances investigated. Thus, the conditions described constitute a compromise between complete hydrolytic release of sialic acids and decomposition by the relatively high concentration of acid. These conditions also lead to almost complete elimination of 0-acetyl groups. Therefore, for preparation of 0-acetylated sialic acids, hydrolysis is conducted10i under milder conditions in 0.01 M hydrochloric or formic acid (pH 2) for 60 rnin at 70"; 70-80% of the 0-acetyl groups are then retained. However, the release of the sialic acid is not quantitative. Based on these observations, the following procedure for quantitative release of sialic acid and optimal preservation of 0-acetyl groups is recoinmended.L07 After the mild-hydrolysis step at 70°, the sialic acids liberated are removed from the sample by dialysis or ultrafiltration at 2", and the niacromolecular material is rehydrolyzed, using, however, the stronger acidic conditions of 0.1 M acid. The dialysis time ranges between 6 ancl 24 h, depending on the volume ancl viscosity of the hydrolysis mixture. Therefore, the optimum dialysis time should be evaluated b y determinations of sialic acid in the eluate, or by addition of a trace of radioactive NeuSAc. The dialyzates, or filtrates, are combined, and processed a s will be described. By using this procedure, the overall yield of purified sialic acids is 70-8070, and the loss of 0-acetyl groupsL0'is 40%. Neu-P-Me is one ofthe earliest Neu derivatives to be prepared," as already mentioned. This compound is still of interest, mainly for preparative purposes; for example, it may be employed in the synthesis of Neu5Ac or Neu5Gc, especially with radioactive labels in the N-acyl groups.69-L"9 Neu-P-Me can readily lie prepared from crude sialoglycoconjugates (for example, edible I>ird's-nest substance, or submancli\>ular-gland glycoproteins) as a homogeneous compound, regardless of the possible presence of a mixture of sialic acids in the native materials, by using a procedure that includes niethanolysis and ion-exchange cliroiiiatography."".'07

-

-

(108) A. Nculwrger and W. ,4. Hatcliffe, Aiochcrn. J . , 133 (1973) 623-628. (109) R. Schauer, F. Wiitz-Peitz, and H. Faillard, Z. P/i!/,yzol. C:hern., 351 (1970) 359364.

SIALIC ACIDS

149

2. Enzymic Hydrolysis of Glycosidic Bonds

A great variety of sialidases (acylneuraminyl hydrolases; EC 3.2.1.18) occur in Nature. These enzymes are involved in the cleavage of the a-glycosidic bonds of sialic acids in oligosaccharides and glycoconjugates. Their occurrence and properties are reviewed in Refs. 55 and 110, and will be discussed in Section V1,l. The sialidases frequently used for isolation of sialic acids are obtained from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens; they may be purchased in partially purified forms having high specific activities. Only a few bacterial and viral sialidases have been purified to high purity or even to protein Complete purification of sialidase on a preparative scale from the culture filtrate of C. perfringens was achieved' '' by using poly(acry1aniide) gel-electrophoresis as the final purification step (see Section V1,l). It is necessary that such purified sialidases be available, as the presence of proteases or other glycosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). Pure sialidases are also needed for studies with immobilized enzymes. The absence of nonenzyinic protein enables a quantitative iinmobilization of enzyme on the particles, as was achieved with sialidases from C. perfrngens112and V. ~ h o l e r a e ~using ~ ~ , "Sepharose ~ 4B and glass beads. The advantages of such immobilized enzymes for the release of sialic acid both from cells and from soluble, complex carbohydrates are their stability, and the possibility of repeated use of the enzyme and of separation of the enzyme either from soluble substrates or treated cells. The high stability of immobilized sialidases is of great advantage for quantitative desialylation of soluble glycoconjugates which, in inany cases, cannot be achieved within a short time. For this purpose, a closed-circuit system has been c o n ~ t r u c t e d "that ~ permits desialylation of soluble material by repeated pumping over immobilized sialidase for some 24 h at 37". A further advantage of'this method is removal, by continuous dialysis, of the sialic acids liber(110) A. Rosenberg and C.-L. Schengrund, i n Ref. 19, pp. 295-359. (111) S. Nees, R. W. Veh, R. Schauer, a r i d I<. Ehrlich, Z. Ph!/sicd. Chem., 356 (1975) 1027-1042. (112) T. L. Parker, A. P. Corfield, R. W. Veh, and R. Schauer, Z. Ph!/siol. Chem., 358 (1977)789-795. (113) A. P. Corfield, J.-M. Beau, and H. Schauer, 2. Physiol. C h e m . , 359 (1978) 13351342. (114) Yu. V. Vertiev, G. K. Beljanskaja, and Y n . V. Ezepchuk, Biokhimiya, 42 (1977) 1736- 1741.

1so

ROLAND SCHAUEH

ated, thus allowing determination of the amount of sialic acids released and also preventing the inhibition of enzymes that is known to be caused by free sialic acids.11s When sialidases are used for the preparation of sialic acids, a iiumthe requirement for metal ions ber of factors must be (for example, V. cholerne sialidase needs 4 m M Ca2+for full activity), the slightly acidic pH optimum, the substrate specificity with regard to the general nature of the substrate (oligosaccharide, glycoprotein, ganglioside, or cell membranes), the nature of the sugar and the position to which the sialic acid is a-glycosidically linked, the position ofthe sialyl group (at the end, or as a branch, of oligosaccharide chains), and the nature of the N-acyl and the position ofthe 0-acyl groups in the sialic acid molecules. Sialic acid residues that are partially or completely resistant towards the action of different sialidases are those that have a 4-0-acyl group or are situated at the internal Gal residue of gangliosides5s,"0 (see Section V1,l). For routine, enzymic hydrolysis of sialic acids on a preparative or an analytical scale in our laboratory, the substrates are incubated with bacterial sialidases in 50 mM acetate buffer at pH 5.5 and 37" for times ranging between a few minutes and several hours, depending on the subst~ate.'"~ After incubation, the sialic acids liberated are separated b y dialysis, ultrafiltration, or protein precipitation at 0-2", and purified as will be described in the following Section.

3. Ion-exchange Chromatography Sialic acids must be purified after enzymic or acid hydrolysis in order to provide exact analytical data on their quantity and n a t ~ r e . " ' ~ Only a short description of' the purification procedure, with inclusion of the most recently acquired knowledge, is given here. All purification procedures should be performed between 0 and 4".As a first piirification step, ether extraction of' the hydro1yzate, especially of acidic hydrolyzates from whole cells or cell iiieinbranes that may contain fatty acids or other lipid material disturbing the periodic acid-thiobarbituric acid assay, is recommended. Thereafter, the solution of the sialic acids is passed through a column of a cation-exchange resin (Dowex 50, H+) which is eluted with water, and the material is adsorbed to Dowex-2 X8 anion-exchange resin (100-200 mesh, fonnate or acetate form). Depending on the quantity of sialic acid and the proportion of impurity, the elution may be performed batchwise on a (11.5) R. Schauer and A. P. Corfield, in F. G. de las Heras and S. Vega (Eds.), Medicirid Chemistry Arlocinces, Pergamon, Oxford, 1981, pp. 423-434.

large or small scale (for exatiiplc, i t r 3-mL Pastrllr pipets, for iitnotillts of less than 100 w g of sialic acitl), o r h y use of a gradicnt. For batchwise elution, 0.8- 1.O .\I foi-qiic acid, o r 0.25 111 pyritliniuin acetate, pH 5.4, is applied. F o r quantitative elutioti of the sialic acids, the needed amount and concentration ofthe elriaiit may be iiiflur.ncetl by the quality ofthe batch oftlie coiiriiiercial resin, as well a s b y other factors, and should therefow I)c checked b y chroinatography of a reference sample, preferably 1)). inclusion of ii trace of radioactive Ne115Ac. Gradient elution effects tilore efficient purification of tlie sialic acid. Usually, a gradient 01' 0 - 1.5 JI foniiic acid or 0-0.5 .I1 pyridinium acetate is applied. H o w ( ~ v ~b~y r use , of a i i 10-fold coltinin volume of a very gradually iiicreastd gradient of O-0.5 ,If formic acid, partial fractionation of a sialic acid iiiixtnre on Dowex-2 X8 resin is obtainetl.R2It is probable that the different sialic acids are separated o n the basis of differences in their ~ J K~ a l r i e s , ~in~hydrophobic ' j ~ ~ ~ ~ iiiteractions due to the presence ot'O-iicetyl groups, ant1 in structural confoiinatioiis attributable to iiitr;iiiiolt~cularhydrogen-l)oiicliii~."~ Thus, sialic acids acetylated at 0 - 7 o i r I > , , o r containing several O-acetyl groups, are eluted at lower coticeirtr;itions of foriiiic acid than sialic acids substituted only at 0-9. I1iisul)stituted NclitEiAc aiid NeufjCc are eluted betweeii these two groiips of 0-acetylatccl sialic iicicls.x2T h i s behavior niay be used for a Iwttc~r,f i l i a l purification of intlividiid sialic acids by cellulose chromatography (see later). The sialic acid eluates froiii ion-(~xchungechroniatograplry may he evaporated by freeze-drying, o r b y careful rotarb, evaporation at ;I bath temperature of 35", and storecl i i i a tleep-freezer. Despite treatment by ion-esc~hangc~ chroinatograpli)--,tht. sialic acid preparation tilay still contain siic.11 iinpurities as proteins, pcpticlcJs, o r other ionic coinpounds that I I iay iirterfere with fiirther analytical o r pie parat ive procedure s . I11 t 1i (, se cii s e s , ch roni iit ( ) g 1-21ph >. of' the 11 re paration on Sephadex G-10, C;-25, 01- Biogel P2 i n water inay prove he l l h 1.

-

4. Chromatography on Cellulose

To obtain individual sialic acitls i t i pure, o r alniost piire, f o t - n i , the sialic acid mixture obtained t;-oin tlie procedures just dt.scril)rcl iire subjected to partition chromato~gr.a~,hv on cellulose powder, using, a s the ~ o l v e n t , 1~: 2: ~ ~1~ ( v"/ v~/ \ , ) Ll)iitatiol- l-i?ropatiol-water. The (less hydrophilic) tri- aiid di-O-ac*et?latc,(l sialic acids are eluted first, foltl acids, NeuSAc, and Neii5Gc, aclowed by m o n o ~ ~ - a c e t y l a t ehialic

ROLAND SCI-IAUEH

152

cording to their increasing hydrophilicity. For further or better fractionation, the batches of sialic acid may be rechroinatographed. After chromatography, the alcoholic solvent should be removed as soon as possible, by freeze-drying or rotary evaporation, to prevent esterificatioii of the carboxylic group of the sialic acids with 1-propanol or 1-butanol, and to pemiit exact, analytical deteniiinations. Carefully purified and freeze-dried samples of sialic acid, including the O-acetylated derivatives, can be stored at - 20" for more than 10 years without appreciable decomposition. During all of these isolation procedures, special care should be taken to avoid contamination of the samples by plasticizing agents (for example, phthalic esters) which may be derived from the ion-exchange resins or plastic vessels, and which niay disturli the following analytical procedures. Thus, use of plastic materials should be largely avoided, and the sialic acids should be processed and stored in glass vessels .

Iv.

ANALYSIS OF SIALIC

ACIDS

The unequivocal determination of sialic acids occurring in low concentrations in many biological materials is rather difficult, and this e x plains the many errors that have been made in this field; for example, the erroneous determination of sialic acid in plants, already mentioned. Substances known to interfere include 3-deoxy-2-glyculosonic acids [for example, 3-deoxy-D-n~c~n~io-~-octulosonic acid*17a (KDO)], a few pentoses and hexoses, polyunsaturated fatty acids, and pyrrole derivatives (sunimarized i n Ref. 107).These compounds, as well as mistakes i n handling the labile sialic acids, may influence the formation of chromophores during aiialytical procedures in the test tube, or the RF values in t.l.c., or may give rise to additional peaks in g.1.c. Plasticizers interfere appreciably with g.1.c. analysis.95 Exact analysis of sialic acid is required in biological experiments where the biological role of sialic acid is frequently studied with the aid of sialidases, and the amount of sialic acids released is determined. This is also important for periodate oxidation studies on biological systems, where modification of sialic acids by periodate is only assumed, but chemical analysis of this effect by isolation and analysis of the modified sialic acids is seldom perfoniied. These uncertainties in determinations of sialic acid can be overcome by the purification procedures already described. Furthennore, it must be stressed that unequivocal determination of the structure of a sialic acid, especially (117a) F. M .Unger, Ado. Corboh!/tlr.Chum. Biochenr., 38 (1981) 323-388.

of the 0-acetylated species, is possible only I)y litass spectrometry and n.1n.r. spectroscopy, to be discussed. With regard to the nature, ti innher, and position of N - and 0 - a c y l groups, the colorimetric and chromatographic methods permit o n l y ii tentative assignment of individual sialic acids.

1. Colorimetric Methods Various methods for the colorimetric analysis o f sialic acids exist; they have been reviewed in Rcfs. 2 1, 107, and 118. Some of the procedures are suitable for the deteriniiration both of free and glycosidically bound sialic acids; these include the ~~-dimethylainiIlol,enziiizal~lehyde -HCI reagent (direct Ehrlich r e ~ t i o n ) , the " ~ orcinol- or resorcinolFe:3+or -Cu'+-HC1 reagents,":'.l2"and the periodic acid-3-methyl-2benzothiazolinone hydrazone reagent.121 In contrast, the periodic acid -thiobarbituric acid reaction according to Warrenz2or Aniinoff122is applicable only to free sialic acicls. At the end of the last century, 1'. Ehrlich observed the fbnnation of a purple color after heating mricins with ~~-dimetliylamiiiol~enzaldehyde in acid solution.123This rt,action has been adapted 1)y Werner and O ~ l i n "to~ a more specific, cluantitative test for sialic acids. i2s 5( ~n ru hi n o -te t ri tol- 1-y l ) p y rro1c-2-carlmx y 1ic acid fonne d froin s ial ic acids under the influence of a strong acid is presumably the irnntediate chromogen in this reaction, prefoi-nied pyrroles occurring in body fluids or tissues interfere with the reaction.2""g However, if purified sialoglycocoiijugates or free sinlic acids are analyzed, the inethod leads to exact values for sialic acid. Significant differences i n the color yield from Neu5Ac, Neu5Gc, and 0-acetylated sialic acids do not exist. The main disadvantage o f the reaction is its lower sensitivity a s compared with those of the following methods (inilliniolar extinction coefficient for Neu5Ac 1.556). For this reason, the Ehrlich method is infrequently used today. In contrast, the diphenol reactioiis leading to a I)lue-purple color are in wide use for sialic acid tleterminations. Sialic acids are heated either with orcinol and Fe"+(the Rial reagent':'), or with resorcinol and Cut+ (Refs. 30 and 120) in coiicc~iitratedhydrochloric acid."'7I n 1903, R. W. Ledeeri a n d R. K. Yu, i n 1 ~ ~ ~ 19, 1 ' . pp. 1-57. I. Werner and L. Odiii, Actci Sot,. .!f[,(/. I'ps., 57 (1952) 230-241. L. Sveir.rrerholm, Ark. Ketni, I 0 ( 1957) 577-586. Y. Slassamiri, 41.Beljean, G . D i i r a l i , J . Fegei-, 41. Pa1.b. and J . .4grieray, Aiicil. R i o c h o n . , 91 (1978) 618-625. (122) 11. Amiiioff, Biochem. I., 81 (19611 384-382. (123) P. Ehrlich, M e d . Woche (Berliii 1, I ( I 901) 151- 153; F. Priiwher, Z . PhI/.vioZ. Chem., 31 (1901) 520-526. (118) (118) (120) (121)

154

ROLAND SCHAUER

Bia1Iz4had introduced orcinol dissolved in HC1 and containing FeCl, , CuSO,, or HgO as oxidizing agents for the determination of pentoses. Klenk',21s'z5was the first to use this reagent, and he observed a characteristic, purple color during heating of brain tissue or rnucolipids. B6hm and coworkersz3developed the orcinol- Fe3+assay into a sensitive and reproducible method for the cletenniiiation of free and glycosidically bound sialic acids. The chromophore fonned can tie extracted into pentyl alcohol, giving a blue-grey color having an absorption maximum at 572 nm. The formation and structure of this chroniophore, which is specific for N e u derivatives, have not yet been fully elucidated; it is probably a iiiethine dye.Iz6 With this method, all natural and synthetic Neu derivatives can be determined. Thus, 0acetylated sialic acids and Neu2enSAc give color yields similar to those of niaternal NeGAc and NeuTjGc (see Table 11). Furthennore, C, (C,-NeuSAc, C7-Neu5Gc)and C , (C,-NeuSAc, C,-NeuSGc) analogs of Neu5Ac and Neu5Gc, namely, 5-acetamido(or glycoly1amido)-3,5dideoxy-L-clmbino-heptulopyranosonic acid and 5-acetamido(or glycolylamido)-3,5-dideoxy-~~-~nZncto-octulopyranosonic acid, respectively (see Section IV, 2 and formulas) obtained from periodate oxidationtiorohydride reduction of sialic acids ciin also be analyzed Iiy this test. However, the colors fomied, and the inillimolar extinction coefficients so far determined for the latter NeuSAc derivatives are different froin those of the natural sialic acids (see Table 11). Minimum amounts of 2-3 pg of sialic acids can be detemiined in, for example, the orcinol-Fe3+ assay, if the volumes of the Bial reagentsz3 are one-fifth of those originally described.I0' By using this method, sialic acids can tie accurately detemiined only if they have been prepurified, as free or glycosidically bound pentoses, hexoses, or alduronic acids interfere with the reaction b y giving green chromophores.'"7 In our experience, an approximate determination of the sialic acid in a complex carbohydrate is only possible if its sialic acid content is > 5%. An increase in sensitivity of 30-50% can be achieved if resorcinol and CuZ+are used."" This increase may be by a factor of 3 to 6 if the sialic acid side-chains are oxidized by periodate prior to application of the resorciiiol-Cuz+ reaction.'*' More sensitive, and most frequently used, is the periodic acid-thiobarbituric acid assay devised by Warrenzz and modified b y Aminoff.107J22 In a reaction sequence initiated by periodate oxidation, the (124) M. B i d , Dtsch. Metf. Wochoischr., 29 (1903) 477-478. (125) E. Klenk and H. Langerbeins, Z. Ph!/siol. Chern., 270 (1941) 185-193. (126) F. Wirtz-Peitz, Doctoral Thesis, University of Bochum, 1969. (127) G. W. Jourdian, L. Dean, and S. Roseinan,J. B i o l . Cherii., 246 (1971) 430-435.

TABLEI1 Colorimetric Determination of Sialic Acids and the C7 and C, Analogs of NeuSAc. Millimolar Absorption Coefficients in the Diphenol and Periodic Acid-Thiobarbituric Acid Assays ~~

~

~~

Diphenol assays

Compound Neu5Ac Neu4,SAq Neu5,7Ao, Neu5,9Acz Neu5,7,9Ac3 C8Neu5Ac C,Neu5Ac Neu5Gc Neu4Ac5Gc Neu9Ac5Gc Neu5Gc8Me Neu2en5Ac Neu-&Me

Periodic acid-thiobarbituric acid assays Periodate-resorcinolOrcinol-Fe3+-HC1" Resorcinol-Cuz+-HClb Cua+-HClr According to Aminoffd According to Warrene (max. absorp. 572 nm) (max. absorp. 580 nm) (max. absorp. 630 nm) (max. absorp. 549 nm) (max. absorp. 549 nm) 5.30 5.30 5.30 5.30 5.30 10.308 8.00" 6.30 6.30 6.30 6.30 4.90 6.78

6.90 -r

63.00 65.00 3.50 30.00 0 51.00 51.00 23.00

0 0

61.00 47.00 61.00 61.00 50.00 -

6.00 0 0

" Refs. 23,99, 107, 109, and 141. Refs. 30, 107, and 120. Refs. 107 and 127. Refs. 89, 107, 122, and 141. Refs. 22, 99, and 107. r-,

not determined. Max. absorp. 562 nm.l4l Max. absorp. 602 nm.I4l

156

R O L A N D SCHAUER

3-formylpyruvic acid formed couples to the thiobarbituric acid, yielding a red chromophore having a maximal absorbance at 549 mi. This assay is 6-10 times as sensitive as the orcinol-Fe3+ assay, thus allowing determination of minimal amounts of 0.5 p g of sialic acids b y using micro-adaptations of the original assays."" The millirnolar extinction coefficients ofdifferent sialic acids are shown in Table 11. According to the original procedures, the chromophore is extracted either into cyclohexanone22 or acidic l - b u t a ~ i o l for ~ ~ )reading. ~ It has been reported12#that replacement of these organic phases by dimethyl sulfoxide appreciably enhances the stability of the chromophore. As a further improvement, extraction of the chroinophore into HC1-acetone or 2-methoxyethanol ("methyl Cellosolve") has been recommended.'2g Hammond and Papermaster'"" reported an increase of the sensitivity of the periodic acid-thiobarbituric acid assay by a factor of -50 (over the micro-assay just mentioned) when the chroinophore extracted into the acidic 1-butanol phase is excited at 550 iim and the emitted light is measured at 570 nm. The periodic acid- thiobarbituric acid test has some disadvantages that may cause severe errors in the determination of sialic acid. ( a ) As only free sialic acids react, acid or enzymic hydrolysis of glycosidic bonds must precede the analysis, which may lead to some loss of sialic acids clue to acid hydrolysis, incomplete enzymic release, and tlie purification procedure already discussed. ( h ) Neu2enfjAc does not react, and some 0-acetyl groups, depending on their position in the Neu molecule, appreciably influence the formation of color. Whereas an acetyl group on 0 - 4 has little influence, the same group on 0 - 7 almost completely prevents color formation, and an acetyl group on 0 - 9 lowers the inillirnolar absorption coefficients74,m,in7of the corresponding Neu5Ac and Neu5Gc by -60% (see Table 11).These effects can be explained by the strong influence of the 0-acetyl groups o f t h e sialic acid side-chains on tlie oxidation rate b y periodateY"and by the mechanism of formation of 3-fomiylpyruvic acid.131According to the latter study, an aldehyde group at C-6 of Neu is required for formation of 3-formylpyruvic acid during heating with thiobarbituric acid. Formation of such an aldehyde is prevented b y an acetyl group on 0-7. Furthermore, oxidation of the sialic acid side-chain is not completely prevented, but is markedly hin(128) L. Skoza and S. Mohos, Biocheiii. J., 159 (1976) 457-462. (129) Y. Uchida, Y. Tsukada, and T. Sugimori, ,/, Riocheiii. ( T o / q o ) ,82 (1977) 14251433. (130) K. S. Hainniontl and D. S. Papernlaster, A r i d . Biochetn., 74 (1976) 292-297. (131) G. B. Paerels and J . Schut, Biochcwi. /,, 96 (1965) 787-792.

dered, if an acetyl group is prcssent on 0-9; this can be explainetl by the tt-nizs-disposition of the h~~tlroxyl groups on C-7 and C-8, recognized in Neu5Ac and Neu5,9AtS, oii thc basis of the results of several physicocheniical studies, includiiig n.1n.r. spectroscopy.’~.”:”””Ill an unsul,stituted sialic acid, however, periodate oxidation of the Dcr!/thro-glycerol-l-yl side-chaiii is iiot restrained, as thc confonnational hindrance at C-7 and C:-8 is abolished b y the formation of an aldehyde group at C-8. For quaiititativc. detenniiiation of sialic acids i n this colorimetric assay, there tor<,,O-acetyl groups must be removed froni the sialic acids by O-deacc~tylationin 0.01 M NaOH for 45 min at 0”. (More-concentrated alkali slioultl he avoided, to oliviate degradation of sialic acids by an aldol-cleavage reactioii.“) ( c ) A further drawback of this a iy is the fact that several coinpounds occurring in biological tiiatc,rials, such a s L-fiicose, ~ - ~ ~ o x ) T - D cr!ythro-pentose, 3-deoxy-2-g1~ciilos01~ic acids, m(1,especially, iiiisaturated fatty acids, lower the yield of the cliromophore derived from sialic acids, or afford other retl(1isli c~hroniophoresthat m a y lead rious errors in the quantitative detc.rtiiination of sialic acids.“’ method for overcoming these prol)leins has heen devised b y Warren by measuring the chromophorr, ;it t\vo wavelengths (549 and 532 nin) and calculating the true sialic acid concentration from the extinction values This inetliotl is practicable only in the prest:nce of low proportions of interfering siilxtaiices. As crude biological niaterials frequently contain large proportions of interfering substances (compared to the sialic acid coiicentr~~tioi~), purification of sialic acids after hydrolysis, b y ion-excliatige cliroinatograpli~,and extraction of lipids b y ether or iilethanol-clilorofoi-in (see Section III), before application ofthe thiobarbituric acid a s s a y , is strongly recommendetl. In our experience, the necessity l o r this mode of purification of sialic acid is much higher after iicitl hytlrolysis than after sialitlase treatment, and this is due to the 1il)eration of further interfering suhstances, mainly fatty acids, especiall?. wheii analyzing cellular (membrane) materials. A further method for estiiixitioii of‘sialic acid using periodic acid has been reported h y Ma miri and coworkers.“’ Foimaldehyde prodiiced b y mild, oxidative clc,avag:e of the D-r.)-!itliro-glycerol-l -yl side-chain of Neil can be detected b y 3-metliyl-2-benzotliiazolinone hydrazone, which yields a grwn-l)liie color (a1)sorbance maxiinuin 625 iini). This test, which is slightly more sensitivc than t h e thiollarbi(132) E. B. Brown, W. S. Brev, J i - . , i i i i ( l b’. Wrltiwr, JI-., H i o c , l i i r r i . Biorh!/,$. Artci, 399 (1975) 124-1:30. (133)S. S. Kuwahara and hl. C . Siirttiiig, Atitil. Hioclzerii., 100 (1979) 118-121.

158

ROLAND SCHAUER

turic acid assay (millirnolar extinction coefficient for Neu5Ac 67.00) has been applied for determinations of sialic acid in erythrocyte rnemb r a n e ~ . As '~~ a variety of compounds of biological origin interfere with this test, we replaced the hydrazone derivative b y 2,4-pentanedione, and fluorometrically nieasured the chromophore formed ( h 410 and 510 nni).13sBy this modification, quantities of free or glycosidically bound sialic acid as low as 1 ninole can be determined. A sensitive, and the most specific method, for determination of sialic acid is the use of acylneuraininate pyruvate-lyase from C . perfrirzgens (10 mU/inL of assay mixture).'07In this enzymic reaction, sialic and p y r ~ v a t e . ' ~ , ' ~ ~ acids are cleaved to ~,O-acy~amino-D-ina~inoses Fomiation of the latter compound is monitored at 366 nrn by using lactate dehydrogenase (EC 1.1.1.27) and NADH. Cleavage of sialic acid and, thus, its determination, is quantitative by use of this coupling of the two enzyme reactions. Neu5Ac and Neu5Gc are split at equal rates. It is recommended that 4-O-acetyl groups of sialic acid be removed with dilute alkali before application of this analytical procedure, as this group lowers'07.'"';the reaction rate of the lyase b y 90%. Neu5Ac4Me, Neu2en5Ac, and glycosidically bound sialic acids are inactive in the lyase test. Because evidence is accumulating for a wide distribution in Nature (see Section 11) of O-acylated sialic acids which influence the quantitative analysis of sialic acids, and (as will be discussed in Section VI) also affect the activity of sialidases, detemiination of O-acyl groups in sialic acids is necessary. This is performed with free or glycosidically bound sialic acids according to He~triii,'"~ with the modification of Ludowieg and Dorfman,13* using alkaline hydroxylarnine and ferric perchlorate. The reddish color is read at 520 nm; minimum amounts of 0.05 pmol of ester in a sample of sialic acid can be determined'07 if only one-tenth of the original volumes of reagents is used. It must be borne in mind that ester groups other than those in the sialic acids may interfere with this reaction. The nature of the O-acyl substitiients can be tentatively determined by t.1.c. as acylhydroxamates. However, the nature and position of the ester groups in sialic acids can only be unequivocally determined by g.1.c.-m.s. or b y n.m.r. spectroscopy (see later). As O-acetyl groups have been found to migrate within the sialic acid side-chaing0 (see

-

(134) Y. Massaniiri, G. Ilurand, A. Richard, J . Fkger, and J. Agneray,Aiid. Riochem., 97 (1979) 346-351. (135) A. K. Shukla and R. Schauer, 2. Ph!/siol. Ch~ni., 362 (1981) 236-237. (136) R. Schauer, M. Wember, F. Wirtz-Peitz, and C. Ferreira do Amaral, 2. Physiol. Chem., 352 (1971) 1073-1080. (137) S. Hestrin,J. B i d . Chem., 180 (1949) 249-261. (138) J. Ludowieg and A. Dorfinan, Riochim. Riophys. Acta, 38 (1960) 212-218.

Section 11), and as this may o('ci1r daring the isolation procediire, an analytical tool is desirable for dt,tcrinii~ationof the priinaiy position of 0-acetyl groups in sialic acitls in native materials, inc~ludingiittact cells or cell membranes. For this piirpose, a niethotl has Iieeii &>vised that entails "freezing" of the 0-acetyl groups b y replacing thein h y inethyl groups, followed b y andysis of' the partially (I-inethylated sialic acids by g.l.~!.-rti.s.,~~!' a s will I)e described in Scction IV.5. Although this method has been siiccessfully applied to sialic acid methyl glycosides, it has not yet I)c.en adapted to the analysis of crude biological materials, isolated coniplex carboh!dnites, or enzyinic, 0ace ty 1-transfe r studies . Another possibility for tentativc localization of 0-acetyl groups i n sialic acids, and also for estimation of tlieir concentratioii in crude biological materials, is the mild oxidation of sialic acid side-chains I)? periodate, and determination of the resulting formwldeliycle 1)y 3-methylo r 2,4-peiitanedione as already 2-l)eiizothiazoliiioiie hydrazoiic.'2'.':%4 descrihed. In the case of ail ac*ct\ilgroup o n 0-7, one niole of formaldehyde is fonned per mole of sialic acid, as with tiot10-acet);lated sialic acids, or those having ;i 4-0-au.tyI group. For acetyl groups at 0-8 or 0-9 (or at both 0 - 7 and 0 - 9 , o r 0-8 and 0 - Y ) , o n l y insigiiificarit amounts of fonnaldehyde are t o r i i i t , d under the conditions choscw, for reasons already explained.'"" \Vhc.n these data are related to thc total amount of sialic acid detemiiiic.tl I)y other colorimetric methocls, the quantity of sialic acids mono- o r di-O-:icetyIatetl at tlie positions in&cattd can be estimated. This (Iiiantity can also be deteniiiiiecl l)y a p plication of the periodate p r o c ~ d u r cbefore a i i d after removal of the 0-acetyl groups. Comparisotr of' t l r e vulues afforded b y this method with those obtained after acid Irytlrolysis showed a good corrc,lation i n studies of a variety of m a i n i t i a l i a i i c,r?throcytes.Thus, 60, 40, and 20% of the membrane sialic acid rc.sic1iit.s were foiind to bc acetylatecl at 0-8 or 0-9 in mouse, rat, and r:ihI)i t erythrocytes, respectively.':'5The corresponding value for piirificacl, I)oviiir, sul~iiiaiiclil~i~lar-glaii~l fil ycoprotein is YS%. Tentative localization of a c t s t ) - l groiips at thc 0 - 4 atoins of sialic acids in native complex carlmli!,drates is possible with tlie aid of'sialidases, which are almost, o r cwnplc,tc,ly, inactive with these coiiipounds or, after isolation of tho sial ic acids, b y ac.yltieiir~iiniiiatepyriivate-lyase, which also shows littlc activity with these srilxtratcs (see Section VI). Chemical determination of' ,Y-ac,yl groups i n sialic acids is no longer routinely perfoiiiied, a s o n l y two N-acyl groups (acetyl and glyco(139) H. van Halheek, J. IIavrrkaiirp. l . l'. lianrerling, J . F. G . Vliegrnthat-t, C . Vt~rsluis, and H. Schauer, Carhoh!/r/r.H e . .60 ( 1978)51-62.

160

ROLAND SCHAUEH

lyl) are known to occur in natural Neu derivatives, and the respective Neu5Ac and NeuSGc can be readily distinguished by t.1.c. and g.1.c. However, if N-glycolyl groups have to be determined in sialoglycoconjugates, they can be hydrolyzed off, and the glycolic acid esterified b y heating the material in 20% p-toluenesulfonic acid in ethanol, followed b y distillation of the resulting ethyl a c y l a t e ~ . ' ~ Glycolyl ~,~~~ esters isolated in this way give a reddish purple color, monitored at 546 nm by heating with 2,7dihydroxynaphthalene in concentrated sulfuric acid. ~ - L a c t ygroups l described as occurring at 0-9 of sialic acids isolated from different tissues (see Section 11) are analyzed either as their hydroxylainine derivatives, by t.l.c., or, in a more specific way,97by L-lactate dehydrogenase and NAD+ in the presence of hydrazine, after 0 deacylation with 0.05 M NaOH.

2. Periodate Oxidation Periodate oxidation of sialic acids had earlier been used for structural determination of 0-substituted sialic acids.89Whereas, for example, one mole of a 4-O-acetylated sialic acid consumes 2 moles ofperiodate within 10-20 rnin at o", the same amount of the 7-O-acetyl isomer is oxidized by only one mole. As already discussed in connection with the periodic acid-thiobarbituric acid assay, 9-O-substituted sialic acids exhibit a very low rate of oxidation as compared with the unsubstituted sialic acids"; this observation originally led to the erroneous assigninenP of the 9-O-acetyl group to 0-8. All other sialic acids having O-acetyl groups on the side chain are expected to be unaffected by periodate. Accordingly, differences in the susceptibility to periodate oxidation must be considered in oxidation experiments on cell-membrane sialic acids conducted in studies intended to elucidate the biological role of sialic acids. Information concerning the success of such experiments on the modification of sialic acids, which usually include a borohydride-reduction step, is seldom available, due to the lack of methods for analysis of the modified sialic acids. However, the following technique is now available for this purpose. Treatment of the periodateoxidized sialic acids (for example, 4 and 5 ) with borohydride leads to the C , and C8 analogs (6 and 7) of Neu5Ac, or of Neu5Gc, shown in Scheme 1. These compounds can be prepared by periodate-borohydride treatment of sialic acid-rich glycoconjugates, for example, porcine submandibular-gland glycoprotein for the N-glycolyl derivatives, (140) H. J. Schoop and H. Failiard, Z. Physiol. Chenz., 348 (1967) 1509-1517

AcNH -t

1

I

OH

HO

HCOH I HCOH

HCOH I CHO

4

I

CH,OH

5

3 NnBH,

J p

AcNH

Ar NH

rro HCOH

I

6

CH,OH

7

SCHEME1.-Formation from NeuSiic (;I~c.ositle(3) o f t h r C 7.41&hyde ( 4 ) and the C , Aldehyde ( 5 ) ,as well as the C, Analog (6)iS-Acetamido-3,5-tiideoxy-~-clrcil,itio-l~ept1tlosonic Acid) and the C, Analog (7)( S - A ~ ~ ~ ~ t ; t n r i ~ i o - 3 , 5 - d i d e o x y - ~ - ~ ~Acid) ~/~~~~~~-~~c Iiy Period~ite-BorohydrideTreatmcwt of CIyc.osidically Bound Ner15.k.

or ec1il)le hird's-nest siiljstancc. for the N-acebl derivatives.'" Based on studies with gangliosides, the oxidation conditions should lie rather mild (molar ratio of periotlatc. : sialic acid = I : I , reaction time 10-20 tnin, O", pH 5.5) in o I t 1 t . r to olitain mainly the C, derivatives, and somewhat stronger for tho C:, compounds (10-fold molar ewess of periodate, 10-20 miti).'-" Aticr acid hydrolysis, the niodific-d sialic acids are purified b y ion-excliange chromatography, a i i d fractionated on ce 11ii lo s e co 1ti inns a s a1rct ad y tlc s cri be d . T h 1atte I' proce d i i re permits separation of the C,, C,, and C, sialic acids of Iioth the h'-acetyl and N-glycolyl series. The Ijehitvior of these coinpoutids in t.1.c. and g.l.c., and their analysis I)\. 111.x. is described later. The availability of, and aiial~.tic.aldata for, these staticlards arc prerequisites for quantitative a n t 1 cluditative ~ ~ n ~ ~ l of y s the i s yiel(1 of t t e ~ ~and, modified sialic acids from p r , r i o d a t e - l i o r o l ~ y ~ l r i ( ~ e - t r e ~cells, correspondingly, for evaluatioti of' the influence of such niodific,'1 t lolls ' on the biological behavior ofcc.lls. In such an experiment, related to a study of the life expectancy of' ra1)I)it crythroc>.tcs,the simultaneous analysis of NeuSAc, Neu5Gc, a t t d thcir C, and C , analogs froin rah(3

(141) H. W. Veh, A. P. Corfield, M. Sutrtler, arid H. Schauer, B i o c l i h . B ~ J J J / I IActu, / , x . 486 (1977) 145-160.

162

ROLAND SCHAUEH

bit-erythrocyte membranes b y g.1.c. - in .s. was described.142 However, more work is necessary for exact, quantitative evaluation ofperiodatemodified sialic acids from cell-membrane and miicous glycoproteins.

3. Thin-layer Chromatography Prior to analysis on thin-layer plates, sialic acids must be purified b y ion-exchange chromatography, its traces of cations and other coinpounds may change their rate of migration. chromatography is conducted either o n thin layers of cellulose prewashed in 0.1 A 1 HC1, or on thin layers of silica gel; in both instances, the layers are 0.10.5 nim thick. The following solvents (v/v), reviewed in Ref. 107, are used for cellulose: 6 : 4 : 3 l-butanol-pyridiiie-water, 4 : 1: 5 l-butanol -acetic acid-water, 1 :2 : 1 l-1)utaiiol-l-propanol-water, and 1 :2: 1 l-1)utanol- l-propanol-0.1 M HCl. In o u r experience, the last ~ y s t e r n ' ~ " gives the best, arid most reproducible, chromatographic results. On silica gel, sialic acids are developed with 7 : 3 l-propanol-water. The spots for sialic acid are stained b y spraying the layers with the orciiiol - Fe:'+-HC1 reagent diluted one-third with water, followed by heating for 15 i-niii at 120", or b y spraying with the periodic acid-thiobarbituric acid reagent adapted for this purpose according to Ref. 107. In Table 111, the behavior of different, natnral sialic acids on cellulose with l-butanol- l-propanol-0.1 h1 HCI is shown. The rate of migration of the sialic acids increases with the number of O-acetyl groups, and is infliienced b y the position of the O-acetyl groups, as may be seen from Table I11 b y coniparison of, for example, NeuS,7Ac2 and Neu5,9Ac2. N-Glycolyl groups generally lead to it decrease of the RF values compared to those of the corresponding N-acetylsialic acids. Similar behavior of N-acetyl- or N-glycolyl-sialic acids is observed in the other chromatographic systems described, with the exception of silica gel, where N-glycolyl groups do not have such an influence.'"' A thin-la y er chromatographic 111e t hod i ii two di in e n s ions , with intermediary treatment of the sialic acids with ammonia, has been clescribecP4Joi; it enables the detection of O-acylated sialic acids b y a change in the RF values during chromatography in the second dimension after alkaline reinoval of the 0-acetyl groups on the sialic acid present on the thin-layer plates, and identification of the O-deacetylated Neu5Ac and NeuSGc. This method can also be applied to gangliosides containing O-acylated sialic acids; the R F values are lowered by deacylation." Two-dimensional, thin-layer chromatography can also be used for (142) G . Pfinnschmidt and I<. Schaiter, Z. P h / s i o / . C : h c , r r l . , 361 (1980) 1683-1695. (143) E. Svennerholm and L,. Sventwrholiii, N a t u r e , 181 (1958) 1154-1155.

sI.4LIc: ACIDS -1ABLb

163

111

Thin-layer Chromatography (R, values) of Natural, N,O-Acylated Sialic Acids and of the C, and C, Analog\ of Neu5Ac and NeuSGc in System A' and in Gas-Liquid Chromatography ( R , Values, Relative to Neu5Ac = 1.00) in Sy\tenis 8''and C'

R , values Compound

R , values System A 0.45 0.60 0.54 -

0.63 -

0.70 0.75 0.80 O.Fi6 -

0.55 0.54 0.58 0.35

0.55 0.70 -

0.38 0.42

System B

System C

1.00

1.00 1.37 1.20

1.18 I .04 1.05 1.13 1.31 1.14 1.19 1.15 2.55 3.01 1.09 0.50 0.29 1.81 2.02 I .83 2.04 2.01 1.99 1.$In 0.95 0.60

-

1.28 1.60 1.53 1.64 1.87 -

1..32 -

1.43 -

1.75 2.06

( I System A: 0.1 iiiiii cellulose t h i i i - l a ~ v r s . Solvelit: I : 2 : 1 (v/v/v) I-butanol1-propanol-0.1 34 lHCl.'4"Data from H<%ts.34,82, 97, 105, 107, 141, and 149. " System B: l l e t h y l esters, trirnethylsilyl r t h e r s of \ialic acids; glass column (0.4 x 200 cm), 3.8% of SE-30 on Chromosorl) W/AM'-I)\I(:S IIP (80-100 mesh), oven teniperature, 215". D a t a from Refs. 34, 82, 94, 95, 97. IOS, 141, 142, and 148. System C : T r i ~ ~ r e t h y l silyl esters, triiiiethylsilyl ethers 01' sialic ac,icls; glass coluilln (0.2 x 220 em), 3.5% of OV-17 011 Chroiii CAW-DMCS (80- 1 0 0 iiic,sh), temperatiire programmiiig f~-otn200280" with 2"/min (Rrsfs. 82 a n d 149). " Hvf's. 141 and 142. K c y : -, not tleterntinrcl.

identification of the 0-acyl groups of sialic acids b y intennediary spraying of the plate with alkalilie hydroxylainine, and use of 6 : 2 : 1 1-propaiiol- 10% aqueous amnrniiiiiin carbonate-5 A 1 aminoniuni hydroxide for chromatography, iir the second diniension, of the acylhydroxamates f ~ n ~ l e dThe . ~ acylhydroxamates ~ . ~ ~ ~ are made visible as purple spots by iiieans of 10%' aqueous FeCl, spray. If individual, purified sialic acids are availal)le, it is, however, recommended that

164

HOLAND SCHAUER

the 0-acyl groups be converted into acylhydroxamates in the test tube, by use of alkaline hydroxylamine in and these chromatographed in one dimension with the ammonium carlioiiate system. reThin-layer chromatography of ~-acy~aniido-2-deoxy-D-mannoses sulting from cleavage of sialic acids with acylneurarninate pyruvatelyase is described in Ref. 107, together with the R, values ofthe different O-acyl-2-acylamido-2-deoxy-~-mannose species.

4. Gas-Liquid Chromatography Gas-liquid chromatography of sialic acids is employed for ( a ) qualitative and quantitative identification of sialic acids isolated from biological materials, ( I J ) control of the presence of contaminating substances in the preparations of sialic acid, and (c) (in combination with m.s.) structural analysis. Sialic acids were earlier analyzed as the per(trimethylsily1) ethers of their methyl (methyl P-glycu1osid)onates after mild methaiiolysis of the complex carbohydrate^.^^^,'^^^^^^ The disadvantage of this method is loss of0-acyl groups and partial loss of N-acyl groups under the acidic conditions of hydrolysis. Therefore, methods have been developed in which the sialic acids are either directly per(trimethylsily1)ated with 1-(trimethylsilyl)imidazole, to yield the per(trimethylsily1) ethers of the trimethylsilyl esters,i46or the sialic acids are esterified with diazornethane before per(triniethy1sily1)ation with hexaniethyldisilazane and chlorotrii-nethylsilane i n pyridine, to yield the per(trimethylsily1) ethers of the methyl esters of the sialic acids.94The procedures are detailed in Ref. 107. Gas-liquid chromatography of the methyl ester derivatives is more in use at present, as these derivatives are more stable than the trimethylsilyl ester derivatives, and impurities can be removed by partition between chloroform and water.94Chromatography of these ester ethers of sialic acids is p e r f o n n e ~ l on ' ~ ~OV-1, OV-17, or OV-22, and the per(triniethylsilyl) ethers of the methyl esters are on SE-30. The relative retention-times of various natural sialic acids, and of derivatives having shortened s i ~ l e - c h a i n s ,are ~ ~ given * ~ ~ ~in ~ Table 111. (144) J. R. Clamp, G. Dawson, anti L. Hoiigh, Biorhini. Rioph!/s.Acta, 148 (1967) 342349. . h e m . Soc., 85 (145) C. C. Sweeley, R. Bviitley, M. hlakita, and W. W. Wells,]. A t ~ i C (1963) 2497-2507. (146) J. Casals-Stenzel, H.-F. Brischer, a r i d R. Schauer,Aticil. Hiocltrrn., 65 (1975) 507524. (147) J. P. Kameding, J. Hnverkainp, J . F. G. Vliegenthart, C. Versluis, and R. Schauer, i n A. Frigerio (Ed.),Rrcrtit L ) c u e / o l ~ i i i c ~ iin t s M m . s Spectmtiwtr!/ i t i Hiocliettiistrt~ a n d Medicine, Vol. 1, Pleiiuiii, New, York. 1978, pp. 503-520.

Analysis of sialic acids b y gas-licliiicl chroiiratoRrapliy is sensitive (the iiiiiiiiriuin amounts required for detection 1x:ing 0.5- 1 p g ) and spec i fic, especially if se ve ra I s 1I pi)()rt s and mod i ficat io 11 p roc^ d ure s are applied, and permits control of t h e colorinietric tests, the errors in which have already been disciisscd. Uiiaiiibiguous identification with respect to structural analysis of sialic acids is only possilllcl i n conil)ination with niass spectrometr! . Qiiantitative deteiiniiiation of sialic acids is also possible with g.1.c. if‘ piire compouirds are a\~ailuble for calibration, a s has I)eeri tlcwril)ecl146 for N e u 5 A c , NeuSC:c, mid Neu5,9Ac2. Increase in the sc,ttsitivity, specificity, and resolution of g.1.c. of sialic acids is to be expected from capill~ir);-col~imn chroiiiatography, which may facilitiite detection of sialic acids occurring only in sinall amounts in sialic acid mixtures. Progrc,ss i n this respect is currently being made.

5. Mass Spectrometry Although the different niethocls for the analysis of sialic acids thus far described give some insight into the structure of‘these conipoiiiids, mass spectrometry pei-niits unambiguous detennination of the cllain length and the number, type, a i i d position of N- o r 0-acyl substituents in N e u derivatives. Thus, a v;ii-it,ty of new and rare sialic acids have been discovered in the past few y e a r s , and the presence of known, niaiiily 0-acetylated, sialic wicls lias Iieen estal)lislied in ;I variety of biological ~ i i a t e r i a l s . ” , ~ ’ , ~ ~ l i . 9 8 . I0.5.10i.14i-l.1H Ideiitificatiorr of sialic acids ;is tlie per(trimetli);lsilyl) ethers of their methyl esters is possible on the, hasis of seven typical fragment-ions, A-G, the formation and interpre,tatioii of which were discussed 11y Kamerling and coworker^.^^^^‘^^ T l i e ) ) I /;. values, or the absence of these fragment-ions, pennit exact structural analysis of’natural o r synthetic sialic acids, a s shown in Talilc. 1V for it variety of natural N,O-acylneuraminic acids, including the, C, : t i i d C, iinalogs of NeuSAc and NeuSGc. The corresponding data for soine synthetic N e u derivatives were suiniiiarized in Ref‘. 147. In ni.s., partially inethylatecl sialic acids also lead to c1iarac:tcristic fragiiients that can be used f o r analysis of natural, o r syiitlietic, Omethylatetl sialic acid^."^"^^':"'^'^^ hlethvl ether derivatives are obtained in methylation analyses for structural studies of sialyl-sialyl linkages, or in experiments for the localization of 0 - a c e ~ groups l in sialic acids. For the identificatioii of such N ~ Lclei-ivatives, I the 0-(tri-

ROLAND SCHAUER

166

TABLE

Iv

Mass Units of Characteristic Fragment-ions A-G Used for the Identification of Natural, N,O-Acylated Sialic Acids, and of the C7 and C, Analogs of Neu5Ac and Neu5Gc as their Methyl Esters, Trimethylsilyl Ethers, by Mass Spectrometry"

Fragments" Compound Neu5Ac Neu4,5AcZ Neu5,7Ac, Neu5,8Acz Neu5,9Ac2 Neu4,5,9Ac3 Neu5,7,9Ac3 Neu5,8,9Ac3 Neu5,7,8,9Ac4 Neu5AcSLac Neu4,5Ac29Lac Neu2en5Ac C8Neu5Ac C7Neu5Ac Neu5Gc Neu4Ac5Cc Neu7Ac5Gc NeuYAc5Gc Neu7,9Ac25Gc Neu8,9Ac25Gc Neu7,8,9A~5Gc C8Neri5Cc C7Neu5Gc

A 668 638 638 638 638 608 608 608 578 740 710 578 566 464 756 726 726 726 696 696 666 654 552

B 624 594 594 594 594 564 564 564 534 696 666 -

522 420 712 682 682 682 652 652 622 610

508

C 478 448 478 478 448 4 78 4 78 448 388 478

D

E

298 298 298 298 298 298 298 298 298 298

317 317 317 317 317 317 317 317 227 317 317 317

F

G

~

-

-

566 536

386 386

-

-

566

386

-

-

566

386

-

-

5fi6 -

386 -

-

317 317 317 317 317 317 317

20.5 205 205 175 175 175 277 277 205 103" -

205 205 205 175 175 103" -

173 143 173 173 173 143 173 173 173 173 143 173 173 26 1 23 1 26 1 261 261 261 261 261 261

" Refs. 94, 141, 142, and 147. I' The nature ofthe fragment ions is shown, for example, in Refs. 94, 107, and 147. -, ion absent. ' I ttt/z, 103 represents fragment F of neuraminic acid minus C-9. methylsilyl) or 0-acetyl derivatives of a set of partially 0-inethylated N-acetyl-N-inethyl-P-~-~ieuraminicacid methyl glycoside methyl esters were synthesized, and the inass-fraginentation patterns of these Based on this method (2+8)-sialic acid linkcompounds sti~died.'"~ ages were identified in GT,,, gangli~side,'~" rat-brain glycoprotein,l5' and colorninic acicl.1sn.1y2 Conversion of ester groups of sialic acids into inethoxyl groups has (150) J. Haverkarnp, J. P. Karnerling, J . F. G. Vliegenthart, R. W. Veh, arid R. Schauer, FEBS Lett., 73 (1977) 215-219. (151) J. Finne, T. Kruisius, arid H. Rauvala, Riochettr. Rioph!/s. Res. Cot,itnun., 74 (1977) 405-410. (152) A. K. Bhattacharjee and €1. J . Jennings, Cnrbohytlr. R e s . , 51 (1976) 253-261.

gained importance for the detcrniination of the priinar-).site of O-acyl groups in natural sialic acids. Rc,cmise O-acetylatetl sialic acids can, at present, only be analyzed aftor a tedious isolatioii-process during which partial iiiigration ofO-acrt).l groups seeins to occur, a metliod for "fixation" of O-acetyl groups \)t,fore hyclrolysis of the sialic acid glycosidic bond would be of grc,at \.slue. This slionld I > e possible b y the vinylation method, tle i h t l i n Hcfs. 139 and L53, ~vhichhas thus far only been applied to synthrtic Neil tleriv,'1 t 1ves. ' Combination of pyrolysis aiicl 111.s.has been found to enable rapid analysis of sialic acids and other c;trbohydrates (for ng quantities of o 1igo sacc ha ride s and po 1y saccl i ari d e s ) . Thus , the pre se ncc' of (2- 8) and (2-9) 1in kage s in N eissc t-ici t )I 4 i i i t ig i t id is caps 11 1ar po 1y s acc harides has been confirmed.'54

6. Nuclear Magnetic Resonance Spectroscopy N.ti1.r. spectroscopy is a fiirt1rc.r tnethocl that h a s markedly contrihuted to o u r knowledge coiicerniiig tlie structures aiicl gl!,cosidic linkages of sialic acids. By use of' lld-n.ni.r. spectroscopy, the pvraiiose fonn both of free Neii5Ac and its glycosides w a s found to exist i n the ~C , ( Lc)o l ~ f o n n a t i o1 4l~~l 4~: 1 . l 6 , l i . I 3 2 It WIS fiit-theiiiwre found, a s mentioned ,-

i n the Introduction, that free sialic acids in aqueous solution comprise a mixture of 7%' of the a and W%, of the p anonic,r.'5-'i 0 1 1 applying 300-MHz, 'H-n.ti1.r. spectroscol)y, Friebolin and deliionstrated that a-Neu5Ac is a cleavage product of sialo-oligosaccharides formed b y the action of C:. pvrjt-it1goti.s a i d A. urrvcifk.ioi.v sialidsses. a-Neu5Ac slowly inutarotates to thtl p tonil, which is in excess i n the equilibrium state. It has l x x x i i toiiitd that 360-%1Hz,IH-it.ii1.r. spectrometers give, for O-acetylatetl sialic acids, characteristic spectra that can be interpreted not only wit11 regard to the nature, nrinilwr, antl position of the N - and O-acyl groups biit also with respect to thc coilforination of the sialic acid iiioIeciilcs.i"~'iThus, the sialic acid structures obtained by m . s . could he coiifirnied h y n.1ii.r. spectroscopy. By use o f 500-;MHz, 'H-n.ni.r. spc,ctroscopy, it w a s also possible to localize acetyl groups exc1usive.ly at 0 - 4 of the N e i l in sialyl-lactose'i (earlier isolated froin Echidna iiiilk 1)y hlesser and coworkerss6)antl to establish the a-(2+3) linkage ol'the sinlic acid residue ofthis oligosaccharide.15s (153) A . N. tle Beltlcr a i i d B. N o r r i i i a i i , (:(ir/Joh!ydr,R r s . , 8 (19(iH) 1-6. (154) J. Haverkamp, H. L. C. Meuzt.laar, K. C:. Heriver), P. \1. H o o n e h i r p , m i d R . H. Tiesjenra, " i t i d . Rioclzeiri., 104 ( IWO) 407-418. (155) J. P. Kainerling, L. Dorlatitl, H.WII Fl;ill)eek, J. F. C;. L'liegeiitlxirt, M. Llesser, antl R . Schauer, Curhohyrlr. R c c . , 1 0 0 (1982) 331 -340.

168

ROLAN I) SC 13AU E ii

The binding of Ca2+to Neii5Gc and Neu5Ac was studied b y 'H- and 13C-n.m.r.spectroscopy, which showed the requirement that the 8-hydroxyl group of N e u and the hydroxyl group of the glycolyl group be free, for optimal Ca*+ complexing.1s6Therefore, Neu5Gc is a much better Ca2+-binding compound than Neu5Ac, a fact also shown by spin-lattice-relaxation studies of the (2-3) and (2+6) isomers of sialyl-lactose.'j' 'H-N.m.r.-spectroscopic studies at 360 arid 500 MHz have given fascinating insights into the position and type of glycosidic linkages of sialic acids in sialo-oligosaccharides and glycopeptides. These studies, perfonned by Vliegenthart and coworkers in the past few years (see, for example, Refs. 158- 160),permitted not only unequivocal determination ofa-(2+3), (2+6), or (2-8) linkages in unbranched oligosaccharides having one, or two, sialyl residue(s),but also in branched oligosaccharides having several, temiinating sialic acids. In oligo-antennary oligosaccharides, different glycosidic linkages of sialic acids can be recognized and also localized. In this respect, 500-MHz, 'H1i.ni.r. spectroscopy has proved to he a much more powerful tool than 360-MHz spectroscopy, especially with regard to elucidation of the location and the type of linkage of terminal sialic acid and L-fucose residues. By using SOO-MHz, 'H-n.m.r. spectroscopy, it was clemonstrated that the internal sialic acid residues of the disialogaiigliotetraose from GD,, [IV5,11"(Neu5Ac)2-GgOse,],or the monosialogangliotetraose from GM, ( I13Neu5Ac-GgOse,), have less confonnational freedom161 than the terminal residues. N.1n.r.-spectroscopic studies have furthermore shown that sialyltransferase from lmvirie colostrum preferentially incorporates sialyl residues into a-(2-6) linkages of glycopeptides from a,-acid glycoprotein.,'j2 Interpretation of the spectra recorded for solutions of oligosaccharides in deuterium oxide is based on chemical shifts and coupling (156) L. W. Jacqucs, B. F. Riesco, and W. Wr:ltnc,r, Jr.. Cnrboh!ydr. Rcs., 83 (1980)2132. (1.57) L. W. Jacques, S. Glant, arid W. Weltner, JI-., Ccirboh!ydr. Res., 80 (1980)207-211. (158) H. van Hallwek, L. Dorlantl, J . I;. G. Vliegeirthart, K. Schmid, J. Montreuil, B. Foumet, and W. E. Hull, F E B S L p t f . , 114 (1980) 11-16. (159) J . F. G. Vliegenthart, 1-1. van Halbec.l\, i i i i d L,. Dorland, Pure AppI. C h m , ~ . 53 , (1981) 45-77. (160) H. van Halbeek, L. l)orland, J. F. G. Vliejienthatt, A,-M. Fiat, and P. Jolli.s, Biochim. Bioph!/.s. Acta, 623 (1980) 295-300. (161) L. Dorland, H. van Halheek, and J. F. G. Vliegenthart, unpitblishetl rcsults. (162) D. H . van den Eijritlen, D. H. Joziasse, L. Dorland, H. van Halbeek, J . F. C . \'liegenthart, ant1 K. Schinid, Biochevi. B i o ~ ~ h ! / .Rcs. v . Coniinuri., 92 (1980) 839-845.

constants of the anonieric protoris of the individual moiiosaccharides in the oligosaccharide chain. Based on these "reporter" protons and other characterizing protons, wliich are clearly separated from most of the other protons, determination of the primary sequence both of N and 0-glycosyl oligosaccharide chains is possible (see, for example, Refs. 163 and 164). This sensitive niethod requires only 100 ninoles of a pure substance, and is rapid and nondestructive compared with the classical procedures for the structural analysis of oligosaccharides. A detailed discussion of this aiialytical tool, and of its application to the structural analysis of a variety of oligosaccharitles and glycoproteins, will lie presented in this '"C-N.m.r.-spectroscopic analyses have been sriccessfull3. made for determination of the position o f O-wetyl groups in sialic acids, using the 9-0-acetyl- and 4,9-di4-acetyl derivatives of Neil-P-hle methyl ester a s model compounds,":' o r in the sialic acid residues of polysaccharide antigens of N . inenirigititlis. 1 6 6 , 1 6 7 N.ni.r.-spectral (13Cand 'H) studies gave infomiation regarding the occurrence of hydrogen bonds in sialic acid^.^^.'^^ Thus, hydrogen bonds in the methyl a- and P-gl>w)sidesofNeu5Ac have been located by Czarniecki and Thornton16H 1)y ':'C-spii~-lattice-rel~~x~itiorl (TI)studies. Accordingly, the arnido N - I I is liydrogen-bonded to the 0 - 7 atom, the 4-hydroxyl group fonns a 11) tlrogen lmnd with the carbonyl group of the N-acetyl group, and the 8-h yclroxyl group is hydrogen-I)oiided to the ring-oxygen atom. The 9hydroxyl group seems to be free. However, a structure, based mainly- on theoretical studies, has been proposed that is not in complete agreenient with this itiodel.1t'9Some data obtained by 360-MHz, 'H-n.m.r. spectroscopy can be interpreted as indicating a hydrogen bond lwtweeti the OH-7 and the ainitle proton .'7 In fonn at io ri concerning g I y cc ) s i t li c 1in kage s , a1s o , ti1 a y be obtai ne d froin '"C-n.m.r. spectra. In this way, the type of glycosidic linkage in lxicterial polysaccharides has l)c:en studied b y Bhattachaijee and co(163) B. Fournet, J . \lontreuil, G . Strc*(kt.r, L. Dorland, J . Hitverk:iiirp, J . F. G. Vliegenthart, J. P. Binette, and K . Sc.hnritl, Bioc~he~nistr!/, 17 (1978) 5206-5214. (164) K. Schmitl, J . P. Binette, L. Dorlaiitl, J . F. G . Vliegenthart, B. Fournet, and J . Montreuil, Biochim. R i o p h l s . Actci, 581 (1979) 356-359. (165) J . F. G . Vliegenthart, L. Dorland, and H. viin Halljeek, Adc. Ccirbcdiydr. C h e m . Rioclzem., Vol. 41, in press. (166) A . K. Bhattacharjee, H. J . Jeiiriiiigs, C;. P. Iicimy, A. Martin, and 1. C. P. Sinith, Car,. J . Biockern., 54 (1976) 1-8. (167) A. K. Bhattacliarjee, H. J. Jeiuriirxs, C:. P. Kenny, A. \laitin, aiid I. C. P. Smith, J . Biol. Churn., 250 (1975) 1926- 1932. (168) M . F. Czamiecki and E. R. Thoriit~iii,]. Atti. Chetri. Soc.., 99 (1977) 8273-8279. (169) K. Veluraja arid V. S. R. Rao, B i o ( , h i i i i . Bzop/i!/.s.Actci, 630 (1980)442-446.

170

R O L A N D SCHAUER

workers,166,16i and the anomeric configuration of the glycosylic linkage of the CMP glycosyl ester of Neu5Ac has been found to be P on the basis of the heteronuclear coupliiig-constaiits.'"

7. Other Physical Methods X-Ray crystallographic studies of Neu-P-Me trihydrate'"' and Neu5Ac dihydrate"' have not only shown the content of water in the crystals of these Neu derivatives, but have also provided valuable information on the orientation of the different parts of their molecules, including the trans-disposition of the 7- and 8-hydroxyl groups. Only a few reports on the circular dichroism (c.d.) of free, or oligosaccharide-bound, sialic acids exist.'i2-1i5The main band obtained is centered at < 200 nm, and corresponds to the acetamido group, showing little confonnational or structural sensitivity.175Spectral features at 225 nm can be attributed to the carboxyl chromophore, and give information about the glycosidic linkage, a-glycosidic linkages of Neu5Ac showing a negative, and P-glycosides, a positive, band. The magnitude ofthe band is increased by restricted mobility ofthe glycosidic bond. Esterification of the carboxyl group has little influence on the spectrum, compared to that obtained at an acidic pH, which is, however, in contrast to the spectra of its salts.175C.d. spectra of Ne~5Ac4Me,C M P - N ~ U ~ A C a13d ~ M CMP-Neu5Ac ~, have bee11 described by Beau.176The last compound exhihits four positive maxima, at 271, 250, 206, and 190 n i n , and two negative maxima, at 220 and 198 nm. Spin-labelling of free, or cell-surface, sialic acids has been used in order to obtain infomiation al)out the rate of rotational orientation of the label after attachment to macromolecules; this knowledge is important in the investigation of the orientation and mobility of sialoglycoproteins in, for example, cell membranes. I n a first approach, the label was introduced into the carlioxyl groups by a carbodiimide-mediated, amidation procedure.LiiThis method is, however, not specific

-

(170) A. Biedl, Naturt ~schufteii,58 (1971) 95-96. (171) J. L. Flippen, Ac r!/stcdlogr., S C C . ~B, . 29 (1973) 1881-1886. (172) G. Keilich, R. Brossiiier, V. Eschei~feltler,antl L. Holmclnist, Carho/z!/t/r.Res., 40 (1975) 255-262. (173) H. R. Dickinsoii and C. A. Bush, Biochcrnistr!y, 14 (1975) 2299-2304. (174) H. J. Jennings antl R. E . Williains, Ccirboh!ydr. R 50 (1976) 257-265. (175) L. D. Melton, E. R. Morris, I). A. Rees, and D. Thorn, ./. Cliem. Soc., Pukiri Trtrrts. 2 (1979) 10-17. (176) J.-M. Bean, Iloetoral Thesis, University of OrlCans, 1979. (177) J. D. Aplin, 1). E. Brooks, C. F. A. Culling, L. 13. Hall, anti P. E. Reid, C a r b o h ~ / d r . Res., 75 (1979) 11-16.

for sialic acids, as the carboxy-l groups of amino acid are also 1al)elled. The specificity for sialic acids c.oiild be increased I)y using mild, periodate oxidation, followed by iittroductio~iof a nitroxide spin-laljel into the side chains of the inodifitlcl sialic acid.Iix From the typical, electron paramagnetic resonance spectra and inotional correlation times obtained, a general entry into spill-laljelling of specific loci on isolated glycoproteins and of cell-surfk,t, glycocoiijugates is to be expected. By use of a nitroxide spin-label, 40%; of erythrocyte-meinljrane sialic acids were spin-labelled, a i i t l :in increased nnotion of sialic acids was observed after the addition of' Phm(w1zis culgar-is phytoheinagglutii-~in."~

8. Histochemical Analysis Because sialic acids, especially those of cell nieinbraiies, are involved in many biological processes, their cellular localization b y histochemical means becomes inore ancl more iniportant. Correspondingly, rapid progress has been made i n the histochemical denioiistrat i m of nonsubstituted, and 0-acetylated, sialic acids in tissues and cells. Staining of sialic acids is possible, either on the basis of their relatively high acidity (for exaiitple, with Alcian B l ~ i e " ~ 'at~ "pH 2.5, colloidal iron, cationized ferritin, or Ruthenium Red'"), o r on the basis of oxidation of their side chains b y periodate in the classical, or modified, periodic acid- Schiff' (PAS), staining procedure."'*'xz The aldehyde groups of the modified sialic acid may also be made visible in a very sensitive way by using such fluorescent reagents ;is dansylhydrazine,Ix:' rhodarnine, or tliioresc.ciii-contaiiiing l i y d r a z i ~ l e s or , ~ ~by ~ reaction with biotin, followed Ijv interaction with ferritin-coiijiigated avidin.lH5The last method is particiilarly suitable for electron-iiiicroscope studies of cell-meinbraire sial ic acids. A prerequisite for the specificity of the staining procedures 1)ased on periodate oxidation is exclusive. oxidation ofthe sialic acid residues by low concentrations of periodate during short reaction-times. The (178) J. I). Aplin, h4. A. B e r n s t e i n , C;. I;. A . (;iiIling, I,. 11. Hall, and P. E. Ht,itl, C:urboh l d r . R e s . , 70 (1979) ~ 9 - ~ 1 2 . B. F r i x ;uid 11. A . Butterfic~ltl,FliUS Lcjlt., 11.5 (1980) 185-188. . S. S p i c e r , R . G. Horn, a n d '1.. 1 . I,t.ppi, in B. hl. Wagner and U. E. S m i t h (Etla.), The C o r i t i c ~ f i c Tissue, c~ Williaiiis a i i ( 1 Wilkiiis, Baltiinore, 1967, pp. 2.51-CO3. (181) Y . S. K a n w a r and M. G. Farcliili~tr,L a / > .I J I W ~ 42 . , (1980) 375-384. (182) R. W. Veh, A. P. Corfield, R. Scliaiic,r, and K. H . Anclrtss, in Ref. 80, pp. 652-653. (183) P. \Veher, F. U'. Harrison, ant1 1,. I l o f , Histoche,rii.str.!/, 45 (1975) 271-277. (184) M .Wilchek, S. Spiegel, and Y . Spit,gc,l, Hiocheni. Biop/i!/,s.R M . C , ' O I J I J I ~ U I I . ,92 (1980) 1215-1222. (185) E. Skutelsky, 1).Uanon, hl. U'ilc~lwl,, ; u i t l E. A . Bayer, / . ~ 7 / ! r m t r u c R(,.r.. t. 61 (1977) 325-33.5.

172

ROLAND SCHAUER

conditions for almost-specific oxidation of sialic acid, leading to the C, and C, analogs of sialic acids if a borohydride step is included for reduction of the aldehyde groups, was first described by Suttajit and Winzler,lX6and was later applied to gangliosides by Veh and coworke r ~In, the ~ latter ~ ~ study, it was found that, in ganglioside GM,, up to 75% of the sialic acid residues are oxidized, and the other carbohydrate groups remain intact if 1-10 nlM periodate is used during 10 miii at 0".These conditions may be for a specific staining of sialic acids having side chains either uiisubstituted or bearing a substituent at 0-7. For similar purposes, Culling and ReidY1used morevigorous conditions, known from the classical PAS reaction, namely, 1%periodic acid during 30 miii of reaction. The specificity for staining ofsialic acids is further increased by the use of sialidases that release sialic acid residues and concomitantly lessen, or abolish, the stainability of the biological materials, either b y basic dyes, or after periodate o ~ i d a t i o n . ~However, l-~*~ errors are possible, as the susceptibility of the sialic acids to sialidases is variable, as will be discussed in Section V1,l. The best example of resistance towards the action of sialidases is that ofthe sialic acids having an aceton 04, arid consequently, alkaline treatment of the mayl groupX9,."' terials before treatment with sialidase is recommended. Tentative infomiation regarding the presence of sialic acids in histological materials may also be obtained by mild treatment with acid, with staining before and after this h y d r o l y ~ i s . ~ ' Histocheniical demonstration of most of the 0-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation ofthis part of the molecule to an extent dependent on the number antl position of the 0-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanolY1)may increase the staining reaction of a sialic acid. For example, the presence of 0-acylated sialic acids has been demonstrated in colonic, epithelial inucin of inaii and various maiiirnals (summarized in Ref. 91), in healthy and diseased, human siiiall-intestiiie,l*x-l~o in bovine submandibular gland,1xzin mouse and and in human lymphocyte^.'^^ rat erythrocyte-membra~ies,~~~ (186) M. Suttajit antl R. J . Winz1er.J. Riol. Clzeni., 246 (1971) 3398-3401. (187) P. Schmitz-Mooriiiaiiii, Histochemistry, 20 (1969) 78-86. (188) M. I. Filipe and C. Fcnger, Histocheni. I., 11 (1979) 277-287. (189) C . F. A. Culling, P. E. Reid, and W. L. Dunn,J. Cli7l. P a t h o l . , 32 (1979) 12721277. (190) P. E . Reid, C. F. A. Culling, W. L. Dunn, C. W. Ramey, A. B. Magi], and M . G. Clay,]. Histochert). C!ytoc.liem.,-28 (1980) 217-222. (191) A. H . Sarris aiid G. E. Palade,J. R i d . Chein., 254 (1979) 6724-6731. (192) J. Makovitzky, i n Ref'. 80,pp. 195-196.

I n a series of investigations, Culling and coworkers e1al)orated a procedure for the localization otO-acetyl groups on the N e i l m o l t ~ ~ t l e on the basis of periodate oxitlation and nomial PAS, or thionincaSchiff; staining-procedures."' \Vliertlas unsu1,stituted sialic acids, and their derivatives acetylated at 0 - 4 o r 0 - 7 , are fully PAS-positive, 9 4 - . acetylated sialic acids do not react under mild conditions of oxidation, and only give a full color if the conditions are appreciably more vigorous (0.5 A4 periodate for 120 miri at rooin 8-0-Acetylated sialic acids, described iis occurring in colon inucin o t i the 1)asis of histochemical experiments, iis ~ l as l7,9- o r 8,9-di(>-~~cetylated arid 7,8,9-tri-O-acetylated sial ic ;tcicls,X2 require (I-deacetylation prior to staining.Y1In contrast to the latter histochemical finding, clwniical investigationy2of the sialic acids froin normal, mid malignant, hutnan large-bowel Inucosa did riot rtiwal tlie existence of sialic acids nionoacetylated at 0-8, but showed small amounts of' 8,Y-di~)-acetvlated Neu5Ac. With the thionine- Scxhiff rcagent, 1)liie o r red colors have been reported to develop with tlie clifferent sialic acids occiirriiig in mucous materials.91 Further di ffere t i t iat i on be tw tie t i I I 11 s u b s t i t ti tc cl s ial ic aci ( 1s , 9-i n o I i o 0-acetylated, a i d di- or tri4-acetylated derivatives, respectively, w a s possible in studies with bovitir, siibrnanclibular glands.1x2,1"3 111 the first step, the unsubstitutetl siitlic acids are oxidized h y 10 tnM periodate for 10 rnin at o",and the proditcts are reduced with borohytlride, a procedure introduced b y Reid a n d coworkers'!"; in this way,these acids are excluded from staininq. For exclusive staining of the 0-acetvlated sialic acids remaining, tlie ester groups iire removed h y alkali, and the product is subjected t o a svcond, mild, periodate treatnient, followed by staining with the Schitf reagent. l a t eor d , 8,9-(li<)-acetyThe group of 7- and 8 - m o i ~ o ~ ~ - ~ t c ~ t y7,Ylated, and 7,8,9-triO-acetylate(t sialic acids (the existence of which i n Imvine, submandibular glands is ell establishedH2)is specifically stained in the following way. TissucJ slices are treated with 0.5 A4 periodate for 120 min at 25", caitsing cotiiplete oxidation of'the side chain to the C , aldehyde of the inisiil)stitittecl and 9-itrono-O-acetyl~Itt.tLsialic acids. After reduction of tlie aldeh>.tlc,with lx)rohydritle, the ester groups of the remaining mono-, di-, atrtl tri-O-acetylated sialic acids that a r e unaffected by these vigorous conditions of oxidation are hydrolyzed by alkali, the products are sul)jvcted to tiiild oxidation with periodate,

174

R O L A N D SCHAUER

and the products are stained with the normal, Schiffreagent. By using this procedure, this group of 0-acetylated sialic acids was found to occur mainly in the mucous, and not in the seromucous, cells of bovine, submandibular glands. Based on this observation, and chemical analysis of the “major” m u c i i ~ from ~ ~ bovine, ~ submandibular gland, which showed that this glycoprotein contains mainly di-0-acetylated sialic acids,Ig6 the site of production of the “major” iniicin was assumed to be the mucous, acinar cells.182In contrast, the “minor” muciiiISs is derived from the seromucous cells, and contains mainly 9mono-0-acetylsialic acids.1wL,196 These sialic acids cannot be directly identified by histochemical means; a tentative, indirect method for specific staining of Neu5,9Ac2 and NeuYAc5Gc, even if they occur together with other sialic acids 0-acetylated in the side chain, is described in Ref. 193. Mild, selective oxidation of the sialic acid side-chain, followed b y the addition of bisulfite and Toluidine Blue has been used for topooptical staining of sialoglycocoiijugates in erythrocyte and lymphocyte membranes. This method, which is based on a polarizationmicroscope technique, permits not only specific, and very sensitive, light-microscope staining of sialic acid residues on cell surfaces but also the study of the spatial arrangement of sialoglycoconjugate molecules on cell ~ u r f a c e ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Sialic acids may also be specifically stained h y using peroxidaselabelled Limulus polypheinus agglutinin in combination with 3,3’diaminobiphenyl or Alcian Blue, and the procedure was tested with various, mammalian tissues by light-microscopy.lY~ A method for selective, radioactive labelling of sialic acids, especially in cell membranes, by mild oxidation with periodate followed b y reduction with lmrotritide, has heen described by Gahmberg and Andersson.200This procedure can be used either for isolation and characterization of the labelled, cell-surface glycoconjugates (see, for example, Ref. 201) or for autoradiography of tissues and cells (for example, erythrocytes)

(195) F. Downs, A. Hcrp, J. Moschera, aiid W. Pigman, Biochim. B i o p h / s . Acta, 328 (1973) 182-192. (196) A. P. Corfield, R. W. Veh, aiid R. Schauer, unpublished results. (197) G. Geyer and J. Makovitzky,/. Microsc., 119 (1980) 407-414. (198) J. Makovitzky, Acta Histochent., 66 (1980) 192-196. (199) K. Yainada a i d S. Shimizu, IIistochem. /., 11 (1979) 457-471. (200) C. G. Gahmberg and L. C. Aiidersson,/. B i o l . Chern., 252 (1977) 5888-5894. (201) L. C . Andersson and C. G. Gahrnberg, Blood, 52 (1978) 57-67.

9. Use of Lectins and Antibodies

The lectin liiiiulin, consitltrc.tl to I)e specific for sialic acids, has 1)een isolated from the haeinolyinph of the American horse-shoe crab Lint zi lu s 1x11!jplzc?n21 s, '02 an (1 s ti1( 1i c~tl with regard to its m o 1ecii 1ar 11roperties."'" It binds strongly to a-gl\n)sitlically linked sialic acids, arid weakly to free sialic acids, a i i d is inactive with Neu-P-lle. Tlic best 1)inding-partner is Neu5Gc, whc~rc~as its 4-O-acetylated clcrivativt, ( a s a component of GM, gangliosicle iiicorporated into vesicles) is inactive, and this is reminiscent of' thc 1)c.liavior of 40-acetylatetl sialic acids towards sialidases and ac!-liie-uraminate pyriivate-llvase."'" In addition to the 4-hydroxyl groiip, the c.arl)oxyl group of N e u sliould also be free, in order to allow binding of liniulin. Shortening of the s i d e chain of sialic acid by periotlatc~-I)oroliydride treatment does not influence agglutinability b y t h e Ic,c.tiii. The Indian horse-shoe cral) C(irc.itio.scori,iu.y r o t t i ~ i dr ~~ i r i t l t r also contains a sialic acid-binding lectiii (carcinoscorpiii), wliic~hwas purified almost to homogeneity, and 1 1 a d ~a ~inolcwilar )~ weight of420,OOO. Whcat-genii agglutinin (WGA), which is k n o w n to interact with GlcNAc, has been found also to iiitc,ract with sialic acid residucs of glycoc0r-?jugates'"4.'"fi.2"~; this is due to configuratioiial similarities between Neu5Ac and GlcNAc, that is, structural arrangement of the acetamido and the hydroxyl groups o n C-5 m d C-4 of Neu5Ac, aiicl on C-2 and C - 3 of GlcNAc, respectiveI\.."'"."'~XC:orresporidiiigly, replacement of the N-acetyl group of sialic. acid b y a N-gl) )1y1 group prevents reaction with WGA. The strength of tlre Neu5Ac-WGA interaction is appreciably increased Iiy shortc,iiing of the N e u side-chain to afford the C, aiia10g.204~*06 It was shown 1)y amidation of the car1)oxylic group of the sialic acid resit1uc.s i i i \ esicle-bound gangliosides that, iri contrast to liniulin, the carl)ox>.l group of Neii is not i ~ i v o l v e d ~in" ~ binding of WGA. Binding o f WGA to glycocorljugates containing Neu5Ac is furthennore infliiencd b y Ca'+, and charge and avidity ef-

(202) A.-C. Roche, R. Schauei-,and XI. \loir\igii>., F E E S L c f f . ,57 (1'375) 245-249. (203) R. Kaplan, S . S.-L. Li, and J. X I . K c i h o t ~ ,Biocheriii.ufr!/,16 (1977) 4287-4303. (204) H. hlaget-Dana, R. W. Veh, k l . Saiitler, A,-C. Rochc, 8. Schawr, a i i d Xl. Ilonsigny, Eur. J . Biochern., 114 (198 I ) I 1 - 16. (205) S . Bishayee and D. T. Uorai, H i o r h i i n H i o p h ~ ~Actcl, , ~ , 623 (1980)8H-97. (206) X4. hlonsigny. A.-C.Roche, C. Scirc~,H . h l a g c + l h i a , and F. Deliriottc~,Eirr. / . Riochrm. 104 (1980) 147-1Tj3. (207) V. P. Bhavairantian and A. W. K i i t l k , , / . H i o l . Chent,, 254 (1979) 4000-4008. (208) B. P. Peters, S. Ebisu, I. J. G o l ( l j t t T i i i , a n t 1 X1. Flashiier, B i w / w n i i , s f r ! /18 , (1979) 5505-551 1.

176

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fects, the last describing the binding of the lectin only to cell-surface glycoconjugates (and not to soluble sialoglycopeptides).zo~~zO~ These complex properties of WGA have been used to distinguish between different sialic acid populations on the surface of various Chinesehamster, ovary-cell mutantszo9and between different developmental stages of Tr!ypanosoma cruzi.26111the latter experiments, it was found that WGA reacted solely with sialic acid residues on epimastigote cell-surfaces. A further heniagglutinin specific for sialic acid residues in glycoproteins has been described as occurring in the tissue fluid of the Pacific oyster Crassostrea g i g n ~ . ~Remarkably, ~” agglutination of erythrocytes b y this compound can be inhibited much inore effectively b y bovine, submandibular-gland glycoprotein than by glycopeptides from this mucin, or by free Neu5Ac or a-NeuSAc-(2+6)-2-acetaniido-2-deoxy-Dgalactitol. The irninunogenicity of sialic acids has not yet been studied extensively. However, it has been reported (see Section 11) that NeuSGc as a component of glycolipids may act as a specific antigeii.i1-81The sequence a-Neu5Gc-(2+3)-P-Gal- has been recognized as the antigen in the formation of “sei-uiii sickness” antibodies.211A Waldeiistriini niacroglobulin that is specific for NeuSAc has also been reported.212Furthermore, antibodies directed against different oligosaccharides bound to proteins were raised, thus distinguishing between positional isomers of sialic acids.211These antisera are applicable for the detection of a-sialyl-(2+3) and -(2+6) linkages down to a concentration as low as SO pmol/mL.

v. BIOSYNTHESISO F

SLALIC ACIDS, AND THEIRTRANSFER

1. Biosynthesis of N-Acetylneuraminic Acid Biosynthesis of Neu5Ac will be only briefly reported here, as several reviews exist concerning this topic,213-216 and inore attention will be given to the enzyme reactions modifying this compound. In Na(209) P. Stanley, T. Sudo, and J. P. Carver,]. Cell Biol., 85 (1980) 60-69. (210) S. W. Hardy, P. T . Grant, nnd T. C. Fletcher, Experietitiu, Sell., 33 (1977) 767768. (211) D. F. Smith and V. Ginsburg,]. B i o l . Ckein., 255 (1980) 55-59. (212) C.-M. Tsai, 11. A. Zopf, R. K. Y u , R. Wistar, Jr., and V. Ginsburg, Proc. N n t l . Acatl. Sci. USA, 74 (1977) 4591-4594. (213) E. J . McCuire, in Ref. 19, pp. 123-158. (214) H. Schachter, in Ref. 41(b), pp. 87-181. (215) L. Warren, in Ref. 42(b), pp. 1097-1126. (216) R. Schauer, Methods Enzc~mol.,SO<: (1978) 374-386.

ture, three pathways b y which Neu5Ac is synthesized are kno\vn. These are ( a ) an aldol reactioii lwtween pyruvate arid hlanNAc, catalyzed by acylnenraminate pyrii~~ite-l vase”‘; ( b )coiidensation of enolpyruvate phosphate and ManNAc, catalyzed by Neu5Ac synthase (EC 4.1.3.19) from N . Irieiiirigititlis”H ; arid ( c ) condensation of enolpyruvate phosphate and ManNAc 6-phosphate, catalyzed b>-NeuiSAcSP synthase (EC 4.1.3.20), which is widely clistributed in inamnialian tissues.219 Originally, the acylneiirariiii~~itt, pyriivate-lyase that catalyzes reaction ( a ) was considered to bc responsible for the biosyiithesis of Neu5Ac i n oioo. This enzyme, discovered by Heimer and M e y e P o in extracts of V. cholerae, is widel!, distributed in bacteria and in iiiaiiiinalian tissues. However, there were doubts concerning its role in the biosynthesis of sialic acids, a s i i o sialic acids were found to be fonned in those bacteria, such as V. cliolcrcic. or C . perfringetis, that produce large amounts ofthe enzyme. Frirthennore, the enzyme is absent trom some tissues that continuously procliice large quantities of‘sialic acids, for example, sulmxmdibular g l m d s . In addition, the position of‘the equilibrium of the enzyme reaction is unfavorable for the synthesis of sialic acid under conditions i t 1 L o.z15The acyliieuraminate pyruvatelyase is, therefore, considered to IF involved iii the catabolic pathways of sialic acid metabolisiii. ‘This matter will be discussed later, in addition to its usefulness for syiitliesis of sialic acid in citro. It has been found that the two other eiizyiiie reactions requiring enolpyruvate phosphate are involved in the biosyrithesis of sialic acid, and they have been found only i i i cells or tissues where sialic acids are synthesized. The equilibrium of thcse reactions is on the side of the Neu condensation-products.2’” Whereas reaction ( h ) ,requiring free ManNAc, has thus far only been described as occurring i n bacteria, for example, N . 77ieiIit2gitidi.~,21s..2‘n reaction (c) is coininonly f b u i i d in inammalian tissues, and has been in\,estigated in rat liver,21Y sheep brain,’21 submandibular glands from diff‘rwnt inaiiiinals,2’”.’”’and sheep colonic~ n i i c o s a The .~~~ latter condensation reaction requires prior action of a specific kiiiase (EC 2.7.1.60) pliosphorylatiiig h 1 a n N k at 0-6. This Neu5AcSP-fonning enzyme has been purified froin several sources (217) D. G. Comb and S. Roseman,/ Hiol. C h c ~ n i . 235 . (1960) 2529-2537. (218) R. S. Blacklow and L. Warren,/. N i o l . Chctir., 237 (1962) 3520-3526. (219) L. Warren and H. Felsenfeltl, B i o d i ( , t i t . B i o p h l s . R ~ TCoti~tnutr., . 4 (1961) 232235. (220) R. Heimer and K. Meyer, Prcrc.. h’:cit/. Arcid. Sci. U S A , 42 (1956) 728-734. (221) R. Joseph arid B. K. Bachhawat,/ , ~ ’ f , i ~ r ( ~ (11 . ~(1964) ~ ~ , ~ 517-526. r~., (222) J. Eichberg, J r . , and M . L. Karno\\ky,/. B i d . Chetn., 238 (1963)3827-3834. (223) P. W. K e n t and P. Draper, Bioc/ic,tri. J , 106 (1968) 293-299.

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(described in Ref. 215). After condensation, the Y-phosphate group of Neu5AcYP is removable h y specific or nonspecific phosphatases.*I5A specific Neu5Ac9P phosphatase (N-acylneusaininate-9-phosphatase, EC 3.1.3.29) has been isolated from human erythrocyte^."^ These phosphorylation and dephosphorylation steps are irreversible, thus pennittiiig Neu5Ac synthesis under in Giuo conditions, with relatively low concentrations of substrate. The synthesis of a 9-azitlo-9-deoxy derivative of NeuFjAc, namely, 5-acetarnido- 9-azido-3,5,9- trideoxy -~-gZycero- ~ - g n l a c t o-2- nonulopyranosonic acid, from enolpyruvate phosphate and 2-acetamido-6azido-2,6-dideoxy-~-maiinose,using Neu5Ac synthase from N . n i e n i i ~ gitidis, h a s been The pathway of the biosynthesis of Neu5Ac demonstrates the origin of sialic acids from the cellular hexose and hexosainine pools. These sugars are, therefore, suitable components for the study of the biosyrithesis of sialic acid. However, only ManNAc has been shown to be a relatively specific precursor of sialic acids, as ntay be seen from the distribution of radioactivity between the individual monosaccharides of glycoconjugates after incubation. Injections of radioactive ManNAc into animals, or incubation of surviving tissue slices or individual cells with this compound, give incorporation of label mainly into the sia]ic aCidS.226,22i Radioactive acetate is a cheaper, readily available precursor for e x periments on the labelling ofsialic acid in tissues or cells, and it effecThis method is tively labels the N - and O-acyl groups of sialic acids.22X of great value not only for the preparation of radioactive sialic acids having high specific radioactivity but also for metabolic studies of sialic acids. However, the sialic acids must be isolated before detennination of the specific radioactivity, a s other acetylated hexosamiiies are also labelled. Because NeiiFjAc has been found to lie the precursor of all other sialic acids in mammalian it occiirs, at least in small amounts, in glycoconjugates of tissues, where other types of sialic (224) G. W. Jourdian, A. L. Swwisoir, 1). \Vatsoil, and S. Rosrinaii,J. Riol. C'/wtn.~239 (1964) PC 2714-2716. (225) R. Rrossmer, U. Rose. 1). Kuspvr, T. L. Siiiith, H. C~rasinrik,and F. hl. Unger, R i o c / w i ? i . B i o p / i I / s . R w . C o i i z t i z t o i . , 96 (1980) 1282-1289. (226) H . J . Schoop, H. Schauer, and H . Faillard,Z. Ph!/.siol.C h c ~ i ~ 350 r . , (1969) 155-162. (227) H. C . Yohe, K. Ucno, N.-C. Chang, G. H . Glaser, a i d R. K. Y I I , . ~.Vctrrochcin., . 34 (1980) 560-568. (228) R. Schauer, Z . P/i!/siol. Chotr., 351 (1970) 595-602. (229) R. Schauer,A i i g c w . C,'hcirr. I i i f . E d . E n g / . , 12 (1973) 127- 138. (230) R. Scliauer, Z . Phpiol. Chem., 351 (1970) 749-758. (231) R . Schaiier, Z . PhI/,kd.Cheiti., 351 (1970) 783-791.

acids prevail, for example, i n I)ovine and porcine sul,inantliI,ular g l a 1 1 d ~ , bovine ~ ~ . ~ ~and ~ ~ porciiics ei3&ocytes,32 and sea-tircliins.'i2 In the pool of free sialic acids invclstigated in subinanclibular glands, the proportion of NeuSAc, aiid also its specific radioactivity, are iiiuch higher than in the completed gl~coooiijiigates,and this is in accord with its role as a precursor."LL , 2 2 R . w . u 0 , 2 : i 2 Synthesis of NeuSAc seeins to occur in the cytosol, as the corrc.spontling enzymes are found i n the supernatant liquor after gentlc homogenization of tissues. The synthesis aiid catabolisni of NeuSAc and its derivatives seeiii to he rigidly controlled, as may l)c delineated froin the low concentrations of sialic acid observed in tissucs, serum, ant1 urine (see Section

11). Several feedback-inhibitioii incchaiiisins i n the overall liiosynthesis of sialic acids are known, arid these are reviewed in Ref. 233. L-Glutamine:fructose 6-phosphate amiiiotransferase (EC i3.3.1.19)i s inhibited b y UDP-GlcNAc, as was shown i i i several t i s s ~ i e s . ~ UDP-GlcNA4c "~ is epimerized to ManNAc b y a 2-cpiiiierase (EC 5.1.3.14) that is feedback-inhibited b y C M P - N ~ ~ ~ A CT.h. i"s: inhibition '~ exerted I>ythe end product of the sialic acid bios!,iithctic pathway seems to control the production of sialic acid most effectively. It has been assumed'"" that this regulatoi-y mechanism docs not fiiiiction i n the sialuria patient, who daily excretes gram quantities ofNeu5Ac in the urine, in addition to ManNAc, GlcNAc, 2-acetaiiiido-1,-gl~ical,~'~~ and Nei12en5Ac."'~ In contrast, other features of sialic acid metabolism, such as the rate of transfer of sialic acid onto glycocoiijugates, and the sialidase and acylneuraininate pyruvate-lyasc activities, have been found to be normal.*3iIf this feedhack-inhibitioii ofthe 2-epimerase b y CMP-NeuSAc does not function, the rate throiigh the whole pathway iiiay increase dramatically, and other enzyir1c.s of the anabolic, sialic acid pathway 111ay become rate - 1i in it in g o r fii I i c t i o I i i 11 abn o 1-111a 1, e (1ti i 1i 1, r i i m i c() I i d i cio11s.~~~ The consequence of tliis would be ail overproduction of free ManNAc and, subsequently, of' sialic acid. There i s pro1)ably also an overproduction of CMP-NeuSAc, which is not totally consumed for sialyltrarisferase (EC 2.4.99.1 a i i d 8 ) reactions, I)ut decoiriposed again. (232) H. Schaucr aiid M. Wrmber, Z. / ' / l f / Y i ( J / . < ; ~ P I I L . ,3.51 (1970) 1353- 1358. (233) A. P. Corfield and R. Schaucr. Bto/, CC,//.,36 (1979)213-22263. (234) P. J. Winterbum and C. F. Plrc.l1,\, i i i S. Prii\iriier a n t 1 E. R. Stiidtiiiiiii (ELIS.), 7'he N e w York, 1973, 111,. :343Eriz!/?nesof C:/rrtcirnine .tlctei/>o/i.ctii.Ac.;itlcmic Prc 363. (235) S. K o m f e l d , R. Kornfeld, E. F. N t ~ i i f e l t l m , t f P. J. O'Brieii, Proc,. Y e i t / , Accitl. Sci. U S A , 52 (1964) 371-379. (236) J . P. Kainerliiig, G . Strecker, J.-1'. Fwrriaux, L. Dorluiid. J . Haverkaiiip, a i i t l J . F. G . Vliegrirthart, Hiochim. H i o ? i / ~ ! / ,..\(.f(i. \ 583 (1979) 40:3-408. (237) J.-C. Michalski, A . P. Corfirltl, ;iii(II<. Schwuer, iiirpiil~lislicdrc>siilt\.

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This assumption comes from the high amount of Neu2en5Ac found in serum and urine of this patient; the compound is considered to be derived from CMP-Neu5Ac in a nonenzymic, elimination reaction found to occur under physiological conditions (see later)176,z3R,239. Thus, lack ofacylneuraminate cytidylyltrans~erase(EC 2.7.7.43) activity as the reason for a diminution ofthe concentration of CMP-Neu5Ac and, therefore, lessened feedback-inhibition of UDP-GlcNAc 2-epimerase, can largely lie excluded. That secretion of 2-acetamidoglucal, which is known to be an intermediate in the complex, 2-epirnerase reaction, hut cannot be isolated under normal conditions, may point to another defect in this enzyme that may be independent of the presence or absence o f a feedback-inhibition receptor site for CMP-Neu5Ac has been discussed.2":' An excess of 2-acetamidoglucal may be converted spontaneously into GlcNAc and this, enzymically, into ManNAc; both compounds are secreted in the urine of the sialuria patient. As a consequelice of these reactions, the cellular concentration of ManNAc may increase to a level leading to the synthesis of additioiial Neu5Ac from this compound b y the action of acylneuraminate pyruvate-lyase. All of these reactions, and known or theoretical interactions, were summarized in Fig. 3 of Ref. 233. Little is known of a n y regulation of the metabolism of sialic acid on a level higher than the enzymic level just described. The few experiments performed with animals point to an influence exerted by hormones. Thus, increase of the synthesis of sialoglycoconjugate (mucus) has heen described as occurring in mouse vaginal-epithelium under the influence of estrogen.24"Similarly, insulin therapy restored the sialic acid content of rat hepatocyte-membrane which was decreased in streptozotocin-induced diabetes.241 Synthesis of sialic acid in the nervous system also exhibits a unique, control mechanism that may be delineated from the observation of an increased formation of sialic acid (ganglioside) in rat brain during training.242 Several observations have been made with regard to an influence of age on the sialic acid content of tissues. A marked diminution of sialic (238) J.-M. Beau, J . Havei-kamp, R. Schaucr, and J . F. G. Vliegenthait, A b s t r . Journ. Chitn. Biochinz. Glucicles, Rth, Chumcrolles, 1978, p. 26. (239) J.-M. Beau, R. Schauer, L. Dorlantl, J . P. Kaiirerling, and J . F. G . Vliegenthart, unpublished results. (240) L. Carlborg, Actu Ericiocrinol. (Copenhagen),62 (1969) 663-670. (241) G. Durand, J. P. Dumont, hl. Appel, D. Durand, J. Davy, J. Fkger, and J . Agneray, Horm Metab. Res., 12 (1980) 247-251. (242) B. L. G. Morgan and M . Winick,]. Nutr., 1 1 0 (1980)416-424, 425-432.

acids in thymus and bursa of Fiil)riciiis of chicken, two impoitant, primary, lymphoid organs of birds, h a s I~eenobserved during age-involution.24':'A decrease of sialic acitl cwiiteiit has also 1)een reported in rat gingiva during aging,244and of tlre siul~~lation-grade in rat-brain ganglioside~.~~~ Nonphysiological compounds liave also been described a s influencing the overall metabolism of sialic acid. Administration of ethanol (2 g/kg) to rats significantly decreases the sialic acid content of brain tissue.246Convulsions induced 11,- peirtylenetetrazole (6,7,8,9-tetrahydro-SH-tetrazolo~1zepine)are accoinpanied by 21 diminution i t i the rate of biosynthesis of polysialogaiigliositles GT,,, and CQ,, i n rat l)raiii.22i Such ManNAc analogs as 2-acet;ciiiitlo-1,3,4,6-tetra-O-acetyl-2-deoxyD-niannopyranose or the 2-(trifluoloacetainido) derivative lead to a marked lowering of the incorporation of radioactivity from lahelled MaiiNAc into glycoconjugate sidic acids of murine, erythroleukeniia (Friend) The inhibitory influence of soiite glycosyl esters of nucleotides o n sialyl transfer will be describetl later.

2. Enzymic Modification of N-Acetylneuraminic Acid In contrast to the biosynthesis of NeuSAc, the pathways leading to the many other sialic acids known have not yet been completely elucidated. Insight has, however, been obtained into the biosynthesis of NeuSGc and the 4 0 - and 9-O-acc:tylated sialic acids. Neu5Gc is fonned from NeuSAc. i n an enzymic hydroxyl,'1t'i o n reaction requiring molecular oxygen, ferrous ions, and L-ascorbate o r NADPH.231The properties of tlre enzyme natned N-acetpliit.urainiiiate mono-oxygenase ( E C 1.14.99.18) were described in Kef. 216. The hest source of the enzyme knowti to date is the su1,mandibiilar gland o f t h e pig. Part of the enzyme can lie extracted in soluble fonn, and the rest remains bound to subcellular r r ~ e r n l ~ r a n emainly s , ~ ~ ~to~Golgi ~~~~ membranes isolated by sucroso tleirsity-gradient c e n t r i f ~ i g a t i o i iIt . ~is~ ~ (243) S . N. Kundu, 13. Ile Adhikari, B. li. Bhattacharyya, a i i t l S. P . Bhattachar pwrietitiu, 35 (1979) 1252. (244) J . Nicolau atid Y. A. S. P a i v a , / . Por-iotloritci/ Res., 14 (1979) 503-504. (245) H. Rahmann, in L. Amaducci, A. N. I h v i s o n , and P. Aiitiioiio (Eds.),Agitig of the Brain uiid Derrieiitiu, Aging, Vol. 1:3, Ravcn, New York, 1980, pp. 75-79, (246) W. R. Klemirr and R. L. Engeii,/. Nwr-oaci. Res., 4 (1979) 371-382. (247) E. L. Schwartz, A. E. Brown, A. F.tladfirld, and A. C. Sartorelli, F e d Pt-oc., Fed. A m . SOC.E x p . H i o l . , 39 (1980) 200:3. (248) R. Schauer and M .Weniber, Z. PIt!/.siol. ChrJrti.,352 (1971) 1382-1290. (249) H.-P. Busclier, J . Casals-Stenzcll, P. \ l e t I r s - V e n t ~ i r a and , R. Schauer, E u r . / Riochem., 77 (1977) 297-310.

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assumed that, in the Golgi apparatus, it belongs to a group ofenzynies, including sialyltransferase and other glycosyltransferases, responsible for the assembly and iiiodification of the oligosaccharide chains of porcine, submandibular-gland glycoproteins. As regards substrate specificity, only Neu5Ac, free or glycosidically bound to glycoproteins or glycopeptides, is hydroxylated to Neu5Gc. Precursors of N-acyl groups in sialic acids, such as acetate, acetylcoenzyme A (CoA), or N-acetylhexosamines, are inactive in this enzyme reaction.226It was concluded from these, and radioactivity, studies of the metabolism of Neu5Ac and Neu5Gc in surviving tissueslices or cell-free systems from porcine, submandibular glands, that about half of the Neu5Ac is hydroxylated in the cytosol before its transfer onto nascent, glycoprotein molecules. The other half is hydroxylated after this transfer that occurs in the Golgi membranes. The free Neu5Ac molecules hydroxylated in the cytosol are believed to be modified before their linkage to CMP, as CMP-Neu5Ac has been found to be inactive with the hydroxylase,229and from porcine, submandibular glands can be isolated a fraction of CMP-sialic acids composed of CMP-Neu5Ac and CMP-Neu5Gc in the molar ratio249of 3 :2. Hydroxylation of free Neu5Ac in the test tube was also demonstrated, using porcine, submaiidibular-gland hon~ogenate.21'",z"L In these two pathways of Neu5Gc biosynthesis, summarized i n Scheme 2, free cytosolic Neu5Ac is assumed to be hydroxylated by the soluble inono-oxygenase, and the glycoprotein-bound Neu5Ac by the Golgi enzyme. After the molar ratio ofNeu5Ac to Neu5Gc has reached 1:9 in nascent glycoprotein inolecules, the completed glycoprotein molecules are released from the Golgi membranes, and stored in the gland vesicles before secretion. The mode, and subcellular site, of biosynthesis of Neu5Gc is similar in bovine, submandibular glands, although the activity is less pronounced."'" Neu5Ac-nioiio-oxygenase activity has also been determined in liver and serum of the pig."fi Because of the wide occurrence o f Neu5Gc in Nature, the mono-oxygenase is, presumably, also widespread. Enzyme systems O-acetylating Neu5Ac and Neu5Gc have been studied in bovine and equine submandibular glands. It was assumed from the favored position of acetyl groups at 0 - 9 of N e u in bovine, submandibular-gland glycoproteins that the 9-hydroxyl group is the main target for the O-acetylating enzyme in this t i s s ~ e .It~was ~ ~fur* ~ ~ ~ thermore considered that either the same enzyme or an additional enzyme is responsible for acetylation of G-7; the occurrence of relatively small amounts of 7-mono- or 7,9-di-O-acetylated sialic acids in bovine, submandibular gland has already been described. Thus, the bovine

-

183

3 4a/--3 NeuSAcNeuSGcI

I

-

y

CMP

ManAcyl

NeuAcyl

SCHEME2.-Anal)olic (-) antl Chtal)olic. (---) Routes of Sialic ilcid Metaholisin. [The iiriiiibers represent the following t.iizqiries the subccllular localization of which is iiidicatecl in brackets: la, acetyl-CoA acql~irriraininate-4-o r -9(7)-O-acet!,ltraris~c'r~is~ (cytoplasm); lb, acetyl-CoA:N-acyliirriI~~iiiiii~~t~-4or -9(7)-O-acet).ltrans~erasc(Golgi ineinbr;ine); 2a, h'-acetylneuramiiiate niono-ouyaen~ise(cytoplasrn); 21). N-acetylnrtirairiiiiate mono-oxygenase (Golgi iiieiribriiiw); 3 , ncyliieuraminate cytitlylyltransterasc (nuclear inembrane); 4, sialyltransferase(s) (Colgi iiiembranc~):5 , sialitlase(s) (cytoplasln antl mrmbrane); 6 , acqlneuraminatc pqrriwtc'-lyase (cytoplasiri'?); 7, C ~ l P - a c e t ~ l n t ~ u r a ininate hydrolase (plasma memlirane ). Tlic' opc'n arrows at tht. left indicate Iiiosyntliesis of Neri5Ac from tlie hexose riionophospli;ctr pool. Key: NeiiAcyl = N,C)-acylneiiraminic acids; ManAcyl = 2-acylainido-2-deor? -I)-iiiiiiiiioses, with, o r withorit 0-acetyl groups;

$ = protein

or lipid core; -[XI

=

oIigo\~iccli~tride cliain a s sialic acicI acceptor (t;iken,

and modified, from Ref. 33).]

O-acetyltransferase has prelimiiiaril y been called"6~2"0 acetyl-CoA:Nacyh~euraminatt.-9(7)~-acetyltr~iiisferase (EC 2.3.1.4s). Based on the observation, described in Section 11, that acetyl groups can migrate from 0 - 7 to 0-9 in NeuS,7Ac2,doubt a s to the primary insertion of the acetyl group at 0-0 has arisen, and the following mechanism of O-acetylation of the sialic acid side-chain appears possible under biosynthetic conditions: the primary position of insertion of acetyl groups is at 0-7; then, the wetyl group m a y migrate to 0-9, where, being a primary ester group, it is relatively stable. It is 1111known whether migration occurs directly to 0-9, or b y w a y of the 8hydroxyl group. The latter possihility cannot be excluded, a s there is niass-spectrometric evidence h i - tlie existence of 8-niono4-acetylated sialic acid in sialic acid niixtures isolated from bovine, submandibular-gland glycoproteins (see Section II), and conversion of Neu35,7,9Ac, into Neu5,8,9Ac3 has been monitored by 'H-1i.ni.r. spectros~ o p y . "After ~ , ~ transposition ~ of the 70-acetyl group, it is conceivable that the 0-acetyltransferase agaiii incorporates an acetyl group on 0 - 7 of the same Neu molecule, leading to 7,,Y-di4l-acetylatecl sialic acids, which are frequently isolated from bovine, siibmandibular-gland gly-

184

R O L A N D SCHAUEK

coprotein. In the case of migration of this second acetyl group to 0-8, the same enzyme could transfer a third ester group to one sialic acid molecule; this assumption is supported by the ideiitificatio~i**~'~* of both Neu5Ac and Neu5Gc derivatives with complete O-acetylation of the D-erytlzro-glycerol-1-yl side-chains (see Table I). There occurs, in equine tissues, a corresponding acetyl-CoA:N-acylneuraminate 44-acetyltransferase ( E C 2.3.1.44) that has been investigated in more detail in submandibular glands.":l.*I6It specifically incorporates acetyl groups froin acetyl-CoA to 0 - 4 of both Neu5Ac and Neu5Gc. Froin the occurrence of very small amounts of 9-mono-Oacetylated and 4,9-di-O-acetylated sialic acids in this tissue, the existence of a low 9(7)-C)-acetyltransferase activity is assumed. No evidence exists for migration of the 4-O-acetyl group of Neu4,5Acz to 0-9. With regard to the subcellular distribution of enzyme activity and substrate specificity (free or glycoprotein-bound sialic acids), both 4and 9(7)-O-acetyltransferases are similar to the inono-oxygenase .*Ifi More-detailed investigations using bovine, submandibular glandszs0 have shown that the 9(7)-O-acetyltransferase activity is also distributed between a soluble-enzyme fraction and a Golgi membraneenriched, smooth meinbrane-bound fraction, the latter obtained with the aid of a discontinuous, Ficoll gradient. Whereas it is considered that the soluble enzyme modifies free sialic acids, the niembraiiebound enzyme presuinably O-acetylates sialic acids of niembranebound, nascent, glycoprotein molecules. Further evidence supporting, for the biosynthesis of O-acetylated sialic acids, a reaction sequence similar to that for Neu5Gc (see Scheme 2) comes from study of the distribution of the specific radioactivity between free O-acetylated sialic acids and those bound to Golgi membranes and soluble cytosol glycoproteins, respectively, using surviving tissue slices and radioactive acetate as the precursor of the O-acetyl groups.""~z16~z2R~zB0~24* In addition, isolation of the CMP glycosyl esters of Neu5Ac, Neu5Gc, and Neu5,9Acz in the molar ratios of 5 : 1 : 4 revealed33O-acetylation of free Neu5Ac before activation of its glycosidic group in the intact cell. However, CMP-Neu5Ac is not a substrate for the O-acetyltransferase. Accordingly, O-acetylation of free Neu5Ac could be demonstrated in oit ro. 33

N-Acylneuraminate-9(7)-O-acetyltraiisferaseseems to be widespread in Nature, as sialic acids O-acetylated in the side chain are found in many animal species, including man (see Section 11). However, enzyme activity has thus far been measured only in bovine, submandihular gland and in bovine serum. The 4-0-acetyltransferase appears (250) J . Casals-Stenzel, A. P. Corfield, R. W. Veh, a n d R. Schauer, unpublished results.

less frequently. From the presence of4-0-acetylated sialic acids (see Section 11), enzyme activity m a y I)e assumed to exist in donkey liver, according to the finding of Nei14,5Ac-,in donkey-serum glycoproteins, and in Echidna mainniary gland, ;IS well as in the (partially iiivestigated) equine tissues. Both the inono-oxygenase a i r (1 ( 1 -acet y 1trans ferase s are p h y 1oge 11et i cally old enzymes, which n i a ) . I)e delineated from the occiirrence of both N-glycolyl mid 9-O-acetpl groiips in sialic acids from Echiriotfernaatci (for example, sea-urchins) and 40-acetyl groups in the monotreine Echidna (see Section I I ) . 0-Rcetyltransf~~rase activity has also been detected in group C meiiiiigocoeci incorporating acetyl groups from acetyl-CoA onto 0 - 7 or 0 - 8 of the Neu5Ac residues of the capsular h ~ i n o l ~ o l y s a c c h a r i ~ e . ~ ~ l Nothing is yet known concerning the forination of lactyl groups found at 0 - 9 of sialic acids from different sources, reported in Section 11. This is also true of the bios!~nthesis of methyl ether ant1 siilfuric ester groups occurring in soinc‘ natural sialic acids. Neu2en5Ac, detected in huiii;iii l)otly-fluids,”””’”is presumed to lie of nonenzymic origin, as foriliatioil of this coinpourid has not heen observed in enzyme reactions of tlrr sialic acid metaliolisin, for e x a n r p l e , in the condensation between ciiolp\ ruvate phosphate and hlanNAc 6phosphate, and in CMP-Neu5Ac. hydrolase ( E C 3.1.4.40) o r sialiclase reactions. It may be fonned, iir an elimination reaction. from CMPNc,u5Ac after nucleophilic attack of‘the axial proton on C-3 undcr in ciuo, as well as under in uitro, conditions. Treatment of this glycosyl ester at increasing pH value.; results in increased fo~iri~ition of Neu2en5Ac, measured b y t.l.c., g.l.c.-in.s,, and 36O-MHz, ‘H-n.ni.r. spectroscopy.23HAt pH 7.2, 2% of the CMP-NeuSAc is degraded to Neu2en5Ac in the course of 24 11 at 37“, whereas, at pH 12, > 20% of Neu2en5Ac was formed. Corrc.sl)oiitliiigly, Neu2eii5Ac a s an iinpiirity is always present in the ChI P-sialate samples prepared 11)- eiiz!mic meail s and purified by us ing‘”’ t r i e t 1 I y 1am i n on i 1111111 y droge ir car1)onatc buffers at pH 7.6-8.0. The aiiioiint of the unsaturated sialic acid fonned at pH 7.2 corresponds to tlica relative aniouirt of this coinpoiiiid nomially occurring in serum mid ririne, and thus fits \\.ell with the mechanism of foimation proposetl for i n uico conditions. It m a y be speculated that the relatively high rate of secretion of Neu2euSAc observed in the saliva of some pvoplc? may be related to a relatively high concentration of CMP-NeiiSAc and a high rate of synthesis of sialic acid, respectively, in the salivary glands.

-

186

ROLAND SCHAUEH

3. Enzymic Synthesis of CMP-Sialates The glycosidic hydroxyl group of sialic acids is activated by CTP leading to CMP-sialates and pyrophosphate before transfer of the sialic acids to oligosaccharides and glycocoiijugates.” With the exception ofthe activation ofKDO, where a CMP glycosyl ester is also f o n ~ l e d this , ~ ~kind ~ of “sugar nucleotide” foniiation involving only one phosphate group in the glycosylic linkage seems to be unique, as all other known sugars are linked to the nucleoside residues through pyrophosphate bridges. CMP-Neu5Ac was discovered in E . coli by Comb and coworkers,255 and has been isolated, in addition to CMP glyoosyl esters of Neu5Gc and Neu5,9Ac2,from porcine’?!‘ and subinaiidibular-gland tissues. The concentration of these nucleotide esters in the latter tissues was found to vary between 5 aiid 14 ph.1. CMP-Neu5Ac has also been isolated from mouse liver aiid kidney.256The occurrence of this nucleotide ester is expected in all tissues wherein sialoglycocoiijugates are synthesized. Its isolation m a y prove to be difficult, because of the low quantities present and its lability. The isolation of CMP-sialates from tissues is possible when the tissues are extracted with triethylainnionium 1iydrogeiic:arl)oiiate buffers of pH 7.6, followed b y purification of the nucleotide esters b y ion-exchange c h r o i i i a t ~ g r a p h y . ~ ~ * ~ ~ ~ Because of the wide occurrence of sialoglycocorijugates, the acylneuraminate cyticlylyltransferase must also be widespread. In several tissues, its activity is sufficiently high that tissue extracts m a y be used for synthesis of CMP-sialates, either directly, o r after enrichment of the enzyme. The cytidylyltransferase sources, and the procedure used for preparation of the nucleotide esters of sialic acid frequently required for sialyltransferase studies, are summarized in Ref. 252. Liver, brain, and sul~mandil~ular glands are the preferred tissues for the preparation of CMP-sialic acids. Frog’s liver has proved to be a very good source for the cytidylyltransferase, perniittiiig synthesis of CMPNeu5Ac in almost quantitative yield h y use ofthe crude tissue-extract or an enzyme preparation partially purified on D E A E - c e l l u l o ~ e . ~ ~ ~ ~ ~ ’ The enzyme was iminobilized on Sepharose 4B, leading to a stable preparation suitable for repeated use .257 CMP-Neu5Ac can be completely purified on a large scale on Dowex-1 X4 ion-exchange resin, using a gradient of0.01-2 hl triethyl(253) E. I,. Kean and S. ROWIII~III, Methotls b ~ t t z ! / m o 8 ~ ,(1966) , 208-215. (254) M . A . Ghalairihr and E. C. Iieath,J. Rial. Cheni., 241 (1966) 3216-3221. (255) I). G. Conit), F. Shiinizu, and S . Koscinai~,J.A m . Chc,vi. SOC., 81 (1959) 55135514. (256) 11. J . Carey a i r t l C. B. Hirschberg, Hiochc,~~iistr!y, 18 (1979) 2086-2092. (257) A. P. Corfieltl, R. Schauer, and M . Weiril)cxr. Riochem. J . , 177 (1979) 1-7.

animoniuni hydrogencarbonatc, pH 7.8. The purity of the coinpound is checked b y the usual chemical and physical nieans, b y t.1.c. on cellulose in 7:3 (v/v) 95% ethanol-M ainmonium acetate, p H 7.3 ( R F valnes: CMP-Neu5Ac, 0.18; Nclu5Ac, 0.52), and b y 360-MHz, 'Hi1.ni.r. spectroscopy.252The high purity achieved permitted iineqiiivocal confimiation ofthe p-anoiiirric configuration, assinned earlier,2i50 f CMP-Neu5Ac by W-n.m.r.spcictroscopy.iR Although cytidylyltransferascs frotii a few tissues had liecn partially purified determination of the molecular parameters was possible only after purification of tlic, rilzyme from frog's liver to honiogeneity b y use of preparative, poly(acrylaniide) jiel-electropliorcsis as the final purification step.72Tlie tiiost striking property of the frog cytid yl y 1trans ferase is its high mo 1w I 11a r weight ( 163,000); s iibu n i t s co id d not be detected. Cytidylyltraiisferases from some tissues exhibit differciices i n s u b strate specificity, especially with regard to the N- and 0 - a c y l substituents of Neu.2i5-z53 An examplc of siich studies is the frog-liver enzyme, which was found to be inactive with 0-acetylated Nei15~4c'~; this is in contrast to the behavior of the correspontling enzymes from bovine, porcine, and equine siibniaiidibular-glaii~s, which were found to activate Neu5Ac, Ncau5Gc, Neu4,5Ac2, and Neii5,9Ac2 at about equal rates, and to exhibit siiiiilar Michaelis- Menten constants with these snbstrates.z58*2s9 In contrast to the frog eiizyiiie, the cyticlylyltraiisferase from equine, sii1)iiii~iidil)iilargland activates the synthetic Nen5Ac4Me, although at a rate lower than that with the natural sialic acids.2ti"Formation of CRlP-Nei15Gc8Me was observed iii the hoinogenate of the starfish Astc.r-icis~forlx:si.99 Localization of cytidylyltransferase i n cell nuclei has 1)eeii reported for liver, spleen, kidney, brain, i i t i d retina, and is summarized i n Rc>fs. 72 and 261. It map be assumed that the formation of ChlP-sialic acids i n 2jit;o occurs in the nuclei. Althoiigh t h i s phenomenon is not yet 1111clerstood, it may be speculated that the spatial separation of the site of CMP-sialate synthesis from thc. site of sialic acid transfer o n t o glycocoiijugates occurring in the Golgi ineinbranes, and from the site of CMP-sialate hydrolysiszti2occrirring i n the plasma ineinbranes, is somehow involved in regulation of the hiosynthesis of sialoglycoconjugates. The cytidylyltransferasc. s e c ~ i n to s be only loosely 1)oiindt o nu(258) H. Schauer, \I. Wember, and C . Fc,rrc.ir;l do Amaral, Z. P/i!~.viol.C h c n i . , 353 (1972) 883-886. , , (1973) 1405-1414. (259) R. Scharirr aiid X l . Weinber, Z. I ' / I ! / , s t o / , C h ~ ~ i ) i354 (260) 1.-M.Beau and R. Schauer, k:ur. /. H i c r l w i i i . , 106 (1980) 5.31-540. (261) S. W. Coates, 1'.G iimey , J r , , I,. W. Soiiiirirrs, M. Yell, an(l C . H . Hirsc.Iilwi.g, /. H i o l . Cheni., 255 (1980) 9225-%2%9. (262) E. L. Kearr a r i d K. J . Bighorist.,]. R i o l . C / w i n . , 249 (1974) 7813-7823.

188

KOLAND SCHAUER

clei, as it is readily solubilized during tissue homogenization. ~-P-DArabiiiofuranosylcytosiiie 5-triphosphate has been reportedz6:'to be a potent inhibitor of the synthesis of CMP-Neu5Ac.

4. Transfer of Sialic Acids onto Complex Carbohydrates Sialyltraiisferases are widely distributed in animals and a few bacteria. A great variety of enzymes seems to exist that differ mainly in their acceptor specificity. In the transfer reactions, different types of a-glycosidic linkages are fomied (2+3,2+4,2+6,2+8, and 2-9 linkages), and various sugars are known to be binding partners of the sialic acid residues (see Section 11). Furthermore, strong influences of the class ofthe complex carbohydrates, as well as of the nature ofthe glycosidic linkage of the penultimate sugar of the acceptor molecule, on the sialyltransferase activity have been described. For example, Gal bound in end positions of glycoproteins or glycolipids, and not those of such relatively small molecules as lactose, is the only substrate found for a sialyltransferase from calf t h y r ~ i d - g l a n dThe .~~~ enzyme from goat co10strum'~~ is less specific with regard to the glycoprotein used as acceptor; but its activity is strongly influenced b y the nature of the sugar to which the accepting Gal residue is bound, and b y the linkage of the latter. For instance, the rate of reaction with P-Gal-(1+4)-GlcNAc is 25 times that with the corresponding (1-6) isomer. With P-Gal-(1-4)Glc, the rate of reaction is 1/8th of that with P-Gal-(1+4)-GlcNAc.z65 Sialyltransferase from calf-kidney cortex was found to have TarnmHorsfall glycoprotein as its best substrate, followed by desialylated serum glycoproteins (for example, a,-acid glycoprotein), and to react very poorly with clesialylated ovine, subniandibular-gland niucin.z66 In a variety of excellent review^,^^"-^^^^,"^*^^^^ the properties and subcellular location of sialyltransferases, together with attempts at their purification, are described. Repetition will be avoided here, and 0111y some new experiments described. In the earlier studies, complete purification of sialyltransferases had not been achieved, but this did not preclude the possibility of the presence of more than one sialyltransferase in an enzyme preparat ion, ' and it prevented detailed study of the molecular properties of these enzymes. This is why the results from the foiiner studies on the specificity of substrates must be interpreted with care. Exact studies of si-

-

(263) hl. W. Myers-Robfogel and A. C. Spataro, Caticrr Rc.F., 40 (1980) 1940-1943. (264) H. C . Spiro, Annu. Reu. Hiochenr., 39 (1970) 599-638. (265) S. Roseman, in E . Rossi and E. Stoll (Eds.), Biocheittktry of GZ!/coproteitrsatid Related Substunces, Part 2, Karger, Basel, 1968, pp. 244-269. (266) W. van Dijk, A.-M. Lasthuis, and D. H. van d e n E i j n d e n , Biochiiti. B i o p h y s . Actcz, 584 (1979) 129-142.

alyltraiisferases require both p u r e cAirzynies and we1 I characterized substrates, and great progress has iiow been made with regard to both of these prerequisites. Hill and coworkers have succeeded in effecting complete purification, with tlie aid of affinity chroinatograph~,,of the following three sialyltraiisf.rases. ( ( I ) p-D-Galactoside-a-(2+~)-sialyltraiisferase was purified o\rer 400,OOO-foltl from lioviiie colost r ~ i i 1 ~ the ~ ~enzyme 7 , ~ ~ ~links ~ ; NeiiSAc to p-Gal-(1+4)-GlcNAc residues of either N-acetyl-lactosainine o r asi~~loglycoproteiiis containing this disaccharide unit at the end ofoligosaccliaride chains. Replacement of GlcNAc by Glc, or isomerization of the GlcNAc glycosidic linkage from p-( 1-4) to p-( 1+3) or p-( l+6) bonds, the rate of sialyltransfer b y > 99%. ( b) p-D-Galactoside-a-(243)-s i a1y 1trails ferase transferring Ne u!jAc to p-Gal-( 1+3)-GalNAc-a-( 1-4))-Ser/Thr residues has I x e n piirified 90,000-fold froin porcine, sul~ni~iii~lil~iilar gl:d,2'i!1,270 a r i d ( c )2-acetaiiiic~o-2-deoxy-a-D-galactoside-cY-(~~~)-sialyltransf~r~ise, catalyzing the incorporation of N e u5Ac i 11t ( CY - Gal N Ac-( I+ 0)-Se r/Th r re s id ties , was enriched 117,000-fold from tlie same t i s ~ t i e . ~ ~ ~ - ~ ~ ' The authors of this elegant work consiclered that these three sialyltransferases, now available i i i Iioinogeneous forin, together with at least two other sialyltransferases n o t yet purified, arc required for the biosynthesis of the major sialic acid linkages obsewed in inaininalian g l y c o p r o t e i ~ i s The . ~ ~ ~last two si~~lyltransferascs are involved i n catalyzing the oligosaccharide seqiieiices a-Neu5Ac-(B+3)-P-Gal-( 1-4)GlcNAc and a-NeuSAc-(2+8)-~-NeiiSAc-(2-+)X-, respectively, where X may be ~ a r i a b l e . ' ~Using " the purified sia1yltraiisf~:rasesin addition to pure fucosyltransferases aiicl antifreeze glycoprotein, or human asialotransferrin, as the substratc-, enzyme reactions involved in the biosynthesis of the terminal part of the oligosaccharide chains were studied. These experiments confirmed that sialylation and fucosylation are often alternative steps in chaiir terinination of inaininalian oligosac~harides.'~* They provided additional insight into the complicated (267) J. C. Paulsoii, W. E. Bcranek, a i i t l I{. L. Hill, J . B i o l . Chcrri., 252 (1977) 233562362. (268) J. C. Paulsoii, J. I. Rearick, ant1 R . L. H i l l , / , Riol. Cherri., 252 (1977) 2363-2371. (269) J. E. Sadler, J . I. Rearick, J. C;. Parilaon. a i i t l R . L. IIiII, ,I. Riol. C'hcJrti., 2.54 (1979) 4434 -4443. (270) J. I. Rearick, J. E. Sadler, J. C . P ; u i l s o i ~ a, i i d K. L. H i l l , / H i o l . C / i c , i r l . , 234 (1C37Y) 4444 -4451. (271) 1. E. Sadler, J . I. Rearick, and K. L. H i l l , / . Hi()/.C h i t i . , 254 (1979) 5934-5941. (272) T. A. Reyer, J . I. Rearick, J. C:. I'arilwn, J,-P. Prieels, J . E. Sadler, and R. L. Hill, J . Riol. Cherii., 254 (1979) 12,531 -12,541. (273) J . F i n n e , T. Kriisius, H. Rauvala, atid K . Hciiiiiiiniki, t.Jitr. J . H i ~ ~ ~ h ~ 77 ? i i(1977) . , 319-323.

190

ROLAND SCHAUEK

problem of the specificity of glycosyltrar?sferases and the control mechanisms involved in the biosynthesis of bi- and tri-antennary, carbohydrate chains. Although asialomuciii from porcine, submandibular gland is a good substrate for the a-(2-+3)-sialyltransferase isolated from this tissue, no function could be ascribed to this enzyme with regard to sialylation in oiuo of mucus glycoproteins containing only a(2+6)-sialyl linkages. It may therefore lie a s ~ u i n e d that ~ ’ ~ this sialyltransferase is responsible for the biosynthesis of a-sialyl-(2-+3)-Gal 1in kage s of gari gl ios ide s in pore ine , submand ibular gland. By using the piire sialyltransferases and CMP-Neu5Ac, it was possible to restore the biological activities of some desialylated glycocoiijugates. The activity of the clesialylated, binding protein of rabbit liver that is responsible for the binding of desialylated serum glycoproteins was almost fully restored by resialylation with the aid of P-D-galactoside-a-(2+6)-sialyltransfera~e.~~~ Similarly, incorporation of a-(2-3)sialyl groups into human erythrocytes b y P-D-galactoside-a-(2+3)-sialyltransferase led to restoration of specific, inyxovirus receptorsiteszi5(see also, Section VII,4). These experiments, and additional sul3strate-specificity studies made by Hill aiid coworkers with the purified sialyltransferases, showed that these eiizyines are not specific with regard to the kind of substrate (oligosaccharide, glycoprotein, or glycolipid), although remarkable differences in the rate of sialyl transfer and other kinetic properties exist, but that they are strongly specific with regard to the position of the acceptor molecule to which the sialic acid is bound, as well as to the nature, or substitution, of the accepting sugar residue. Strong specificity with regard to the accepting sugar residue, but not with regard to the overall nature of the acceptor molecule, may also be delineated from studies made in our laboratory, w i n g synthetic, glycosylated lysozyme derivatives as substrates and particulate sialyltrans ferase s froi n 1ive r and su b m an dibular glands Where a s GlcN Ac residues did not serve as sialyl acceptors at all, Gal or lactose residues were active with sialyltransferases from frog or bovine liver, and froni porcine or bovine subinaiidibular-glands, as was expected from the oligosaccharide structures of the glycoproteins synthesized in these tissues.2i6 Only bovine sialyltraiisferase reacted with GalNAc residues; the unexpected inactivity of the porcine enzyme, known to incorporate sialyl residues a-(2~6)-glycosidicallyinto GalNAc resi(274) J. C. Parilson, R . L. Hill, T. Tanal)e, a i i d <.: Ashwell,]. Biol. ChcJttr.,252 (1977) 8624 -8628. (275) J . C. Paulson, J . E. Sadler, and R. L,. Hill,]. Aiol. Chert)., 254 (1979) 2120-2124. (276) R. Schaiier, E. Moczar, and hl. Weniher, Z. P h y s i o / . <;hem.,360 (1979) 15871593.

s 1A I I c: AC I 11s >

191

dues , with 1y sozy me-phen y lazc )-/3-&1 N Ac may lie explained 1) y the hydrophobic group linking the sugar to the protein molecule, or b y the short distance of GalNAc froin the protein. The experiments, however, clearly demonstrated a striking difference between the 2-acetamido-2-deoxy-cu-~-galactoside-tu-(2~6)-sia~ytransferases of porcine and bovine subinandibiilar-glands, respectively. Substrate-specificity studies on microsomal, frog-liver sialyltransferase revealed the presence of(2-3) and (2-6) activities.2ii This enzyme system readily sialylates oligosaccharides, but is alinost inactive with asialofetuin, which is in contrast to the sialylation of oligosaccharides, as well as asialofetuiii, ti). rat-liver sialyltra~isferase.~~~ The conclusion from this observatioir is that acceptor specificity of sialyltransferases isolated from liver of evolutionary distant animals is similar for substrates of low molecular weight, but differs for compounds of high molecular weight.279 Donor-specificity of sialyltransferases is not limited to Neu5Ac, but extends to other sialic acids. Mic.rosome-bouud sialyltransferases from bovine, porcine, and equine siil)niantlibular-~l~~ii[ls were found to transfer radioactive NeuSAc, NeuSGc, Neu5,9Ac2, and Neu4,SAcz from their CMP glycosyl esters to inembratie glycoproteins at aliout Correspoiidingly, the ratio equal rates having similar K,, valiies.25H,2s9 of the different sialic acids incorporated into porcine, h v i n e , and equine microsonies correlated with the ratio of thc various CMP-sialic acids present in the incubatioir niediuni. For exainple, this is especial 1y s t ri k in g for ni icrosoni e s fro i n po rcin e , s ulm iandibu lar g 1and, which, in Nature, do not contain detectable amounts of 0-acetylated sialic acids, but transfer O-acc,tylated NeuSAc at the same rate a s Neu5Gc, the main sialic acid i l l this tissue. This property of the submandibular-glaiitl sialyltransferases investigated fits well with the mechanism of biosynthesis of the different sialic acids, with partial O acetylation or hydroxylation occurring before their transfer to nascent glycoconjugate inolecules, as alr~atiydiscussed. In contrast to the behavior of sut~mandiliular-glandsialyltransferases, reports exist in the literature describing niarked differences in the rate of transfer of different sialic. acids to glycocoiijugates. For instance, in embryonic chickell-brain, the occiirrc’iice of at least four si(277) E. B. Lnpiiia, N. D. Gabrielyan, ~ i i r t lA . Y a . Khorlin, B i o k / i i n t i ! / u ,44 (1979) 16481656. (278) E. B. Lapina, N. D. Gabrielyari, : i i i ( I A . Y a . K h o r l i n . Rioorg. H i i i t t . , 5 (1979) 17201727. (279) A . Ya. Khorlin, N. D. Gabrielyiiii. S. E. Zrimbyan, and 11. L. Shiiliiiiin, i i r S. h‘. Anancheiiko (Etl.), Frontiers of Bioorgciriic. ChcJmi,ytr!gc i i i t l .\folc’r.tilur Riolog!y, Pergainon, Oxford, 1980, pp. 73-7:).

192

ROLAND SCHAUER

alyltrarisferases exhibiting specificity either towards Neu5Ac or Neu5Gc has been observed.2H"Furtheiiiiore, Neu5Ac4Me is active with sialyltransferase from equine, submandibular glands only at a rate markedly lower than that for the corresponding, 4G-acetylated Neu5Ac derivative.'j' As compared to oligosaccharide or glycoproteiii sialyltransferases, less knowledge is available concerning ganglioside sialyltransferases. The involvement of specific sialyltransferases in the biosynthesis of gangliosides was reviewed in Refs. 213, 281, and 282. These enzymes transfer sialic acids only to P-D-linked u-galactosyl residues of glycolipids, and not to those of oligosaccharides or glycoproteins, and to ganglioside sialyl residues. A ChlP-Neu5Ac:heniatoside (GM,) sialyltransferase involved in the biosynthesis of GD, was solubilized, and partially purified, from rat-liver, Golgi iiienilmines.283 Sialyltraiisferases are situated in the membranes of the smooth, endoplasmic reticulum, together with other glycosyltraiisferases, forniing multiglycosyltransferase complexes involved in the biosyiithesis ' ' ~ . "highest, ~ of the oligosaccharide chains of g l y ~ o c o n j ~ g a t e s . ~ ' ~ .The specific sialyltrarisferase activity has been found in the Golgi inembranes after fractionation of subcellular membranes from various tisthe Golgis u e ~ . ~In ~porcine " ~ ~and * bovine ~ ~ ~ subiiiaiidibular-glands, ~ ~ membrane sialyltransferase has been shown to be associated with the Neu5Ac mono-oxygenase and 0-acetyltransferase activities, respect i ~ e l y . ~Sialyltransferase *~~~~" activity also occurs in outer, initochondrial membranes, where it provides autononiy of this cell organelle with regard to glycocoiijugate sialylation.28x" Sialyltransferase activity has also been described in plasma membranes from different cells, including blood platelets.285The function of the enzyme on cell surfaces is unknown, as resialylation of inembrane components directly on the cell surfice is improbable, owing to the presence of CMP-Neu5Ac hydrolase in the plasma membrane, and the lack of CMP-sialic acids in the extracellular space. Furthermore, evidence has never been obtained for a role of cell-surface glycosyltransferases in cellular interaction according to a hypothesis of Roseman's.zx' (280) B. Kaufnian, S. Basu, and S. Roseinan,J. B i d . C h e r n . , 243 (1968) 5804-5807. (281) S. Rosernan, Chern. P h y s . Lipicls, 3 (1970) 270-297. (282) G. D a w s a n , in Ref. 41(b), pp. 255-284. (283) C. M. Eppler, D. J. Marrk, and T. W. Keeiian, Biochim. Bioph!/s.Actu, 619 (1980) 318-331. (284) 0. Gateau, M. Rocha de Morillo, P. Louisot, and R. Marelis, Biochirn. Bioph!/s. Actu, 595 (1980) 157-160. (285) K. K. WLIand C. S. L. Ku, Thronih. R u . , 13 (1978) 183-192.

s I *\I > Ic \<:I 11s

193

Sialyltraiisferases can be so1iil)ilizetl from tlieir subcellular site by using detergents, and be piirifircl l,!, affinity chromatography o n , for example, CUP-6-aminoliexaiiol - a g a r o ~ e , ' ~ ' a s alrendy descri1,ed. Solubilization of frog- and rat-liver sialyltrarisferases b y iiieaiis of Triton X-100 has been d e s ~ r i b e d . "Soluble ~ sialyltransflrase occurs in colostrum, and is also present in siiiall qiiaiitities in 1101-1iial blood-serum. From the latter source, the c'iizy-iiie was purified 300-foltl b y poly(acrylarnic1e) gel-electrophor~:sis."" Interest in serum sialyltransfi~r~tse h a s risen rapidly, because its activity has been described as lieing higher in the sera from patients suffering from some kinds of c a i i c ~ ~especially r, of the gastrointestinal tract and the mammary g l a 1 1 d . ~Determination ~~,~~~ of the activity of this enzyme may become of diagnostic and therapeutic value, as its serum level has 1)eeii found to increase coiicoiiiitaiitly with tumor growth and di s semi nat ion of 111 t't ;i stiwe s , and to t lecre ase aft c>r s 11 rgery or chemotherapy. Its reappearance rnay iiiclicate fiiitlier growth of metastases. The tissue origin of thc si:ilyltrai~sferase activity is not yet known. Its secretion from t u m o r ccalls is assiiiiiecl, although reports concerning increased sialyltraiisfc.~tsc.activities i n human iieoplastic tissues are conflicting.zx9 In the case of rat and 1iuni:iti-I)reitstcancer, i i i i increase in seriiiiisialyltransferase activity is consitlc~reclto be the coiiseqiiei>ceof Imth increased production and release, tlre latter perhaps through cell-surface sheddiii g of the enzyme f rc )II r t 11 c, i i i etas ta s i zing , m am ni ary- t uiii o r cells.2Yo Accordingly, release o f Iitrgr ainoiints of sial yltransferase from he pa toma ce 11-1ine s derived fro 11I pat i c'n t s h av i t i g 11e pat(w e 11ti 1ar carc i iionia was observed, in contrast t o cell lines derived from nomial liiitiian-liver.z91An increasecl l c ~ c of ~ l sialyltransferase has been 01)served i ii regenerating rat-1i ve I . "I)'' A unique , me nib raiie -a s soc i i t t c d , s i a 1y 1trans fe 1-as e system in E . c'o i has been described that catalyzcls t h v synthesis of sialic acid pol>mers with the aid of s ial y 1-inonopli o s p 110 I i o I in tlec tip re 11o 1.29,'1 I 11 m a n 1111 a1i aii systems, no evidence was oI,taiiic,tl for the involvement of lipid iiiterlinkmediates in sialic acid transfer, o r in the fonnatioii ofsiaIy-l-(2-+8)

-

(286) S. N c e s and R. Schaiier, u l l p l l l ) l i \ l l c c l rta\lllts. (287) U . Ganzingel- and E. I k r i t s c ~ l i (, ' ( i t t ( , ( , r - HcJc.,40 (1980) 1300-1303. (288) 1. P. Durham, D. Gillies, A. 13;1ut<.l-.; i ~ r t lH . 0. Lopc o l i a , Clirr. cliirti.:\ctr~. 95 (1979) 425-432. (289) H. A. Ingraham aiitl J. A. A l h a t l t ~ l I ~ ,. /V. a t / . (,'un(wr I t r s t . . 6 1 (1078) 1:371- 1374. (290) R. J. Bertiacki aiid U. Kim, Sc.ic.trc.c,, 19.5 (1977) 577-580. (291) C.-K. Liu, H. Schinied, and S \%'~IYIII~II, C/iit. C h i ~ r iAct(i. . 98 (1979) 223-2333, (292) F. Serafiiri-Cessi. Biockcwr. /., 166 (1977)981 -386. (293) F. A. T roy and h1. A. 3 l c C l o s k c ~. ~/ . f l i o / . ( , ' / t o r i i . , 255.1 (1979) 7377-7387.

I94

ROLAND SCHAUEH

ages in gangliositles.2H2 The same is true for ineiiiiigococcus group C sialyltraiisferase.~sl Studies with regard to regulation, stimulation, or inhibition of sialyltransferase activity are just beginning. It is considered that insulin maintains the activity- of this enzyme at norinal levels; in diabetic mice, a significant tlecrease has I x e n observed that could lie an important, pathogenetic factor in diabetic g l o i i i e r u l ~ n e p h r i t i s .Human, ~~~ chorioiiic gonadotropin was shown to enhance sialyltransferase activity in the immature r a t - o ~ a r y Sial . ~ ~yltransferase ~ activity in the serum and mammary gland of rats increases during proliferation of the maininary gland under honnonal Significant changes of sialyltrans ferase activity i 11 h uin an, ce ivical e p i the 1i urn throughout the menstrual cycle were Furthermore, sialyltransferase activity increases in mammalian skeletal-inuscle after cknervatioii, which may indicate de iiouo synthesis of glycoproteins during the regeneration process.29H Stimulatioii of sialyltransferase activity was ohserved in blood platelets b y platelet-aggregating in rat serum and tissues by colchiciiie,29gaiid in microsollies b y lysolecithin.:'"" Stimulation of enzyme activity b y various, synthetic detergents will not be described here. CMP, CDP, CTP, antl synthetic derivatives of these nucleotides Interest in have lieen found to inhibit sialyltransferase such inhibitors is increasing, a s they may lie expected to serve as anticancer agents.2"',3"i,3n3 Therefore , re&rulation of Golgi sialyltransferase activity appears possible Iiy nucleotides as products of sialyl- and other glycosyl-transferase activities.:"" Interestingly, Epstein-Barr virus infection of human B, lyrnphohlastoid cell-lines leads to a dimit i u t ion of s ial y 1trans ferase activity .:'04 (294) P. Bardos, h l . Lacortl-Botitit.a~i,J , Hakotoarivoiry, J . P. Muh, and J. Weill, I r i t . J . Hiocherrl., 12 (1Y80) 505-507. (295) W. C. Freiich ;utd 0. P. Rahl, Eritloc~ririoloUy,106 (1980) 559-566. (296) C . I p , Bioehini. Rioph!/.s.Acta, 628 (1980) 249-254. (297) E. Chantler and E . l l e l ~ r i i y n c ,in M . Elstein antl 1).V. Parke (Eds.), Mucus i i i H e a l t h cintl / l i s c c ~ s u Pleriuiri, , N e w York, 1977, pp. 131-141. (298) P. L. Jeffrey ;ind S. H . Appcl, E x p Ncurol., 61 (1978) 432-441. (299) I. H . Fraser, S. Hatnani, 1. 51. Collins, atid S . llookcrjea,]. B i d . C h i n . , 255 (1980) 66 17-6625. (300) W. T. Shier and J. T. Trottei-, FERS L o f t . , 62 (1976) 165-168. (301) W. 11. Klohs, R. J . Benracki, a t i d IV. Koryttiyk, Cnticrr R w . , 39 (1979) 1231-1238. (302) C . M. Eppler, 11. J. l l o r r 4 , and T. W. Keeiran, Biochim. Bioph!/s.Actu, 619 (1980) 332 -343. (303) W. Korytnyk, N. Angelino, W. 1).Klohs, ;mtl H. Bernacki,Ror.J.M e t / . C,'hem. Chitii. ?'hc:r., 15 (1980) 77-84. (304) S . W. L. Lai and M. El, Ng, AI-ch.Virol., 63 (1980) 31-41.

VI. ENZYMIC RELEASEOF- SIALIC ACIDS FROM GLYCOSIDICLINKAGES..4NI> FURTHER DEGRADATION 1. Action of Sialidases These enzymes were originally called neuramitiitlases.'j"~However,

the teiiii "sialidases" is reconiiiieiitletl, a s neuraniinic acid itself is iiot their substrate, but various sialic acids are. Sialitlases are the most iiiiportant enzymes for initiation ot' the catnbolic metabolism of sialoglycoconjugates and sialo-oligos~u.cli~iricles b y hydrolytic release of the aglycosidically bound sialyl rc.sic1iic.s. It has been mentioned in Section IV, 6 that the primary product of the ljacterial-enzyrne reaction is the a-anomeric form of free sialic acid, which, in the case of NeuSAc, mutarotates in aqueous solution, yielding mainly the p a n o ~ i i e r . ~This ~-I~ type of cleavage was found to I)c independent of the chemical nature of the ketosidic group of the siil)strates.:"'5The authors provided evidence that hydrolysis of the ketosidic group affords a carbotiiuni ion at C-2; this interniediate is locket1 i n the active site of the enzyiire in c be fbnned by the attack such a conformation that only ~ ~ - N e l l , 5 Acan of a hydroxyl group at C-2. Because sialoglycocoiijugates ;ire essential components of cells and body fluids, and are frequently involved in specific, biological fiinctions, sialidases may become "toxic" enzymes when present in nonphysiological amounts. On the other hand, absence or lessening of the nonnal amount of this enzyme i l i a ) ' lead to diseases, a s I~ecameevident in studies of some fonris of iiiricolipicloses ant1 sialitloses, respectively. Sialiclases have a wider distril)iitioii in Nature than have the sialic acids. T h e y have been found in ;I variety of viruses (mainly oitho- and para-myxoviruses) which tlo i i o t iisually contain sialic acids,1i0 in in several many pathogenic and noiipathogc,iiic l~a~teria,iiO,:j')fj~:ln" strains of the fungus Strcptoni!/cc.v ( I ~ ~ I U in S , such ~ ~ ~ protozoa as Tric1ionioriu.y fot'tus,:"O'"lland in aniiii;il, including huinan, (305) A . Gottschalk, Biochittt. BiojiA!/.\ k t n , 2 3 (1957) 645-646. (306) S. J . Mattiiigly, T . W. Milligair, .4.A . Pierpoiit, aiitl I>. C ; . Straria,/. Cliti. 3fict-o/ > i d . , 12 (1980) 633-635. (307) H . llrzeiiiek, Curr. Top. M i c r o / ) i ( i / .Z ) i t t n u t i o / . , 59 (197%)35-74. (308) Y. Uchida, Y. Tsukada, and T. Siigiiii(iIi, H i ~ h i t ? iBioj)h!/s. . Actci, 350 (1974)425431. (309) X I . Myhill aiitl T. hl. Cook, C:
196

ROLAND SCHAUER

This field has been extensively reviewed by Kosenberg and Schengrund,”O D r ~ e n i e k Gottschalk ,~~~ and D r ~ e n i e k ; ” ~ and Corfield and coworkers.ss Owing to the existence of these collections of sialidase data, oiily the most iinportant aspects of sialidase biochemistry, and developments therein, will be discussed. To date, only a few bacterial and viral sialidases have been purified (although V. cholerue sialidase has been crystallized) and their niolecular parameters investigated, as was reviewed in Ref. 110. We have succeeded in achieving complete purification of the enzyme from the incubation medium of C. perfringem, b y using preparative, poly(acry1aniide) gel-electrophoresis as the final purification-step,”’ in a yield of 65% and with a purification factor of380,000. The specific activity of the enzyme was 600 U/mg of protein, and the molecular weight was determined to be 64,000 b y four independent methods. This high yield of enzyine was possible after maximal stimulation of enzyme production by glycopeptides prepared from edible bird’s-nest substance.314A study of the process of iiiduction of sialidase was inade possible by use of a synthetic incubation-medium lacking sialic acidcontaining substances (in the presence of which the production of sialidase by the bacteria declined to zero). The sialidase from A. ureufuciens has been purified to homogeneity by using affinity chromatography on coloininic acidlZ9;the enzyme consists of two fornis, having molecular weights of 39,000 and 51,000, respectively.315Purification, with the aid of affinity chromatography on N-(4-nitrophenyl)oxarnic acid, of two sialidases from Trichomonus foetus exhibiting rnolecular weights of 38,000 and 320,000, respectively, has been repoited.”I Successful isolation from Propionibncteriurn acnes of a sialidase having a molecular weight316of 33,000, and, from Streptococcus uiridans, of two sialidase fonns having molecular weights of 45,000 and 86,000, was described.”j An extracellular sialidase from group A Streptococcus was partially purified, and its molecular weight estimated”* to be 90,000. Purification of a variety of viral sialidases (influenza and paramyxovirus) after solubilization by proteases or detergents has been exten-

(313) A. Gottschalk and R. Drzeniek, in Ref’. 42(a), pp. 381-402. (314) S. N e e s atid R. Schauer, Behriiig I n s t . k f i t t . , 55 (1974) 68-78 (315) Y. Uchida, Y. Tsukada, and T. Sugimori, J . Biochein. ( T o k y o ) , 86 (1979) 15731585. (316) H. von Nicolai, U. Htiffler, a n d F. Zilliken, Zentmlbl. Rakteriol. P ( i r ( d t e r t k . 171f e k t i o n s k r . H!yg. Alit. O r i g . Reihe A , 247 (1980)84-94. (317) H. von Nicolai, H. E. Miiller, and F. Zilliken, F E B S L e t t . , 117 (1980) 107-110. (318) L. Davis, M. M . Baig, and E. M.Avoub, Infect. Inztnu?l., 24 (1979)780-786.

sively reviewed in Refs. 110 a t i t l 307. Based on the sialidase component, purification of influenza viruses is possible b y adsorption o n aand p-ketosides of N e u bound t o Scpharose, anti elution with N t > i i 5 k benzyl a-ketoside."19 Affinity clar0iIiatography of' Newcastle-disease viruses (NDV) is also possible 0 1 1 Neil-/%Me o r "Neu2en" hound to Se p h aros e through the am i i i ( g r()11p s .:I2" Compared to microbial sialitlases, much less progress h a s been inade in the isolation of tnatiIitialiati sialitlases, the occurrence of which has been widely studicltl atid reviewecl."~"" The highest grades of purification thus far repoitecl were 4000-fold aiid 3400-fold eitrichre men t s of s ial idase s from ral )I,i t k it l ne y:%2and rat he a t-t-miisc 1e spectively. The latter enzyme is gatiglioside-specific. Great efforts, attended by limited success, havc. I,een made in the purification of brain sialidases, but, although the eir~yttrc.from pig brain w a s enriched 600fold,323corresponding experinleiits with hutnail brain Progress has been made in tlte c1i;iracterizatioti o f huinan-liver sialidases. The lysosomal activit?, \vas shown to consist of at least two, clifferent sialidases, one remaiiiit ig fii-iiily bound to the lysosomal meinbranes after short ultrasonicat ion, and the ot1ic.r being solubilized. The insoluble enzyme provcbtl t o I)e oligosaccharide- and glycoprotein-specific, whereas the soliil)ilizcti enzyme w a s active with gangliosides and mucus glycoproteiiis. The latter eitzyme was purified 4000-fold, alinost to hoinogc.nc.ity, b y affinity cI.1roinatoFr~~ph~ on Sepharose-bound, equine subunantlibrilar-gl~an~l iniicin.:"4The presence of a glycoprotein- and a glycolipicl-specific sialiclase, distri1)rited hetw ee 11 different , s ubce ll i t 1ar- t I I t,t i i 1)rai ic> tract ic)t I s , w a s a 1s o de 1110 nstrated in horse liver.3z2" Although most of the sia1itl;ises have been piirified b y classical methods of enzyme isolation, affinity chromatography is iiow coming more and more into use, as is itrtlicated b y the foregoing exainples o f purification of sialidases. The aclsorhent first used for chrotuatogl-aphy of sialidases was sialic acid h o r i i i ( 1 to the surf'ace of intact erythrocytes;

-

198

ROLAND SCHAUER

this pennitted binding, and specific elution, of sialidase (earlier called recei~tor-destroyingenzyme") from V. clzolerc~e.~~","~' Later followed the use of derivatives of oxamic acid3z28 which, however, did not always lead to satisfactory Affinity chromatography with this material seeins to be Ixised on hydrophobic interaction with the enzyme at sites other than the active center, which results in preservation of the enzyme activity but with some change in the kinetic data.32gBetter purification results, and more-specific binding of sialidases can be achieved, l i y using iininobilized fetuin, coloniinic acid,IzYor subinandibular-glaii~lniuciiis.:'2n The enzymes m a y be desorbed b y alteration of both the pH and the salt concentration. The disadvantage of these natural supports is, however, release of sialic acid by the bound sialidase, even at lower temperatures, and this may limit repeated u s e ofthe coliimns. Affiiiity chromatography is also possible h y direct binding of cw-Neu5Ac to Sepharose, as studied with V. cholercie sialidase; the enzyme could be desorbed b y Neu5Ac benzyl a-ketoside.:{:{" In contrast, Neu-p-Me or Neu2en bound to Sepharose proved to be less-suitable compounds for affinity chromatography.:'") Owing to the difficulties in isolation of sialidases, especially of those from inaniiii a1ian ti ss ue s , know 1edge of the r n olecul ar parameters of most sialidases is still scanty, or even lacking. For bacterial sialidases, the molecular weights thus far detennined (see earlier) lie in the range of 30,000 to 100,000, but inay be 200,000 for viral sialic~ases."" For the latter, subunits m a y tie obtained having molecular weights in the range of those of bacterial sialidases."" The molecular weights of mammalian sialidases are unknown, with the exception of the ganglioside-specific, lysosomal sialidase from human liver, the molecular weight of which was estimatedJz4to be 70,000. In no case is the amino acid coinposition of a sialidase yet known, and complete insight into the amino acids involved in enzyme catalysis is unavailable. There is one report demonstrating a possible involveinent of tryptophan residues in enzyme catalysis."3' Establishing o f t h e complete, amino acid sequence of sialidases from different subtypes o f influenza virus is to be expected from the determination of the nucleotide sequence of the viral genome b y using a plasmid technique.:332Preliminary results revealed the identity of the first 12 amino ' I

(326) G. L. Ada and E. L. French, Nutirrc, 183 (1959) 1740-1741. (327) G. Schranini and E. Mohr, Nafrtrc., 183 (1959) 1677-1678. (328) P. Cuatrecasas and G. Illiano, Hioclietti. B i o p l i y s . R r s . Comniun., 44 (1971) 178184. (329) D. Ziegler, G. Keilich, and R. Brossmcr, Z. P h ! / s i o / .Clzein., 361 (1980) 1361. (330) L. Holniquist, Actci [,'herti. Sccitd., Ser. B , 28 (1974) 1065-1068. (331) H. Bachmayer, F E R S Lctt., 23 (1972) 217-219. (332) J. Blok and G. M. Air, Virolog!/, 107 (1980) 50-60.

acids of several enzyme ~nolc~cules having markedly different sequences, thus explaining the appreciable, antigenic differences observed hetween the different, influenza virus sialidase-subtypes L. More is known as to the pH optimum (usually, p H 4.5-5.5), the requirement for ions, the substrate specificity, and kinetic data on sialican also lie obdases, as such results, extensively revie~ed,","",'l"~.~j~:~ tained b y use of crude or partially purified eiizynies. The data obtained with impure enzymes iirust be interpreted with care, as the presence of various sialidascs, of other glycosiclases modifying the sulxtrate during incubation, o r of Iiypothetical activators and inhibitors may lead to large errors. (:orrc.spoiidingl?., such studies require well characterized substrates, mr(1 identification of the enzyme reaction-products. In the following, studies o n the sulistrate specificity of various sialidases, adhering, as fir a s possible, to the foregoing criteria of purity will be described. Since the stuclies of Rrossmer, Faillard, and Tuppy, the strong influence ofthe size otthe aniino substitilent of Neil on bacterial and viral sialidase-activity lias become known. A suhstituent having two carbon atoms, as i n Nc2uFjAc, is the best sutxtrate coinpared with those having one' (or three) carbon atom(s),as i n N-fonuyliieuramiiiic acid or N-propaiio~lnc~riraminic acid, respectively. Glycosides of N-butanoyl-, N-l)enzoyl-, and N - ( I~enzyloxycarl~ony1)iieuraminic acid are completely resistant towards sialidase action .334,335 Although some differences Iwtween the natural sialic acids having N-acetyl or N-glycolyl groups a s regards the action of sialidase had n o detailed study with well defined subearlier been strates had been made. Therefore, a series of substrate pairs were prepared that contained either NeiiSAc. or Neu5Gc: (1) a-~ialyl-(2+=3)-lactose (the N-glycolyl derivative was obtained b y ozonoly~is':'~ of GM,), (2) a-sialyl-(2+6)-GalNAc (isolated from bovine, submaiidi1)ulargland mucin b y alkaline elimiiiation, followed h y fractionation, and and ( 3 ) GM, (the purification, b y several chronr~tographic glycolyl derivative was iso1atc.d froin horse erythrocyte-meni~~r~~iies,"""

(333) R . Brossiner a i d E. Nebelin, F I : H S Lrtt., 4 (1969) 335-336. (334) P. Meintll aiid H. Tuppy, A f o t i t r t . ; / i . C / w t i i , , 97 (1966) 1628-16.17. (335) H. Faillard, C. Ferreira tlo Aiir;iral, a i i t l M . Blohm, Z . P h ! / , ~ i oChvtti.. l, 350 (1969) 798-802. (336) P. Janiesoii and A. S. Leviiic,,/ Hoctc,rio/.,90 (1965) 563-569. (337) H. Wiegaiidt mcl H. W. Biickiiig, Eur. /. Hiochevi., 15 (1970) 287-292. (338) A . P. Corfield, R. W. Veh, 51. Wt,inl)rl-,J.-C;. Xlichalski, and R. Scharier, Hiochent. /., 197 (1981) 293-299. (339) S.-I. Hakoiiiori mid T. Saito, Hioc./~r,ttii.vlr!/,8 (1969) 5082-5088.

200

ROLAND SCHAUER

and the acetyl derivative from human lives4")).Bacterial sialidases (C. perfringens, V. cholercbe, and A . ureafuciens) showed lower activity with the glycolyl derivatives of these compounds compared with the corresponding acetyl derivatives, as reflected in higher K,, and lower V,,, values for the substrates containing N e ~ 5 G c . ~ ~ ' Favored cleavage of NeuFjAc was also observed in 0-deacetylated bovine, submandibular-gland inucin containing both Neu5Ac and Neu5Gc, or in a mixture of human and porcine erythrocytes containing about equal amounts of Neu5Ac and N e i 1 5 G c . ~Siniilar ~~ differences were observed with NDV, fowl-plague virus (FPV), and influenza A, virus (IA,) sialidases. In contrast to bacterial sialidases, however, the K , values were similar for Neu5Ac and Neu5Gc substrates, and the V,,, values were usually higher for the Neu5Ac cornpounds. Human-liver sialidase also hydrolyzes Neu5Gc from GM, at a lower rate compared with N e ~ 5 A c . " "The ~ relative cleavage-rates of some substrates with various sialidases are shown in Table V. 0-Acetylation of both Neu5Ac and Neu5Gc strongly influences the hydrolytic release of these compounds b y sialidases. This influence is most pronounced if the acetyl group is on 0 - 4 of neuraininic acid, rendering the respective sialic acid almost completely, or completely, resistant towards the action of sialidases. This phenomenon was first observed with bacterial sialidases (V. clzolerae and C. perfriigens,HYand A. ul^e~lfuciell.s"~~), with viral sialidases (NDV, FPV, and IA2),:143 and with mammalian sialidases (human brain and heart, and horse liver).144Whereas the bacterial enzymes studied were completely inactive with 4Q-acetylated sialic acids, the viral enzymes released these sialic acids very slowly, at rates 2-570 of those found with 1111substituted Neu5Ac or Neu5Gc. There is no principal difference in this respect between glycoproteins (suhmandibular-gland glycoprotein or &,-acid glycoprotein from horse), ganglioside (GM, from horse erythrocyte-meiiibrai~es),and sialyl-lactose containing Neu4,5Ac,. It is interesting that 44-acetylatecl sialic acids, which are typical for horse glycocorjugates, are inactive with the sialidases from horse liver.344 From the latter observation, the presence in equine tissues of an esterase capable of hydrolyzing the sialic acid ester groups, before further degradation of sialoglycoconjugates, was suspected. Such an en(340) T. N. Seyfried, S. Airdo, m i d H. K. YII,]. Lipid R r s . , 19 (1978) 538-544. (341) A. P. Corfield, R. W. V e h , M. Wrmlx~r,and R. Schauer, 2. P l i p i o l . Chern., 361 (1980) 231-232. (342) A. P. Corfield, J.-C. Michalski. M . Sander, R Schaiier, and R. Rott, unpublishrd results. (343) R. Schaiier, M. Sander, R. W. Veh, aiid hi. Weinlwr, in Ref. 80, pp. 360-361. (344) M . Sander, R. W. Vrh, and R. Schauer, i n Hcf. 80, pp. 358-359.

TABLEV. The Substrate Specificity of Sialidases, and Their Inhibition by NeuPenSAc Sialic acid structural features" Side chain 0N-Acyl group GM, PentaInhibition Acetyl group Unsub- Acetylwithout saccharide by a-(2+8)" at 0-4' stitutcd' ated" AcetyP Glycolyl" cholate from GM, NeuPen5Ac

Glycosidic linkage Sialidases of Clostridium perfringens Vihrio chol e rcir A rthrobacter tircci.facieti.s u c\\~c:Istl e t l I\ea>e L irus

Fowl plagiie virus Influenza A, virus Horse liver (I ysosomal) Horse liver (plasma i i i e i i i hrane) Human liver

a42-3)"

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100

44 53

44

-

100

31

-

60

100

32

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50 l1.d.~

100 100

20 25

(+) -

+ +

100

7

+

+

+

50

100 IISI.

+

l1.J.

I1.d.

100

(lysosomal) Huinaii heart Hrimaii Ixaiti

0.2 2 0.4

SO

100 100

n.d.

1l.d.

l1.d.

I1.d

-i

IlSl

d.

l1.d.

l1.d.

+A

+

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27

l1.d.

l1.d.

IISI.

l1.d.

n.d.

l1.d.

n.d.

1l.d.

1l.d.

l1.d.

l1.d.

25"'

100'

II

-. i + h

+

+

'' 11, the columiis 2-4 and 6-9, the relative rate5 of cleavage are indicated (100 = tull activity). For i i i o r r details, see the text, ;tnd Refs. 55 ;cnd 115. a-Neu,5Ac-(\7-.3)-1actose. ' a-Neu5Ac-(2~fi)-Iactose." a - ~ e ~ i S A c - ( ~ ~ ~ j - a - N e ~ 1 5 A c - ~ ~ ~": GM, 3 j - ~ containing ~tctos~~:'~~ Ner14Ac,5Cc, and l i o ~ - s c ~ - s ~ a,-acid r u ~ ~ i Qlycoprotc,in containing Neu4,5Ac, De-r.;terifietl, bovine submandihular-glaii~glycoprotein. * Native, bovine s u l ~ m a ~ i t l i l ~ u l a r glycoproteiii - g l ~ ~ ~ ~ l containing sialic acids acetylated niainl?; at 0 - 9 (the values are approximate, as well defined substrates are not available). a-Neu5Gc-(\7-t3)-lactose. ' n.tl., not detennined. Glycoprotein-specific sialidase. Cangliosidespecific sialidase. ' CM3 containing NCL115Ac. GM, coiitairiing Nei15Cc. 'I

,

'

J

'I'

202

ROLAND SCHAlJER

zyme activity was not found in equine or bovine tissues. Enzymic 0-deacetylation of Neu4Ac5Gc as a component of GM, was, however, possible by a human-liver, lysosomal preparation.345 No explanation for the resistance of the 44)-acetylated sialic acids towards sialidases is available. Because Neu5Ac4Me is hydrolyzed both by viral and bacterial sialidases, although at an appreciably lower rate by the V. cholerne enzyme comparedz6" with unsubstituted Neu5Ac, a steric influence of the 4G-acetyl group, less pronounced for the Lie-methyl group, on sialidase activity appears more likely of the hythan disturbance, described by Czarniecki and Thornton,IfiH drogen bonding between the 4-hydroxyl group and the carbonyl group of the N-acyl group. 0-Acetyl groups on the sialic acid side-chain lower the sialidase action by - 50%; this was studied with bacterialx9and NDV43enzymes and bovine, subinandibular-gland mucin as the substrate. Chemical p e r a ~ e t y l a t i o nof ~ ~the ~ sialic acid molecule, and e~terification~~' or a m i d a t i ~ n of , ~ its ~ carboxylic group, block sialidase action completely. Diminution of the length of the sialic acid side-chain by mild periodate-borohydride treatment also lowers the rate of cleavage by sialidases, as was first described by Suttajit and Winzler.Ix6 A strong influence is exerted on the kinetic properties of sialidases b y the chemical nature of the glycosylic linkage. Sialidases can only cleave a-glycosylic linkages of glycosides, but not the corresponding, synthetic N-glycosyl compounds or 2-thiogly~osides,:'~~ which are sialidase inhibitors.lIs As regards the type of glycosidic linkage, (2-3) bonds are rapidly split, whereas (2-6) linkages are hydrolyzed much more slowly by most sialidases. Such differences were earlier described for viral neuraininidases by Dr~eniek.~"~."'","" (2+6) Linkages are cleaved so slowly that viral sialidases have been recommended as tools for structural analysis of sialic acid bonds.35" By using highly purified (2-3) and (2-6) isomers of ~ i a l y l - l a c t o s e a, ~variety ~~ of urine oligosaccharides and glycopeptides or glycoproteins sialylated by use of either (2-3)- or (2-6)-sialyltransferases, this general feature of (345) A. P. Corfield, J.-C. Miclxdski, and H. Schaiier, unpublished results. (346) 13. Faillard, G . Kirchner, mid M. Blohm, Z. Ph!/siol. Cheiti., 347 (1966) 87-93. (347) J. D. Karkas and E. J . Chargaff,/. B i o l . Client., 239 (1964) 949-957. (348) A. K. Shukla, A. P. Corfieltl, R. Schauer, L. Dorlantl, and J . I;. G . Vliegenthart, unpublished results. (349) A. Ya. Khorlin, I. M. Privalova, L. Ya. Zakstelskaya, E. V. Molil)og, and N.A. Evstignceva, FEHS Lett., 8 (1970) 17-19. (350) R. Drzeniek, Hiutochetti. /., 5 (1973) 271-290. (351) R. W. Veh, J.-C. Michalski, A. P. Corfieltl, M. Sancler, D. Gies, and H. Schauer,j. Chroniufogr., 212 (1981) 313-322.

viral sialidases was confirnicd..'"2'Ihc. iiiost i i i a r k c d differences were observed with NDV and FPV c'iizyines, exhibiting up to 1/100th the rates ofcleavage ofthe (2-6) isoiircrs ;is ofthe (2-3) isoni examples are shown in Table V. Hydrolysis of(2-3) arid ( and resistance of (2-6) 1inkagt.s has 1)eeii reported:'5':'for the paraniyxovirus Duck - Mi ssissippi -75. Bacterial sialidases follow the. sanie trend, although the differences are less pronounced, with thc vxccption of the '4. urcyfiicic>ti.s sialiclase, which c I e : i ~ e s ~ ' ~(2-6) - : j ~ ~linkagcs inore efficiently than (2-3). (2-8) Linkages of disialyl-lactose ;ire cleaved 1)y all vim1 and bacterial sialidases at rates lying I)etwec>ii those for the two other linkagetypes.':js2In this respect, huniaii-livc,r, lysosomal sialidase behaves exceptionally, as it splits (2-8) litiknges :it the lowest rate; (2-3) linkages are the most readily hydr01)ized:j~~ (see Table V). Apart from the 4-0-acetylatctl sialic acids, another "sialidase-rcsistant" sialic acid exists in Nature, namely, the internal, Gal-bound, sialic acid residue o f GM,. In cotitrast to this side-positioned sialic acid, the sialyl residues bound to tht, pt~ripheralGal of gaiigliosicles, or to both the peripheral and the intcriial sialic acid residues fonning oligosialyl chains i n several metiil)rsrs of' the large ganglioside can be readily removed b y sialitlases, a s w a s tested with viral, bacterial, and iiiain t i i a1i an enzymes .35"Thc internal, sialyl residut. of G M , is not completely resistant to some sialidases, so long as the oligosaccharide chain is \)ound to the ceramide part of GM, : it is slowly cleaved b y C . perfrittgetis sialidase in the presence of bile ~alts.~'.:':'~-:"'!' Siirprisingly, it is a relatively good siibstrate for the A. urmfacictts ~ i d i d a s e . Hapid ~ ~ " hydrolysis of GM, sialic acid has also been observc~dwitli Sentlai viriis sialidase, i n contrast to the enzymes from NUV or iufliienza virus to wards iiiamn i a1ian s i a1idas tl s h ;L s a1so bee I 1 re po .:{wZ However, it (352) A. P. Corfield, M .Wemlxr, I<. Si.li;iiic>t-,a r i d R. Rott, E u r . J . Riodicrti., 124 (1982) 521-525. (353) N. Kessler antl M .Aytiiard,,/. Gcii, \ ' i ~ / . ,45 (1979) 745-749. (354) K. Suzriki, i i i Ref. 19, pp. lS9- 181. ( 3 5 5 ) G. Ihwsoir, i i r Ref. 41(b),pp. 2x7-336. (356) B. Cestwro, Y. Barenholz, ant1 S.Gatt, H i o c , / i c i t i i s t l . ! / , 19 (1(-)80)61a5-619 (357) D. A. Weriger and S. Wardell,/. , 2 ' c , i t r - o ( , l i c , , ~ i . 20 , (1973) 6 0 7 4 1 2 . (358) H. Rauvala, FI.:RS Lett., 65 (1976'1 22SO-233. (359) A. P. Corfieltl, R. Scharier, G . S ( , l i ~ ~ ~ t i - ~ i r r ~arid i i i i iH. , \ V i c q i i i d t , Z. P/i!/.siol. [ : / I V I I I , , 361 (1980)231. rigarm, and Y. N;igai./. Biol Chcnr., 254 (1979) 784;3-7854. hforioka, antl hl. \latsitttroto, B i o ( , h i t t i , H i o & ~ . A<.tcl, 610 (1980) 632-639. (362) E. H. Koloduy, J. Katifer, J . h l . ( J r i i t k , a i r d R. 0. Bratl\,,J. B i o l . ( . ' l i c t t l . ,246 (1971) 1426- 1431.

204

ROLAND SCHAUER

does not react at all with the V. cholerue enzyme."" As with GM,, the side-chain-positioned, sialic acid residue of GM, has also heen reported to be resistant to various sialidases of viral, bacterial, and mammalian origin.' In order to obtain more insight into these conflicting observations made with different sialidases (partly under the influence of detergents) and to improve understanding of the unusual hehavior of the internal Neu5Ac residue, the pentasaccharide 113Neu5Ac-Gg0se4 has been prepared337from GM, . Remarkably, the compound is completely resistant to the action ofC. perfringetis sialidase, even in the presence of bile salts. It is furthennore inactive with viral sialidases."j9 In contrast, the monosialogangliotetraose can be desialylated by the A. urecrfucien,s enzyme.:360 This resistance (to the action of most sialidases) of the Neu residue linked to cis-3,40-substituted Gal, with sialic acid on 0 - 3 and GalNAc on 0-4, is presumed to be due to steric hindrance caused by this structural feature. 'H-N.1n.r.-spectroscopic studies of GD,, and GM, revealed that the glycosidic oxygen atom of the internal Neu lines a cage-like cavity together with other oxygen atoms, including the carbony1 oxygen atom of GalNAc."6:3As has been discussed in Section IV,6, in mono- and di-sialogangliotetraoses, the (2-3) linkage of the peripheral Neu5Ac is more flexible than the corresponding linkage of' the internal Neu5Ac resi~lue."~ This orientation around the glycosidic linkage of the internal Neu is considered not to be easily accessible for the active center of sialidases. It may be assumed that the conformation ofthe internal sialic acid is influenced by the lipid moiety of GM, in such a way that C. perfriiigens sialidase can slowly act on the sialic acid glycosidic bond of this compound, in contrast to the isolated oligosaccharide. This lipid effect seems to be enhanced b y cholate. Gel filtration of pure, 12sI-labelled, C. perfriiigens sialidase with GM, on Sephadex G-200 showed co-migration of the enzyme with the GM, or GM,-cholate micelles, giving support for the assumption of an interaction of the enzyme with the ganglioside in a way facilitating hydrolysis of the glycosidic linkage.359 The action of the Arthrobacter enzyme, even on monosialogangliotetraose, may be similarly explained by a conformational influence of the enzyme protein, or of impurities, on the nonterminal sialic acid residue. A disruption of the confonnation by ionic interaction of the carboxyl groups of the sialic acid with amino groups appears possible, as bis(monosialogangliotetraityl)amine,a synthetic diiner of GM,-oli(363) P. L. Harris and E. R. Thornton,J. A m Chetn. Soc., 100 (1978) 6738-6745. (364) A. P. Corfield, L. Dorland, H. van Hallleek, H. Wiegandt, J. F. G. Vliegenthart, and R. Schauer, unpublished results.

gosaccharides liuked b y an atnitio group, is s l o w l y active with C . \wrfriiigeiis sialidase.364

The internal, sialic acid residiirx of GM, or GM, is reaclil?-siisceptible to sialidase action after enzyiiiic release of the peripheral Gal and GalNAc residues, leading to C1lc3 having a terniinal sialyl residue, and this represents the natural patliwny of the catabolism of ganglio110,334.355

The substrate-s]?ecificity stii(1it.s cltxscribecl i n this Sul)section show striking similarities t~etweeiithe sialiclases from various soiirces. but also characteristic differencxs \\Tit11 regard to the N - and O-sul)stitutions of Neu, the sialic acid glycosiclic linkages, and the oligosaccharide, glycoprotein, or gangliositle nature of thc, Whereas some sialidases o c c * i i rtiaturally in soluble fomi, others are firmly h u n d to particles. In I,ac.teria, siwlidases appear a s soluble c x o enzymes; in viruses, they are particle-ljound; and, i n most maniiiialian tissues, most of the sialid activity is attached to lysosoine Enzyme activity has also been t l e s c r i l d in G o l g F and plasma tiwinbralles.110.325.366,:Ei In horse livc,r, i t was demoirstratetl that the lysosoinal sialidase is specific for gl, coproteins, and the microsoma1 o r plasina-membrane enzyme, fbr ~ ~ ~ t r g l i ~ j s i ~'The l e s . specificit? :j~~ (for gan gl io s i de s) of the s ial idase s( ) 1 I I 1) i 1i ze d fro1n 11 ti i n an -1i ve r 1y sosoI tie s has already been mentioned. S\-tiaptosotnal niembranes are especially rich in this enzyme activity."".:1fiiIn addition to these ~netnljratreIiound enzymes, low, soluble-sialitlase activities h a v e I ~ e detected n in hiiriian serum and ;mcl are regularly found in the s u p e r natant liquor oftissue hoiirogeti~ites.'"'~'"2~"6" In the latter, it is unknown whether these activities occiir naturally in so1ul)le foilti within cells, or have I ~ e n solubilized from iirc~tril,rane sites during the homogeni za t i o t i . Sial id a s e of iioiiiial , 11i i I II ;I II sc ruin has liee 11 e n r iche tl :300- fo 1 d b y iise of poly(acry1ainide) gcl-c,lec.troplioresis."X" More insight into the suljcc>lliilardistribution of sialidases it1 intact cells is possible b y histochetiiical means. For this purpose, 2-(S-broeu5Ac moindol-3-yl)-a-Neu5Ac and ~ - ( ( i - l j r o r n o - 2 - r i ~ ~ ~ ~ l i t l i y l ) - a - ~were synthesized, and the enzyniicul Iy lilwrated 5-\jro11ioinotit~tnc,oiisly o r couplcd to (365) G. S. Kishorr, D. R. P. T u l \ i i u l i , V. P. B h a v a ~ i a ~ ~ t,mtl l a ~ R. ~ , (;iiriil)elli, J . H i o l . Cheu!., 250 (1975)265552659, (366) C. L. Schengrund and A. Roscii1)c.i-p../. H i o l . Chertl., 5-15 (1970) 6196-6200. (367) B. Veirerando, .4.Preti, A. Lointxirilo, B. Cestaro, a n d G. Tettanlanti, Rioc.lLin1. Riophys. Acta, 527 (1978) 17-30. (368) R. Schauer, R. CV. Veh, 11. WwnlwI, i i t l ( l H.-P. B u s ~ l r c ~%. r , P h / ~ . i o l<.; ~ I C W . , 3,57 (1976) 559-566. (369) R. Gossrau, V. Eschenfeltler, d i i i l H . Brossnrer, Ilistochc,i,iistr!/, 5.3 ( 1 9 7 ) 189192.

206

ROLAND SCHAUEH

an azo dye,"O respectively. These Neu5Ac derivatives are also useful for sialidase staining in poly(acrylaniide) gels. Because sialidases have been recognized to be of great pathophysiological significance (see later, and Section VII), estimation of their concentration in serum and tissue in diseases may be of special interest. A dramatic increase of serum sialidase has been observed in gasedema patients; the bacterial origin of the enzyme is Partial purification (200-fold) of this enzyme activity on equine, submandibular-gland glycoprotein bound to Sepharose was p o s s i b l ~ . "There ~ is also indirect evidence for an increase of this enzyme in pneuinococcal infections of children, as erythrocytes from such patients expose inore Gal residues than do erythrocytes from normal persons, as was made visible by use of fluorescent, peanut a g g l i ~ t i n i n . " ~ ~ Direct evidence for the presence of sialidase in serum in this disease was presented by Seger and coworkers.374Appreciable sialidase activity was also found i n the seruin of a patient with a Citmbacter freuiidii infection."72Elevated levels of extracellular sialidase were observed with clinical isolates of Type 111, Group B S ' t r e p t ~ c o c c i . ~ ~ ~ Sialic acid-depleted, red cells after acute, myocardial infarction point to the possibility of an increase of sixlidase activity in this disease.376 Enhanced sialidase activity has been denionstrated in the blood of arthritic rats."77No significant increase in serum sialidase activity was observed in cancer diseases, as studied with 250 patients suffering from different kinds of cancer, using tritium-labelled, a,-acid glycoprotein as the substrate.37X This observation together with reports of an increased sialyltransferase activity in sennn from cancer patients, already described, leads to the suggestion that only anabolic, sialic acid metabolism is stimulated in some neoplastic diseases. Interest in sialidases has been further stimulated in hunian me&cine b y the observation that a variety of mucolipidoses are in-

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(370) V. Eschenfelder, Habilitationsschrift, University of Heidelberg, 1980. (371) R. Schauer, J . M.Jancik, and M . Wernber, iii Ref. 80, pp. 362-363. (372) R. Schauer, A. P. Corfield, and M. Wember, unpublished results. (373) K. Fischer, A. Poschmaii, and H. Oster, Morzcitsschr. Kiiiderhcilktl., 119 (1971) 2-8. (374) R. Seger, P. J o l l e r , K. Baerlocher, and W. H . Hitzig, Schtueiz. .\.led Wocheiischr., 110 (1980) 14.54-1456. (375) T. W. Milligan, C. J . Baker, 1). traus, aiicl S. J. hlattiirgly, Zitfect. Z n i ~ i z t ~ i i21 ., (1978) 738-746. (376) V. A . Hanson, Jr., S. A. Laiidaw, X I . Flashner, S . 11. Wax, a n d W. R. Webb, A7ii. Hecirt J . , 99 (1980) 483-486. (377) N. W. Marchand, G. S. Kishore, and R. Canlbelli, E x p . M o l . Pnthol., 29 (1978) 273-280. (378) M. Schiiidelhauer arid R. Schaaer, iinpublished results.

SIALIC ACIDS

207

born errors of sialic acid metabolism caused by a sialidase deficiency.55,379,380 These diseases (the first case being discovered in the children’s hospital in Kiel, in 1968, by Spranger and coworkers381) have been called379“sialidoses,” because of the excretion of a great variety of sialylated oligosaccharides in the urine.40In various case-reports, several syndromes with deficiencies in sialidase and, partly, other glycosidase activities also, have been described. The extent of the sialidase deficiency reported seems to be dependent on the type of disorder and on the substrate used for sialidase estimation. In earlier studies, enzyme deficiency in Mucolipidosis I was demonstrated only by using substrates having (2+6)-sialyl groups.382In the meantime, the existence of two different sialidases, one acting on glycoproteins and oligosaccharides, and the other, on gangliosides in human tissues, became evident. These sialidases seem to be under a different genetic control (see, for example, Refs. 379 and 383). Correspondingly, fibroblasts from patients with Mucolipidosis I show deficient sialidase activity with fetuin and sialyl-lactose, and normal activity with g a n g l i o s i d e ~ .On ~ ~ the ~ , ~other ~ ~ hand, sialidase activity was significantly lessened with GM,, in contrast to glycoproteins and oligosaccharides as substrates, when investigated in fibroblasts from Mucolipidosis IV patient^."^ As many conflicting observations have been made in this field, better characterization of human sialidases, especially those from the readily obtainable fibroblasts, is needed, as well as standardized, natural substrates and sialidase-assay conditions. It should be noted that sialidase deficiency has been observed386in the liver of SM/ J mice tested with 4-methylumbelliferyl a-Neu5Ac. Variable sialidase activities are known to occur physiologically during ontogenesis.”O In chick retina, the lowest enzyme activity was observed in the 8-day-old embryo, whereas the highest activity was measured at hatching. Gangliosides were shown to be the best substrates (379) J. A. Lowden and J. S. O’Brien, A m . J . Hum. Genet., 31 (1979) 1-18. (380) A. T. Hoogeveen, F. W. Verheijen, A. d’Azzo, and H. Galjaard,Nature, 285 (1980) 500- 502. (381) J. W. Spranger, H. R. Wiedemann, M. Tolksdorf, E. Graucob, and R. Caesar, Z . Kinderheilkd., 103 (1968) 285-306. (382) G. Strecker, J.-C. Michalski, J. Montreuil, and J.-P. Farriaux, Biomedicine, 25 (1976) 235-239. (383) G . Thomas, L. W. Reynolds, and C. S. Miller, Biochim. Biophys.Acta, 568 (1979) 39-48. (384) M. Cantz and H. Messer, E u r . J . Biochem., 97 (1979) 113-118. (385) G. Bach, M . Zeigler, T . Schaap, and G. Kohn, Biochem. Biophys. Res. Conrmun., 90 (1979) 1341-1347. (386) M. Potier, D. Lu Shun Yan, and J. E. Womack, F E B S Lett., 108 (1979) 345-348.

208

ROLAND SCHAUER

in these experiii~ents."~' Changes in gaiiglioside sialidase were also observed in the developing trout-brain."88 Sialidase activity in the sinall intestine of rats increases 5-fold between birth and 8 days of age, and then declines. In addition, a positive correlation between this sialidase activity in the suckling animals and the sialic acid content of milk was Several natural arid synthetic substrates are available for the determination of sialiclase activities. Among the natural substrates, sialyllactose and fetuin, glycopeptides from edible, tjird's-nest substance and from hovine, or porcine, sulimandibular-glaiid glycoproteins, and the gangliosides GD,, or GM,, are in wide Because the sialic acid liberated is quantitated b y the periodic acid-thiobarbituric acid assay, application of these coinpounds is recoinmended only if high sialidase activities, or purified enzymes, are present. If low activities have to be determined in crucle, biological materials, exact colorimetric measurement of the sialic acids liberated may be difficult, and may lead to errors dut: to reasons discussed i n Section IV,1. In the latter case, such radioactive substrates asXYo( a ) sialyl-lactit[3H]ol, ( b )a,-acid glycoprotein, fetuin, and GM, and GD,, gangliosides specifically labelled in their sialic acid r n ~ i e t i e s , ' " , ' ~ ~ . ' ~ the case of GD,, , tritium-labelled in the sphingosine part,", or (c) such chromogenic substrates as:392 p-methoxyphenyl a-NeuSAc 01393~"4 4-111e thy 1umbe 11i a-NeuSAc, may be used for sensitive, and mostspecific, sialidas ys. When using glycoconjugates tritium-labelled in the sialic acid moieties Ijy periodate-borotritide, conditions should tie ~ e l e c t e d ' leading ~' to an optimum relative amount of the C, analog of Neu5Ac (which is susceptible to sialidase at rates similar to those for unmodified NeuSAc, in contrast to the C, analog, which is hydrolyzed appreciably more slowly'XG), whereliy this effect varies with the sialidase studied. For elucidation of the specificity of' sialitlase action in biological ex-

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(387) .4.Preti, .4.Fiorilli, H. Dreyfiis, S. Haith, P.-F. Url)an, and P. Mandel, E x p . E y e Rrs., 26 (1978) 621 -628. (388) H. Schiller, K. Segler, G. Jeserich, 11. H(isnrr, and H. Rahmann, Lift Sci., 25 (1979)2029-2033. (389) J. J . Uickson and X I . Messer, Biochcin. I . , 170 (1978) 407-413. (390) V. P. Bhavanantlan, A. K. Yvh, and H. Cariil)elIi, Ancil. B i o c h c w . , 69 (1975) 385394. (391) G. Schwarzmann, Biocliini. H i o ~ i h ! / . s A. c t u , 529 (1978) 106-1 14. (392) H. Tuppy and P. Paleac, F E B S L e t t . , 3 (1969) 72-75. (393) 11. Pntier, L. Xlameli, k f . Helisle, L. Dallaire, and S. B. , \ l e l a n p i , Anul. B i o d w m . , 94 (1979) 287-2963, (C94) R. W. Xlyers, R. T. Lee, Y. C. Lev, G. H. Tlioiiias, L. W. Heynolds, and Y.Uchicla, Anal. Riocheni., 101 (1980) 166-174.

periments, a specific inhibitor of' this enzyme is required. Although a large variety of natural and splithetic sialidase inhibitors is known (as reviewed in Refs. 110 and 115), only Neu2en5Ac and its derivatives are relatively suitable As already mentioned, Neu2en5Ac occurs in low quailtities in human fluids; modern methods for its synthesis are described in Refs. 396 and 397. Neu2en5Ac fully and competitively inhibits sialidase at 1 to 5 rrlM concentrations,:'" exhibiting a i l inhibitor constant of 10 FLV.It inhibits all mammalian, bacterial, ant1 viral sialitlases so far investigated, with the exception of the glvcoprotein-specific sialidases of h ~ i r n a n " ~and h o r ~ e 3 ~liver. j 'The N-trifluoroacetyl derivative of Neu2en5Ac has been descrilietl 11s 1)eiiig an even inore effective, sialidase inhibitor, exhibiting"gsa K i value of- 1 p M . The diastereoisomer of Neu2en5Ac, namely, 5-acetamitlo-2,6-anhytfro-3,5-dideoxy-D-glycero-D-tu~o-non-2-e1loilicacid (4-epi-Neu2en5Ac), and its methyl ester, were also found to be sialitlasr inhibitors, as tested with the enzyiiie from Artlzrohucter ~ ~ i u l ~ ) ? ) l i i l i i , ~ . ~ j ~ i A further valuable inhibitor is N-(4-nitropheriyl)ox~~iiiic acid,:'gxused for the affinity chromatography of sialidases as already described. Application of this compound and of Neu2en5Ac eiiabled discrimination between at least two sialidases in human liver. Whereas Neu2enSAc o n l y inhibits sialidase action on gangliosides and small oligosaccharides (for example, sialyl-lactose), N-(4-nitrophenyl)ox~i1r1icacid only inhibits enzyme action 011 glvcoproteins (fetuin) and large oligosaccharides [for example, cu-Neu5Ac-(2+6)-/3-Ga1-(1+4)-/3-GlcNAc(1+2)-cu-Man-( l+€i)-P-Man-( 1+4)-CIcNAc].":' The inhibition of s i d i dases by Neu5Ac itself was mentioned in Section III,2. Application of sialidase inhil~itorsfor iiiedical use is still in a premature state. It is iinaginable that inhibitors would 11e useful drugs in infections, caused b y micro-orgaiiisms, that lead to extensive production of sialidase, for example, in gas ~ ' d ~ ' n i a In . ~ 'the ~ oral cavity, plaque formation and dental caries may IN> influenced by desialylation of salivary glycoconjugates,"~ and I)acterial sialidases may play a role therein. This process may he retarded by secretion of the inhibitor Neu2en5Ac in saliva at concentrations which, i n some cases, were fouiid to be close to the K i value for sialidases."4

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(395) P. hleindl, G. Bodo, P. Palest., 1. Sclirilman, and H . Tiippy, Virolog!y. 58 11974) 457-463. (396) J.-M. Beau arid R. Schauer, in Hc.1'. 80. pp. 356-357. (397) V. Kumar, J. Kessler, M . E. Scott, B. 11. Patwardhun, S. W. Tanerrl~auirl,and M. Flashner, Carbohydr. Re.v., 94 (1981) 123-130. (398) R. Brossmer, G. Keilich, and U. Zicglri-,2. Physiol. C / w r l i . , 358 (1977) 391-396. (399) 1 4 . J. Perlitsch and I . Glickinan,/ Pcriotlorifol., 37 (1966) 368-373.

210

ROLAND SCHAUEH

Although viral sialidases seem to play a role, still poorly understood, in the mechanism of infection, and Neu2en5Ac derivatives inhibit virus replication in cell culture, iio protective influence against influenza or paraiiiflueiiza virus infections in the intact host could be demonstrated b y application of these inhibitors.4onThis failure could be due to the rapid, urinary excretion of Neu2en5Ac observed after oral or intravenous application to rats and I n contrast, the presence of influenza-sialidase antibodies was shown to inhibit, specifically, influenza virus infection in man and to lessen expulsion of the virus into the environment and the successive spread of an epidemic.4n2 Various strains of influenza virus can be distinguished from one another in a sensitive manner by their different sialidase antibodies from serum and nasal fluids, using a sialidase-inhibition test on a micro scale, thus enabling a full kiiowledge of variation in, for example, influenza A viruses.4o3 Partial inhibition of Arthrohncter sialidase (almost complete inhibition when tested with substrates of high molecular weight, and no inhibition with sialyl-lactose) is possible by a monospecific antibody raised in guinea-pigs "~ from several influenza against A. siulophi1zr.s s i a l i d a ~ e . ~Sialidases viruses, non-Arthrohacter bacteria, or human fibroblasts failed to react with this antibody. The antibody furthermore allowed the measurement of nanograms of the A . siuloplailus enzyme. The use of iinmobilized sialidases for desialylation both of soluble sialoglycocoiijugates and cell surfaces continues to become increasingly important, as mentioned in Section III,2. Purified C. perfringens sialidase has been immobilized on Sepharose 4B, or on controlledpore glass-beads,L'2and V. cholerae sialidase has been linked to Sep h a r o ~ e " or ~ , Nylon ~ ~ ~ tubing,4°5 either covalently or by adsorption to oxaniic acid derivative^.^"^ Such iinniobilized sialidases have the following advantages: repeated use of the enzyme; separation of the de-

(400) P. Palese and J . L. Schulman, in J . S. Oxford (Ed.),ChEnioproph!iZaxis and Virus 1rifection.s in the, Respircitor!/ Tract, Vol. 1, CRC Press, Cleveland, 1977, pp. 189205. (401) U. Nhhle and R. Schauer, 2. P l r y s i o l . Chetn.. 362 (1981) 1495-1506. (402) N. Yainane, T. Odagiri, J . Arikawa, hl. Kumasaku, and N. Ishida, ;24icrohiol. 1rnrnunol., 23 (1979) 565-567. (403) J. Werner, 0. Thraenhait, and E. K u w e r t , M d .L4ficrohio/.Zinniuno/., 166 (1978) 165-171. (404) R. Huchzeriineyer, S . W. Tanciil)aiim, antl M. Flashiier, Aiicil. Bioclzcm., 105 (1980) 454-460. (405) E. R. Bazariiun antl I,. B. Wingartl, J r . , J . Wistochcrii. C!/tochwi., 27 (1979) 125127. (406) R. Rrossmer, 11. Ziegler, and G. Keilich, 2 . Pliysiol. Clwm., 358 (1977) 397-400.

sialylatetl compounds, or cells, ti-otii the enzyme; no adsorption of sialidase to cell surfaces; and coitiplclte desialylation of so1ul)lc coinpounds by recycling over the iniinobilized enzynre, with concomitant dialysis of the sialic acids rt.lwscd.":( Application of such inimobilized sialidases in cell biology1':'o r i t ) the treatment of Ieiikeniia4"5will be discussed in Section VII. In the present Section, on sialidases, another enzyme should IK, included that cleaves sialic acid glycosyl ester Imnds hyclrolyticallv i n manitiialian cells, namely, ChlP-NeiiSAc hydrolase (CMP-NviiFiAc phosghodiesterase, EC 3.1.4.40).~''2,1"7 As the glycosyl ester bonds in CMP-Neu5Ac have the p coiifigiir;itioir,lxthis enzyine n i a y be considered to be a "p-sialohydrolasc-." Its siilxellular location is plasnra membrane. No data are a s yet ~ivai1:il)leconcerning the exact role of this enzyme; its involveinelit i n regulation of the celliilar CMPNeu5Ac level, and possibk prc~vc~ntion of a i r oversialylation of cellsurf& glycoconjugates h y tlestrnction of CMP-NeuSAc arriviiig at this site, should also be taken i i i t o consitleration, a s w a s discussed in Section V,3. The hydrolase provides :I link with the initial part of'the amino sugar pathway, a s it is f ~ ~ c ~ d l ~ a u k - i t i l i i lb~yi tUDP-GlcNAc.'"' e~l

2. Action of Acylneuraminate Pyruvate-Lyase

The reversible cleavage of sialic iicitls into 2-ac)ilamido-2-deoxv-I~inannoses and pyruvate by this eiizyme, the influence of N e u 0substituents o n this reaction, a t i d its use for analysis and synthesis o f sialic acid were descrilwtl i n Section IV,1 and have heen reviewed.107~21~~229~2"" Glycosidicallb, linked sialic acids are inactive. The enzyme has beeii found to ouciir i i i a variety of bacteria, lrequcutly together with sialidases,:io6atic I generally in tiiiiint iialiarr tissues. The subcellular site ofthe enzynie that can be extracted from tissues without the use of detergents is iitikttown. In C:. p ~ r f r i t i g e n s it , was shown to be an endoenzyme liberated iiito the incuhatioti medium only after cell l y ~ i s . ~ Production ~* of the. r n z ! ~ n e in this lxicteriiiin can he induced b y free o r glycosidical Iy \ ) o i i i i d Neu5Ac, siinilarl\r to the sialidase.:"4 The l y a s e from C. p m f r i i i g c t i , ~W;IS purified to honiogc,treity b y use of preparative gel-electrop1iorc.sis a s t h e final purification step, thus allowing investigation of its tiiolc~cularparaineters."OXThe average nro-

212

R O L A N D SCHAUEH

lecular weight was estimated to be 99,200; the molecules are composed of two sulmiits of molecular weight 50,000 each. The diineric form of the native-enzyme molecules was made visible by electron and piglo Partial purification of the lyase from kidney has also been achieved. The pig-kidney lyase could be purified by affinity chromatography on Neu-P-Me bound to S e p h a r ~ s e . ~ ~ ~ This isolation procedure includes a heating step41o wherein appreciable inactivation of the enzyme may occur. This inactivation can largely be avoided b y the addition of 40 in114 pyruvate."'2 In the catalysis of the lyase from C. perfririgens, the participation of lysiiie residues fonning iiitemiediary Schiff bases between enzyme and substrate molecules, and of histidine residues, has been clemonstrated with the aid of photooxidatioii, reagents for histidine inodificati on, and lioro h y d r ide re duct io 1.1 in the pre se lice of s ub s trate .40H ,41s Thus, according to Frazi and coworkers,414the lyase belongs to the class I lyases (aldolases). The catalytic mechanism proposed is outlined in Scheme 3 . Evidence has lieell educed for the existence of a similar mechanism of cleavage of sialic acid b y the lyase enriched froin pig kidney.4" The function of the acylneuraminate pyruvate-lyase is still soiiiewhat speculative. It may be assumed that, in bacteria, its role is degradation, to pyruvic acid and 2-acylamido-2-deoxy-~-mannoses, of sialic acids liberated b y sialiclase, and taken up by the cell with the aid of Ne~5Ac-permease"'~ (see Scheme 4). The degradation products may be consumed by the cells as a source of energy. In mammalian tissues, the lyase may have a regulatory role in the metabolism of sialic acid by preventing recycling of sialic acids liberated by sialidase action, as is shown in Scheme 2. Experiments on oral or intravenous application of double-labelled NeuSAc have shown that the minute amounts of sialic acids taken up b y tissues, other than those of the intestine, are probably cleaved by the action of the intracellular l y a ~ e . ~ (Most "' of the sialic acid is rapidly excreted in the urine.) Whereas pyruvate is con-

-

(409) D. A. Sirhasku and S. B. Binkley, Riochitti. Biophys. Actu, 206 (1970) 479-482. (410) P. Brunetti, G. W. Joiirdiaii, antl S. Roselnm,]. B i o l . C h e m . , 237 (1962) 24472453. (411) R. Schauer, A. P. Corfield, and bl. Wember, unpublished results. (412) F. N. Kolisis, T. G . Sotiroudis, and A. E. Evaiigelopoulos, F E H S L c f t . , 121 (1980) 280-282. (413) R. Schauer antl M .Weiiibei-, Z. Ph!/siol. Chettr., 352 (1971) 1517-1523. (414) E. Grazi, H. Melochc. G. Martinez, W. A. Wood, and R. L. Horecker, Biochrtn. R i o p h ! / s . Res. Commutr., 10 (1963) 4-10.

SIALIC ACIDS

213

702-

co;

c= 0

C

I

I HCH I

- H,O h

7

HCOH

+

@

N-

I

H-CH CI f-

+H,O

I

AcHNCH

ACHNCH

I

I I HCOH I

HOCH I HCOH

H

HOCH

I

HCOH I CH,OH

HYOH

CH,OH

41

CO,

I

C=O

I

CH3

H,C$O _. H

I I

AcHNCH HOCH

I

HCOH I

HCOH

I

CH,OH

SCHEME3.-Proposed Mechanism of Reversible Cleavage of NeuSAc by Acylneuraminate Pyruvate-lyase. (Taken, and modified, from Ref. 408.)

surned in the energy metabolism, the 2-acylamido-2-deoxy-~-mannoses may be re-used for the synthesis of sialic acid after phosphorylation, or converted into other amino sugars by way of G I ~ N A c . ~ ~ ~ Remarkably, only the enzymes of sialic acid catabolism, namely, sialidase and acylneuraminate pyruvate-lyase, are significantly influenced by N - and 0-acyl, or 0-methyl, groups of sialic acids in the mammalian, bacterial, and viral systems investigated (described in the preceding Sections). (Knowledge in this regard concerning CMPNeu5Ac-hydrolase is not yet availalile.) This observation associates modification of sialic acid, especially acetylation at 0-4, with an irnportant, regulatory role on the catabolic site of the metabolism of sialic acid. The low influence of these niodifications on the anabolic enzyme-reactions, also shown in Schenie 2, has already been described.

214

ROLAND SCHAUER

SCHEME4.--Induction and Cooperation of the Sialic acid-specific Enzymes Sialidase, Acylneuraminate Pyruvate-lyase, and Neu5Ac-perinease (hypothetical) in Clos, sialoglycoprotein; 0 , free sialic acid; tridiurn perfringens. [ ~ e y : , cleavage of glycoproteins by clostridial proteases (taken from Ref. 455).]

4

VII. BIOLOGICALSIGNIFICANCE O F

SIALIC

ACIDS

A great variety of biological phenomena that can be ascribed to the sialic acids is known. This was shown after removal of sialic acid by sialidases, or after modification of sialic acid, for example, by periodate oxidation. However, it has not yet been possible to explain all of these phenomena uniformly, that is, to attribute one general role to the sialic acids. Furthermore, the numerous, biological phenomena ascertained to be due to the presence of sialic acids may be influenced by the compounds to which the sialic acids are bound. These are oligosaccharide chains, and protein and lipid residues having various biological roles. The functions of these compounds may be modified, enhanced, or lessened by linking the relatively large, hydrophilic, and strongly acidic sialic acid molecules, usually to the exposed end of the oligosaccharicle chains. It is also imaginable that the structure of macromolecules and cell membranes, and, correspondingly, the behavior of cells, is influenced by these anionic compounds. In the following Sections, the biological functions of sialic acids will be divided into four groups, and described on the basis of some well established examples selected from the flood of publications in

this field. (For detailed descriptions, see the reviews in Refs. 229 and 415-419.) This classification is tviitativc., and overlappiiig prc~siunably cannot be avoided.

1. Function D u e to the Negative Charge of Sialic Acids On the basis of the accumulation of the iiegatively charged sidic acid residues on cell ineml,raiies,"i6 it may lie expected that these compounds strongly influence, t h e behavior of cells. To indicate the density of sialic acids on cell iiic>inl)raiies,it m a y lie calculated, on the basis of data published in Refs. 8Fi and 106,that > 10' N e u residues are bound to the surface of a single liiiiiiaii-eiythrocyte. This figure agrees well with the 1.8 x lo7negative elcwentary charges due only to sialic acids on the surface of one huinan erythrocyte, detemiined b y the electrophoretic mobility of tlic cells i n dependence on the ionic ~treiigth.~'" On the surface of guinea-pig granulocytes, 3.2 iiinoles of sialic acid per 10' cells have 1 ) w t i f o 1 1 n d . ~ * ~ As regards the function of this electronegative shield in some celltypes, membrane sialic acids prrveiit aggregation clue to electrostatic repulsion in, for example, blood platelets, erythrocytes, and carciiionia cells in c i i l t i ~ r e whercus, ,~~~ i n others, for example, chick, ernbryonic muscle-cells,422aggregation is facilitated, most probably by Ca2' bridges. Sialic acid was a l s o shown to be involved in tlw attachment both of endotheliiim m t l epithelium to glomeriilar Ixisementmembranes of rat kidney.4z::' The repulsive, electrostatic. forces of sialic acids contribute to the rigidity of the cell surfice, a s W;LS shown b y an increase i n the crlcforinability of sarcoiiia cells after rnzyniic removal of sialic acid resid ~ eEnzymic ~ . ~ release ~ ~of sialic. acids from the zona pellucida of rabbit ovum lesseiis the rigidity o f this cell, and spemiatozoa can no longer penetrate it.42sGlycoprotc,iiis o n the siirface of sea-urchin eggs

(415) H. Fnillartl aiitl R. Schaiier, i l l 1 ~ c . l ' .,42(l)),pp. 1241i- 1267. (416) K . W. Jeanloz and J. F. Codiiigtoti, i i i Ref. 19, pp. 201-2:38. (417) A . Rosenberg and C.-L. Schc.iigIriiitl. i n Kcf.. 19, p p 2775-294. (418) R. ScIiauc,r, CJiri,ytiatiu Alhertitici, 9 (1978) 35-45. (419) E. Kiittgen, C . Bauer, W. Rciittc,I-, i i i i ( l W. Gerok, K l i l r . \t'oc./ic,trrc,lir-., 57 (!979) 151-159, 199-214. (420) E. Donath and D. Lerclw, H i o c . / ~ . c ~ / t - e ~ c ~ l r Rioc~tiorg., c~tti. 7 (1980) 41 -.53. (421) J . W. IIePierrc, J . Lazdiiis, a i i c l \ I . 1,. K m i o v s k y , B ~ o c ~ P J J J102 . . [(1980) ., 54.3-550. (422) R. B. K e i n p , / . Cell Sci., 6 (1970) 7.51-7Wj. (423) Y. S. Kaiiwar aiitl hl. C . Farcliili;ii, f,ci/). Zttt;c..c.t., 42 (1'380) 375-384. (424) L. M'eiss./. C:c,l/ Riol., 26 (1965) 7335-739. (425) P. Soupart and T. €1. Clewe, Z d ~ c * i - / i / S f c > r i / . ,16 (1965) 677-689

216

ROLANII SCHAUER

bind spermatozoa species-specifically, due to the high content of sialic Sialic acids seem to facilitate binding of cationic compounds to macromolecules and cells. Thus, sialic acids on the surface of L1210 mouse-leukemia cells have been found to influence transport ofpotasThe iiptake of 2-amino-2sium ions through the cell methylpropanoic acicl by HeLa cells is decreased after treatment with sialidase; the authors42xinferred from this a general role of sialic acid at the cell surface in the transport of amino acids. It is imaginable that the acidic glycocalyx on cell surfaces acts like an ion-exchanger. Correspondingly, sialic acids in iiiuco~islayers, and on epithelial cell-surfaces, of fish are assumed to be generally involved in ion t r a ~ i s p o r t . ~ ~ ~ The passage of several substances through the mucous layer on the gut wall was shown to be influenced b y the electrical charge of the ~ n u c u s . ~The ~ " positively charged, serotonin molecule is bound by membrane sialic acids in rat s n i o o t h - ~ n u s c l e .Correspondingly, ~~~ extensive desialylatioii of human platelets led to a significant decrease of the rate of uptake of this compound.43*Plasinapexin, a sialic acidcontaining glycoprotein of blood plasma, binds the base histamine with the aid of sialic Sialic acid residues are important Ca2+-binding sites in muscle cells.434Reports in the literature with regard to an influence of sialidase treatment on muscle contractibility are, however, conflicting. No change in the strength of contraction of stimulated, guinea-pig, atrial muscle-cells occurred after enzymic reinoval of sialic acid from cardiac s a r c o l e ~ n n i a .Similar ~ ~ ~ observations were made with frog Sartorius-muscle cells; only shifts of the cell-surface potential to more positive values were n i e a s i ~ r e c lIn . ~ contrast, ~~ treatment of nerve cells with sialidase led to changes in their activity. Intracellular injection of the enzyme into presynaptic neurons of squids was shown to block

(426) S. Isaka, K. Hotta, a n d M . K~irokawa,E X T JCell . Res., 59 (1970)37-42. (427) J . L. Click and S. Githens, N a t u r e , 208 (1965) 88. (428) D. M. Brown and A. F. Michael, Proc. Soc. E x p . B i d . Merl., 131 (1969)568-570. (429) H. HPntschel and M. Miiller, Covip. Biochcni. Plzysiol. A, 64 (1979) 585-588. ~ . ,(1980) (430) F. Nimmerfall a n d J . Rosenthaler, Biocheiii. Biophys. Res. C m i i i ~ i t ~ i94 960-966. (431) W. Wesenrann and F. Zilliken, 2. P h y s i o l . Clzem., 349 (1968)823-830. (432) V. M. Masters, J. Wehster, and G. h l . W.Cook, Bioclzeiii. Phcirnwcol., 29 (1980) 3189-3201. (433) J . Labat, B. Lebel, G . Parrot, J. L. Parrot, a r i d J. E. Courtois, C . R . Acnd. Sci. Ser. L), 263 (1966)2050-2063. (434) S. E. Hardiug and J . Halliday, Nattrrc, 286 (1980) 819-821. (435) M. Dorrscheidt-KBfer, Pjluegei-s Arcli., 380 (1979) 171-179.

synaptic t ran sill i s s ion ,416,437 al t 11( ) I igh the se an inial s seem to 1ack s ia 1i c acids.*S Injection of sialidase into spinal-cord segments or the optic tectuni of frog and fish, respectivel!., caitsed a significant, initial increase of ne tiro ne activity .43x These alterations ofthe activity art' Iwlieved to be related to the degree of sialylation of gangliositles, which have been shown to bind C.d?- ions,4:39This ion can be 1il)c~rutcdb y nioiiov;dc~ntcations and act.tylcholine, thus causing spiiaptic transmission. Tubocurarine and serotonin also iiifluence calciiiiir-ganglioside interactions,"') a s can t e i i ~ p e r a t i i r e .In ~ ~the ~ last expc.riiiiclirts,it was shown that a higher polarity of neiironal gangliosidcs 1c.atls to i~ lower, theniial sensitivity of the Ca" binding. This m a y lie the I'WSOII why more-polar, polysialylgangliosides are foiiiietl clriring the accliiriatizatioii of poikilotheiiiiic vertebrates to ~ O W W t:~rvironmental te~riperatiires.""~~~~ Based on these and other obserwtioiis, it was assumed that the Ca"+ganglioside complexes, and, t h u s , the electroiiegative charge of sialic acids, play an important role ill the activity of nerve cells; the forniation of such complexes might "tighten" the presynaptic menibrane, whereas dissociation may "open" it.442Corresponding models have been discussed in the publications ~ i t e c l ~ ~ and " . " ~i n others. Another niotlel describes the probable role of the enzymic sialylation-desialylation cycle of gangliosicks at the nerve-ending inemljranes involved in the activity of the nervous t i s ~ ~ . ~ ~ ~ Sialoglycocoiijugates also seem to be involved i n calcium binding of bone tissues.415Neu as a coniponent of an acidic glycoprotein is directly involved in the formation of ii spei-ni reservoir in the vagina of the pig by salt-like coagulation with a Ijasic protein of the Ijoar ejacu+

(436) L. Tauc and D. H. Hinzen, B i n ! i r ficr.. 80 (1974) 340-344. (437) F. X . Hipp, Lf'. Gielen, M. A . ll;i\,iv\, a i i t l 11. H. Hinzeii, Pj/ucg:ri-.r .4rc/i.. 385 (1980) 45-50. 1438) H. Riimc:r a i d H. Rahnraiin, E.v/J. Bruiri Hca.. 34 (1979) 4Y-58. (439) W. Prolist, H . Riisner, H. Wiegair(It, and H. Hahiiiann. Z. P/i!/.siol. C / i c ~ i i r . ,360 (1979) 979-986. (440) 11. Miihleiseir, W. Probst, H. \Vic.gaiitlt, a i i d H. Kahirianir, Life Sc,i., 25 (1979) 791 -796. (441) W. Probst and H. Rahiiiarrn,/. ' ! ~ / i v i - r r i .B i o l . , 5 (1980)243-2447, (442) H. Rahmann,]pn. /. E x p . M c d . , 48 (1978) 85-96, (443) G. Baux, M . Siiiionneau, and L. l'uric, / . P / u J Y ~ ( J291 / . , (1979) 161-178. (444) H. Rahinaiiir, i n H. Matthies, hl. K w g , ,urcl N . Popov (Etls.),Aiologiccil Aspect., of Lenrrririg, Mevior{/ Forrncitioir c i r i t l O t i t ~ g ~ i ~ i ! /f t CNS, h ~ Abh. Akad. M. 1979, pp. 83-1 10. (445) G. Tettanranti, A. Preti, B. Cestaro, 11. Xlasserirri, S. Soiririiro, atid R. Gliitloiri, i n C. C . Sweeley (Ed.),Cell S u r f o w C;[!/(v/ijiids, ACS S ! / i t r / J . Ser. N o . 128, Ainerican Cheinical Society, Washington, I).(:., 1980, pp. 321-:343.

218

ROLAND SCHAUER

late, thus guaranteeing f e r t i l i ~ a t i o n .Similarly, ~~~ evidence was obtained for an increase of the viscosity of mucus in cystic fibrosis by crosslinking of the polyanionic, mucous glycoproteins with an as-yetunidentified, polycationic protein.447

2. Influence of Sialic Acids on Macromolecular Structure Although the influences of sialyl residues on the confomiation of macromolecules and cell niembranes are considered to be mainly clue to their negative charge, and some of the phenomena discussed here may therefore overlap with those treated in the preceding Section, the following effects of sialic acids justify a separate grouping. It was first shown by Gottschalk and that partial removal of sialic acids from submaiidibular-gland glycoproteins drastically lowers their viscosity. This discovery was confirmed in an investigation demonstrating that sialic acid residues increase the intrinsic viscosity of all glycoproteins studied thus far.44yTherefore, sialic acid and, in some niucins, sulfuric ester groups also, are considered to contribute markedly to the high degree of viscosity of the inany mucous secretions, from, for example, the respiratory, digestive, or urogenital tracts, the e y e socket, and the body surface of fish and eels.229,41a,41x This effect is considered to be due to the mutually repelling sialic acid residues extending oligosaccharide chains from the protein core, thus giving these molecules (having high molecular weights) a rod-like structure, and facilitating gel-formation in water.415s450 It should, however, be noted that, in several mucous glycoproteins (those in the stomach being especially well studied) that are relatively poor in sialic acids, disulfide bonds between peptide chains have a strong influence on the visco~ity.~"' The mucous secretions are vital, as they act as lubricants and defensive agents, in cavities of the body communicating with the environment, or on body s u r f ~ ~ c e s . z 2 y ~ 4 ~ 5 ~ 4 1 * ~ 4 s 2 The influence of sialic acids on the macromolecular conformation seems to be the reason for the proteolytic resistance of several sialoglycoproteins. The first example of such a role for sialic acids came from studies by Faillard and P r i l ~ i l l that a ~ ~demonstrated ~ a loss ofthe (446) J. C. Boursnell, E. F. Hartree, and P. A. Briggs, Biochein.J., 117 (1970)981-996. (447) R. W. Lewis, Tex. Rep. B i d . Mcd., 36 (1978) 33-38. (448) A. Gottschalk arid hl. A. W. Thomas, Biochirn. B i o p h y s . Actu, 46 (1961) 91-98. (449) F. Ahmad and P. McPhie, I n t . J . Biochem., 11 (1980) 91-96, (450) A. Herp, A. M. Wu, aiid J. Moschera, M o l . Cell. Biochem., 23 (1979) 27-44. (451) A. Allan and A. Gamer, C u t , 21 (1980) 249-262. (452) L. Reid, in W. M. Thurlbeck and M. R. Abele (Eds.),The Lung, Structure, Function und Ilisense, Williams and Wilkins, Baltimore, 1978, pp. 138- 150. (453) H. Faillard a n d W. Pribilla, K l i i i . Wochenschr., 42 (1964)686-693.

proteolytic resistance of the intrimsic, factor and, concomitantly, of its binding capacity for vitamin B,* after release of sialic acid. Similarly, sialoglycoproteins of the jelly coat of frog's eggs resist proteolytic attack, allowing development ot' tlic embryos, even in piitref>.ing water.41" Protection, b y sialyl residues, of tlopamine p-hyclroxylase ( E C 1.14.17.1)against proteases h a s I)een In that publication, a few inore examples of siic*li;I protecting effect on enzyme protein, and also on fibronectin, w ( : inentioned. ~ On the basis of this anti-proteolytic effect of sialic acids, a hypotlictfor the role of sia1itl;ise in clostridial intectioiis is shown ical 111oclel~~~ in Scheme 4 . It is considered that t h e Ixicterial enzyine releases sialic acids from cell-surface glycoproteins of the infected tissue, which thereafter can be readily attack(d h y proteases. This cooperation between sialidase and protease niay slipport the spreading of the I)acteria. Acylneuraminate pyruvate-l!we, also shown in this n ~ o d e l ,degrades sialic acids for energy supply, and growth, of the bacteria. Conformations1 changes of c.c.ll-siirface components after the removal of sialic acid, leading to loss of tlie rigidity of rabbit ova, and to inhibition of the passage of sperniatozoa through the zoiia pellucida, have been mentioned in the preceding sub-Section."2" The frequent occurrence of sialylated enzyines, o r e v e n of niiiltiple foiins, which are sometimes tissiie-dependent, with a varying n u n i b e r of sialyl residues as, for example, in y-glutamyltranspeptidase ( E C 2,3.2,2),456*4si is riot yet fully unclerstood. Although the activity of inost of these enzymes is not infliiencecl I)y removal of sialic acid,454the activity of monoamine oxidase A (EC 1.4.3.4)of outer mitochondria1 membranes of rat liver has bee11 shown to be destroyed by treatment with sialidase45*;the substrate specificity of acetylcliolinesterase (EC 3.1.1.7)is the kinetic properties of human acid and alkaline phosphatases ( E C 3.1.3.1and 3.1.3.2)are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firin evidence. A role of sialylated, oligosaccliaritle chains i n the achievement of

(4.54) D. Aquino, R. Wong, R. U. Margolis, and R. K. Margolis, FI.;BS Lptt., 112 (1980) 195- 198. (455) R. Schauer, Instrum. Forsch., 3 (1975) 21-41. (456) Y. Matsuda, A. Tsuji, and N . Kntuiruina, J . Riocliem. ( T o k ! / o ) ,87 (1980) 12431248. (457) N . 11. Das and H . Shichi, Life S c i . , 2 5 (1979) 1821-1828. (458) M .D. Houslay and R. J. Marchinont,./. PAcirni. Phcirniacol., 32 (1980) 65-66. (459) U . Brocibeck, R. Gentinetta, and S . J . Liindin, Acta Chern. Scciritl., 27 (1973) 561 572.

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the final confoimation of glycoproteins during biosynthesis was cliscussed by Gibson a i d

3. Anti-Recognition Effect of Sialic Acids The anti-recognition effect is one of' the most fascinating functions of Neil, and it has given research on sialic acids an enonnous stimulus. Such an effect was clearly recognized, and established on a molecular basis, initially by Ashwell and Morell,"l when they discovered sialic acids masking the D-galactosyl residues of various seruin-glycoproteins and thus protecting the survival of these molecules in the blood stream. After enzyiiiic removal of sialic acids, Gal residues are e x posed on these glycoproteins, arid the products are then rapidly recognized b y D-Gal-specific receptors on the surface of mammalian hepatocytes; this is followed by fast clearance of the desialylated molecules from the circulation, and decomposition in the parenchyilia1 cell, reviewed in Refs. 461 and 462. This "lectin" has been isolated, and characteri~ed.~".~"j It is a I,rlycoprotein that also contains sialic acids a s essential residues. The specific involvement of this lectin in the uptake of desialylated glycoproteins has been demonstrated b y raising an antibody against this receptor, isolated from rat liver, which markedly lessened the uptake of, for example, asialo-orosomucoitl in the perfused liver.464Studies have shown that the receptor occurs not only on the surface of rat hepatocytes (6.7 x lo4 receptor molecules per cell, corresponding to 5% of the total cellular a i n o u n P ) but also, inainly (95%)intracellularly, on the external or cytosolic surfiice of lysosoines and the inner or liiniinal surface of membranes from the Golgi complex and the encloplasmic r e t i c u l ~ i n . These ~ ~ ~ ~ experiments ~"~ showed the stability and recycling of the receptor, with an average residency-time on the cell surface of - 3 inin under conditions where the ligand is continually transported to the lysosomes and Evidence has been obtained that an additional site for elimination

(460) H. Gibson, S. Kornfeld, and S . Sclrlesinger, TZBS, 5 (1980) 290-293. (461) G. Ashwell and A. G. Morell, A d o . E t i z ! / m o l . , 41 (1974) 99-128. (462) E. F. Neufeld and G . Ashwell, in W. J. Leiinarz (Ed.),The Biocherni.stq ofCl!ycoproteins u d Proteoglycaris, Plenuin, New York, 1980, pp. 241-266. (463) P. H. Weige1,J. B i o l . C h e m . , 255 (1980) 6111-6120. (464) R. J . Stockert, U. Giirtner, A. G. Morrll, and A. W. Wolkoff,]. R i o l . Chc.nr., 255 (1980) 3830-3831. (465) C. J. Steer and G. Ashwell,]. B i o l . Client., 255 (1980) 3008-3013. (466) C. Ashwell, in Ref. 80, pp. 2-3. (467) T. Tanabe, W. E. Pricer, and G. Ashwell,./. B i o l . C h e m . , 254 (1979) 1038-1043.

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of desialylated glycoproteins b y way of the D-Gal-specific pathway is the bone marrow of rabbits.46X Optimal uptake and degradation by this tissue requires the presence of hiantennary glycans, as was studied with human asialotransferrin, whereas, in liver, glycoproteins having tri- and tetra-antennary glycans are more readily bound. These studies demonstrating a protective effect of sialic acid residues on serum glycoproteins provide an explanation for earlier, conflicting observations about the 1)iological effect of, for example, desialylated erythropoietin, which stimulates erythropoiesis only after direct application to bone-marrow cell-cultures, and not after injection into the blood stream.469In the latter experiment, only the native, sialylated hormone was active. Rapid clearance and inactivation of follicle-stimulating or interferon,4" after treatment with sialidase may be explained by uptake into liver cells. An increased rate of metabolic clearance has been observed after removal of sialic acid from human, low-density lipoprotein in u i ~ j o . ~ ~ ~ Sialic acid controls the receptor-mediated uptake of this lipoprotein b y fibroblasts. Removal of sialic acid residues accelerates the rate of internalization of the lipoprotein and, subsequently, the regulation of the metabolism of cellular Sialic acid seems to be involved not only in regulation of the lifetime of soluble, serum glycoproteins but also of mammalian bloodcells. It was observed by Wooclruff and G e ~ n e Pthat ~ ~desialylated lymphocytes are reversibly trapped in liver; they recirculate to the blood stream after about 24 h. This phenomenon was confirmed with Listeria-specific, mouse T lymphocytes, which accumulated in the liver for one day, in contrast to the control cells.H"Reappearance of these cells in the circulation after one day may be explained by resialylation of their membrane gl ycoconjugates. This time period is in the range observed for the turnover of sialic acid in cell membranes, lasting, for example, for 33 h i n rat-liver h e p a t o c y t e ~ . ~ ~ ~

(468) E . Regoeczi, P. A. Chindemi, M . W. C. Hatton, and L. H. Berry, Arch. Biochein. Rioph!/.s.,205 (1980) 76-84. (469) P. P. Dukes, Biochern. Bioph!/s. H c s . Cnmmun., 31 (1968) 345-354. (470) E. F i n d , Eitdokrinologie, 72 (1978) 365-366. (471) V. Bocci, A. Pacini, G. P. Pessina, V. Bargigli, and M . Russi,]. Gem Virol., 35 (1977) 525-534. (472) C. L. Malmendier, C. Delcroix, and M . Fontaine, Atherosclerosis, 37 (1980) 277284. (473) I. Filipovic and E. Buddecke, Eur. J. Biochern., 101 (1979) 119-122. (474) J. J. Woodruffand B. M . Gesner,]. E x p . Metf., 129 (1969) 551-567. (475) W. Kreisel, B. A. Volk, R. Buchsel, and W. Reutter, Proc. Natl. Acud. Sci. USA, 77 (1980) 1828-1831.

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Desialylation o f t l ~ r o ~ n l ~ o c y t and e s ~ erythrocytes ~"~~~ also leads to their tl i sappearance from ci rcii 1at io n . Treatment of e iyth rocy te s from different manin~al s , i n cl ud in g i n an, with s ial i dase re s ul t s in rapid clearance of these cells fi-om the blood stream within a few hours, a phenomenon first observed b y Peronu and and later investigated in various laboratories; it was reviewed in Ref. 479. In man, the life-time of red blood-cells decreases from the normal 120 days to - 2 h after sialidase Complete desialylation of erythrocytes is not necessary for eiythrocyte sequestration; liberation of 15-5" of membrane sialic acids was found to be sufficient for a sigiiificaiit cliniinution of the life-time of erythrocyte^.^^^.^^^ For an in vitro system with rat-peritoneal niacrophages (see later), this threshold value was found to be only 10% when using soluble V. clzolertic sialidiise, litit it was 30% when the erythrocytes were treated with the same enzyme immobilized on Sepharose.":' In contrast to serum gl y co pro te i n s , de sial y 1ate d erythrocyte s are trapped, and pliagocytosed, b y liver Kiipffer cells and spleen macrophages; this was investigated b y direct measurement of the radioactivity incorporated into these tissues, o r hy sciiitigraphy of rabbits after re-injec t ion of 51 C r-I abe 11ecl , tl e s i d y 1ate d e ryt 11roc y te s .4x4 Bin di n g of sialidase-treated erytlirocytes to hepatocytes was observed only in citro by mixing the eiythrocytes with isolated hepatocytes.4X5 Zit v i w , however, such interaction seeins not to occur, owing to the inaccessiliility, for erythrocytes, of the hepatocytes, which are lined b y epithelial cells.4x4Accordingly, the 1)inding of desialylated erythrocytes by phagocytes, obseivetl during i t , o and i n citro experiments, is con-

-

(476) S. C h i , 1. \'. Siiiioiie, a n d L. J . J o u r n e k , Br. ]. Ilucnwtol.. 22 (1972) 93-101. (477) J. P. Greriil)erg, \I. A. Packhain, 1 1 . A. Giiccione, 51. L. R a n d , H.-J. Reinrera, a n d J. F. \Iiistai-d, B l o o d , ij3 (1979) 916-926. (478) G. Perona, S. Cortesi, P. Xotlo, C . Scantlellari, G . Ghiotto, and G . l>e Sandrr, A c f n I s Y J4~(1964) ., 287-285. (479) R. Schaiirr, in T. Schewc~ancl S. Riipopoit (Eds.),.\lo/cc.tr/ur D i . y e c l u ~ sPrrganron, , O x f o r d , 1979, pp. 31-40. (480) J. M . Jancik, R. Schauei-, m t l H.-J. Streicher, Z. Ph!/.sio/.( ; / I C J J I . , 3.56 (1975) 13291331. (481) C . L. Baltluiiii, (;. Riceviiti, hl. C. Sosso, E. Ascari, A . Brovelli, m d C. Halduini, AC~H Oc ~ c ~ l J l ~ l t o57 l . , (1977) 178- 187. (482) L. Gattcgno. 11.Blatlier, a i i t l P. <:ornillot, Z. Ph!/.viol. C h ~ i i t .356 , (1975)391-397. (483)M. Franco, F. J . Nordt, A . P. Corfield, a i i d R . Schaiicr, clctci R i o l . M e t / . C a r . , 40 (1981) 409-412. (484) J . hl. Jancik, R. Schauer,K. 13. Aiitlres, and \1. von Iluring, Ccll ?'i.r.sucRes., 186 (1978) 20<J-226. (485) H. Kolb, (7. Schrltlt, V. I
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223

sidered to correspond to the cellular site of natural eliniination of red blood-cells in the intact organisiii. The mechanisms of elimination of sialidase-treated erythrocytes aiid glycoproteins, respectively, show similarities, inasmuch as lectins recognizing P-D-galactosyl residues appear to exist on the surface of macrophages and hepatocytes. ‘This assumption is tiased on the finding that D-Gal, or compoiinds containing D - G residues ~ in a terminal position, such as lactose, desial ylatetl orosomucoitl, desialylated glycophorin, or such synthetic glycoproteins as galactosyl-albumiii or galactosyl-lysozyme, inhibit adhesion of desialylated erythrocytes to liver cells or to peritoneal ~ n a c r o p l i a g e s . ~A~detailed ” ~ ~ ~ investigation of the binding of‘desialylatetl erythrocytes both to rat hepatocytes and Kupffer cells, and the inhibition of this phenomenon, revealed similar binding-characteristics of the lectin on the two cell types.487 Knowledge of the molecular paranieters of the phagocyte lectin is not yet available. Although hinding of sialidase-treatetl, rat erythrocytes to honiologons Kupffer cells or peritoneal iiracrophages occurs in buffer only, no significant phagocytosis of the cxrvthrocytes occurs under these concliti on^.^^^ However, after the addition of homologous serum, the adherent erythrocytes are rapidly engulfed by the inacrophages. This could lie monitored under the microscope, or b y lysis of the adherent erythrocytes in hypotonic h f f e r followed b y lysis of the remaining macrophages in sodium hydroxicic and counting of the j’Cr radioactivity ofthe two lysates. Whereas the radioactivity of the hypotonic lysate represents the quantity of adherent erythrocytes, the alkaline lysate indicates the amount of the erythrocytes i n g e ~ t e d . ~ ~ ~ ~ ~ ~ ~ From these results, it may he suggested that phagocytosis of partially desialylated, rat erythrocytes in the in zjitro system occurs in two steps: first, adhesion of the erythrocytes to macrophages, mediated b y new 1y exposed P-D-galactos y 1 rc, s i diie s , and second, e n gul fine n t in oderated by serum components. The nature of such serum compounds is largely unknown; the involvcincnt of immiinoglobulins aiid complement is a s s u ~ i i e d .The ~ ~requirement ~ ~ ~ ~ ~ of ~ complement ~ ~ ~ , ~ ~ap~ pears likely, as heat inactivation ofthe serum leads to an appreciable lowering of the rate of phagocytosis of sialidase-treated, rat erythrocytes that can tie restored b?. t h r . addition of guinea-pig comple(486) J. Schlepper-Schiifer, V. Koll,-H;ic.liofc.ti, and H . Koll,, Rioche7n. J , 186 (1980) 827-831. (487) H. Kolb, D. Vogt, L. Herbeitz, A . ( ; o r f i e l d , R. Schauri-, ant1 J. Schlepper-Sc.li~iter, Z . P h y s i o l . C h w i . , 361 (1980) 1747- 1750. / . h e m . , 361 (1980) 291. (488) J . hl. Kiister and R. Schaner, Z. P h ! / , ~ i o C

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r n e i ~ tIt. ~was ~ ~found that this two-step mechanism requiring the macrophage lectin for erythrocyte phagocytosis also operates under in v i w conditions: injection of lactose or desialylated fetuin into rabbits prevents sequestration of clesialylated erythrocytes at concentrations required for inhibition of eiythrophagocytosis in uitro."(' In rabbits, erythrocytes are protected froin sequestration not only by siirface sialyl hut also b y terminal a-D-galactosyl residues.142In contrast to treatment with sialidase, the action ofa-D-galactosidase o n the erythrocytes does not result in phagocytosis by macrophages, but in rapid, intravascular hemolysis. Treatment of rabbit eiythrocytes with a-D-galactosidase leads to uncovering of P-D-galactosyl residues of glycolipids, but in a molar concentration almost 10 tiines that demasked by ~ialidase.'~' It is at present unclear whether the different clearing-iiiechanisins of rabbit erythrocytes observed after treatment with sialidase and a - ~ galactosidase, respectively, are mediated by two different antibodies, one causing sequestration b y macrophages after depositioii of C3b complexes on the erythrocyte surface as the second step in the aforementioned mechanism, and the other leading to intravascular destriiction of erythrocytes b y activation of the whole complement cascade. The other possibility would be that there is involved oidy one type of antibody, which binds both to sialidase- and a-D-galactosidase-demasked, b-D-galactosyl residues, although to different extents, depending on the different quantities of b-D-galactosyl residues demasked by the two glycosidases. For a-D-galactosidase-treated, rabbit erythrocytes, a high density of antibodies on the erythrocyte surface may cause complete activation of the complement cascade up to the factor C9, re sultin g i 11 in t ravasc u lar hem ol y si s . With sial idase-t reatecl erythrocytes, a low density of ailtibody may result in a lower, and slower, activation of the complement system up to C3b, leading to phagocytosis. In this connection, it is interesting that an inverse relationship between the content of sialic acid arid the capacity of n ~ o u s and e ~ ~sheep4g' ~ erythrocytes to activate the human, alternativecomplement pathway has been observed. These, and other, mechanisms possibly involved in erythrophagocytosis will have to be elucidated in the future. Also should be investigated the matter of whether antigenic sites (proteins or lipids), other than the desialylated glycoproteins and glycolipids having penulti(489) M . Franco, E . Miiller, and R. Schauer, unpiiblished results. (490) E. Miiller, M . Franco, and H. Schauer, Z . Plqsiol. Chern., 362 (1981) 1615-1620. (491) U. E. Nydegger, D. T. Fearon, and K. F. Austen, Proc. N a t l . Ac(zd. S c i . USA, 75 (1978) 6078-6082. (492) D. T. Fearon, Proc. Nnfl. Acud. S c i . USA, 75 (1978) 1971-1975.

mate P-D-galactosyl residues, eiirc.rgc. froni the erythrocyte inembrane after the treatment with sialiclase or cu-D-galactosidase, and may thus contribute to the destruction of'erythrocytes. Another "antigen" possibly responsible for the sequestration of sialidase-treated erythrocytes is the sialidase protein itself; V. c*liolerae sialidase molecules were shown to remain attached to c x ~ l ls i ~ r f i c e s . ~ In~ additioii, ~j antibodies against V. cholerae sialidase ha\.e been found i n a relatively large 11 umbe r of hiiiii an se ra.4y4 '1'1i c' s(' co ul d bin t l to s i d i das e-t rea t e d erythrocytes, and lead to coinp 1e iI 1e 1it act ivat io 11, fo 11owed by 1111 agocytosis of the erythrocytes. The following experiments i t r ( ~ at variance with such a clearance iiiechanism. Inculxition of' raI)hit erythrocytes with V. cholcrne sialidase in the presence of inhihitiiig concentrations of Neu2enSAc p r o tects surface sialic acids and, corresl,oiidingly, survival of the cells in circi 11at ion. Treatment of e ry t 1 I r()cyte s by V. cl io 1e rcie s i a1i tlase i m mobilized on Sepharose 4B, allowing complete separation of the enzyme froin the cells after inculxitioii, resulted i n engulfinent of these cells h y macrophages at a ratc. similar to that olisewed with cells treated with the soluble enzynic~:'"'~ Knowledge concerning structiiral features of sialic acids necessary for their anti-recognition effect in erythrocytes is still scanty. The sialic acid side-chain seems not to l)e involved in this function, 1)ecause shorten i ng by pe ri odate -bo ro 11 y d r i tl e treat me 11t doe s not s igii i ficaiit 1y influence the vialiility of the ~~r!dirocytes.'~' It is considered that the carlioxyl group of Neu plays tlic. main role in the protective effect of sialic acid residues. The question as to whether old erythrocytes iire eliminated b y iliacrophages b y a niechanism similar to that for si~ilidase-treated cells has not yet been satisfactorily aiiswered.~48 As was shown for ra1ibiP6 and r a P 3 erythrocytes consicleretl to be old on the basis oftheir higher density, these cells have a shorter life-time in the circulation, o r are more rapidly bound and phagocytosed b y peritoneal inacrophages, compared with the lighter, y o u n g cells. Most researchers i n this field have assumed that the mechallisin o f sequestration of old erythrocytes differs froin that of sialidase-treatt.cl cells, despite the o1)sei-vat I' o n (made in various laboratories) that 1O-20% of inembrane sialic acids (493) G. Liiben, H. H. Sedlacek, a i i t l 1'. I<. Sc,iler, Bchriitg l i n t . A f i t t . , 59 (1976) 30-.37. (494) R. Johannsen, H. H. Setllacek, I{. Sclimicltl)erger, H. J . Schick, : t i i d F. 11. Seiler, J . RTnt/. cancc,i- Z P l S t . , 62 (1979) 7:3:3-732. (495) C. Tannert, (2. Schmidt, D. KIatt, atid S. hl. Rapoliort, A c t n B i d . ,Zlc,t/. C k r - . . 36 (1977) 831-836. (496) C:. L. Balduini, F. Sinigaglia, E . Aac.utt, and C. Baltlrlini, Z. P h ! / , ~ i o lC. / I ~ I359 ., (1978) 1573-1577.

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have been lost per cell, or ineinbraiie weight-unit, during aging.4s7-49s The proportion of other membrane-carbohydrates also decreases in the course of the erythrocyte life-ti,me.”O From this and other ~ b s e r v a t i o n s , ’ ~it’ .was ~ ~ ~concluded that an old erythrocyte that has undergone multiple surface-changes is not necessarily identical with a glycosidase-treated cell, and that the chemical signal(s) leading to its sequestration may be different. One signal seeins to be ankyrin, a transinembrane protein, which emerges at the surface of senescent, human and mouse eiythrocytes and leads to phagocytosis of these cells after binding of an IgG a~ito-antibody.~~’ It remains to be eliicidated whether treatment with a glycosidase also results in deniasking of this, or similar, erythrocyte antigens. The observation made by Kolb and coworkers that sialidase-treated lymphocytes bind to hepatocytes and Kupffer cells, both i i L vitro and in v i m , is of great pathophysiological iniportance.502,so0” Inhibition of this interaction by moleciiles having terminal b-D-galactosyl groups suggests mediation of this phenomenon by the D-galactose-specific lectin on liver cells. The autoimmune, cytotoxic activity of the lymphocytes increases as a consequence of this hinding.jo3 Further evidence for the masking of antigenic sites on cell membranes b y sialyl residues conies from reproductive systems. A glycoprotein layer, rich in sialic acids, on the surface of trophoblast which constitute the boundary zone between maternal and fetal tissue, is considered to form an iinmunobarrier between the two organisms, thus preventing the formation of antibodies by the mother against the child. This assumption is based on the observation that antibodies against mouse trophoblast-cells can be formed in the maternal mouse only after enzymic reinoval ofthe acylneuraininic acid from the siirface of these cells. As a result of this treatment, transplanted tissue from young animals is no longer tolerated by the maternal organism and is rejected,50s or the frequency of abortions is increased in (497) A. Baxter and J. G. Beeley, Biocherti. S O C . T r u n . ~ .3, (1975) 134-136. (498) G. V. F. Seaman, R. J. Knox, F. J. Nortlt, and D. H. Regan, Blood, 50 (1977) 10011011. (499) H. U. Lutz and J. Fehr,J. B i o l . C h e m . , 254 (1979) 11,177-11,180, (500) L. Gattegno, D. Bladier, M. Gamier, and P. Cornillot, Curboh&. R e s . , 52 (1976) 197-208. (501) M. M. B. Kay, Actu B i o l . Med. Ger., 40 (1981) 385-391. (502) H. Kolb, A. Kriese, V. Kolb-Bachofen, and H.-A. Kolb, Cell. Zrtrrnunol., 40 (1978) 457-462. (503) V. Kolb-Bachofen and H. Kolli, J . Z n ~ n ~ u t i o123 ~ . , (1979) 2830-2834. (504) L. K. Kelley, B. F. King, L. W. Johnson, and C . H. Smith, E x p . Cell Re.s., 123 (1979) 167-176. (505) G. A. Currie, W. van Iloorninck, and K. U. Bagshawe, Nature, 219 (1968) 191192.

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subsequent pregnancies.jo6Although other investigators failed to confirm these results, Taylor and coworkersso7have shown, in a careful investigation, that transplantation immunity can be increased by sialidase treatment of mouse trophoblast cells, presumably b y disruption of cell-surface glycoconjugates masking the transplantation antigens. Protection of the fetus from maternal, immunological attack was also discussed b y Rajan and coworkersSoHon the basis of an increase of bound sialic acids in the serum of pregnant women. There is evidence for a similar masking-effect of the glycoprotein sialic acids in kidney glomerular-niein\)ranes, and the decrease in the sialic acid of the glomerular membranes that is observed in some renal diseases is presumed to be related to immunological injuries to the g l o m e r ~ l i .It~ has ~ ~ generally * ~ ~ ~ l ~ e e nol)served that cells and tissues rich in sialoglycoconjugates tend to be the cause of autoimmune cliseases.419 Evidence is accumulating that sialic acid residues mask tumor antigeiis.229~41s,419.511514 Accordingly, several chemically induced, malignant tumors of animals can be hrought to regression, or even to disappearance, after re-injection of sialidase-treated tumor-cells. Similar observations have been made with spontaneous breast-tumors of the dog.s12A method of “chess-boarcl vaccination” with sialidase-treated tumor-cells has been devised, and its results, the theoretical background, the probable involvement of sialidase molecules, and the use of an optimal number of cells have all been extensively discussed. Theoretically, this method of cancer treatment appears promising, as evidence is accumulating, from studies in several laboratories, that, in a variety of tumors, the amount of membrane sialic acids is higher than in the cells of the corresponding, normal t i s s ~ eThis ~ . (506) A. Nista, M. L. Sezzi, and L. Bcllelli, O?icology, 28 (1973)402-410. (507) P. V. Taylor, K. W. Hancock, aiitl G. Gowland, Tramplantation, 28 (1979) 256257. (508) R. Rajan, U. C. Hegde, S. S. Rae, H. N . Purandare, and M . C. Purandare, Ztidicin J . Med. Res., 70 (1979) 733-740. (509) J . Chiu and K. N. Drummonrl, A m . J . Pnthol., 68 (1972) 391-406. (510) E. B. Blau and J . E . Haas, Lab. Itioest., 28 (1973) 477-481. (511) P. K. Ray,Ado. A p p l . Microbid., 21 (1977) 227-267. (512) H. H. Sedlacek, M. Weise, A. Lemmer, and F. R. Seiler, Caticer Zttttnu~io/.Z n i munother.,6 (1979) 47-58. (513) R. L. Simmons, G. V. Aranha, A. Gunnarsson, T. B. Grage, and C. F. McKhann, in W. D. Terry and D. Windhorst (Eds.),Zrtttnunotherapy of Cancer: Present Status of Trials in Man, Raven, New York, 1978, pp. 123-133. (514) H . H. Sedlacek and F. R. Seiler, C:awer Inimunol. Imnaunother., 5 (1978) 153163. (515) E. E. Lengle,J. Natl. Cancer Z t i , ~ t . ,62 (1979) 1565-1567. (516) W. P. van Beek, L. A. Smets, P. Emnielot, K. J. Roozendaal, and H. Behrendt, Leukemia Res., 2 (1978) 163-171.

~

~

228

ROLAND SCHAUER

fits well with the observations of higher sialyltransferase activities in the blood serum from patients suffering from some kinds of cancer (see also, Section V,4). Thus, some tumor cells, or their tumor-specific antigens, seem to be efficiently masked by sialic acids, enabling the transformed cells to escape from immunological attack. It can at present only be speculated that this oversialylation, and a probable decrease of cell-surface D-Gal or GalNAc residues in tumor cells, may be one of the reasons for the lack of contact inhibition observed with these cells, fortifying the invasive growth of cancer tissue. One of the observations pointing in this direction is the lessening ofthe invasiveness of tumor cells in mouse-heart explants after sialidase treatment, which lasted for a few days (until replacement of the sialyl residues had ~ c c u r r e d ) . ~ " The preceding knowledge in this aspect of oncology is increasingly being applied in the diagnosis and therapy of human cancer. For example, the presence o f Thomsen- Friedenreich (T) antigen in the serum of patients having breast cancer may be used for diagnosis and prognosis.51sAlthough some favorable results were obtained with, for example, human gastric51gor co1on"O neoplasm, acute leukemia,"l or melanoma,513it is too early to judge the importance of cancer treatment with the aid of sialidase. These problems have been extensively and critically reviewed by Sedlacek and S e i l e ~ - . ~ ' ~ An important role of cell-surface sialic acids in the regulation of normal cellular-interaction inay be assumed, because lectins are widespread in tissues that recognize P-D-galactosyl residues, and thus may participate in cell a d h e s i o ~ i . ~ It ~is~conceivable -~~~ that variations in the sialylation of cell-surface, D-galactosyl residues modulate the strength of cellular binding. Sialic acids on cell surfaces seem to be involved in the masking of immunoglobulin receptors. Thus, incubation of human, or rat, bloodinonocytes with sialidase exposes cryptic IgM (Fc) and (517) M. M . Yarnell and E. J. Ainhrose, Eur. J. Cancer, 5 (1969) 255-263. (518) G. F. Springer, M. S. Murthy, P. R. llesai, and E. F. Scanlon, Cancer, 45 (1980) 2949-2954. (519) T. Akiyoshi, M . Kawaguchi, S. Miyazaki, and H. Tsuji, Ortcology, 37 (1980) 309313. (520) W. A. F. Tompkins, J. D. Schniale, R. Mock, N. T. Tick, T. Lock, N. Sidell, and J. L. Palnier,J. Natl. Cancer I n s t . , 62 (1979) 503-511. (521) G. J. Bekesi, J. F. Holland, and J. P. Roboz, M e d . Clits. North Am., 5 (1977) 10881100. (522) J. T. Powell, Biochens. J., 187 (1980) 123-129. (523) E. B. Briles, W. Gregory, P. Fletcher, and S. Kornfeld, J . Cell Biol., 81 (1979) 528537. (524) S. Kadowaki and T. Osawa,Jpti.J . E x p . Med., 49 (1979) 397-404. (525) D. G. Haegert, Clin. E x p . Inirnunol., 35 (1979) 484-490.

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229

IgC receptors on cultured, human l y ~ n p h o c y t e sSialidase .~~~ treatment of lymphocytes from sensitized subjects increased the responsiveness to viral, bacterial, and fungal antigens.527The same treatment of human, Herpes simplex virus-infected cells was found to enhance the sensitivity of the cells to lysis mediated by antibody and complemen t .528

4. Sialic Acid as a Component of Receptors In this Section, a variety of specific interactions between sialic acids and biologically active compounds and micro-organisms will be described for selected examples. This field was reviewed in various publ i c a t i o n ~ . ~Agglutination ~ ~ , ~ ~ ~ , of ~ ~native, ~ in contrast to desialylated, erythrocytes by myxoviruses, including influenza viruses, was first observed by Burnet and c 0 w o r k e r s . 1 ~This ~ ~ ~phenomenon ~~ is due to a specific interaction between cell-surface sialyl residues and viral sialidase molecules, and it seems to play an important, but not yet fully understood, role in the mechanism of infection of host cells by the micro-organisms. It is considered that at least one function of the viral sialidase is to prevent aggregation of progeny virus by removing potential virus-receptors from their own g l y c o p r ~ t e i n s Such . ~ ~ ~ aggregates would limit spreading ofthe virus. It has been found that viruses do not bind equally to all sialyl linkages. Polyoma virus adsorbs only to sialyl-oligosaccharide chains having (r-Neu5Ac-(2+3)-Gal linkages, and not to the corresponding (2-6) groups on the erythrocyte surface.531This was shown by complete desialylation of erythrocytes, followed by specific incorporation of sialyl residues with the aid of the p-D-galactoside-(2-+3)-sialyltransferase,leading to binding of the virus, or by selective removal of (2+3)-sialyl groups from the erythrocytes with the aid of NDV sialidase, destroying the binding capacity of the erythrocytes for polyoma virus. Interestingly, erythrocytes that had lost (2+3)-sialyl residues were still agglutinable by influenza viruses.531In similar experiments with Madin-Darby, bovine kidney-cells and Sendai viruses, (2+3)-sialyl residues as components of the oligosaccharide sequence a-Neu5Ac(2-+3)+-Gal-( 1-+3)-GalNAc constitute specific receptors for these (526) K. Itoh and K. Kumagai,J. Inintunol., 124 (1980) 1830-1836. (527) T. Han, Clin. E x p . Irnrnunol., 13 (1973) 165-170. (528) W. A. F. Tompkins, P. Seth, S. Gee, and W. E. Rawls, /. I n t n i u n o l . , 116 (1976) 489-495. (529) C. H. Andrewes, F. B. Bang, and F. M. Bumet, Virologfl, 1 (1955) 176-184. (530) P. W. Choppin and A. Scheid, Rel; Infect. Dis., 2 (1980) 40-61. (531) L. D. Cahan and J. C. Paulson, Virology, 103 (1980) 505-509.

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niyxoviruses.""' Whereas (2+S)-sialyl linkages are inactive, (2-8)sialyl groups bind Sendai virus even iiirich more effectively than (2+3) groups. The latter phenomenon was shown b y Holmgren and coworkersss3by using various, plastic-at1sorl)etI gangliosides. The best receptors were GT,, , Go,,,, a r i d GP,,. , having a-Neu45Ac-(2+8)-a-Neu5Ac(2+3)-p-Gal-( 1+3)-P-GalNAc- structures. Several Mycoplasmu species were also found to bind to eukaryotic, cell-surface membranes through sialidase-sensitive, cell-surface receptors (cited in Ref. 534). In a stucly with several gl ycop rote in s , M co 1, lu ,Y111 u gull ise p ticui n was found to bind most strongly to human glycophorin so long as this molecule was sialylated.":'4 Sialic acids, mainly as components of gangliosides, have been recognized a s being involved in the binding, to cells, of a variety of b o t ~ i l i n u s , ~and " ~ cholera toxins, such as tetaiiiis,416r535 as well as (+)-tul)ociirarine":'~ and colchiceine.:'"" In these studies, some binding specificity between the kind of toxin and the nature of the gaiiglioside has been observed, cholera toxin, for example, exh i b i t i ~ i g "strong ~' 1)iiiding-activity with GM, . The 13-siibunit of cholera toxin binds to the sialoglycoconjugate receptor of the cell menibrane, and this is followed l,y dissociation of the A-subunit from the native, toxin molecule, which penetrates into the meni1,rane and stimulates541 the synthesis of cyclic AMP. This mechanisni provides an explanation for the striking observation that addition of, for example, cholera toxin to cultured, rat-adrenal cells,544' or ovarian cells,s44" leads to stimulation of the synthesis ofcorticosterone or progesterone, mediated h y cyclic AMP. Further examples of this toxin's causing a biological response i n a wide variety of (532) M.A. K. Markwc.11 antl J . C. Paulson, Proc. .Vuf/. Accid. Sci. USA, 77 (1980)56935697. (533) 1 . Holnrgren, L. Sveiinerholm, H. Elwiirg, P. Frctlman, and 6. Strannegird, Proc. N u h . Acnd. Sci. USA, 77 (1980) 1947-l9SO. (534) L. R. Glasgow and R. L. Hill, I n f i c f . I i t i n i u i L . , 30 (1980) 353-361. , (1977) 194-198. (535) T. B. Helting, 0. Zwisler, antl H . Wiegandt,]. B i o l . C h e ~ n . 252 (536) Y u . V. Vertiev and Y u . V. Ezepchuk, B u l l . E x p . Biol. M c d . ( U S S R ) ,ij (1978) 549551. (537) M. Kitamura, M . Iwanrori, and Y. Nagai, Biochirn. Bioph!/s.Actu, 628 (1980) 328335. (538) W. E . van Heyningen, Nnfure, 249 (1975) 415-417. (539) H. Riisner, G. Mcrz, and H. Rahniann, Z . Physiol. C h e m . , 360 (1979) 413-420. (540) H. Hiistier and M. Schiinhartiiig, Z . Ph!/siol.Clzem., 358 (1977) 915-919. (541) P. H. Fishnran and R. 0. Bradv, Sciriiw, 194 (1976) 906-915. (542) A. Haksar, D. V. Mandsley, a n d F. G . Peron, Nuture, 251 (1974) 514-515. (543) S. Azhar, P. Fitzpatrick, and K. M. J . hlenoii, Biochcni. R i o p h ! / s .Res. C ~ ~ ~ I U J I . , 83 (1978) 493-500.

1nammalian cells in culture art' cited in Ref. 541. The occurrence of a relatively high amount of gangliosides in the palatal epithelium ofthe human oral-cavity suggests that these gangliosides might participate in binding and inactivation of 1)acterial toxins produced b y invading micro-organisms .544 Evidence exists for a similar interaction of glycoprotein hormones with membrane glycoconjugates. 'The honnone most satisfactorily studied in this respect is the thyroid-stimulating hormone (TSH), and it was found that its binding to the target cell-membranes is inhibited most effective1y"I by G D I h .This hoiinone, as well as related glycoprotein hormones, is composed of a- and 6-suhunits, the a-subunit being common to these horniones, and the p-subunit conferring target-organ specificity. Similarities to the mechanism of action of cholera toxin were delineated froin the observation that the p-subiinit binds to a membrane receptor (of the target tissue) probably coiisisting both of glycoprotein and g a 1 i g l i o s i d e , 4 ~ and ~~~ this ~ ~ interaction, studied with TSH and thyroid ineinbranes, can be inhibited b y cholera toxin.541As ii consequence of alterations i n the nieiiibraiie fluidity, the a-subunit activates the intracellular, adenylate-cyclase system. This stimulation, together with effects on the translation antl transcription level, is the molecular h s i s of the honnone effect. The specificity of hormones acting on individual target-tissues is considered to be due to the glycoconjugate composition of the respective hornionerecept~r.~~~."~ The receptor for insulin in liver has also been shown to be a glycoconjugate, although involvenivnt of sialic acid in the function of this receptor has not yet been uiieqiiivocally elucidated.s45~s44" D - G seeins ~ to be an essential constituent of' this receptor.547Evidence was obtained for the binding of intertcron to cell membranes by way of gang1iosides. 541.548

As so many physiological c.oiiiponnds regulating cell growth and iiietabolism appear to interact with gangliosides as components ofcellular receptors, it has been suggested that the loss of growth control in certain transformed cells may pnitially lie based on the altered gang1i o side coni po s it ion ob s erne d i I 1 I 1 e opl ast ic ce 11s.54 ' Further examples of the iiivolvenient of sialic acids in the function of receptors are ( a )guinea-pig niacrophages, which hind the migration(544) U. Lekholm and L. Svennerlrolnr, Atu.h. Oral Biol., 24 (1979) 47-51. (545) M. Caron, J . Picard, antl P. Kei-11,BiodLirtt. B i o p h / s . A c t u , 512 (1978) 29-40. (546) 11.Tsutla, S. l a k e t o m i , ant1 M. Iw;tt\rlka, A m . J . P h y s i o l . , 239 (1980)E1X6-191. (547) J . Picard, 11.Caron, arid J . ( ; a p - a i ~ ,i l l Rrf. 80, pp. 469-470. 1,548) F. Hesanqon ;md H . Ankel, N t r t r r r - ( 2 , L52 (1974) 478-480.

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inhibition factor ( M I F ) with the aid of sialylated glyc~lipids~";( h ) huinan lymphocytes in culture, the IgM receptor of which was alinost completely inactivated by sialidase, in contrast to the IgG receptor, the expression of which was increased b y this eiizyiiie treatineiit526; and (c) terminal Neu5Ac on the surface of human eiythrocytes, which recognizes a inonoclonal, cold agglutini~i."~ Evidence is accumulating that surfice glycoproteiiis and sialic acid residues of lymphocytes and macrophages are critically involved in the activation of 1ym p hoc y te s by i n itoge i i ic s t i i n uli , including anti geii s , plant 1ectins, and oxidation b y periodate or D-galactose oxidase.55'-55:3 The way in which these inodifications, for exainple, the aldehyde groups from the oxidative treatments, influence the production of growth and other stimulating factor^,^^".^^" is not, however, yet understood.

VIII. CONCLUDING REMARKS Although, in several biological systems in which sialic acids have been shown to be directly involved in biological phenomena, more experiments are needed in order to establish such a role unequivocally, the participation of these compounds in the regulation of niauy physiological processes is undoubted, arid this is why sialic acids are considered to be impol-tant components for the protection of life. There is only one exception to this general trend, and sialic acids seem to have been "misused" with regard to this function in the preservation of noniial life, namely, in cancer tissue, where they seem to contribute to a better chance of survival of inalignnnt cells, based on their anti-recognition effect. The part of the sialic acid molecule that is principally responsible for this biological fiinctioii is still uiiknowii; the supposed, main coii(549) D. Y. Liu, K. D. Petscliek, H. G . Reirioltl, and _I. R. Uavic1,J. Irnniurto/, 124 (1980) 2042-2047. (550) 11. Roelcke, W. Pruzanski, W. Ebert, W. Riimer, E. Fischcr, V. Lenharcl, and E. Rauterberg, Blood, 55 (1980) 677-681. (551) J . H. L. O'Brien, J. W. Parker, J . A. Frelitiger, J. F. P. Dixon, hl. L. Phillips, and 1. L. Gordon, in J. G. Kaplan (Ed.), The Moleculur Basis of Iriirnurte Cell Furictiort, Elsevier, Amsterrlarn, 1979, pp. 87-98. (552) J. L,. Pauly, M . J. Cerinain, and T. Han,J. Med. (Wesfbur!/,N Y ) , 9 (1978)223-236. (553) G. Durand, hl. C;ueuorinou, J. F(.gcr, and J . Agneray, Bioclieni. B i o p h y s . Res. Co~iirnutt., 83 (1978) 114-123. (554) .4. Novogrodsky, 14. Suthaiithiran, B. Saltz, D. Newinan, A. L. Rubiit, and K. H. Stenzel,]. E x ) , . Med., 151 (1980) 755-760. (555) D. K. Greinetler, H. E. Hockliii, and J. R. David,]. 1rnmurtol., 123 (1979) 28042807.

tribution of the c.arboxy1 group is only itn assuinption antl lras not yet heen studied. The sialic acid sick-c,haiii is probal,l\, of little infliicnc.e, a s can be delineated froin the fk\v oxperiinents wherein thc citle chain was shortened b y periodate-l~oroIi~.tlritle treatiiir,nt without causing drainatic 1)iological-effects. (Tho strong effects of periodatcl oxidation alone on lymphocytes, alreatlh. citcd. s w i n to 1)e due not to shorteniiig of the sialic acid side-chains hiit to the 1i)rniation of aldch!~tle groups.) Little information is availa1,lc its to thca role of modifications of‘sialic acid resulting in a species- a i i t l tissue-specific distribiition of different N,O-acylated and O-metliyl;ttcd sialic acids (see Section 11). With the exception of the 4-0)-acetyl groiip’s rc,ntlering t h e respective Neii dcrivatives resistant to the actioii of sialitlases antl the sialic. acid 1! asc, the role of the other O-acet>.l groups is still not k i i o m 7 n . It nia!~ be speciilated that they influence t l i c s iiiiinunological bchavior of glycocoiijiigate s . The oh s e i-vation t l I ii t t h c’ ;t n t i gen i ci t\ of the 0 -ace t y 1ated coloininic acid fi-oni E . coli K 1 is iiicreased, coniparetl with the corrosponding non-O--acetylatetl l)olI\.saccharitle, points iii this direction.4i Although similar ol )sc~rvationswere miicle with the O-itcetylated, type 1 pneumococcal capsular polysaccharide, the O-acetylnegative, group C meningococc.al polysuccharidt. variant is niorc’ ininiiinogenic than the O-ac.c.t?l-positive derivative. Thc. authors4i assiunetl from these stlitlies that tlic O-acetyl group is not itself a i l a i l t ige n , I n t mod i fie s the i nim ti n ( ) I ( )gi cal react i \.i !t- ( )f the 1,act e rial po 1y saccharides. It is unknown wlictlieI the wide occiirrence of 0-acetyl groups on sialic acids i n inairiiiialian tissues plays a similar role. It is possible that 0-acetyl groiips sciisitively iiiflueiice the physicochemical propeities of glycoc.oi!jiigat~,s,especially i n such amphiphiWhercas 0-acetyl groups lie cell-coinpartinelits a s cell iii(~inl>rai>es. may increase the hydrophobicity, e.specially of t h e s i d e chain of the rather hydrophilic, sialic acitl rc.sitlues, N-glycolyl groups m a y evoke the contrary. Influence on hyclrog:c~ii-l,oiidiiigand, c.orresl~ontlingly, on the conforination of the oligosac.cliaride chains in iiiacroinolecules, ancl on cell membranes, b y 0-;ind 1\’-acyl groups is fc.asihle, biit iiiust await proof. ‘The antigenic rffcsct of the N-gl~7col>.lgroup i n inan has heen mentioned; its biological sigiiificance is still u n k n o w n . I n view of the appearance ol‘iic~iiraniiiiicacid i n evoliitioii, the cliicstion must be posed as to whicli iiiolecrile pre-existed, and m a y still I)e found, in those primitive aninids unable to s),nthesize sialic itcids where comparable functions iiiiglit I ) e expected, for c a x a l n p l e , i i I cell rneinl>ranes, or i n the nervous system. Are such molecules uronic acids, or neutral sugars, substitutetl l ) y such acids a s phosphoric o r sillfiiric acid? At this point, the prt’cciit e iy may he concli.itlr~1b y point-

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ing to the future, where not only this last question remains to be answered, but also many problems in the physicocheii~ical, I~iochemical, and biological fields of research o n sialic acids.

ACKNOWLEDGMENTS I should like to thank my coworkers at the Universities of Bocliuni and Kiel who have contributed to the progress in the chemistry and biology of' sialic acid over the past 14 years, and also the many colleagues, all over the world, who have given m e advice, and participated in fruitful collaboration. Thanks are also due A. P. Corfield, G. Reuter (University of Kiel), and H. Rahniann (University of Stiittgart- Hohenheirn) for assistance and criticism dnring the preparation of the nranuscript.

ADVANCES I X CARBOHYDRATE (:tiEhllb'I'RY A N D BIOCHEllISTRY, VOL 10

BIOSYNTHESIS AND CATABOLISM OF GLYCOSPHINGOLIPIDS

BY

Yu-TEH

L I AND SU-CHEN

LI

Depurtment of Biochemistr!y, Tidune Unioersit!y School of Medicine, New Orleans, Louisianu 711112, Deltu Regioncil Priniute Reseurch Center, Tulane Unioersity, Cooington, Loui.riuna 70433

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 1. Scope of Article 2. General Structur 11. Biosynthesis of Glycosphingolipids 1. Enzyme Preparation and Enz 2. Biosynthesis of Neutral Glycosphingolipids 3. Biosynthesis of Gangliosides . . . . . . . . . . . . 4. Biosynthesis of Glycosphingolipids in Pathological Cond 5. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Catabolism of Glycosphingolipic 1. Enzyme Preparation and Enz 2. Degradation of Cangliosides by Sialidase . . . . . . . . . . . 3. Catabolism of Glycosphingolipids Containing b-Linked D-GalactOSe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274 4. Catabolism of GM2, Asialo GM2, and Globotetraosylceramide . . . . . . . . .276 5. Catabolism of Glycosphi a-Linked D-Galactose 279 6. Protein Activators for the Enzymic Hydrolysis of Glycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 7 . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,285

I. INTRODUCTION The major landmarks in the field of glycosphingolipids are the discoveries of cerebroside by Thudichum' in 1884, of ganglioside by Klenk2 in 1935, of hematoside by Yaniakawa and Suzuki3 in 1951, and (1) J . L. W. Thudichum, A Treatise o i i the Chemical Constitution of the Bruin, Bailliere, Tindall, and Cox, London, 1884. (2) E. Klenk, Hoppe-Seyler's Z. P h y s i o / . Cheni., 235 (1935) 24-36. (3) T. Yamakawa and S. Suzuki,]. Biochem. (Tokyo), 38 (1951) 199-212.

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Copyright 0 1982 b y Academic Prew, lnc. All rights of reproduction in any lomi reserved ISBN 0- 12-007240-8

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YU-TEH LI AND SU-CHEN LI

of globoside by Klenk and Lauenstein4in 1951 and also by Yamakawa and Suzuki5 in 1952. From the original dozen or so glycosphingolipids,6 which were isolated mainly from neural tissues and erythrocytes, the number of glycosphingolipids that have been isolated and characterized from different tissues and organs has increased dramatically during the past two decades. Interest in the isolation, structural elucidation, biosynthesis, and catabolism of this group of compounds was stimulated by the discovery of various lipid-storage diseases attributable to inborn errors of glycosphingolipid catabolism. Moreover, recognition of the participation of glycosphingolipids in many membrane phenomena now provides additional impetus for investigators in many branches of experimental biology to elucidate the biological roles of these molecules. 1. Scope of Article As in the case of other biological compounds, the development of our knowledge of glycosphingolipids started with an initial discovery, followed by the isolation and characterization of structurally related compounds, their biosynthesis and degradation, and, finally, the delineation of their biological function. Although we have already entered the era of the investigation of the biological roles of glycosphingolipids, our knowledge of the biosynthesis and degradation of these molecules is still far from complete. The present article concentrates mainly on the advances made in the biosynthesis and catabolism of sugar chains in glycosphingolipids during the past two decades. No attempt will be made to provide an exhaustive compilation of the literature pertaining to these two topics that has been published to date. Neither will the biosynthesis and catabolism of ceramides (a generic term for the lipid moiety), nor the sulfation and desulfation of glycosphingolipids, be discussed.

2. General Structural Features of Glycosphingolipids Glycosphingolipids are made up of a hydrophobic ceramide and a hydrophilic, complex-carbohydrate moiety. Ceramides consist of sphingosine [trans-4-sphingenine or (2S,3R,4E)-2-amino-4-octadecene-1,3-diol] or an analog, such as dihydrosphingosine (sphinganine) or phytosphingosine (4-~-hydroxysphinganine)which is N-acylated with a long-chain fatty acid ranging from C,, to Cz6. Glycosphingo(4) E. Klenk and K. Lauenstein, Hoppe-Seyler’s Z. P h y s i d . Cheni., 288 (1951) 220-228. (5) T. Yamakawa and S. Suzuki,J. Biochenz. (Tokyo),39 (1952) 393-402. (6) H. E. Carter, P. Johnson, and E. J. Weber,Annu. Reo. Biochern., 34 (1965) 109-142.

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237

CH,OH

I

HCNH, I HYOH

Sphingosine (a reramide)

lipids are believed to be anchored in the lipid bilayer of the membrane through the hydrophobic, ceramide moiety. The length of the complex carbohydrate chains in glycosphingolipids may range from one sugar residue to more than thirty. The sugar chain is attached to the 1-hydroxyl group of the ceramide. All of the sugar units found in glycosphingolipids are of the D configuration, with the exception of Lfucose. The diverse nature of the saccharide moiety in glycosphingolipids may reflect their specific functional roles. Glycosphingolipids may be divided into acidic glycosphingolipids, which contain sialic acid or sulfhe, and neutral glycosphingolipids, which contain only uncharged, carbohydrate moieties. Sialic acidcontaining glycosphingolipids (sialosylglycosphingolipids)are named gangliosides, whereas glycosphingolipids carrying a sulfuric ester group are called sulfoglycosphingolipids (formerly, they were known as sulfatides, but this terminology is not recommended by the Lipid Document7).In general, acidic glycosphingolipids are more abundant in neural tissues than in visceral organs. It is extremely difficult to maintain uniform nomenclature or abbreviations for these molecules due to the diverse nature of the saccharide moiety and the continual discovery of new glycosphingolipids. A semi-systematic nomenclature for glycosphingolipids has been recommended b y the IUPAC-IUB Commission on Biochemical N ~ m e n c l a t u r e and , ~ has been reviewed in some detail.*-*OReaders are urged to consult these articles in order to become familiar with the no(7) IUPAC-IUB Commission on Biochemical Nomenclature, Lipids, 12 (1977) 455468. (8) R. M. Burton, in L. A. Witting (Ed.),Clycolipid Methodolog!l, American Oil Cheinists’ Society, Champaign, Illinois, 1976, pp. 1-11. (9) C. C. Sweeley and B. Siddiqui, in M. I. Horowitz and W. Piginan (Eds.), The Cl!ycoconjugates, Vol. I, Academic Press, New York, 1977, pp. 459-540. (10) H. Wiegandt, in L. Svennerholm, P. Mandel, H. Dreyfus, and P.-F. Urban (Eds.), Structure und Function of Canglio,sides, Plenum Press, New York, 1980, pp. 3-10.

YU-TEH LI A N D S U - C H E N LI

238

Ceramide

Galpl-

1'Cer

Glcol--1'Cer I

,

4

I

t

Glucosylceramide family

Galactosylceramide family

SCHEME 1. The Attachment of Different Monosaccharides to t h e C e r a m i d e ( C e r ) Moiety.

menclature in this field. The carbohydrate moieties of the glycosphingolipids provide the basis for the classification and the structure (and often the metabolic) interrelationship of these complex lipids (see Schemes 1-3). The four monoglycosylceramides shown in Scheme 1 have been isolated: galactosylcerarnide from human brain by Thudichum'; glucosylceramide froin the spleen of a patient with Gaucher's disease"; fucosylcerainide from huinan colon tulnorsl2; a i d xylosylceramide from the salt gland of the herring gull.'" The attachment of

Gal (31- 1'Cer

NeuAccuZ -3

Gal 01-1

HS03--3

'Cer

Galljl--1'Cer

Galactosylceramide-13-sulfate (ISS03-GalCer)

N-Acetylneuraminosylgalactosylc eramide (I?NeuAc-GalCer)

-

-

Gal a1 4 Gal 131 1'C e r Galactobiosylceramide (GaOse,Cer) SCHEME 2. Glycosphingolipids D e r i v e d from Galactosylceramide N. Halliday, H . J. D e u e l , Jr., L. J. T r a g e r m a n , a n d W. E. Ward,]. B i d . Chem., 132 (1940) 171- 180. K. Watanabe, T. Matsubara, a n d S.-I. Hakoniori,]. Biol. C h e m . , 251 (1976) 23852387. K.-A. Karlsson, B. E. Samuelsson, a n d G. 0. Steen, ]. Lipid Res., 13 (1972) 169176.

Glc p l jl’Cer,

~

NeuAcaZ -6Glc Ol-I’Cer

Man 131-4

N-Acetylneuraminosylglucosylceramide (PNeuAc-GlcCer)

Glc (31-1‘Cer

Mannosylglucosylceramide

t

NeuAca2-3Gal~1-4Glc

(31-1’Cer

II3- N-Acetylneuraminosyl-

GalNAc $1-4Galp1-4Glcpl

-

Gal p1-4Glc

Mannose-containing glycosphingolipids

01-

1‘Cer

-1‘Cer

Gangliotriaosylcerarnide (GgOse,Cer)

-

HSO, -3Gal!31-4Glc

pl-

1‘Cer

Lactosylceramide- 113-sulfate (n3-sq-L a c c e r )

Galal-

3Galpl-4Glc

0 1 -1

Globotriaosylceramide

I I

(Ganglio s e r i e s )

Galo1-4Galol-

4Glc (31--1’Cer

t

(Isoglobo s e r i e s )

GlcNAc pl-3Galp1-4Glc

01-l’Cer

Mucotriaosylceramide (McOse&er)

Lactotriaosylceramide (LacOsqCer)

I I

I I

I

v .

(Muco s e r i e s )

’Cer

Isoglobotriaosylceramide

i

(Lacto and neolacto s e r i e s )

SCHEME3. Glycosphingolipids Derived from Glucosylceramide.

240

YU-TEH LI AND SU-CHEN LI

additional sugar units to Galpl .+ 1'Cer and Glcpl -+ 1'Cer affords glycosphingolipids of the galactosylceramide family (see Scheme 2) and the glucosylceramide family (see Scheme 3 ) . Table I lists some of the coniinon neutral glycosphingolipids isolated from vertebrates. In the glucosylceramide family (see Scheme 3 ) , lactosylcerainide is further glycosylated into ii multiplicity of glycosphiiigolipids, including the ganglio, lacto, neolacto, niiico, globo, and isoglobo series (see also, Table I). In addition to these simple, neutral glycosphingolipids, numerous L-fucose-containing, neutral glycosphingolipids have been isolated from various sources.9J0J4In general, lactotetraosylcerarnide and neolactotetraosylceraniide constitute the coininon, core structure of fucoglycosphingolipids. Some gaiiglio~ides'~-'~ have also been found to contain fucose. I n contrast to the glycosphiiigolipids of vertebrates, several mannose-containing glycosphingolipids have been isolated from fresh-water Some of the mannose-containing glycosphingolipids also contain xylose."," Also noteworthy was the finding of glycosphingolipids ofthe lacto series containing long sugar The backbone of these glycolipids consist^^^,^^ of N-acetyllactosamine repeating-units at-

(14) B. A. Macher and C. C. Sweeley, Methods Etzzyrriol., SO (1978) 236-251. (15) S. Ando and R. K. Yu, in J . D. Gregory and R. W. Jeanloz (Eds.),Glycoconjugnte Research, Vol. 1,Proc. Zut. S y r n p . Glycocorijugnte.s,4th, Academic Press, New York, 1979, pp. 79-82. (16) S. Sonnino, R. Ghidoni, G. Galli, and C. Tettaiiianti,]. Neurocliem., 31 (1978)947956. (17) K. Watanabe, M . Powell, and S.-I.HakomoriJ. B i d . Cheni., 253 (1978)8962-8967. (18) M. Sugita, S. Shirai, 0. Itasak,i, and T. Hori,]. Biochem. (Tok!yo),77 (1975) 125130. (19) 0. Itasaka, M. Sugita, H. Yoshizaki, and T . Hori,J. Biochem. (Tokyo), 80 (1976) 935-936. (20) T. Hori, M. Sugita, J . Kanabayashi, and 0. Itasaka,]. Biocheni. (Tok!yo),81 (1977) 107-114. (21) T. Hori, H. Takeda, M .Sugita, and 0. Itasaka,]. Bioclzem. (Tokyo), 82 (1977)12811285. (22) 0. Itasaka and T. Hori,]. Bioclzem. (Tokyo), 85 (1979) 1469-1481. (23) A. Cardas, Enr. ]. Biocher~i.,68 (1976) 177-183. (24) M. Dejter-Juszynski, N . Harpas, H. hi. Flowers, and N . Sharon, E u r . J . Hioclzenz., 83 (1978) 363-378. (25) J . Koscielak, E. Ztlebska, and H. Miller-Podraza, in H. Schauel-,P. Boer, E. Buddecke, M. F. Kramer, and J. F. C . Vliegenthart (Eds.),Proc. Z t i t . Sym?).C~l!~c.ocotijugates, 5th, Ceorg Thieme, Stuttgart, 1979, pp. 49-50. (26) H. Nakagawa, T. Yarnada, J.-L. Chien, A. Cardas, M. Kitamikado, S.-C. Li, and Y.T. Li,]. B i d . Chem., 255 (1980)5955-5959.

TABLEI Structure, Nomenclature, and Abbreviations of Some Selected, Neutral Glycosphingolipids Abbreviation Structure

Name ~

~~

Gala 1+4Galpl+4Clcp l+l’Cer GalNAcp1+3Gala 1+4Calpl+4Clcpl+ 1’Cer

GalNAcal+3GalNAc~1+3Gala1+4Galpl+4Glcpl-t 1‘Cer Gala 1+3Galp 1+4Glcpl+l‘Cer GalNAcp1+3Gala 1+3Galp 1+4Glcp 1- 1‘Cer Galpl+4Galpl+4Glcp 1+l’Cer Gal~1+3Gal~1+4Galp1~4Glcp1+1‘Cer GlcNAcp1+3Galpl+4Glcp 1+1 ‘Cer Galp 1+3GlcNAcp 1+3Galp 1+4Glcp 1+ 1’Cer Gal@l+4GlcNAc~l+3Cal~l+4Glc~l+l‘Cer GalNAcp1+4Galp 1+4Glcp 1- 1‘Cer Galp1+3GalNAcp1+4Calp1+4Glcp l-t 1’Cer Gala l 4 C a l p 1- 1’Cer

Symbol

Short symbol

GbOse,Cer GbOse,Cer GbOse,Cer iCbOse,Cer iCbOse,Cer McOse,Cer McOse,Cer LcOse,Cer LcOse,Cer nLcOse,Cer GgOse,Cer GgOse,Cer GaOse,Cer

Gb,Cer Gb,Cer Gb,Cer iGbsCer iCb,Cer Mc,Cer Mc,Cer LcsCer Lc,Cer nLc,Cer GgCer Gg,Cer Ca&er

~~

Globotriaosylceramide Globotetraosylceramide Globopentaosylceramide Isoglobotriaosylceramide Isoglobotetraosylceramide Mucotriaosylceramide Mucotetraosylceramide Lactotriaosylceramide Lactotetraosylceramide Neolactotetraosylceramide Gangliotriaosylceramide Gangliotetraosylceramide Galabiosylceramide

YU-TEH LI AND SU-CHEN LI

242

TABLE

11

Structure, Nomenclature, and Abbreviations of Some

Structure

NeuAca2+3GalpI-+ 1’Cer NeuAca2+3Calpl+4Glcp 1 41’Cer NeuAca2-+8NeuAca2-+3Galp 1+4Glcpl+I’Cer GalNAcP 1+4Gal(3+2aNenAc)p 1+4Glcp 1 4 1’Cer

GalNAc~l+4Gal(3+2aNeuAc8+2aNeuAc)pl+4Glc~l+

1’Cer

Galpl-+3GalNAcp 1+4Gal(3+2aNeuAc)p 1+4Glcp I+ 1’Cer NeuAca2-+3Galp 1-+3GalNAcp 1-+4Ga1(34a NeuAc)p l+4Clcp 1+ 1’Cer Galp 1+3GaINAcp1+4Ga1(3+2aNeuAc8~2aNeuAc)p 1-+4Glcp 1-+1’Cer

tached to lactosylceramide, +3Galp1+

4GlcNAcp+3Galpl+

4Glcpl

-+

1‘Cer.

In polyglycosylceramides, there can be as many as twenty N-acetyllactosamine repeating-units. The internal P-D-galactosyl linkages in these glycolipids can be hydrolyzed b y the endo-p-D-galactosidase isolated froin Escherichia freunclii.26It is of interest that the nature of the sugar chains in these types of glycosphingolipids is analogous to that of the N-acetyllactosamiiie repeating-units found in keratan sulfate. Most of the gangliosides can be divided into ganglio and lacto types, with the exception of the gangliosides NeuAca2 + 6Glcpl + 1’Cer and NeuAca2 -+ 3Galpl -+ 1’Cer. Table I1 lists the striictures and names of some common gangliosides. The isolation of the following gangliosides is noteworthy: IV”NeuAcGgOse,Cer, NeuAca2 + 3GalP1 + 3GalNAcP1 + 4Galp1 + 4Glcpl + 1’Cer (GMlb) from rat

BIOSYNTHESIS AND CATABOLISM OF GLYCOSPHINGOLIPIDS

243

Selected, Acidic Glycosphingolipids Abbreviations SvennerName

Lipid document

13-a-N-Acetylneuraminosylgalactosylceratnide 13aNeuAc-GalCer L13-a-N-Acetylneuraminosyl-lactosylceratnide I13aNeuAc-LacCer I13-a-N-Acetylneuraminosyl-a2+8-NII%( NeuAc),-LacCer acetylneuraminosyl-lactosylceraniide I13aNeuAc-GgOse,Cer I13-a-N-Acetylneuraminosylgangliotriglycosylceramide IISa(NeuAc),-GgOse,Cer I13-a-N-Acetylneuraminosyl-a2+8-Nacetylneuraminosyl-gangliotriglycos y Iceratnide 1I"aNeuAc-GgOse,Cer I13-a-N-Acetylneuraminosylgangliotetraglycos ylceramide 1I"aNeuAcIV"aNeuAcI13,1VS-a,a-Di-N-acetylneuraminosy1gangliotetragl ycos ylceramide GgOse,Cer II"a(NeuAc),-GgOse,Cer I13-a-N-Acetvlneurariiiiiosvl-a2~8-~acetylneiiraminosyl-gangliotetraglycosvlceraniide IV3-a-N-GlycolylneuraminosylIV3aNeuGc-nLcOse,Cer neolactotetraglycosylceramide V13-a-N-AcetylneuratninosylVI%xNeuAc-nLcOse,Cer neolactohexaglycosylceramide

holm GM4 GM3 GD3 GM2 GD2 GM 1

GDla

CDlb

-

ascites hepatoma cellsz7 and human erythrocyteszx; NeuAccu + Galp + GalNAcp + Gala -+ Gal@+ Glc + Cer, a ganglioside containing a galactosylglobotetraosyl sequence, froin chicken musclez9; and a ganglioside, containing arabinose and an internal sialic acid in the sugar chain, isolated from a starfish, Asterinci pectiniferu.30A comprehensive listing of various glycosphingolipids may be found else~ h e r e . ~ J "Previous J~ abbreviations proposed by Svennerhol~n"~ for brain gangliosides have been adopted to designate the names of such ganglioside-storage diseases as GM1-gangliosidosis and GM2-gangliosidosis. For the sake of simplicity, Svennerholm's nomenclature (27) Y. Hirabayashi, T. Taki, and M . hlatsumoto, F E H S Lett., 100 (1979) 253-257. (28) K. Watanabe, M. E. Powell, and S.-I. Hakomori,]. B i o l . CIzem., 254 (1979) 82238229. (29) J.-L. Chien and E. L. Hogan, Fed. Pro(.., 39 (1980) 2183. (30) M. Sugita,]. Biochem. (Tokyo), 86 (1979) 765-772. (31) L. Svennerholm,J. Neurochem., 10 (1963) 613-623.

244

YU-TEH LI A N D S U - C H E N LI

will be used in this article, along with the new nomenclature for gangliosicles having gaiiglio-type sugar chains.

11. BIOSYNTHESIS O F GLYCOSPHINGOLIPIDS Glycosphingolipids in animal cells have been shown to reflect tissue and species ~ p e c i f i c i t y . : $Careful ~ * ~ ~ consideration must be given to the age, organ, and species of the animal under study, a s these are factors that inay greatly affect the rate of synthesis, a s well a s the rate of degradation of glycosphingolipids. Radioactive tracers have been used to study the biosynthesis of glycosphingolipids i n uivo.34However, due to the inherent difficulties involved with i n oiuo studies,34 most of the work concerning the biosyiithesis of glycosphingolipids has been carried out in oitro. In 1958, Burton and coworkers:’J showed that the microsomal fraction of a young rat brain contained an activity which transferred a galactosyl group from a glycosyl donor, UDP-Gal, to an endogenous, glycolipid acceptor; this probably constitutes one ofthe earliest descriptions of the i n oitro biosynthesis of glycosphingolipids. In general, the liiosynthesis of glycosphingolipids i t i oitro is detected by observing the transfer ofa radioactive sugar unit from a “sugar nucleotide” donor to a glycolipid acceptor, with the formation of new, radioactive glycolipid( s). The enzyme preparation that catalyzes this transfer is .usually particulate in nature. It has been postulated that the elongation of sugar chains iii glycosphiiigo1ii)ids occiirs through the stepwise addition of monosaccharide units to the nonreducing end of the growing oligosaccharide chain. Hoseinan:”’proposed that the sugar chains in gangliosides are synthesized by a “niultiglycosyltransferase complex,” a s all of the enzymes are found in the same particulate fraction, and each glycosyltransferase is specific not only for a “sugar nucleotide” but also for the acceptor niolecule. Based on the substrate specificities and location of each glycosyltransferase, the different glycosyltrarisferase complexes would he responsible for the synthesis of different gangliosides. The sugar chains i n glycoproteins were originally assumed to be synthesized by the same mechanisin.”v3’ However, it is now firmly established that a lipid-linked, oli(32) T. Yamakawa, in E. Schiitte (Ed.), Lipoide, C:o//ocl. Ce.5. Ph!/sio[. Cheni., 16th, Springer, Berlin, 1966, pp. 87- 111. (33) H. Wiegandt, Adc. Lipid Hes., 9 (1971) 249-289. (34) R. M. Buitoii, Lipid,y, 5 (1970) 475-484. (35) R. hf. Burton, hl. A. Sodd, and R. Brady,]. Bio/,Chem.,233 (1958) 1053-1060. (36) S. Roseman, Chen7. Ph!/.r. Lipids, ,5 (1970) 270-297. (37) R. G. Spiro, A d c . Protc:in C/icw1.,27 (1973) 349-467.

BIOSYNTHESIS A N D CATABOLISM OF GLYCOSPHINGOLIPIDS

245

gosaccharide intermediate is involved in the biosynthesis of sugar chains in glycoproteins that contain the asparagine-2-acetamido-2deoxy-D-glucose linkage.38,39Behrens and coworkers40 investigated the possibility that dolichol (D-glucosyl monophosphate) might serve as an intermediate for the D-glucosylation of ceramide by brain extracts of young rats, but obtained negative results. However, in view of the discovery of complex glycosphingolipids having long sugar chains (namely, polyglycosylceramides), it may be premature to conclude that the biosynthesis of sugar chains in glycosphingolipids does not involve any other mechanisms. Study of the biosynthesis of glycosphingolipids in uitro also has many difficulties and limitations. I n order to facilitate the transfer of a glycosyl group from a water-soluble “sugar nucleotide” to a lipid-soluble acceptor by a particulate-enzyme preparation, a detergent is often needed in order to disperse both the glycolipid acceptor and the membrane-bound enzyme. So far, it is still not possible to solubilize and purify the membrane-associated glycosyltransferases. Furthermore, the in uitro, biosynthetic product is predetermined by the nature of the added acceptor. Therefore, it is not unusual that an enzyme preparation obtained from a certain tissue may synthesize a glycosphingolipid product that is not nomially present in that tissue. For example, even though GMlb is not normally found in rat brain, nevertheless, rat-brain homogenates transfer41an N-acetylneuraniinosyl group from CMP-NeuAc to 0-3 of the terminal D-gdlaCtOSyl residue in gangliotetraglycosylceramide (asialo GM l), to form IVNeuAc-GgOse,Cer (GMlb) instead of I13NeuAc-GgOse,Cer (GMla). The results of in vitro study may indicate the presence, or absence, of a particular glycosyltransferase activity in the tissue examined. However, extrapolation of results obtained from in vitro studies to in uivo reactions must be essayed with extreme caution.

1. Enzyme Preparation and Enzyme Assay The incubation mixture for the biosynthesis of glycosphingolipids usually includes an enzyme preparation, a glycosyl donor, a glycolipid acceptor, a detergent, and metal ions. Most of the glycosyltransferases are membrane bound and are usually associated with the microsomal (38) C. J. Waechter and W. J . Lennarz,Atitiu. Reo. Biochem., 45 (1976) 95-112. (39) D . K. Struck and W. J . Lennarz, in W. J . Lennarz (Ed.), The Biochemistry of Glycoproteins und Proteoglycuns, Plenum Press, New York, 1980, p. 35. (40) N . H . Behrens, A. J. Parodi, L. F. Leloir, and C. R . Krisinan, Arch. Biochem. B i o p h y s . , 143 (1971) 375-383. (41) P. (J.) Stoffyn and A. Stoffyn, Corbohydr. Res., 78 (1980) 327-340.

246

YU-TEH L,I ilND SU-CHEN LI

fraction or a subfraction of mitochondria. A Golgi-rich f r a c t i o i ~has ~~ been shown to be abundant in glycosyltransferases. Whole tissue hornogeiiates have also been used a s the enzyme sources. I n most cases, the enzyme preparation and the glycolipid acceptor are not prepared froin the saiiie tissue. The same glycolipid acceptor isolated from different sources m a y contain different fatty acid compositions, which niay affect acceptor specificity. l:i UDP glycosyl esters serve a s the donors of D-glucosyl, D-galactosyl, 2-acetamid0-2-deoxy-~-gliicosyl,and 2-acetamido-2-deoxy-D-galactosyl groups. CMP-NeuAc a i d GDP-Fuc are the donors of N-acevlneuraminic acid aiid L-fucosyl groups, respectively. There are two ways in which to employ the glycosyl donors. ( u ) The most widely used method is the transfer, h y ;I particulate-enzynie preparation, of a radioactively labeled sugar unit from a “sugar nucleotide” to a nonradioactive, glycolipid acceptor. With this method, the newly synthesized glycosphingolipid contains a radioactive glycosyl group at the (nonreducing) end of the sugar chain. ( / I ) The alternative inethod, developed b y the Stoffyns;“ uses a glycosphiiigolipid acceptor labeled at the (nonreducing)teriniiial glycosyl group and an unla1)eled ‘‘sugar nucleotide” donor; in this way, the penidtimate sugar unit of the newly synthesized glycosphiiigolipid is labeled. This method fiicilitates the determination, b y permethylation analysis, of the linkage between the added sugar unit and the radioactive glycosyl group of the acceptor. I n addition, this method can distinguish between the product derived from the exogenous acceptor arid those derived from the endogenous acceptors. The principle of the method is shown i n Scheme 4.The limitation ofthis method is that the glycolipicl acceptor labeled at the nonreducing terminal is not always available. This method has been extensively applied with glycolipid acceptors having a D-galactosyl group at the nonreducing terminal; this group can be labeled h y treatment with D-galactose oxidase, followed h y reduction with sodium borotrititle. The detergents that have commonly been used in assay systems i r i uifro are sodium taurocholate, Tween 20, Cutscum, aiid Triton X-100. Such metal ions :IS Mg2+ or Mn“ are required by many glycosyltransferases. The reaction is usually conducted for 1-2 h at 37”. When a donor nucleotide containing a radioactive glycosyl group is used, the newly synthesized, radioactive glycosphingolipid can he separated from the radioactive sugar donor, and quantified. Because of the high radioactivity of the glycosyl groiip in the donor, it is essential that the (42) T. W. Keenan, U. J. hlorr6. and S . Basu,.\. B i o l . C h e i n . , 249 (1974) 310-315 (43) S . F. Kemp and A. C. Stoolmiller,j. Neurocheni., 27 (1976) 723-732. (44)P. (J.) Stoffyn and A. Stoffyn, Curliohydr. He.s., 74 (1979) 279-286.

BIOSYNTHESIS AND CATABOLISM O F GLYCOSPHINGOLIPIDS

247

SHCHOH

XDP-Glyc

+

“Q

,ql\~cosyl-

OH

1

+

t )-nn.yfmwse

2

XDP

................... 3

where Glyc is any monosaccharidic glycosyl group

SCHEME4. Glycosyltransferase Reaction Using a Non-radioactive “Sugar Nucleotide” Donor (1) and a Radioactive Glycolipid Acceptor (2). [The biosynthetic product (3) is labeled in its penultimate glycosyl unit. It is, therefore, possible to determine the position of attachment of the new sugar residue to the radioactive glycosyl unit of the glycolipid acceptor by permethylation analysi~.~‘]

newly synthesized glycolipid be completely free from the donor. This can be achieved by: (i) Folch partition45to recover the glycolipid in the lower layer while leaving the radioactive, “sugar nucleotide” in the upper layer, or (ii) separation of the two by paper chromatograpliy:6 high-voltage e l e c t r o p h ~ r e s i s or , ~ ~column c h r ~ m a t o g r a p h y . ~ ~ Needless to say, it is of the utmost importance to establish the structure of the newly synthesized glycosphingolipids. Unfortunately, many of the newly synthesized glycosphingolipids have been identified solely on the basis of their mobilities in thin-layer chromatography (t.1.c.); however, t.1.c. mobility alone is not sufficient to establish the structure of a glycosphingolipid with any certainty.

2. Biosynthesis of Neutral Glycosphingolipids a. Biosynthesis of Galactosylceramide and Glucosylceramide .-In 1960, Cleland and E. P. Kennedy49reported the synthesis of psychosine (galactosylsphingosine) from UDP-Gal plus sphingosine by microsomes isolated from the brains of guinea pigs and young rats. Since then, several reports have shown the synthesis of galactosylceramide and glucosylceramide through galactosylsphingosine and glucosylsphingosine, followed by a c y l a t i o ~ i j ~with - ~ ~ acyl-CoA. Subsequent (45) J. Folch, M . Lees, and G. H. Sloane-Stanley,J. B i d . Chem., 226 (1957) 497-509. (46) J.-L. Chien, T. Williams, and S. Basu,J. B i d . Chem., 248 (1973) 1778-1785. (47) M. Basu and S. Basu,J. B i d . Cheni., 247 (1972) 1489-1495. (48) M. A. Wells and J. Dittmer, Biochemistry, 2 (1963) 1259- 1267. (49) W. W. Cleland and E. P. Kennedy,]. B i d . Chem., 235 (1960) 45-51. (SO) J. N. Kanfer, Lipids, 4 (1969) 163-170. (51) J. Hildebrand, P. (J.) Stoffyn, and G. Hauser,]. Ncurochem., 17 (1970) 403-411. (52) S. Hainmarstrom, Biochem. Bioph!ls. Res. Cornmun., 45 (1971) 459-486. (53) J. A. Curtino and R. Caputto, Lipich, 7 (1972) 525-527.

248

YU-TEH LI AND SU-CHEN L1

studies b y inany lalioi-atories5'+sxrevealed that ;.eramide is a better acceptor for galactose and glucose than sphingosine, and that psycliosine can be non-enzymically acylated59b y acyl-CoA. It is now widely considered that in citro glycosylation of ceramide is the major syiithetic pathway for the synthesis of galactosylceran~ideand glucosylceraniide. The D-galactosylatiorr of ceraiiiide b y if P-D-galactosyltransferase prepared from rat and chicken embryonic showed a marked preference for the cerarnide containing 2-hydroxy fatty acids over tlie one with non-hydroxy fhtty acids, although both types ofcerarnicle are found in the I)rain in siinilar quantities. On the other hand, P-D-glucosyltransfer~isedid not show such p r e f e r e i ~ c e . ~ ~ Tlie - " ~galactosyltransferase that transferred galactose to the non-hydroxy fhty acids ceraniide was shown to be inore iiustable than the enzynie that transferred galactose to the hydroxy fatty acids cerainide.6zConstantino-Ceccarini and Morel16"found that the synthesis of galactosylceramide and glucosylceraniide in mouse kidney was influenced not only by the age of the anirrial but also b y its sex.

b. Biosynthesis of Lactosylceramide and Digalactosy1ceramide.Lactosylceraniide is the coininon, biosynthetic precursor for the glycosphingolipids of the globo, lacto, and ganglio series. The lactosylceramide-synthesizing enzyme, namely, UDPGa1:glucosylcerainide P-D-gal~ictoSyltraiiSferase,has been detected in a wide variety of tissues, including rat l>rain,sleiiibryonic-chicken l)rain,5i spleen,64-66 and k i ~ l i i e y . Tlie " ~ activity ofthis enzyme in rat brain was found to be highest at bii-th, decreasing gradually In contrast to the wide distribution of the lactosylceraiiiicle-synthesiziiig enzyme, tlie digalactosylceramide-syiitl~esiziiigenzyme has been detected only in (54) Y. Flijino and M . Nakmo, Bioc/ic.rrr. I . , 113 (1969) 573-575. (55) P. Morel1 and N . S. Hiidin, Biochertri.Tfr!/,8 (1969) 506-512. (56) P. Morell, E. Costantino-Cecc;irini, and N . S. Kadin,Ar.ch. Bioclaenr. B i o p h y . ~ . ,141 (1970) 738-748. (57) S. Basu, B. Kaufinan, and S. Hoseinail,,/. H i o l . Chevr., 243 (1968) 5802-5804, (58) S. Basu, B. Kaufinan, and S . Rosenrm,,/. H i d . Chent., 248 (1973) 1388- 1394. (59) S. Hammarstriim, F E B S Lett., 21 (1972) 259-263. (60) A. Breiikert and N. S. Radin, Bruiti He.,., 36 (1972) 183-193. (61) S. Hasu, A. 51. Schultz, M. Basu, and S. Hoseinan,J. B i d . C h n . , 246 (1971) 42724279. (62) P. Morel1 and P. B r a u n J . Lipid Hea., 13 (1972) 293-310. (63) E. Constantino-Ceccarini ant1 P. Morel1,J. B i d . Chetir., 248 (1973) 8240-8246. (64) C. Hauser, Hiochem. H i o p l i y s . Res. Courmiin., 28 (1967) 502-509. (65) E . Xldrtensson, K. Ohiiim, hl. Graves, and 1,. Svennerholm, ,/. H i o l . Chem., 249 (1974) 4132-4137. (66) J. Hildel)rand and G . Haiiser,J. B i d . Cheni., 244 (1969) 5170-5180.

BIOSYNTHESIS AND CATANOLISll OF CLYCOSPHINGOLIPIDS

249

kidney.65*fi7-69 This finding is corroborated b y the fact that Gala1 + 4Galpl -+ 1'Cer was detected o n l y in kidney and siiiall intestine."' c. Biosynthesis of Glycosphingolipids of the Globo and Isoglobo Series.-The two galactosyl residues in glol>otriaosylcerariii~leare linked a1 -+ 4,whereas, in isoglobotriaosylceramide, this linkage is a1 + 3 . Globotriaosylceramide" and glol~otetraosylceramicle~,~,i~ have been isolated from a number of sources. Isoglobotriaosylceramide has been isolated from rat spleen," and isoglobotetraosylceraii~ide from rat l y m p h o s a r ~ o m aand ~ ~ rat kidney.79 Stoffyn and coworkers reported that rat-kidney microsomesx" synthesized both globotriaosylceramide and isoglobotriaosylceramide, whereas rat spleen and bone-marrowM' synthesized only isoglobotriaosylceramide. UDPGal : lactosylceramide a-galactosyltraiisferaseactivity has been detected in rat rat kidney,'j5 hainster BHK and NIL cells,x' and hornogenates of nomial adrenal cloned Y-1-K culture.X"In only two rep o r t ~ ~ "was * ~ 'the linkage detennined between the two galactosyl residues in the newly synthesized trimsylceramide. I n the other reports, this linkage was not detennined. The enzyme for the biosynthesis of globotetraosylcerarnide, UDPGa1NAc:globotriaosylceramide p-N-acetylgalactosaniinyltraiisfer~ise, L. Cole and G. hl. Gray, Bioclzerir. Biop/ii/.u. Hea. Cor)irtiun., 38 (1970) 520-526. G. M. Gray, B i o c l ~ i m Biophys. . Actci, J. R . Hay and G. ?(I. Gray, B i i d i e m . Bioph!/.u. Res. Coinr~iciti.,38 (1970) 527-532. C. Suzuki, A. Makita, and Z. Yosizawa, Arch. Riockein. B i o p h y s . , 127 (1968) 140149. C. C. Sweeley and B. Klionsky,]. B i d . C\ietti., 238 (1963) pc3148-pc3150. A. Makita and T . Yarnakawa,]. t3ioc/ieiii. (Tokyo), 55 (1964) 365-370. J. Kawanami,]. Biochenz. (Tok!/o),62 (1967) 105- 117. E. P. Adanis and G. M. Gray, Cheiii. Ph!/.c..Lipids, 2 (1968) 147- 155. T. Miyatake,Jpti. ]. E x p . Meil., :39 (1969) 35-45. S.-I. Hakomori, B. Siddiqui, Y.-T. I i , S.-C. Li, and C. G. Hellerqvist, ]. Biol. Clzern., 246 (1971) 2271-2277. P. (J.) Stoffyn, A. Stoffyn, and G . Hauser, Biochini. Biop/iys. Actci, 306 (1973)283286. R. Laiiie, C . C. Sweeley, Y.-T. Li, atid M . 51. Rapport,]. Lipid Rcu., 13 (1972)519524. B. Siddiqui, J . Kawanami, Y.-T. Li, and S.-I. Hakomori, J . Lipid Res., 13 (1972) 657-662. A. Stoffyn, P. (J.) Stoffyn, and C;. I - I a i i w i - , Hiochini. B i o ) h ! / s .Actcr, :360 (1974) 174178. P. (J.) Stoffyn, ,4.Stoffyn, and G . Haiisei-,]. B i d . Cheiii., 248 (1973) 1920-1923. S . Kijimoto and S.-I. Hakomori, Biricheni. HicipIzy.~.Res. C o n z t n i ~ t ~44 . , (1971)557563. K.-K. Yenng, J. R . Moskal, J.-L. Chieii, D. A. Gardner, and S. Basu, Biochc,ni. Bioph!/s. Res. Conitnuti., 59 (1974)252-260.

250

YU-TEH LI AND SU-CHEN LI

has been detected in embryonic c h i c k e n - t ~ a i nguinea-pig ,~~ kidney,x4 and mouse adrenal Y-1 tumor cells.x“The biosynthesis of Forssman hapten, globopentaosylceramide, b y UDP-Ga1NAc:globotetraosylceramide a-N-acetylgalactosaminyltransferasehas been studied in mouse-adrenal tumor-cellsx3and guinea-pig k i d n e ~ . * ~This J ’ ~ enzyme was solubilized froin dog-spleen microsomes by a combination of Triton X-100 treatment and sonicationH6;the solubilized enzyme was partially purified by use of calcium phosphate gel, ammonium sulfate fractionation, and chromatography on a column of DEAE-cellulose. Kijimoto-Ochiai and coworkersH7used cultured hamster NIL-2K cells to study the biosynthesis of Forssman hapten (globopentaosylceramide) from its precursor (two steps removed), namely, globotriaosylceramide. They found that the globotetraosylceramide formed by fl-Nacetylgalactosaminyltransferase in the enzyme complex served as the substrate for the a-N-acetylgalactosaminyltraiisferase. However, exogenously added globotetraosylceramide did not serve a s the substrate. These observations led them to propose that the intermediate product remained bound, and served as a bound, transient product-substrate for the subsequent reaction. Scheme 5 summarizes the various glycosyltransfer reactions related to the biosynthesis of glycosphingolipids having a globo-series sugar chain. d. Biosynthesis of Glycosphingolipids of Lacto and Neolacto Series.-Tetraglycosylceramide of the neolacto series constitutes the common, core structure of a number of blood-group-active glycosphingolipids. Our knowledge of the biosynthesis of the lacto and neolacto series is still quite fragmentary. The transfer of 2-acetamido2-deoxy-D-glucose from UDP-GlcNAc to lactosylceramide to form a triglycosylceramide was reported by Basu and coworkers,HX who used an enzyme preparation isolated from rat bone-marrow. Chroniatographic mobility was used to characterize the nature of the newly synthesized triglycosylceramide. The B a s i ~ sreported ~~ the galactosylation of lactotriaosylceramide, GlcNAcPl .+ 3Galpl + 4Glcpl -+ l’Cer, b y a galactosyltransferase from rabbit bone-marrow. It should be pointed out that the tetraglycosylceramide synthesized by this (84) T. Ishil)ashi, S. Kijimoto, and A. Makita, Biochim. Biophys. Actci, 337 (1974) 92106. (85) S. Kijiinoto, T. Ishibashi, and A. bfakita, Aiochem. B i ~ ~ i h yRes. . ~ . Conimnr~.,56 (1974) 177-184. (86) T. Ishibashi, I. Ohkubo, a n d A. Makita, Biochim. Hioph!/.y.Actu, 847 (1977)24-34. (87) S.Kijimoto-Ochiai, N. Yokosawa, m d A . Makita,]. R i d . Chem., 255 (1980) 90379040. (88) S. Basu, M. Basti, H . Den, and S . Roseman, Fed. Proc., 29 (1970) 410.

I

I

+,-1

Gal p1-4Glcp

I I

UDP-Ga1:GlcCev 11- ~ a l a c l o s y l t r o n s f r v a s ~ ~

7

~

Galal-.

1 ‘Cer

?Gal p1-4Glcpl

-l’CeIbS-sS

~al-4Galpl-4Glcpl~’Cerm Galal-3Gal

pl--4Gl~p1--1‘Cer~~*~

UDP-Gal: LocCer o-~alqcloseltr-ans.ferase

t‘

#-

Galcrl-4Galgl-4GicBl-l~er

GalNAcpl-

I

?Galal-

4Gal p1-4Glcp1-

1’Cer46*m*B4

I

,I -- 4

]

UDP-GalNAc:GbOse,C e r p-N-acelyl~alactosan~in~~llransferase

I

I GalNAc pl-3Gala1-4Gal~1-4Glcp

1 -1’Cer

I 1

,---I

GalNAcol-

?GalNAcp1-3Galal-4Galpl-

4GlcB1-1

’Cer“ ,67

I

UDP-GalNAc :GbOse4Cev a-N-acelplgnlaclosaminyltvansfe~ase

f 1 I

GalNAcal-

3GalNAc 131 -3Gala

1-

4 G a l e 1-

4Glcpl-

1 ’Cer

SCHEME5. Various Glycosyltransfer Reactions Related to the Biosynthesis of Glycosphingolipids having Globo-series Sugar Chains. (Broken arrows indicate the hypothetical, biosynthetic pathway.)

YU-TEH LI A N D SU-CHEN LI

252

reaction could be a mixture of lactotetraosylcerainide, GalP1 + 3GlcNAcPl -+ 3Galpl + 4Glcpl += l’Cer, and neolactotetraosylceramide, G a l P l + 4GlcNAcpl-+ 3Galp1- 4Glcpl-+ 1’Cer. The BasusHSalso described an a-galactosyltransferase from rabbit bone-marrow that catalyzed the transfer of a D-galactosyl group to both lactotetraosylceraniide and neolactotetraosylceramide. Although the anomeric linkage of the newly added D-galactosyl group was determined to be a by using fig a-D-galactosidase,!’Othe location of the linkage to the penultimate galactose residues in the newly synthesized peiitaglycosylcerairiide was not determined. An a-L-fucosyltransferase activity was detected in a purified membrane preparation from bovine spleen that catalyzed the transfer of Lfucose from GDP-L-[’-’C]F~~ to neolactotetraosylceramide to afford an H-active glycosphingolipid.Y1The L-fucose in the newly synthesized pentaglycos ylcerainide was susceptible to CIzuroiiia lanipas a-L-fucosidase.s2The radioactive product inhibited the hemagglutination reaction of 0-type cells against eel anti H ( 0 ) globulin, and formed a precipitin line with Ulex europeus lectin. The exact linkage between the L-fucose and the terminal D-galactose was not established. In addition to neolactotetraosylceramide, B-active pentaglycosylceramide, namely, Gala1 + 3Galpl

-+

4GlcNAcp1

-+

3Galp1 + 4Glcpl

+

l’Cer,

was also a good substrate for this fu~osyltransferase.~~ Stellner and coworker^^^ found that HI-glycolipid, namely, C a l ( 2 t l a F u c ) p l -+ 4GlcNAcp1 -+ 3Galp1 + 4Glcpl -+ 1’Cer, was converted into “A“”glycolipid, GalNAca1 + 3Gal(2+laFuc)pl -+ 4GlcNAcpl- 3 G a l p 1 - + 4Glcpl + 1’Cer by an a-N-acetylgalactosaminyltransferase of A serum, or by that prepared from gastrointestinal inucosal epithelia. HI-Glycolipid was also converted into B-I glycolipid, Gala1 -+ 3Ga1(2 + 1aFuc)pl +. 4GlcNAcpl + 3Galpl -+ 4Glcpl +. I’Cer, (89) M. Basu and S. Basu,]. Biol. C h e m . , 248 (1973) 1700-1706. (90) Y.-T. Li and S.-C. Li, Methods E n z y n i o l . , 28 (1972) 714-720. (91) S. Basu, M . Basu, and J.-L. Chien,]. B i d . Chem., 250 (1975) 2956-2962. (92) Y. Iijima and F. Egarni,]. Biochem. ( T o k y o ) ,70 (1971) 75-78. (93) K. Stellner, S.-I. Hakoinori, and G. A. Warner, Bioche?n. Biophys. Res. Coinn~un., 55 (1973) 439-445.

1j y an a - ~ - g a l ato c s y 1tran s fe r;is e ()I'

gas t roin te s t inal I 11iico s a1 e p i the 1i a. Interestingly, in carcinoma clerivetl from gastrointestinal epithclia, the activities of these enzymes wcrc found to I x only a fifth ( o r less) of the iioniial activity. Aiiother f~icosyltransferase that transferred fucose to the terminal G l c N A c group of lactotriaosylceratiii~~e, GlcNAcP1 + 3Galpl + 4Glc,!j1 + l'Cer, has been detected b y Basu and coworkersY4in cultures of African green-monkey, kicltiey cells (Vero) and in neuroblastoma IhlR-32 cells.95This glycolipid may be the intermediate for the syntliesis of the glycolipid

GalBl + 4GlcNAc(3+lcuFuc-)Pl

+ 3GalPl

+

4Glc,!j1 + 1'Cer

reported h y Yang and Hakoiiiori.!"' A sialosyltransferase that catalyzed froni CMP-NeuAc a i ~ d the synthesis of sialosylneolactos~lcer~i~iiide neolactosylceraniide has been tletected in em1)r)miic chicken-l)rain and bovine splee11,~'a s well a s i n SV-40 transformed, glial cell-culture derived from the cerebrum of ;I fetus with Tay-Sachs disease.sxAgain, the linkage between sialic acid and galactose in the ncwly synthesized ganglioside was not estahl ishecl. Oljviously, v e r y little is k i i o w i i about the biosynthesis of gaiigliosicles having lacto- and neolactotype sugar chains.

3. Biosynthesis of Gangliosides The biosynthesis of lacto- ; i t id neolacto-type gangliosides was briefly discussed in the previous Section. The present Section deals with the hi o sy 11t 11e s i s of s ial o s y 1 gal act o s y Ice ram i de , s i a1o s >rllactc )s y I ceram ide, di s i a10 s y 1lacto s y lce ran I i de , and gan gl i o s i tle s 11 iiv i n g a g a n g lio-type sugar chain.

a. Biosynthesis of Sialosylgalactosylceramide (NeuAccuZ + 3GalP1 + l'Cer).-Sialosylgalactosylceramide, first isolated and characterized as a minor gangliositl(~of human I~raiii,~" Io2 has also Ijeen (94) h l . Rwsii, J . H. Moskal, D. A. (;ar[lii(ir, ; u i d S. Basu, Hio m u t t , , 66 (1973) 1380-1.388. (9S) K. A . Prcspei-, X I . B ~ S Land I , S. I h \ r i , I ' i o c . . .\'ut/. A u l t l . 293. (96) H.-J. Yang and S.-I. Hakoinori,/. B i o l . C/i<>rii., 246 1,1971) 1192- 1200. (97) S. Basu, M .Basu, and ].-I2. Cliicii, i i r Ref. 10, pp. 213-2:39. (98) hl. Basu, K. A . Presper, S . Basu, L. 11. Hoftllran, ant1 S..:b brook^, P r o c ~.Vnt/. . .\cd. Sci. U.S.A., 76 (1979)4270-427-4. (99) R. Kuhn aiid H . Wieganclt, Z. N ~ i t i i r / o r ~ \ c /Teil i , , H, 19 (1964) 256-257. (100) E. Kleiik a i i t l L. Georgias, Ho/,/,(,-.S~,!//f,r'~ 2 . P h y s i o l . C/wrri., :348 ( 1967) 12611267. (101) B. Siddiqui aiid R. H . hIcClurl-,/. /,ipid H c s . , 9 (1968) 366-370. (102) H. W. Ledeeir, R. K. Yu,and I,. 1 . Eirg. / . Setrr-oc/ic,,,i.,21 (1973) 829-839.

254

YU-TEH LI A N D SU-CHEN LI

found in hen egg-yolkl'yJ;it has heen shown to be specifically localized in myelin and in the oligodendroglia of the central nervous system.Io4 A sialosyltransferase that catalyzed the transfer of sialic acid from CMP-NeuAc to galactosylceramide was detected in the microsoriles of mouse brain and liver.1osThe enzynie also catalyzed the synthesis of sialosyllactosylceramide. In this study, the linkage between sialic acid and galactose was not deterniinetl. The K,,, value for galactosylcerainide leading to sialosylgalactosylceralriide was estimated to be 870 p M , whereas that for lactosylceraiiiide leading to sialosyllactosylceramide was estiniated a s 89 p M . Mixed-substrate experiments showed that the addition of lactosylceraiiiide spared the synthesis of s ial os y 1gal act o s y Ice raiii i de , itii cl that ni o s t of the radioactivity which was incorporated appeared in sialosyllactosylcer~~iiii~le. The synthesis of sialosylgalactosylceraiiii~~e in iiiouse liver is higher than that in the brain, although mouse liver is completely devoid of this ganglioside; this could ineaii that the synthesis of this ganglioside is an i r i uitr-o phenomenon reflecting the lack of specificity for the enzyme which synthesized sialosyllactosylceramide. On the other hand, the Stoffyns""' used different cell-lines to show that the sialosyltransfe ras e act i vi ti e s involved i n the I) io s y n t he s i s of s ial o s y 1galacto s y Ice rainide and sialosyllactosylceramide in astrocytoma cells are different from each other. They further showed that, in both cases, sialic acid was linked to 0 - 3 of galactose.

b. Biosynthesis of Sialosyllactosylceramide (IPNeuAc-LacCer, GM3) and Disialosyllactosylceramide (I13NeuAcz-LacCer,GD3).Sialosyllactosylceraiilide (GM3) is one of the major gangliosicles found in visceral organs. However, in thecentral nervous system, the concentration of this ganglioside is so low that it can only be considered to be the precursor for various coniplex gangliosides. A sialosyltransferase that catalyzes the transfer of sialic acid from CMP-NeuAc to lactosylceraniide was first descriljed in the embryonic chickenbrain b y B a ~ u ' ~a r' i~d Kaufiiian and coworkers,1oX arid in the brain of

(103) S.-C. Li, J.-L. Chien, C . C. Wan, and Y.-T. Li, Hioclieni. I., 173 (1978) 697-699. (104) R. K. YU and K. I q l d , ] . Nenroclic.tri., 32 (1979) 29.3-300. (105) R. K. Y u and S. H. Lee,]. B i o l . C h i n . , 251 (1976) 198-203. (106) P. (J.)Stoffyn arid A. Stoffyn, i n A . Varinavuori (Ed.),Z i L t . Cotigr.Pure A p p l . Chetn., 27th, Pergainon Press, Oxford and New York, 1980, pp. 225-231. (107) S. Basu, Ph.D. Thesis, University of k4ichigai1, 1966. , S. Roseinan, in S. hl. Aronson and B. N. Volk (Eds.), (108) B. Kaufnian, S. B ~ I S Uand Iriborri Disorders of S p l i i r ~ g o l i p i dMetciboli,sn~,Proc. I r l t . Syrnp. Cerebrul S p h i n golij)idosc?s,3 r d , Pergarnori Press, Oxford and New York, 1966, pp. 193-213.

BIOSYNTHESIS A N D CATAHOLISLLZ OF GLYCOSPHINGOLIPIDS

2.55

young rats b y Arce and coworkers."'!' This enzyme was also found i n rat liver,33mouse liver and brai~i,I"~ chicken retina,"" and astrocytoma ce1ls.ln6In all cases, compositional analysis, chromatographic behavior, and susceptibility to sialidnse were used to characterize the newly synthesized gangliosides, but the linkage between N-acetylneuraminic acid and galactose was not determined. For the synthesis of disialosyllactosylceralnide, the sialosyltransferase which catalyzes the reaction CMP-NeuAc

+ NeuGca2 -+ 3GalP1 + 4Glcpl-

-

1'Cer NeuAcc~2+ 8Nei1C;cd! + 3Galpl + 4Glcpl

+

1'Cer

was studied in embryonic chickeii-hrain by Kaufinan and coworke r ~As. the ~ glycolipid ~ ~ acceptor contained N-glycolylneuramiiiic acid instead of N-acetylneuraminic acid, they were able to distinguish the newly synthesized sialic acid fioni the sialic acid i n the acceptor. They used periodate oxidation to show that the lJC-lalieledN-acetylneuraininic acid was linked to the N-glycolylneuraniiiiic acid at 0-8. The authors also found that phospliatidylethanolamiiie stiniulated the sy n the s i s of d i s i a1os y 1lact osy 1ce r ii i i i i (1 e . The s am e s i a1o sy 1t raii s fe ras e was also detected in chick retiixi and cultured neurons.110 Sialosyllactosylceramide and disialosyll~tctosylcerainideare both key intennediates for the biosynthesis of higher gangliosides of the ganglio series.

c . Biosynthesis of Ganglio-type Gang1iosides.-If various gangliosides of the ganglio series are synthesized from cerainide b y the stepwise addition of a inonosaccliuride uiiit to the elongating sugar chain, several hypothetical pathways citii be drawn, hecause of the existence of the branched structures. For e x m i p l e , GM2 can be synthesized b y adding a 2-acetamido-2-deoxy-r~-gitlwtosylgroup to GM3, or it caii be synthesized b y adding this group to lactosylcer~tiiiidehefore the addition of sialic acid. Kaufnian and coworkerslOXwere the first to show that GM2 was synthesized by t h e d d i t i o n of sialic acid to lactosylceraniide prior to the addition of a ~-wcetamido-2-deoxy-D-g~i~~~ctosyl group by using the particulate preparation o1)tained froin embryonic chicken-brain. This conclusion was based on their oliservation that sialosyllactosylceraniide (GM3) wiis niuch better than lactosylceraniide as an acceptor for 2-acetaniido-2-deoxy-u-galactose. In addition,

(110) H. Dreyfus, S. Harth, A. N . K . Yu\iifi, 1'. F. Urban, ; i i i d P. klantlel, i n Het'. 10, pp. 227-237. ( 1 1 1 ) B. Kaufinan, S. Basu, and S. K o w ~ i n a n , / . B i o l . C/ieiii., 243 (1968) 5804-S806

256

YU-TEH LI AND SU-CHEN LI

they showed that GM2 served as an acceptor of galactose to forni GM 1. By analyzing the acceptor specificities of various glycosphingolipids, they proposed the following pathway for the biosynthesis of GDla. lactosylceramide

- - - CMP-NeuAc

GM3

UDP-GalNAc

GM2

UDP-Gal

GM1

CMP-UeuAc

GDla

(i) Biosynthesis of GM2 (I13NeuAc-GgOse3Cer) from GM3, and GD2 from GD3.--Steigeiwald and coworkers1I2studied an enzyme in the particulate preparation of embryonic chicken-brain that catalyzed the transfer of 2-acetamido-2-deoxy-~-galactosefrom UDP-GalNAc to N-glycolylneuraminosyllactosylceramide (the N-glycolyl analog of GM3) isolated from horse erythrocytes. They identified the product as GM2 containing N-glycolyliieun~minicacid. Their enzyme had a low, but detectable, activity towards lactosylceramide. The filct that sialosyla2 + 3lactose and sialosyla2 -+ Glactose were not good acceptors suggested that the enzyme required the lipophilic portion of the ganglioside. Cumar and coworker^"^ showed that the N-acetylgalactosaminyltransferase activity in their rat-brain preparation catalyzed the transfer of 2-acetamido-2-deoxy-~-galactosefrom UDP-GalNAc to GM3 and GD3, respectively, but not to lactosylceraniide. Also, they found that the GM2 and GD2 synthesized could further accept galactose when UDP-Gal was present i n tlie system. They pointed out that the similarities of kinetic parameters for tlie corresponding steps suggest that an analogous sequence of reactions was involved in the synthesis of GM2 and GD2. DiCesare and Dain"-l3'l5 also studied the same N-acetylgalactosaminyltransferasein rat brain. They found that the GM3 with N-acetyliieuraminic acid was a better acceptor for 2acetamido-2-deoxy-~-galactose than the GM3 analog with N-glycolylneuraiiiinic acid or with 4-0-acetyl-N-glycolyliieuraniinicacid."' Cumar and coworkers,11:'on the contrary, found that GM3 with N-glycolylneuraminic acid was a slightly better acceptor. They also found that lactosylceramide was not a good acceptor. Both Cumar and coworkers"" and DiCesare and Dain".* failed to detect tlie transfer of 2-acetamido-2-deoxy-D-galactose to globotriaosylceramide, suggesting that the N-acetylgalactosaniinyltransferaseinvolved in the synthe-

(112) J. C. Steigerwald, S. Basu, B. Kaufiiraii, and S. Rosemail,]. B i d . C h e m . , 250 (1975) 6727-6734. (113) F. A. Cumar, P. H . Fishniaii, and R. 0. Brady,]. A i d . Chem., 246 (1971) 50755084. (114) J. I,. DiCesare and 1. A . Dain, R i m h i i n . B i o p h p . Actcr, 231 (1971) 385-393. (115) J. L. DiCesare m d J. A. Dain,J. Neurochein., 19 (1972) 403-410.

BIOSYNTHESIS AND CA1'41301.14hl OF C~1,YCO~l'IIINGOLIPIDS 2.57

sis ofGM2 was different from that responsible for the synthesis of gloI~otetraosylceramide. By using competition studies, Chien m d c ~ w o r k e r s -showed '~ that the same N-acetylgalactosamii~yltr~~iisferase of cwi1)ryonic chickeiibrain transferred 2-acetainido-2-tleosy-~-gal~~ctos~ to Loth GM:3 and globotriaosylceraii.lide. Interestingly, Cumar arid coworkers ll'i did not detect a significant transfer of GalNAc to G D l a , although t h e ganglioside GalNAc-GDla has been isolated froin human This gnnglioside has also been found to acciiiiiiilate in the brain o f a patient with Tay-Sachs disease.117As the teriiiinal portions of G M 2 and GalNAcG D l a have the s a n e striicture, i t i s difficult to imagine that the terminal 2-acetamido-2-deoxy-~-g~i~~i~~tosyl groups i n these two ganglios i de s are s yn t he s ized b y two tl i ffk re 11t N-acet y 1gal ac t o s it in in p 1t riiii sferases. Keinp and Stoolmiller.'2 1i)und that a cell-free enzyinepreparation of cultured, inousc~-neurol~lastoma cells catalyzed the transfer of N-acetylneuraminic acid froin CMP-NeuAc to lactosylceramide to afford GM3. Asialo C;M2 was neither an acceptor nor a competitive inhibitor for this si~ilos).ltransferase.The enzyme preparation also contained a UDP-GalNAc:GM3 N-acetylgal~~ctosanlinyltransferase which further convc.rtt4 GM3 into GM2. Lactosylceramide was not converted into asiulo GM2 h y this enzyme. Again, these results support the concept that the first sialic acid of braiii gangliosides is introduced into lactos).lceramide, and that GM2 is synthesized from GM3. By the doul)le-I;ilieliiig technique using UDP-GalNAc labeled with 14C and tritiuin-labeled ChlP-NeuAc, Arce and coworkers,118were able to conclride that, quantit:itively, Gh13 was a more important precursor than a s i a l o Ghl2 for the synthesis of GM2.

(ii) Biosynthesis of GM1 (I13NeuAcGgOse,Cer) from GM2, and G D l b (I13NeuAc2GgOse4Cer)from GD2.-By using the enzynie from embryonic chicken brain, h s r i and coworker^"^ showed the synthesis of GM 1 froiii GM2 catalyzed hy ii UDP-Gd:GM2 P-galactosyltransferase. This galactosyltratisfer~~sewas also detected in frog braiiilz0and rat l)rain.115J21,122 Cuniar and coworkersIz2showed that the P-galactosyltransferase prepared from rat brain transferred galactose to both GD2 and GM2, to forin G D l h and GM1, respectively. Studies (116) L. Svenuerholiir, J.-E. blansson, , c ~ r t lY:T. L,i,/. H i o l . Cltcatr,., 248 ( 1973) 740-742. (117) M . Iwamori and Y. Nagai,]. Ncirr-oclicrn., 32 (1979) 767-777. (118) A. A w e , H. J. Maccioni, and R. C.il)lltto, B i o c / ~ e mI.. , 121 (1971) 483-493. (119) S. Basil, B. Kaiifiiian, a n d S. Roseni
258

YU-TEH LI AND SU-CHEN LI

of the substrate specificity of this enzyme showed that, aniong the various glycosphingolipids tested, GM2 and GD2 were the best acceptors. Their results suggested that the syntheses of GM1 from GM2, anti G D l b from GD2, were catalyzed by the same galactosyltransferase. 'The synthesis of lactosylceramide, on the other hand, was catalyzed by a different galactosyltransferase, because glucosylceramide could not serve as an acceptor for this galactosyltransferase. Hildebrand and coworkers5' also reported that, in rat brain, there appear to be three different galactosyltransferases that catalyze the synthesis of lactosylceramide froin glucosylceramide, GM 1 from GM2, and psychosine from sphingosine.

(iii) Synthesis of G D l a (I13NeuAcIV3NeuAc-GgOse,Cer) from GM1.-A sialosyltrarisferase that transfers N-acetylneuraminic acid to GM1 (to form GDla) has been demonstrated in embryonic chickenbrain."' A similar enzyme was also detected in young rdt-brain.'*'3*'24 The sialosyltransferase that synthesizes G D l a from GM1 has been shown to be different"' from the two sialosyltransferases that synthesize GM3 from lactosylceraniide, and GD3 from GM3. There is, however, still some uncertainty allout the biosynthesis of GM la, G D l b , and other polysialosylgangliosides. Although most of the work suggested that the synthesis of G D l a was from GMl, and of G D l b from GD2, Arce and coworkers,11Husing endogenous acceptors, showed that GM1 was the precursor for both G D l a and G D l b . (iv) Synthesis of G T l b [IV3NeuAcI13(NeuAc)zGgOse4Cer]from GDlb, and of G T l a [IV3(NeuAc)2113NeuAcGgOse4Cer] from GD1a.Mestrallet and demonstrated in embryonic chick-brain a sialosyltransferase activity that transferred N-acetylneuraminic acid to exogenously added G D l b , to form G T l b . The results from substratecompetition and thermal-inactivation studies suggested that the same sialosyltransferase catalyzed the synthesis of G T l b froin G D l b , and G D l a from GM1. Pacuszka and found a similar enzymeactivity catalyzing the synthesis of GTlb from G D l b in the Golgi membranes from bovine thyroid. Yohe and Y u ' * ~demoiistrated a sialosylosyltransferase activity that synthesized G T l a from G D l a in a par-

(123) M . C. M. Yip, B i ( ~ c h i nB~i.o p h ! y ~t.i c f a , 306 (1973) 298-306. (124) J. A. Dain and S . 3 . Ng, in Ref. 10, pp. 239-245. (125) M. G. Mestrallet, F. .4.Cumar, and H. Caputto, M o l . Cell. Bioclzem., 16 (1977) 63-70. (126) T . Pacuszka, R. 0. Duffard, R . N . Nishimura, R. 0. Brady, and P. €1. Fishman, ]. B i d . Cherri., 253 (1978) 5839-5846. (127) H . C. Yohe and R. K. Yu,]. B i o / . Cheni., 255 (1980) 608-613.

BIOSYNTHESIS A N D CATAH )I,lbhl O F GLYCOSPI-IINGOLIPIUX

2.50

ticillate fraction of embryonic chicken-brain. When GM 1 was used as the acceptor, 6570 of the radioactivity was foiind in G D l a , whereas -50% of the reniainiiig radioactivity was found i n GTla. The results suggest that the synthesis of G'I1;i c:ould proceed b y way of the sequence GM1 + G D l a + GTla.

-

(v) Asialo GM2 Pathway.-In contrast to the pathway proposed b y Kaufiiian and coworkers,lo8which involves Gh13 a s the ol~ligateintermediate, an alternative pathway that proceeds through asialo Gh12 has usiilg of UDP-[14C]GlcNAcand also been p r o p o ~ e d ,By ~~ ~ , ~a ~mixture ~ UDP-[14C]GalNAc as the glycosyl donors, Handa and Burton'2x showed that a particulate preparation isolated from rat brain transferred only 2-acetamido-2-deoxy-u-galactose to lactosylceraiiiide to form asialo GM2. Based on the cliroinatographic mol,ility, the authors concluded that the product was asialo GM2. In addition to lactosylcerarii ide , galacto s y lcerain ide iii I d g 1uco s y lce riii11i de we re a1s o good acceptors. However, GM3 isolated from horse erythrocytes was a poor acceptor. For further elongation of the sugar chain, Yip and Daiii129reported a galactosyltransferase tliat catalyzed the transfer of galactose from UDP-Gal to asialo GM2 iii frog h i i n , and nit brain and liver. On the basis of chromatographic mol)ility, the authors concluded that the newly synthesized glycolipid was asialo GM 1 (g~tngliotetraosy1cel.amide). Dawson and coworke (I' reported that a mouse neuroblasto~na cell-line which is virtually devoid of GM3 coriltf synthesize the characteristic gangliosides, such iis Gh12, GM1, GD2, and GDla, of nervous tissues. Based on this ol)servation, they proposed that asialo GM2 might be an intermediate i n the synthesis and the catabolisin of gangliosides. The transfer of sialic acid froiii ChlP-NeuAc to asialo GM 1 was first observed by Basulo7 and K a u f i i i m and coworkers1oXin embryonic chicken-brain. In contrast to ordinary GM 1 isolated from brain, the sialic acid in this newly synthesized ganglioside was susceptible to the action of sialidase. A similar reaction was also found in young ratThe structure of this ganglioside was determined to be NeuAca2 + 3G:ilpl + 3GalNAcP1 + 4Galpl + 4GlcCer (GMlb).lr2 As G M l b is not a normal constituent of brain, or of extraneural tissues, (128) S. Handa and H. M. Burton, Lip id $ , 4 (1969) 589-598. (129) M. C. M. Yip and J. A. Dain,Lipidv, 4 (1969) 270-277. ~. (130) G. Dawson, S. F. Kemp, A. C . Stoolniiller, and A. Dorfman, B i o c h e ? ~Bio&~. Res. Comrnun., 44 (1971) 687-694. (131) M. C. M. Yip, Biochem. Bioph!y~.R c s . Co?t~mrrn., 53 (1973) 737-744. (132) A. Stoffyn, P. (J.) Stoffyn, and M . C. M . Yip, Biochini. Hiophys. Actu, 409 (1975) 97- 103.

260

YU-TEH LI A N D SU-CHEN LI

this biosynthetic product had been widely regarded as an artifact, until the isolation of this ganglioside from rat ascites hepatoniaZ7and human erythrocytes.2H Hirabayashi and coworker^^"^'"^ made a very interesting observation on the composition, as well as the biosynthesis, of glycosphingolipids in the adhesive, and the non-adhesive, rat ascites hepatoma cells. They found the presence of asialo-GM2 and asialo-GM1 in the non-adhesive cells, whereas GM3 was the sole ganglioside detected in the adhesive cell-lines. They also detected an N-acetylgalactosaminyltransferase which synthesized asialo GM2 froin lactosylceramide in the non-adhesive cell-lines, but not in the adhesive cells. The galactosyltransferase activity which catalyzed the synthesis of asialo GM1 from asialo GM2, as well as G M l a from GM2, was demonstrated in both cell-lines. The presence of a sialosyltransferase which transferred the sialic acid from CMP-NeuAc to asialo GM1 was also detected in both cell-lines. The Stoffyns applied their newly developed method4?to a study of the biosynthesis of gangliosides in transformed and nontransformed cells.41They found the transfer of N-acetylneuraminic acid from CMPNeuAc to asialo GM1 (to form GMlb) to occur in homogenates of C2, rat-brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV-40-transformed 3T3 cells, chick-embryo fibroblasts, Rous sarcoma virus-transformed chick-embryo fibroblasts, and 9-day-old rat-brain. In addition, the NIE cells and normal and RSV-transformed chick-embryo fibroblasts synthesized a disialosylganglioside (GD1) differing from G D l a and G D l b , and bearing only one substituent at 0-3 of the terminal D-galactosyl group. Kaufnian and coworkers10xhad also detected the synthesis of a similar disialosylganglioside from asialo-GM1 with embryonic chicken-brain. The G D 1 was also biosynthesized from G M l b and CMP-NeuAc by NIE and chick-embryo cells, but not b y C2, cells, or rat brain. However, C2, cells and rat brain were capable4,of synthesizing G D l a from GM la. These observations suggest that the sialosyltransferase involved in the biosynthesis of GD1 from G M l b is different from the one synthesizing G M l b from asialo-GM1, and G D l a from GMla. The linkage of two sialic acids in the new disialosylganglioside (GD1) remains unknown. These observations may indicate the existence of a pathway involving asialo-GM2 as an intermediate. Schemes 6-8 summarize various gly(133) Y. Hirabayashi, T. Taki, M. Matsumoto, and K. Kojima, Biochim. B i o p h y s . Actu, 529 (1978) 96- 105. (134) T. Taki, Y. Hirabayashi, Y. Suzuki, M. Matsumoto, and K. Kojima, /. Bioclzem. (Tokyo),83 (1978) 1517-1520. (135) T. Taki, Y. Hirabayashi, Y. Ishiwata, M . Matsumoto, and K. Kojima, Biochim. Biophys. Actu, 572 (1979) 113-120.

BIOSYNTHESIS AND C A l h l 3 0 l , l S X l OF GLYCOSPHIS(;OLIPII)S

GalNAc~31-4Gal,3-4Glc~~l3

1'Cer

7

Gal?l-

?GalNAr il-

Galgl-

3GalNAcOl-

2 NeuAc

I I

4Gal813

4Glcpl-

2 NeuAc

I

4Gal,:1-4Gl~,il-l'Cer~~~~~~ 3

ta

Iff

2 NeuAc

261

1'C'er

Gal 131-

3GalNAc 11-

4Gal l ~ l - 4 G l c l ? l -1'Cer"'?'*'

1.

GalOl-3GalNAcpl3 ta

2

NeuAc

G a l /31-3Ga1NAc~Il3 to

2

NeuAc 8 ta

4Galpl--4Glcgl-1 3

NeuAc

'Ccr

Gal01-3GalNAc~31-

4Gal,113

4Gl~~31-1'Cer'~~

to

2 NeuAc

4Galgl-

NeuAc

I I

4Glc/jl-

2 NeuAc

?

1'Cer

3 to

2 NeuAc

2

NeuAc

SCHEME 6. Glycosyltransferase Heaction.; Leading to the Synthesis of GM3, GM2, GM 1, GDla, and GTla, Starting fronr I~.uctosylceramitle.(Broken arrows indicate the hypothetical, biosynthetic pathway.)

262

YU-TEH LI AND SU-CHEN LI

sialos vltrans ferase

-

Gal p 1 3

-

4Glcg 1

1‘C e r

Gal 81-4Gl~gl-l’Cer‘~’ 3

la

to

2 NeuGc

2 NeuGc8 -2aNeuAc

Gal 01-4Glcpl-1’Cer 3

Galp1-4Gl~pl-l’Cer~~o

ta 2 NeuAc

I

NeuAc 7-2oNeuAc

GalNAc71 -?Gal % a pl-

Galgl-4Glcgl;l’Cerr3

4Glcpl-l’Cer1’3

tff

2 NeuAc8-

2ofNeuAc

,

NeuAc8 -2aNeuAc

k- UDP-GalNAc:GD3 I

8-N-acetylgalactosaminyltransjerase

4Gal pl3

Gal?l-7GalNAcpl3

4Glcp1-

l’Cerlzz

ta

la

2 NeuAc8-2aNeuAc

2 NeuAc8 -2aNeuAc

I

I { IJDP-Gal: G D 2 p-ealnctosyltransferase

-

Gal p 1

3

t.

2 NeuAc

-

3GalNAcP 1

4 Gal p l

3

-

i‘ -

4 Glcp 1

I

la

2 NeuAc

3 G a l N A ~ o l - 4Galp1-4Glcpl3

t.

2 Neu Ac 8

G;lgl-

I1

t.

2 Neu Ac 8 -2aNeuAc I

Galpl3

1’Cer

3GalNAcpl-

4Galgl3

la

ta 2

NeuAc

4 CMP-NeuAc:GDlb I

sialosyllransferase

4Glcp1-

1’Cer’zs

-

NeuAc8 -2olNeuAc

I

1’Cer

-

20 NeuAc

SCHEME 7. Glycosyltransferase Reactions Leading to the Synthesis of GD3, GD2, GDlb, and GTlb, Starting from GM3. (Broken arrows indicate the hypothetical, biosynthetic pathway.)

BIOSYNTHESIS A N D CATAHOLISXl OF GLYCOSPHINGOLIPIDS GalB1-

263

4G1c131I

I

r-- I

GalNAr/tl-4Gal;il-4Glc~~l-l’Ccr

Gal?l-?GalNAc~~l-4Gal,ll-4Gl~~~l-l’Cer’~~~’~~

I

I

[I/]/’-

G a l 131

-

3GalNAc,jl-

4 G a l 191

-

V

4 Glc/31

-

1 ’Ccr

I

-

3GalNAc,31-

t (Y

2 NeuAc

GI!&!

~ ~ - ~ ~ / i / / l c / o S ~ ’ / / J ~ / l l l ~ i l ~ ~ ~ . ~ l ~

Gal 131 -3GdlNAclll-4Gal

ijl-4Glcfil

-1’Cer”,’32

NeuAc

I I

G a l $1 3

( ; / I / : il 51/1/o

C,ZII‘-,VCIIAr : .4\ inlo G.W 1 ~ i r i / o . s \ ~ l l ~ n1.11.5 n v 1i ~ ~

f

1’Crr

4 G a l ljl-4Glc,llI I I I

Gal jjl-

3GalNAc1il-4Gal,31-

4Glc1ll-

NeuAc ?-ZnNeuAc

I I

G a l [jl-

-

3 G a l N Acl3 1

3

-

4 G a l 13 1

I

-1

4Glclj 1

t N

2 N e u A r ? -2aNeuAc

SCHEME 8. Glycosyltransferase Heaction\ Leading to tlic Synthesis ol Asido GM2, C;M 1, G M l b , and GD1. (Broketi u i - o w \ indicate the hypothetical, biosynthetic pathway. Handa and Bitrton 12x used a i i i i \ t i i i - c , of UDPGlcNAc and UDPGalS.4c a s the glyco\yl donor.)

cosyl-transferring reactions related to the hiosynthesis of ganglio-type gang 1ios ides . In order to avoid alterations of inemlirane structure caused Iiy tletergents, Arce and coworkers1IXconducted a study on the i n uitro incorporation of radioactive sugars fro111 1al)eled “sugar nucleotides” to the endogenous acceptors present i n particulate preparations. They made the assumption that the particles would contain gangliosides which were in the path to completion when the aninxi1 was sacrificed; these incomplete intermediates will pass to the next step, provided that the appropriate “sugar nucleoticle” is supplied. Their kinetic studies indicated that the enzyme-acceptor system was strictly conipartmentalized, in the sense that the e n z y m e of one particle did not react with the acceptors from other particles. T h e y found that exogenous acceptors and endogenous acceptors did not conipete for the enzyme, and

1’Cer i1.108

YU-TEH LI A N D SU-CHEN LI

264

that precursor-product relationships between the main pools of each ganglioside did not exist. Their results also indicated that GM3 is the preferred intermediate for the synthesis of GM2, GM1, and GDla, as had been proposed by Kaufman and However, they found that G D l b is synthesized from GM1, not from GD2. (vi) “Transient Intermediate” for the Biosynthesis of Gangliosides. -Maccioni and coworkers136examined the rate of labeling of the rat brain gangliosides after intracerebral injection of [3H]2-amino-2deoxy-D-glucose, and found that the N-acetylneuraminic acid from the relatively small pool of GM3, which is supposedly the precursor of other complex gangliosides, was not labeled as expected. T h e y also found that two sialic acids in G D l a had the same specific activity after being labeled from [3H]2-aniino-2-deoxy-D-g~ucose. Similar results were also obtained for the specific radioactivity of proximal and distal galactoses labeled from [14C]glucose.Based on this observation they proposed the existence of “transient intermediates” for the biosynthesis of gangliosides (see Scheme 9). According to this model, the gangliosides originate, at specific sites, from a limited pool of transient intermediates. Once the products are formed, they pass to other positions in the inemlxanes from which they cannot return to the posi-

Site for

/

Hematoside

Monosialoganglioside

Trisialoganglioside

Gal-Glc -Cer

Gal- Glc - Cer Gal- Glc- C er

I

Glycosyltransferase

sialosyl 2-aretamido- 2deoxy-D -galactosyl

GM2

GM2

I End product

GM3

GM1

galactosyl

GT1

SCHEME9. Schematic Illustration of the Biosynthesis of‘ Cangliosidesl”6 by way of “Transient Intermediates.” ( I t should b e noted that the biosynthetic products were identified by composition analysis and t.1.c. niobility only.)

(136) H. J. Maccioni, A. Arce, and R. Caputto, R i o c h e m . J . , 125 (1971) 1131-1137.

BIOSYNTHESIS AND CAI'XROI .ISXI OF GLYCOS~HING0LIPII)S

265

tion of intermediary sulxtrates. 'This scheme explains I d ] the intlependent rate of labeling of all o f t h e end products and the non-existence of partial turnover indicated Iiy the similarities found in the specific activities of repeated components, such a s galactose and sialic acid in the labeled gangliosides. This aspect of the biosynthesis of brain gangliosides has been re\iewed b y Caputto and coworkers.':" I n contrast to this work on whole brain, K e m p and Stoolmillerl:'8 used cultured iiiouse-iieurol,lastom~icells for their study of incorporation of ['H]2-acetaniido-2-deoq -I)-iniiiinose into the sialic acid moiety of gangliosides. They were able to cleinonstrate clearly the precursorproduct relationship among GRf3, GM2, and GMl. They found tliiit cultured NB41A cells incorporated [~H]~-acetamido-~-deoxy-D-mannose into the sialic acid moicJty of GM3 in less than 10 minutes. Labeled GM2 was not detected i n cells incubated for less than 30 niinUtes, and measurable activit). t l i t l not appear in GhI 1 until after 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of gangIii)-type gangliosides proceeds b y w a y of

GM3 -+ GM2 -+GM1. 4. Biosynthesis of Glycosphingolipids in Pathological Conditions a. A Novel Human Sphingolipidosis due to a Deficiency in Canglioside Biosynthesis .-It has l)een well dociimented that the coniposition of glycosphingolipids i i i I)rain changes during developnieiit.1:19-142 The correspoiidiiig changes in various glycosyltransferase activities, especially before and after my el in at ion,"^^^"^^"' suggest that glycosphingolipids are essentiiil to the development of l m i i n . Irngairment in the synthesis of gangliosides should have a detrimental effect on the developing brain. Fishmiin and coworkers'.':j reported a tleficiency in UDP-GalNAc:GM3 N-acetylgalactosiiininyltransferase i n the tissues of a n unusual case of' lipid-storage disease"" characterized (137) I<. Caputto, H. J . Maccioni, iiiid A . Arc.?, ,lfo/. Cell. Hiorlietn., 4 (1974) 97- 106. (138) S. F. Kemp and A. C . Stoolinillt~i-,,/H i d . Clwm., 251 (1976) 7626-7631. (139) K. Suznki,/. Neurochetii., 12 (196S) 969-979. (140) M. T. Vanier, M .Holm, H. <)hinail, ~ c I , . l Svennerholrii,,/ N v t r r o c ~ l i c ~ i i i18 . , (1971) 581-592. (141) A. Merat and J. W. T. Dickersoil,/. ~ ' c ~ r ~ r o c h e r20 t z .(1973) , 873-880. (142) H. Dreyfus, P. F. Urban, S. Edt~I-H~ii-th, and P. MuideI,J. ~\lc~trroc/ie~ii., 25 (1975) 245 - 250. (143) P. H. Fishnran, S. R. Max, J . F T , i l l i i i i u i , R. 0. Brad!-, N . K. ;2laclareir, ,iiid X l . Cornblath, Science, 187 (197S) 68- 7 0 . (144) S. H. Max, N . K.Maclaren, R.0 .Rr;ttl!~, 11, 11. Bradley, 11. B. Reiinels, J . T a d w , J. H. Garcia, and M . Coriiblatlt, R'czrc; E t t g / . /. Med., 291 (1974) 929-931.

266

YU-TEH L1 .4ND SU-CHEN LI

b y the accumulation of GM3 in the patient's liver and brain. They proposed the name "anabolic sphingolipidosis-type GM3" for this disease.I4"The effect on ganglioside biosynthesis in this disease is similar to that observed in certain virally transformed cells (see later). The devastating effects due to the impairment in ganglioside synthesis emphasize the importance of gangliosides in the development of the central nervous systeni and visceral organs.

b. Biosynthesis of Glycosphingolipids in Virus-transformed Cells. -The simplification in glycosphingolipid pattern of eukaryotic plasma membranes after transformation b y oncogenic viruses, chemical carcinogens, or X-ray irradiation has been widely recognized. This topic has been the subject of several r e v i e ~ s . ~ ~ Simplification "-'~~ of patterns b y deletion of more-complex glycosphingolipids, with accumulation of the simpler, precursor glycosphingolipids, has been shown to be due to specific blocks in glycosyltraiisferases (for reviews, see Refs. 145- 147). According to the biosynthetic pathway proposed by Kaufman and coworkers,"'* UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase is the key enzyme in the pathway leading from ceramide to complex gangliosides. This enzyme has been found to be either absent from transformed, tissue-cultured fibroblasts, or present at a very low level,14s+153 thereby explaining the simplification of the ganglioside pattern in the transformed compared with the nonnal cells. Alterations in the biosynthesis of glycosphingolipids (besides the gangliosides) after transformation have also been However, the exact mechanism leading to the changes in the glycosyltransferase activities after transformation remains unknown.

5. Concluding Remarks It should be emphasized that in vitro detection of the transfer of a glycosyl group from a "sugar nucleotide" to an exogenous, glyco(145) H. 0. Brady a n d P. H. Fishman, Biochitn. B i o p h y s . Actu, 355 (1974) 121-148. (146) P. H. Fishman, Chem. P h y s . Lipids, 13 (1974) 303-324. (147) S.-I. Hakornori, Biochim. Biojhy.c. Actu, 417 (1975) 55-89. (148) C. L. Richardson, S. R. Baker, D. J . MorrC., and T. W. Keenan, Biochirn. Biophys. Actu, 417 (1975) 175-186. (149) P. H. Fishman and R. 0. Brady, Science, 194 (1976) 906-915. (150) F. A. Cumar, R. 0 . Brady, E. H. Kolodny, V. W. McFarlantl, a n d P. T. hlora, Proc. Nutl. Acud. Sci. U.S.A., 67 (1970) 757-764. (151) P. H. Fishman, V. W. McFarland, P. T. Mora, and R. 0. Brady, Biochetn. Biophys. Res. Commun., 48 (1972) 48-64. (152) P. T. Mom, P. H. Fishman, R. H. Bassin, R. 0. Brady, and V. W. McFarland, N a t u r e (London)N e w Biol.,245 (1973) 226-229. (153) V. N. Nigam, R. Lallier, and C. Brairlorsley,]. Cell. B i d . , 58 (1973) 307-316.

sphingolipid acceptor b y an etizyiiic. preparation does not necessarily imply that such a reaction actuall!. occurs in u i u o . Using this method, t h e enzyme m a y tie providetl with a glycolipid acceptor that is not present in t h e tissue where t h c c’nzynie is isolated. The same glycos ph i I 1go1i p i d s i so 1at ed from d i tte r e 1 it so I 1rce s ma)‘ tl i ffc: I con s i tle ral) 1y in their fatty acid coinposition, i i i i d consequentl?-, the use ofglycolipid acceptors isolated from soiircc\ other than t h e tissues eniployetl for the exam inatio t i of gl y co s y 1t ra I i \ fc, r ~ ies ;IC tiv i t i e s 111a!‘ give fa 1s e i 1I tormation. Schemes 5 - 8 represeti t tht, collection of ~ l y c o s ? , l t r ~ I n s f e r r i i ~ g reactions detected in clifferetit tishiic>s and organs. The stepwise vloirgation of sugar chains in gl!.c.osl)hiirgolipids has i ~ e v e rI w x n d e n ~ o n s t rate d . The re fo re, t h e 1) i os y 1I t h c’t i c pit t h w ay s in t 1i C R t e d 1, >, ve rt i cal , broken m-1-ows should be r e g a r t l r ~ l;IS hypothetical. Our concepts concerning the. I)ios>wtlieticpathwa golipids are based niainly oil t h e acceptor specific t ra t i s fe rases . Tli e d i fficu 1t y i t i s ( ) 1I I 1) i 1i z i iig a n d p I I ri fl-i t i g t 11 cl g l >.cos y 1transferases has led to the us(’ of indirect techniques to estwl)lish enzyme specificities. These tc~c~hiiiqiies have Iargel?. coiisisted it1 the use of differential stabilities o f (liffbrent enzymc-s towards heat or inhibitors, a n d of sul,stmte-coiiIpttitioti studies. €3). using ti pure sialosyltransferase isolated froin I)o\iiic sul)niaxillar~~ gland, Rearick and coworkers showed that t h e s ;ti I 1e t’i i z y nie trat 1s fe rre d s i a 1i c aci tl fro 111 CMP-NeuAc into t h e seqiieiict. NeuAccu2 + 3Galpl + 3GiiINAc, which is found both in glycoprott,iiis arid gaiigliosides. However, it is still not clear a s to whether gl!.c.os).ltransferases that catalqrze t h e synthesis of sugar chains in gl~~cospliingolipicls can also catalyze the synthesis o f t h e similar sugar chains in glycoproteins. At present, v e r y little is known about t h e control and regulation (that is, activators and inhibitors) of glycosyltransfer~is~!s. I t is noteworthy that Diiffkrd and Capiittolss detected a protein i tihil)itor for C~lP-r\;euAc:lactosylceraniide sialosyltransferase in rat Ilrai 1 1 . The level of this inhil,itor increased with the age of the a t i i i r i n l , and the inhibitor apparently acted b y increasing t h e K,,&value of liictos?-.lceraniide.F u t u r e work on t h e regulation a n d control of t h r 1)iosynthesis of glycosphiiigoligids shou 1cl yield fi-ui t fu 1 results . In most cases, the small amorint ofiiewly synthesized glycosphingolipids can only be identified I)?. t h e ;tnalysis of sugar coinposition, irnmunological activity, or t.1.c. tnol)ility. T h e s e methods, however, camet unequivocal 1y d i s t i n gii i s 11 t 11 e posit i on a1 i s o n 1 e rs . The h i ghl y (1.54)J. I. Rearick, J. E. Sadler, J , C. I’aulwii, ;mtl R. L. Hil1,J. H i o l . C h e i i t . , 254 (1979) 4444-445 1. (155) H. 0. Duffard and R. Caputto, I ~ i o c , h c , t i i i s ~ i - !11 / , (1972) 1396- 1400.

268

YU-TEH LI .4ND SU-CHEN LI

sensitive method introduced by the S t o f f y n ~ is~of~ great importance in determining the structure of the newly synthesized glycosphingolipids. It is noteworthy that 'H-n.in.r. spectroscopy has been applied to characterize newly synthesized g l y c o p e p t i d e ~ . 'Future ~~ progress in this field depends on the development of new approaches, a s well as on new analytical techniques.

111. CATABOLISM OF GLYCOSPHINGOLIPIDS It is not an overstatement to say that much of our knowledge concerning the degradation of sugar chains in glycosphingolipids was derived directly or indirectly from studies of the various sphingolipidoses. From the relationship between the niissing glycosidases and the accumulated glycosphingolipids in various sphingolipid-storage diseases, it appears that these compounds are norinally catabolized through sequential hydrolysis of glycosyl groups from the nonreducing end of the saccharide chains by a series of exoglycosidases. For studying the degradation of sugar chains in glycosphingolipids, the following considerations are pertinent. ( a ) Although isolation and characterization of exoglycosidases have been facilitated by the use of such chromogenic substrates a s p-nitrophenyl or 4-methylunibelliferyl glycoside, a glycosidase whose activity is monitored by using a synthetic substrate during the isolation may not be active with natural substrates. ( b )Glycosidases of the same category (for example, a - ~ g a lactosidase and P-D-galactosidase) isolated from different sources often differ considerably in their specificities towards different substrates. (c) Due to the lipophilic nature of glycosphingolipids, their hydrolysis in vitro by exoglycosidases requires the presence of a detergent in the reaction mixture. The rate of hydrolysis is greatly affected by the concentration and the nature of the detergent used. (cl) The promotion of the hydrolysis of glycosphingolipids by detergents is nonphysiological. Several protein activators isolated from various tissues have been found to replace detergents for stimulating the enzymic hydrolysis of glycosphingolipids i n vitro. This part of the article discusses the irt vitro catabolism of sugar chains in glycosphingolipids by mammalian exoglycosidases, with the foregoing points kept under consideration.

(156) 1).H. van den Eijnden, D. H. Joziasse, L. Uorland, H. van Hallleek, J. F. G. Vliegenthait, and K. Schmid, Riochern. H i o ~ h y s Hes. . Cornrnrrn., 92 (1980)839-845.

HIOSYNTHESIS AND CAI’AHOI,ISXl OF GI,YCOSPHINC~OLIPI1S

269

1. Enzyme Preparation and Enzyme Assay According to the concept of “iiilmrn lysosomal disease” proposed 157,15X catabolism of ~lycosphiiigolipidstakes place in Iysosomes. Lysosoines prepared froui rat liverlSs a n d brairitti0have I ~ e n shown to catabolize glycosphiiigolipids. It is, however, too early to e x clude the possibility that glycosiclases residing in other organelles participate i n the degradation of gl ycosphingolipids. For example, some mammalian sialidases arc> associated with cytosol and plasma membranes, as well as with lysosoiiies.161Owing to the fragility of lyso s omal i n em l n m e s , nio s t g1yc.c)s i ( la s e s in the 1y s o s om al vesicles c m be set free b y mechanical disrription, such ;is homogeiiizutiori iir a Waring Blendor. For large-scale isolation of glycosiclases froin iiiitiiinialian sources, tissues are risiiall y homogenized directly with water, or an appropriate buffer, to release the enzymes, presuina1)ly froin lysosoines. For hrain tissues, treatiiic,iit with acetone is often needed hefore extraction with aqueous I n d i a . The solubilized glycosidasc,s can subsequently he purified b y a ri umbe r of in et h ods . The LI sLI a1 p 1t ri fi cati on steps i 11cl u de am 111 o I 1 i uiii s ul fite fraction at i on, ge 1 fi 1t ra t i ( ) t 1 , ;i 11 d ch roni atograp h y on ii col L I ~11I of an ion-exchange resin, or concaiiavdin A- Sepharose, and other affinity columns. Because of their gl ycoprotein nature, lysosoiiial h>drolases have been found to b e at1sorl)ed to concanavalin A-Sepharose162.163; this property has 1 ) r ~ ~extensively ii exploited for the purification of glycosiclases from ~rrammalian sources. Although different media for affinity clirom~ttogr~tpliy, such ;is inimobilized l-thioglycosicles, glycosylamines, aiid aniiiiophenyl g l ~ ~ c o s i d e s , ’ ” ~have ~lf”’ been used for the purification of glycosidases, the sanie affinity media

by Her., L

,

(157) H. G . Hers, C:cistroenterolog!/, 48 (1965) 625-633. (1.58) H. G. Hers, in H. G. Hers ;itid F. Vaii I l o o f ( E ds.) ,L!yso,omo.~criitl S t o / u g c Di.5rcises, Academic Press, New York, 1973, pp. 147- 171. (159) N. J. Weinreb, R. 0. Brady, i i n d A . L.. ‘rappel, Biochinl. B i o ) h / . Y .A r f c i , 1.59 (1968) 14 1- 146. (160) J. F. Tallnian and R. 0. Bratly,/ N i o l . Chrni., 247 (1972) 7570-7575. (161) A. Rosenberg and C.-L. Schengt-iiiitl. 111 A. Roseillxi-g and C.-L. Scheirgruntl (Eds.),Biologicnl Roles of S i t i l k . / k i d , Plriinin P i - r s , New York, 1976, p p 295359. (162) S. Bishayee ;ind B. K. Bac1iaw;it. .l‘c,tr,-ol,io/og!/,4 (1974) 48-56. (163) S. Hickman, L. J. Shapiro, a i i t l C:. F. Nerifc-ltl, Riocherti. H i o ) i h ! / c . f1c.r. CotrirtIun., 57 (1974) 5 5 6 1 . (164) N . Harpaz ant1 H. M . Flowers, ,\I(~tltod.cE t i z y n i o l . , 34 (1974) 347-:350. d~ 34 (1974) 350-3.58. (165) E . Steers, Jr., and P. Cuatrec;isas, A l ~ t l i ~Etiaytnol., (166) H. Bloch and M. M .Burger, FI.:RS / , ( I f f . , 44 (1974) 286-289.

YU-TEH LI A N D SU-CHEN LI

270

may not be effective for the same enzyme from different sources, and no affinity medium is free from nonspecific bindings. Although several lysosomal glycosidases have been isolated in honiogeneous foml, 167 170 it is still a formidable task to isolate lysosoinal glycosidases in pure form. Development of simple, reproducible schemes for the isolation of pure, lysosomal glycosidases remains one of the foremost challenges in this field. Because of the simplicity and the high sensitivity that chroinogenic substrates provide, p-nitrophenyl and 4-methylumbelliferyl glycoside have been widely used for assaying various glycosidase activities. However, such a synthetic substrate provides the enzyme only with the right glycon of proper anoineric configuration; it does not provide the specific linkages and the characteristics of the aglycon that exist in natural substrates. For exaniple, p-nitrophenol, or 4-methylumbelliferone, is totally different from ceramide. It is, therefore, not unreasonable that a galactosidase diat hydrolyzes p-nitrophenyl p-Dgalactoside may not hydrolyze galactosylceraniide. In order to increase the sensitivity, the sphingoglycolipid sulxtrates are usually labeled radioactively at the sugar chain or the ceramide portion. There are two methods most commonly used for such labeling of the sugar chain. The one introduced by Radin171labels the ~ - g a lactosyl or 2-acetainido-2-deoxy-~-galactosyl group at the nonreducing teiiiiinal of the sugar chain b y treatment with D-galactose oxidase followed by reduction with sodium borotritide. The other involves the labeling of sialic acid b y mild oxidation with periodate, followed b y reduction172with sodium borotritide. Also, glycosyltransferases have been used to synthesize glycosphingolipid sulxtrates labeled in spe~ " ~method ~~' commonly used for lacific portions of the ~ n o l e c u l e . ~The beling the cerarnide moiety is the catalytic reduction of the double bond in the sphingosine b y means of tritium gas.175 Usually, a detergent must be added, to facilitate the enzymic hydrolysis of sphingoglycolipids i n vitro. Such bile salts as taiirocholate ~

(167) S . K. Srivastava, Y. C. Awasthi, A . Yoshida, and E. Beutler,]. H i d . Clzem., 249 (1974) 2043-2048. (168) B. Gieger a n d R. Arnon, Bioclierni.stry, 15 (1976) 3484-3493. (169) J. T. Lo, K. Makerji, Y. C. Awasthi, E. Hanada, K. Suzuki, and S . K. Srivastava, ]. B i d . Claeni., 254 (1979) 6710-6715. (170) E. Beutler and W. Kuhl,]. B i d . Clienz., 247 (1972) 7195-7200. (171) N. S. Radin, Metlzods Em:ymol., 28 (1972) 300-306. (172) L. V. Lenten a n d G . Ashwell, Methods Enzymol., 28 (1972) 209-211. (173) J. M. Quirk, J. F. Tallman, and R . 0.Brxfy,]. Labelled C o m p d . , 8 (1972)483-494. (174) J. F. Tallman, P. H. Fishman, and R. C. Henneberry, Arch. B i o c l i ~ ~ tBt i. o p h y . ~ . , 182 (1977) 556-562. (175) J. L. DiCesare and M. M. Rapport, Cheni. Ph!/,s. Lipids, 13 (1974) 447-452.

BIOSYNTHESIS AND CATAROL,IS.L1 OF GLYCOSPHINCO1,IPIDS

271

and taiirodeoxycholate are the niost effective and frequently used detergents. In some instances, crude bile salts work better than highly purified preparations. Sometimes, bile salts may stimulate a partially purified enzyme, but inactivate ii highly purified enzyme-preparation. Partial protection against inactivation can be provided by the addition of bovine serum albumin to the reaction mixture. When 21 neutral glycosphingolipid labeled at the rionreducing terminal is used as substrate, the radioactive sugar liberated can be separated from the remaining substrate and the product by Folch partition.ls When a radioactive ganglioside is used ;IS substrate, the radioactive monosaccharide liberated can be separated from the substrate and the glycolipid product by dialysis,”” or b y using an ion-exchange resin to adsorb the gangliosides selectively. 177 When a glycosphingolipid labeled at the ceramide moiety is used as suhstrate, the radioactive product is first separated from the siillstrate by thin-layer chroniatography, and then measured for radioactivity by nieans of a s ~ a 1 1 n e r . ~ ~ ~ Also, the product on a t.1.c. platc call be scraped off, and its radioactivity measured with a liquid sciiitill.d t’ion counter.

2. Degradation of Gangliosides by Sialidase The reports in 1963 by Korey arid on the degradation of brain gangliosides by a “gangliosidase system” prepared from rat and human brain probably constitiite t h e first description of the degradation of gangliosides by rnanirnaliiin sialidases and glycosidases. In the same year, Morgan and La~ii-ell l X 1 also showed that homogenates of human brain converted trisialosylganglioside, G T l b , into GM1. There is little doubt that the first step in the degradation of gangliosides is the removal of sialic acids h y sialidases. Sialidases have been detected in mammalian tissues, but they are extremely difficult to purify, and our knowledge concerning their specificities towards different gangliosides is very limited. The most interesting finding on the action of sialidases on polysialosylgaiigliosides having ganglio-type sugar-chains is that the end product is always GM1 which contains a sialic acid attached to the internal, D-galactosyl residue of the sugar cliaiii.1(i1J82 If the sialic acid is attwchetl to the terminal galactose of the (176) S.-C. Li arid Y.-T. Li,J. B i d C h p t t t . , 251 (1976) 1159-1163. (177) T. Miyatake and K. Suzuki, B i o d i i t t i . Riophys. Actci, 337 (1974) 333-342. (178) K. Sandhoff, E. Conzelmanii, and H. Nehrkorn, Hoiipe-Sey[er’.r. Z. P h y s i o l . Cheni., 358 (1977) 779-788. (179) S. R. Korey and A . Stein, L i f e S c i . , 2 (1963) 198-203. (180) S. R. Korey arid A. Stein,]. Ncrrropcithol. Erp. N c u t d . , 22 (1963) 67-80. (181) E . H. Morgan and C.-B. Laurell, N(itrtrr, 197 (1963) 921-922. (182) H. Drzeniek, Histochem. J . , 5 i197:l) 271-290.

272

YU-TEH LI A N D SU-CHEN LI

gangliotetraosyl Chain, it can be cleaved readily by bacterial, as well as main in a1i an, si a1i das e s . The resistance of the internal sialic acid in GM1 and GM2 to sialidases has been postulated to be due to the steric hindrance of the 2acetamido-2-deoxy-~-galactoseresidue, a s the sialic acid in GM3 (but not in GM2) can be readily cleaved by sialidases. However, the reason for such resistance remains to be explained. In 1968, Leibovitz and GatP" partially purified a membrane-bound sialidase from calf brain, and showed that it hydrolyzed tri- arid di-sialosylgangliosides to form inonosialosylganglioside GM 1. This enzyme was not able to cleave the sialic acid from GM 1 and GM2 gangliosides. Tettamanti and Zamb~tti'~ also * found that sialic acids in GM1 and GM2 were not hydrolyzed by the soluble sialidase purified from porcine brain. Based on their studies of brain glycosidases,18s+18H Leibovitz and G a W 3 formulated a possible degradative pathway for \)rain gangliosides as follows.

sialidase

p-D-gnluctosidase

Di- and tri-sialosylgangliosides monosialosylganglioside, GM 1 GM2 P-N~cetylhexosafniiiidase,GM3 sialidnse, lactosylceramide P-wwloctostdase, glucosylcerarnide

0-u-glucusiiluse

,ceramide

r.eraniiilase

sphingosine

+ fatty acid

Although this scheme emphasizes the importance of removing 2-acetamido-2-deoxy-~-galactosefrom GM2 before the hydrolysis of sialic acid, an unusual sialidase capable of hydrolyzing sialic acid from GM2 to form asialo GM2 has been reported to exist in several mammalian tissues.160~189~190 Li and found that the hydrolysis of sialic acid from GM1 and GM2 by clostridial sialidase in the presence of taurodeoxycholate was greatly inhibited by the high ionic strength of the buffer. Clostridial sialidase was able to cleave sialic acid from GM1 and GM2 slowly, hut distinctively, at low ionic strength, even in the absence of taurodeoxycholate. made a detailed study of human-brain siaOhnian and lidase, and found that this enzyme occurred mainly in a particulate (183) Z. Leilmvitz and S. Gatt, Biockim. Biophys. Actu, 152 (1968) 136- 143. (184) G. Tettamanti and V. Zambotti, Etizynrologiri, 35 (1968) 61-74. (185) S. Gatt,J. Biol. Cheni., 241 (1966) 3724-3730. (186) S. Gatt and M . M. Rapport, Biochem. J . , 101 (1966) 680-686. (187) S. Gatt, Biochern. J . , 101 (1966) 687-691. (188) Y. Z. Frohwein and S. Gatt, Biochemistry, 6 (1967) 2783-2787. (189) E. H. Kolodny, J. N. Kanfer, J. M. Quirk, and R. 0. Brady, J . Biol. Cliem., 246 (1971) 1426-1431. (190) J. F. Tallman and H. 0. Brady, Biochinz. Biophys. Actrr, 293 (1973) 434-443. (191) Y.-T. Li, M.-J. King, and S.-C. Li, in Ref. 10, pp. 93-104. (192) R. Ohman, A. Rosenberg, and L. Svennerholm, Biochenzi.stry, 9 (1970) 37743782.

UIOSYNTHESIS AND CATAHOI,ISXl OF GLYCOSPI-IIN(~OLIPIDS 273

foiiii. This enzyme hydrolyzed gangliosides very rapidly. However, when the concentration of gaiigliosides exceeded 100 p M , which coincided with the critical, micell;ar concentration of gangliosides, the enzyme activity was inhibited. 'Their particulate, sialidase preparation contained gangliosides which sei-vecl a s endogenous suhtrates for the enzyme. After depletion of the eiitlogenous substrates, the enzyiiie hydrolyzed such exogenously ;ttltletl gaiigliosides as di- and tri-sialosylganglioside, and converted them into G h l l . GM 1 and GM2 were not susceptible to the enzynie. They also found that G T l b

[NeuAccr2-+3Galpl+3GalNAcp 1+4Ga1(3+2cuNeuAc8+ 2aNeuAc)plj4Glcpljl'Cer] was preferentially converted into G l l l l )

1[GalP1 ~ 3 G a l N A c p 1 ~ 4 G a 1 ( 3 c 2 eiiAc8+-2aNeiiAc)P ~yN 4Glcpl+l'Cer], indicating that the sialic acid i n the disialosyl linkage was more resistant to the action of sialidase than the sialic acid attached to the terminal D-galactosyl group. Schengriind and Rosenberg'x:'provided evidence showing the concentratioii of bovine neuronal-sialiclase activity in the synaptosomal meiii1)raire. As gangliosides also reside in the same membrane, and can s e i ~ ciis ' endogenous sulwtrates, the authors postulated the presence of sialidase-gangliosicle complexes in the presynaptic iiieinbraiie. Sandhoff and coworkers194.1"5 fouiid that such general anesthetics its xenon, nitrous oxide, Halothaiic (BrClCHCF,), and ether enhanced the degradation ofexogenousl?. ~ l d e G d D l a and endogenous gangliosides by the membrane-bountl siiilidase. They postulated that the activity of menibrane-l>ound sialitlase on gangliosides of brain menibraiie was regulated b y the fluidity of these menil>ranes. It is iiow generally considered that polysialosylgangliosides having gangliotype sugar chains are first converted into G h l l b y sialidase, and that the GM1 is then catabolized I)y P-u-galactosidase, followed by P-Nacetylliexosamiiiidase, before the removal of sialic acid ( a s first suggested by Leibovitz and Gatt'X:').The findinglye of accumulatiori of GM3 in the fibroblasts of a patient with mucolipitlosis Type IV, due to (193) C . L. Schengrund and A. RoseiiI)erg,j. Niol. Chmti., 235 (1970) 6196-6200. (194) K. Sandhoff, J. Schraven, and G . Nowoczek, F E B S Lett., 62 (1976) 284-287. (195) K. Sandhoff, B. Pallnran, H. Wieg:aiitIt, i i i i d W. Ziegler, i n S. Catt, L. F r e y z , and P. Mandel (Eds.),Enzymes ~fL i l ~ i dMettrbolism, Pleiiiiin Press, N e w York. 1978, pp. 463-483. (196) G. Bach, M . 11. Cohen, iind G. K o l i n , t3iodienr. B i o p h ~ / ~Ru.s. \ . Conirritrtr., 66 (1975) 1483- 1490.

274

YU-TEH LI AND SU-CHEN LI

a deficiency of GM3-sialidase,Ig7may suggest that the sialidase for the hydrolysis of GM3 is different from the sialidases responsible for the hydrolysis of sialic acids in ganglio-type gangliosides.

3. Catabolism of Glycosphingolipids Containing /%Linked D-Galactose Galactosylceramide, lactosylceramide, GM1-ganglioside (and its asialo derivative), and lacto- or neolacto-tetraosylceramide are the major glycosphingolipids containing a terminal, P-D-galactosyl group. Much of the work has been directed towards the catabolism of galactosylceraiiiide186'1YX-200 and GM l - g a n p l i o ~ i d e , 2 ~ )because ~ - ~ " ~ of their relevance to sphingolipidoses related to the deficiency of P-D-galactosidase. Very little is known about the catabolism of lacto- and neolacto-tetraosylceraniide. In huiiian tissues, there are at least three types of p-D-galactosidase, two acidic and one neutral; these are separable b y gel filtration,z08gel e l e c t r o p h o r e s i ~ , ~isoelectric "~ focusing,206,210 and ion-exchange chromatography.20" The neutral and the acidic P-D-galactosidases have a basic difference in their polypeptides, and they are coded separately by different So far, only the acidic forms of P-D-galactosidase have been shown16g~2"x~212 to hydrolyze GM1. Studies on Krabbe's disease and GM 1-gangliosidosis have contriba great deal to our understandiiig of the c&bolisin of glycosphinG. Bach, M. Ziegler, T. Schaap, and G. Kohn, Biocherri. Biopliys. R e s . Conzmun., 90 (1979) 1341-1347. A. K. Hajra, D. M. Bowen, Y. Kishimoto, and N. S. Kadin,]. L i l ~ i dRes., 7 (1966) 379-386. D. M . Bowen aiid N. S. Radin, Biochiril. B i o p h y s . Actu, 152 (1968) 599-610. N. S. Radin, L. Hof, R. M .Bradley, and H. 0. Brady, Bruin Res., 14, (1969) 497505. M. W. Ho, P. S. J . Cheethanr, and D. Robinson, Biocheir~.I., 1.36 (1973) 351-359. M. Meisler, Alethod., Enz!/mol., 28B (1972) 820-824. A. G. W. Norden and J. S. O'Brien,Arc/i. Biochern. Biopkc~s.,159 (1973) 383-392. A. G. W. Norden, L. Tennant, and J. S. O'Brieii,]. B i o l . Chenl., 249 (1974) 79697976. H. Tanaka and K. Suzuki,]. B i d . Cheiri., 250 (1975) 2324-2332. Y. Suzuki a n d K. Suzuki,]. B i d . C k e m . , 249 (1974) 2098-2104. D. A. Wenger, Chern. Phtys. lipid.^, 13 (1974) 327-339. P. S. J. Cheetham and N. E . Dance, Biocheni. ]., 157 (1976) 189-195. M. W. Ho and J. S. O'Brien, Clin. C l z i n ~Actcl, . 32 (1971) 443-450. P. A. Ockerman,]. Pedicrtr., 75 (1969) 360-365. J. Butteiworth, A. D. Hain, and W. bl. McCrae, Clin. Chim. Acta, 41 (1972) 367373. Li,Carbohydr. Res., 34 (1974) 189(212) S.-C. Li, C.-C. Wan, M Y. Mazzotta, a n d Y.-T. 193.

golipids containing a terminal, 1)-galactosyl group. Kru1il)e’s disease (globoid leukodystrophy) is characterized by a deficiency of galactosylceramide p-D-jialactoSidaSe~”’;and GM 1-gaiigliosidosis, \ ) y a deficiency of t h e P-D-galactosidase that hydrolyzes c;hll-ganglioside, The asialo GM 1, a n d certain artificial, chromogenic sril>strutes.211-”ti activities for lactosylceraniide p-u-galactosid~isein these two disord e r s w e r e q u i t e controversial f o r h ( > i l ) t > time. The results o1)tained in Suzuki’s lalmratory showed tliat tlie activity of li~ctos).lceratiiiclep-Dgalactosidase was relatively normal in t h e liver of ii patient with Kra1)lie’s disease, b u t this acti\.it>*\v;is only 12% of noriiial in the liver of patients with GM l-gaiigliosidosis.””i On the other hand, Wenger aiitl coworkers reported that the s a n e enzyme was normal in t h e b r i i i i i a i i d t h e liver of a patient with GhI1gangliosidosis, 1)ut deficient in tissues of patients with Krabbe’s disease . Tanaka i in cl cow orke is”I ’ ti t rth e r i lives ti gate (1 this p rol)1e 111 1) y using an acidic P-D-galactosicl~ist. purified 250-fold froni hunian liver, and found that t h e discrepancy \v;is diie to t h e differences iii t h e system used in t h e two 1al)oratoric.s. HI‘ Suzuki’s assay s>,sten patic, acidic P-D-galactosidase w;is ecliially active towards synthetic sul)strates, rjMl-gangliosidc., I;ictosI.lcerarnicle, antl asialo C h f l . However, it was inactive towxcl.; I;ictosylcerainide b y Wenger’s assay system. Careful investigatioiis oi I thc variables that affected P-~-giiliictosidase activities revealed th;tt Wenger’s a iy conditions determined the lactosylceraniide clea\,iiig activity (1 tosylcerainidase I ) of gal acto s y lce rain i de p-D- galactc)s i t ;ise , whereas siiz u k i ’s method was optimal for lactosylceramide 1. clrol yzing activit). (lactosylceramitlase I I) b y GM l-P-D-galactosidase.~“~,~i~’ Wenger’s indicated t l i a t human-brain luctosylceramitl~iseI was identical to galactosylcc,r~iiiiidep-D-gahctosidase activity, and both activities w e r e deficient i n all tissues tested froni patients with Kralibe’s disease. Tanaka and SiiziikiZ19also provided evidence for two genetically distinctive, acid P-u-galactosidases, Iloth present in liver antl brain. One of t h e ~ - o - g ; i l ~ i c t o s i d a s eiss deficient i n tissues froni patients with Krabbe’s discase, and the other is inissing in pa-

-

(213) K. Suzuki and Y. Suzuki, Pro(.. ,Vcit/. : I ( ~ i t lSci. . t (214) S. Okada antl J . S. O’Brien, S(,ic,frc,c,, 160 (1968) 1 (215) H. R. Sloan, B. W. Uhlcwrlorf; C:. 13. lacolj\on, antl D. S. Fretirickson, Pctlicltr. Res., 3 (1969) 532-537. (216) Y. Suzuki and K. Suzuki,J. B i o l . ( : / t ( , ) t t . , 249 (1974) 2105-2108. . Actcl, 56 (1974) (217) D. A. Wenger, M. Sattler, C. Clni-h. i u i t l H . h l c K e l v e y , C l i ~ tC‘Itini. 199- 206. (218) H . Tanaka, X I . hleisler, and K. Suzriki, Hiochini. Bio])/ul,s.Actu, 398 (1975) 452463. (219) H . Tanaka and K. Soziiki, Arcli. H i o c h c , r j i . B i o ) h y s . , 175 (1976) 332-340.

276

YU-TEH L1 .4ND SU-CHEN LI

tieiits with GM 1-gangliosidosis. Callahan and Gerrie’2‘J*22’ purified the major P-D-galactosidase from rabbit tx-ain over 400-fold, and reported that they were unable to resolve activities toward GM 1, asialo-GM 1, lactosylceramide, and synthetic substrates. Their data suggested that a single P-D-galactosidase hydrolyzed all three substrates tested. This P-galactosidase was inactive towards galactosylceraiiiide. Miller and coworke purified an acidic p-D-galactosidase from human liver to apparent homogeneity by using affinity chroniatography on an immobilized 1-thio-P-D-galactopyranoside; this preparation showed a single protein band h y disc-gel electrophoresis at pH 8.3. Their studies with this pure p-D-galactosidase revealed that 4-metliyluinbelliferyl P-D-galactosidase and GMl-P-D-galactosidase are identical to lactosylceramidase 11. Ben-Yoseph and separated, into four forms having different molecular weights, the g.‘1 1actocerebrosidase activity of the brain and the liver from norinal individuals and patients with Krabl)e’s disease. They found that the form of high molecular weight possessed higher specific activity towards the natural substrate, and suggested that the enzyme of high molecular weight is the active form in oi2;o. On the other hand, Nishimura and AniaiioTZ4 isolated from porcine thymus a P-D-galactosidase specific for the hydrolysis of lactosylceramide, and they purified it over 3600-fold; this enzyme was not able to hydrolyze GM 1-ganglioside or galactosylceramide. The lactosylceraiiiide-specific P-D-galactosidase has not been detected in other tissues. Depending on the assay conditions, the i n uitro activity of P - ~ - g a lactosidase, as well a s other glycosidases, towards glycolipid substrates varies significantly. It is essential to consider very carefully the fol 1owing factors when exam in in g the specificities of P-galact o sidas e s toward natural substrates: the presence of pure or crude taurodeoxycholate, the presence or absence of oleic acid, the substrate concentration, the nature of the buffer used, and the effect of chloride arid other ions.

4. Catabolism of GM2, Asialo GM2, and Globotetraosylceramide are the best known GM2-Ganglioside and glo~)otetraosylceral1?~sylceraniide glycosphingolipids that contain B-D-linked 2-acetainido-2-deoxy-~-galactose. Owing to the accumulation of GM2-ganglioside (Tay-Sachs (220) J. W. Callahan and J. Gerrie, Biochirii. B i ( ~ p h ~Actci, , ~ . 391 (1975) 141-153. (221) J. W. Callahan and J. Gel-rie, Cuit. J. Hiocliem., 54 (1976) 803-812. (222) A . L. Miller, R. G . Frost, a i d J. S . O’Rrien, Biocherii. j . , 165 (1977) 591-594. (223) Y. Ben-Yoseph, hl.. Hungerford, i t n d M. L. Nadler, Rioclieni. j , , 189 (1980) 9- 15. (224) K. Nishimura and R. Amarro, J . Hioclwrri. ( T o k y o ) , 80 (1976) 209-215.

BIOSYNTHESIS A N D CATAROl,lS,bl OF CI,YCC)bPIIIN~;OL,IPII~S 277

ganglioside) in various types 01 a y - S x l i s disease , the cat a1) o 1i s I I I of this gaiiglioside has been a suliject of’ interest to mmiy Globotetraosylceraiiiide (gloliositle) was one of the first neutral gl?,cosphingolipids isolated from ei->dirocytes5;it also accuinulutes i i i the visceral organs of patients with type 2 Tay-Sachs tlisewse (Sandhoff’s disease) .226 I n 1968, Robinson arid Stirliiig227clescribed the presence of two p-Dhexosaiiiinidase isozymes (p-lieaosaininidase A and B) iit human spleen. Soon thereafter, Okada a i i t l O’Brien,228Hriltl,erg,229mid S a n d hoff2:”’found that p-hexosaminitiusc.~A isozyme was missing from the tissues of classical Tay-Sachs paticiits. These two findings constitute the two most important events ill ititlocking the etiology of Tay-Sachs disease. The quest to understancl this disease has provided a d d c d impetus to investigations of the dc.gradatioii of hesosaniine-coirt~~iiiiiig glycosphingolipids b y p-hexosami tiidases. Numerous reports on the isohtion and characterization of P-hexosaininidases have now appeared.’:” IIowever, irrost of the work was carried out b y using synthetic sul)strates, thus providing very little information coiiceriiing the specificit?. of this enzynie towards hexosaiiiine-coiitainirig glycospliiirgolipids. The enzyme h a s I ~ e n ~L6’”’wx’’’and purified to appare iit homoge n e i t y fro I11 I i timan 111 ace 11t from human 1 i ~ e r . I The ’ ~ study 011 t h e hydrolysis ot‘ GM2-gaiiglioside was initially conducted by using crudc, or pai-tiully purified, enz)wie preparations 188.294.ZS5 in the prescticc~of bile salts. Tnllinan and Brad?.’”) showed a significant hydrolysis of’C;hl2-ganglioside b y a crude, lysosoma1 preparation obtained froiii rat hrain without the aid of detergents. Tallinaii and coworkers”:”;tiii-ther showed that I)oth hesosaminidase A and B isolated fi-on) hiitnan placenta could llytlrolyze (225) J . S. O’Brien. in J. B. Stanliury, J . 13. \V!-tigaarden, mtl 1). S . FretlricLwii ( E d . ) , 7 ’ 1 1 ~lZfet(ibo/icBnsis of Iriheritcd />i\c,u\c, hicCra\%,-fjill, N e w Y o A , l%8. pp. 841-865. (226) K. Sandhoff, h l . Andreae, antl 11. J . t t L k ( S w i t z , Lifv S(.i., 7 (1968) 2K3-288. (227) I). Robinson and J. L. Stirling, H i o < , / i ( , r i i . / , . 107 (1968) 321-327. (228) S. Okada and J. S. O’Brien, Scic,rlr,c,. 16.5 (1969) 698-700. (229) H. Hultberg, Z,ciricef, (1969) 1195. (230) K. Sandhoff,F E H S Lett., 4 (1969) ,151-354. (231) H. 11. Flowers m d N. Sliiirnii,.-I~/i~. K r i z ! / t i i o l . , 48 (1978, 29-95, (232) J . E. Lee arid A . Yoshida, Bioc:lic,rii. J , 151-)(1976) 535-539. (233) A . Hasilik and E. F. N e u f e l d J H i o l . C ~ I W I 255 . , i 1980) 4037-4945. (234) E. H. Kolodny, K. 0. Brady, antl H . W. \.elk, /3ioc/wrtt.H ~ ~ J / J / LH! /c,. 5\ ,. ( : o r i i r r i i i r i . . :37 (1969) 526-531. (23.5) K. Sandhoff, F E B S Lett., 11 (1970) :31.’-344. (236) J . F. Tallman, H. 0. Brady, J. h l . Q i t i i k , h i . Villall)a, and :I. E. C ; a l , J . Riol. C / t c , r i t . , 249 (1974) 3489-3499.

278

YU-TEH LI AN11 SU-CHEN LI

GM2-ganglioside at the same rate, whereas p-hexosaiiiiiiid~,seB was the more active towards asialo GM2. Srivastava and coworker^'^' reported that a crude extract of human placenta could hydrolyze GM2; however, no activity toward GM2 was detected in the purified hexosaminidase A or B. Seyaina and Yamakawi,2:i7 studied p-liexosaminidases from equine kidney; they found that the oligosaccharicle o1)tained from GM2 was hydrolyzed only by isozyine A, and not by B, but the oligosaccharide derived from asialo GM2 or globotetraosylcerami[le w a s hydrolyzed b y both isozyines. The rates of hydrolysis of these oligosaccharides were very low, only about one hundredth of that of the hydrolysis of synthetic substrates. Li and c o ~ o r k e r s ~reported ~ ~ . ~ :that ~ ~ isozyme A, but not B, hydrolyzed GM2 in the presence of a protein activator. Rach and S u z ~ k ialso ~~'~~ reported that asialo Gh42 and globotetraosylceramide were hydrolyzed b y both isozymes, whereas G M 2 was hydrolyzed only b y P-hexo s am i 11i d a s e A. In 1977, Sandhoff and coworkers17xstudied the substrate specificities of these two isozyines purified to apparent homogeneity from huiiian liver. They reported that, in the presence of sodium taurodeoxycholate, the efficiency for isozyine B in hydrolyzing GM2 was only 12% of that of isozyine A. Both isozyme A and B readily hydrolyzed asialo GM2; however, isozyme B worked three times faster than isozyine A. Despite soiiie controversy, it is generally considered that phexosaminidase A is the enzyme responsible for the hydrolysis of GM2. As pointed out b y Li and coworkers,'-"' the hydrolysis ofGM2 by p-liexosaniiiiid~,se A in citro was extremely sensitive to the ionic strength of the medium, whereas the hydrolysis of asialo GM2, globotetraosylceramide, aiid synthetic sullstrates was not affected. This m a y explain why the results ol,tained in various laboratories for the enzymic hydrolysis of GM2 are so different from one another. Hasilik and Neufeld2":'detected the precursors for both a- and p-chain subunits of p- hexos am i n i das e in t h e intact , h 11 rn an d ipl oid-s ki 11, fi brob I as t cu 1ture. These precursors were subsequently converted, to produce the intermediate chain, aiid a- or p-sulmiiit chains. Fibroblasts from patients with classical Tay-Sachs disease contained only p-precursor, p-

(237) Y. S e y a m a and 1'.Y;iin;iknwu,J. Z ~ ~ O C / I C ? J I .('I'okyo), 75 (1974) 495-507. (238) Y.-T. Li, M . Y. hlazzotta, C. C . Wan, H. O r t h , and S.-C. Li,J. B i d . C h e n t . , 248 (1973) 7512-7515. (239) G. Bach and K. Suzuki,J. Bio/,C/ien1.,250 (1975) 1328- 1332. (240) S . C . Li, T. Nakamiira, A . O g a m o , iind Y.-T. L i , / , H i o 2 . Chenz., 254 (1979) 10,59210,595.

BIOSYNTHESIS AND CATABOLISM OF GLYCOSPHINGOLIP!DS

279

interniediates, and p-chain; cells from a patient with Sandhoff" disease contained only a-precursor and a-chain; and cells from a patient with the AB variant had the norind pattern of precursor and processed a- and @-chains.It would be interesting to examine the activity of the proenzyme, if there is any, towards the hydrolysis of various hexosamine-containing glycosphingolipids. The available evidence shows that both p-hexosaminidase A and €3 can hydrolyze globotetraosylcerainide and asialo GM2. The reason why only isozynie A can hydrolyze GM2 remains to be explained. In contrast to glycosphingolipids containing 2-acetamido-2-deoxy-Dgalactose, virtually nothing is known allout the catabolism of glycosphingolipids containing 2-acetaiiiitlo-2-deoxy-~-glucose.

5. Catabolism of Glycosphingolipids Containing a-Linked D-Galactose

The existence of glycosphingolipids containing a-D-galactosyl units was recognized through the study of Fabry's disease, an X-linked glycosphingolipidosis characterized by the accumulation of glycosphingolipids containing a-D-galactosyl residues. The major glycosphingolipid accumulated in the kidney of a patient with Fabry's disease was first isolated by Sweeley and K l i o ~ i s k yin~ ~ 1963, and characterized a s being a triglycosylceramide, Gal(1 + 4)Ga1(1 +4)Glc -+ Cer. Through the work of many laboratories, this glycolipid was found to contain an a-linked D-galactosyl group at the (nonreducing) ter~iiinal.'~;,""'?':~In addition to globotriaosylceramide, tissues of Fabry's patients also accumulate several glycolipids coiitaining terminal a-D-galactosyl unit^."^ By using synthetic suhtrates, K i n F was the first to show a deficiency of a-D-galactositfasc i n the leukocytes of patients with Fabry's disease. In normal, h u i i i m tissues, two isozymes, namely, aD-galactosidase A and a-D-gal~~ctosidase B, have been detected b y using synthetic sul,strates.2.'fi,2.'iThe role of a-D-galactosidase A and B

(241) J. T. H. Clarke, L. S. Wolfe, arid .4.S. PerIin,/. Biol.Chetii., 246 (1971) 5 5 6 3 3 5 6 9 . (242) S. Handa, T . Ariga, T. Miyatakr, a i r t l T. Yarrl;ikawa,/. A i d ~ e l l t (Tok!yo), . 69 i 1971) 625-627. (243) Y.-T. Li and S.-C. Li,J. A i d . C : h c , r i r . , 246 (1971) 3769-3771. (244) R. J. Desnick. B. Klionsky, ant1 (:. C . Sweeley, in Ref. 225, pp. 810-840. (245) J. A. Kint, Science, 167 (1970) 1268-1269. (246) E. Beutler and W. Kuhl,/. Lnh. (,'/in.Metl., 78 (1971) 987. (247) J . A. Kint, Arch. Znt. Ph!lsio/.H i o d r i i u . , 79 (1971) 633-634.

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YU-TEH LI A N D S U - C H E N LI

in the pathogenesis of Fabry's disease has been the subject of intensive study in several l a b o r a t o r i e ~ . ' ~ ~ Immunological , ~ ~ ~ - ~ ~ ~ studies showed that there was no cross-reactivity of antisera made against the a-D-galactosidase two i s o z y ~ n e s .The ~ ~ main ~ ~ ~difference ~ ' ~ ~ ~ between ~ A and B was finally revealed by Shrani arid coworker^"^ and Dean and coworkers,z56who independently identified a-D-galactosidase B from Kusiak and coworkhuman liver as a-N-acetylgalactosaminidase.25335ti e r also ~ reported ~ ~ that ~ a-D-galactosidase B from human placenta hydrolyzed a 2-acetamido-2-deoxy-a-~-galactoside.Thus, human a-Nacetylgalactosaininidase hydrolyzes both 2-acetamido-2-deoxy-a-~galactosides and a-D-galactosides. The a-N-acetylgalactosaniinidasei solated from the limpet was also found to catalyze the hydrolysis of a - D - g a l a c t o ~ i d e s .Dean ~ ~ ~ and S ~ e e l e y " ~showed that highly purified a-D-galactosidase A from human liver catalyzed the hydrolysis of globotriaosylcerainide and galactobiosylcerainide in the presence of sodium taurocholate. From the fact of the missing enzyme, and the accuniulation of glycosphingolipids containing a-D-galactosyl units, in Fabry's disease, there is little doubt that a-D-galactosidase A is responsible for the catabolism of alinked D-galactosyl groups in glycosphingolipids, and that a-D-galactosidase B is inactive toward these glycolipids. However, a-D-galactosidase B (a-N-acetylgalactosaminidase) was found to hydrolyze globotriaosylceramide i n ~ i t r - 0demonstrating ,~~~ that the results of in uitro studies do not always reflect the reaction taking place i n vivo.

(248) P. J. C . M. Rietra, F. A . J. T. M . Van D e n Bergh, and J. M . Tager, Clin. Chint. Actu, 62 (1975) 401-413. (249) W. G. Johnson a n d R. 0. Bradp, Methods Enzyrnol., 28 (1972) 849-856. (250) C. Romeo, C . DiMatteo, M. D'Urso, S.-C. Li, a n d Y.-T. Li, Biochim. Biophys. Actu, 391 (1975) 349-360. (251) P. J. C . M . Rietra, J . L. Molenaar, M.N . Hamers, J. M. Tager, a n d P. Borst, Eur-.J. Biochem., 46 (1974) 89-98. (252) A. W. Schram, M. N . Hamers, B. Brouwer-Kelder, W. E. Donker-Koopnian, a n d J. M. Tager, Biochim. Biophys. Actci, 482 (1977) 125- 137. (253) A. W. Schram, M. W. Hamers, a n d J. M. Tager, Biochinz. Biophys. Actu, 482 (1977) 138- 144. (254) K. J. D e a n a n d C . C. Sweeley,.\. B i o l . Chem., 254 (1979) 9994-10,000. (255) K. J . D e a n a n d C . C. Sweeley,J. B i d . Chent., 254 (1979) 10,001-10,005. (256) K. J. Dean, J . S.-S. Sung, and C . C. Sweeley, Biocheni. Biophys. Res. Contmun., 77 (1977) 1411-1417. (257) J. W. Kusiak, J. M. Quirk, a n d H. 0. Brady,J. B i d . Cheni., 253 (1978) 184-190. (258) Y. Uda, S.-C. Li, Y.-T. Li, a n d J. M . McKibbinJ. B i d . Cheni.,252 (1977) 51945200.

6. Protein Activators for the Enzymic Hydrolysis of Glycosphingolipids One of the most exciting developnients in the field of glycosphingolipid catabolism was the discovvry of protein activators that stimulate the enzymic hydrolysis of glycos~,liiiigolipids. Because of the hydrophobic nature of glycospliingoli~~itls, the enzymic hydrolysis of t h e s e molecules in uitr-o is greatly enli~uicedljy the d d i t i o n of such detergents as sodium taurodeolcycholate""" or Cutscuin'"" [isooctylphenoxypoly(oxyethanol)] in the reaction mixture. As these detergents do not e x i s t naturally i n oiuo, it is co1iceiv;il)le that enzymic h ~ d r o l y s i sof glycosphingolipids i n uico is stiiiiiilatcd I)y substances other than detergent s. Tal lrnan an tl B ~-ady'"~ ( )I) s e r v e d s i gni ficun t 11 y d r o 1y s is of C; h l 2 b y rat-brain, lysosomal preparations, hiit not with the purified ,B-hexosaminidase A. The Lis and coworkers2:IX found that crude liver glycosidase preparations could hydro1yztb gl ycosphingolipids better than the purified enzymes. These observations strongly siiggest the existence of naturally occurring, stimulating tiictors, or activators, for the hydrolysis ofglycosphingolipids. The progwss on the work concerning activator proteins was reviewed by B r a t l ~ ~ 2 'i "n 1978. Activators for the reactions catalyzed b y the followiiig euzymes have lieen descriljed: galactosylceramide sulfate srilflitase, glucosylceraiiiide p-D-glucosidase, GM l-P-D-galactosidase, a i i d ,B-il'-acetylhexosan~iiIi~l~ise A for the hydrolysis of GM2. The activator for galactosylc~,rainidesulfate siilfatase was first cletected by Mehl and Jatzkewitz.2f"2 This is the earliest description of an activator for the enzymic hydrolysis of glycosphingolipicls. Fischer and Jatzkewitz""" subsequently isolated this activator, and characterized it as a protein having an apparent molecular weight of 21,500. They further showed that the activator was localized i n lysosoiiies,264 and suggested that the hydrolysi\ of glycosphiiigolipids takes place in these Their studies on the mode of action ofthe activator

(259) S. Gatt,J.Bio/. Clzern., 238 (1963) ~ ~ 1 3 1 - ~ ~ 1 3 3 . (260) H. 0. Brady, J . N. Kanfer, and D. Shapiro,,\. B i d . C / i c i i i . , 240 (1965) 39-4:3. (261) K . 0. Brady, Aiiriu. Rec. B ~ O ~ W J17 I I .(1978) , 687-713.

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YU-‘TEH LI AND SU-CHEN LI

indicated that “activator- lipid substrate complexes” were the “true substrate” for the siilfiitase.266They failed to detect association of the activator with the enzyme. The activator for the hydrolysis of galactosylceramide sulfate was also found to stimulate the hydrolysis of psychosine sulfate, and seminolipid and its deacylated derivative (lysose~ninolipid).~~’ The activator for the hydrolysis of glucocerebroside was first demonstrated by Ho and c ~ w o r k e r swho , ~ ~found ~ ~ ~that ~ ~the spleen of a patient with adult G a ~ i c h e r ’disease ~ contained a heat-stable, glycoprotein factor that stimulated the hydrolysis of glucocerebroside carried out b y p-D-glucosidase. The glucocerebrosidase activity was at least 70-80 times higher i n the presence than in the absence of the activator.26gSimilar activators were isolated, and characterized, from normal and Gaucher’s spleen b y Peters and coworker^,^^" who found that the activator from norinal spleen differs from that isolated from Gaucher’s spleen in regard to amino acid composition and sugar composition, and in activating capacity. The activator isolated from Gaucher’s spleen was also found to lie capable of stimulating the hydrolysis of GM1-ganglioside catalyzed by human-liver P-D-galactosidase.”] The specificity of this activator remains to b e clarified. The subcellular localization of the activating factor for glucosylceramide P-D-glucosidase was studied b y Chiao and coworkers,27’using sucrose-gradient centrifugation. Electron-microscope examination of fractions from the sucrose gradient demonstrated that the activator was located in the fraction rich in acid phosphatase (lysosomal), which also contained the characteristic Gaucher deposits. Kanfer and coworkers2is reported that a glycopeptide isolated from the spleen of a Gaucher’s patient could stimulate the hydrolysis of glucosylceramitle, as well as the trans-D-glucosylation from 4-inethylumbelliferyl D-glu-

(266) G . Fischer aiid H. Jwtzkewitz, Hiocliirtt. B i o p / i ! p . Actci, 481 (1977) 561-572. (267) G. Fischer, S. Reitei-, and H. Jatzkewitz, Ho/)pe-Se!/[er’.s Z . Phg/siol. Clzern.. 359 (1978) 863-866. (268) M. W. Ho and J. S. O’Brien, Proc. N ( i t l . Accid. Sci. U.S.A., 68 (1971) 2810-2813. (269) M. W. Ho, J. S. O’Brien, N . S. Hadin, and J. S. Ericksoil, Biochei,~.1.. 131 (1973) 173-176. (270) S. P. Peters, P. Coyle, C. J. Coffee, R. H. Glew, M. S . Kuhlenschmidt, L. Rosenfeltl, and Y. C. Lee,]. H i o l . L‘Iwnt., 252 (1977)563-573. (271) S. P. Peters, C . J. Coffee, H. H. Glew, R. E . Lee, D. A. Wenger, S.-C. Li, and Y.-T. Li, Arch. Biocheni. Bioph!/s., 183 (1977) 290-297. (272) Y. B. Chiao, J . P. Chambers, H. H. Glew, R. E. Lee, and D. A . Weriger,Arch. Biod i e m . Biophy.r.., 186 (1978)42-51. (273) J. N . Kanfer, S. S. Raghavan, and R. A. .Ilumford, Biochiin. Biop/ig/s. Actci, 391 (1975) 129- 140.

HIOSYNTHESIS A N D CA‘TAHOI,ISnLl OF GI,YCOSPHINC:OI,IPIUS

283

coside to ceramide, to form filucosylceraiiiide, catalyzed b y the same 0-glucosidase. Whether or iiot this glycopeptide activator is related to other activators for g1ucocerel)losicI~~se remains to lie determined. ~’~ ;I “coglricosidase” for glucocere1,rosidase Berent and R a d i ~ i isolated from bovine spleen. They reportod that this protein was similar to the u-filucosidase-stiiiiulatiiig proteins isolated from Chucher and iiorinal huiiian-sl?leens. This protein h a s i i rnolecular weight of 20,000. ‘The activity was completely destroycd 1)). pronase, ant1 yartially destroyed b y sialidase or periodate. Hanada and S ~ z i i k i reportcd ‘~~ that phosphatidq~lserinefrom l)o\ine brain was a relatively specific activator for human galactosylceramide P-D-galactosidase in the absenct. of sodium taurocholate. Previously, no effective activator had lxen reported for t h i s enzyme, except the 1ow - i n 0 1ecul ar-we ight, 1y s o s o ilia 1 g 1y coprote in, w h ich showed ( ) n 1y slight activating effect on galactosylcer~~micle hydrolysis.”“ in ii preparaAnother activator was reportcd I)y I,i and coworkersL:iX tion obtained from a crude, hiiiiimi-liver extract. This activator was heat-stable and nondialyzable. Siil,sequently, they found that this activator stimulated the hydrolysis of GM 1 b y P-D-galactosidase, GM2 b y P-N-acetylhexosainiiiidase A, and glol)otriaos),lcer~iiiii~le I)y cu-u-galactosidase.z12They further isolated, and characterized, this activator a s a glycoproteiii having a moleciilnr weightIi6 of21,000. After careful examination of the specificit). of the activator, they found that this activator worked best for the hyclrolysis of GM1, but, to a lesser extent, also stimulated the hydrolysis of g1ol)otriaosylceramide and GM2. After cross-examining the spc.cificities of various activators, they found that this activator w a s ver), siniilar, if not identical, to the activator for galactosylceramide sultiite sulf:,itase,but different from that for glucosylceramide P-D-glucositlase. ’The former two did not stimulate the hydrolysis of synthetic suhtrates, and the latter stimulated the h y drolysis of 4-methylumbelliferyl glucoside. The activator for the hydrolysis of Gh12 catalyzed b y p-hexosaminidase A was described by Hechtrnan”’ and Hechtinan and L e B l a ~ i c . ” ~ The GM2-specific activator was piirified over 100-fold froin huinan liver, and was identified a s a 1ieat-lal)ile protein. This activator did not stimulate the hydrolysis of asialo-Ch12 catalyzed either b y hexosaniinidiise A or B. T h e molecular wc,ight of this activator was determined to (274) S. Berent ant1 N . S. Ratlin, Fctl. PI.OC..,39 (1980)2185. . 57.5 (197‘3)410-420 (275) E. Hanada and K. Suzuki, B i ~ i d i i i i /~~. i o / i / i y s Actu, (276) H. Jatzkewitz, in Ref. 195, pp. 5131-571. (277) P. Hechtman, Con. J . Bioche?r~., 5.5 (1977) 315-324. (278) P. Hechtnian m d D. LeBlanc, R i o c ~ / ~ t ,./., ~ t ~167 . (1977) 693-701.

284

YU-TEH LI AND SU-CHEN LI

be 36,000 to 39,000. Conzelinann and S a i ~ d h o f fand ~ ~ ~HechtmaiPO presented evidence to show that the stimulating factor for the hydrolysis of GM2 was defective in the Type-AB GM2-gangliosidosis. However, Li and coworkerszX'examined two cases of Type-AB GM2gangliosidosis,2xzand found that one was deficient in the activator and the other had an elevated level of the activator. The latter case constitutes a new variant of Type-AB GM2-gangliosidosis which was not due to the deficiency of the activator,2x3but to a defect in phexosaminidase A. Conzelmann ancl Sandhoff 2H4 purified the activator protein for the degradation of GM2 and asialo GM2 b y hexosaminidase A 2500-fold from noimal human kidney. This activator was heatstable up to 60" and its inolecular weight was found to be 25,000. They suggested that this activator formed with lipid sulxtrate a complex that facilitated enzyme-substrate interaction. Interestingly, the activator isolated from human liver b y Hechtman and LeBlancz7#did not stirnulate the hydrolysis of asialo GM2, whereas the activator isolated from human kidney by Conzelinann and SandhoffZx4 stimulated the hydrolysis of asialo GM2 better than GM2. Li and coworkers also purified a similar activator from hurnan liver'HH" and brain2*' over 105-fold. They found that the activators from these two sources were identical in their specificities, and physical, chemical, and immunological properties. This activator is very specific for stimulating the hydrolysis of GM2, but only slightly effective in stimulating the hydrolysis of asialo GM2 or globotetraosylceramide catalyzed by p-hexosaminidase A. This activator does not stimulate the hydrolysis of the aforementioned three glycosphingolipids catalyzed by p-hexosaminidase B. The concept of the catabolism of glycosphingolipids by glycohydrolases has entered a new era with the discovery of protein activators, but there are many questions remaining to be answered. What is the physiological role of the activators? What is the mode of their action'?

-

(279) E. Conzelmann and K. Sandhoff, Pi-oc. h'citl.Accitl. Sci. U.S.A., 75 (1978) 39793983. (280) P. Hechtinan, Annu. ,%feet.A m . Soc. H u m . Genet., 31 (1980) 42A. (281) Y.-T. Li, Y. Hirabayashi, and S.-C. Li, in T. Yainakawa, T. Osawa, and S. Handa (Eds.),Glqcoconjug!cites, Proc. Zut. Syiiip. Gl!/eoco,ijugntes,6th, Japan Scientific Societies Press, Tokyo, 1981, pp. 48-49. (282) J. E. Goldman, T. Yainanaka, I. Rapin, M. Adachi, K. Siizuki, and K. Suzuki, Actu Netiropcrthol., 52 (1980) 189-202. (283) S . C . Li, Y. Hirabayashi, a n d Y.-T. Li, Riochem. Aioplu/.r.. R e s . Corrimut1,, 101 (1981) 479-485. (284) E. Conzelmann and K. Sandhoff, Hoppe-Seyler'.~ Z. Physiol. C h e m . , 360 (1979) 1837-1849. (285) S.-C. Li, Y. Hirabayashi, and Y.-T. Li,J. Biol. Cheiii., 256 (1981) 6234-6240.

RIOSYNTHESIS AND CATAH01,IShl OF GLYCOSPHINGOLIPIDS

285

Does each glycosidase require ii specific activator, or can one activator serve for the hydrolysis of several glycosphingolipicls? Li and coworkers240 educed evidence for the presence of two separate, protein activators for the enzymic hydrolysis of GM1 aiid GM2 gangliosides in norinal, human liver; they also suggested the presence of an inhibitor which somehow inhibited the hydrolysis of GM2 b y p-hexosaminidase A in the presence of GM2-specific activator.”” The nature of this inhibitor remains to be characterized. These results suggest that the catabolism of glycosphingolipids is more than simply a series of reactions iiivolving just glycosidases a i l t i glycolipid substrates. Complete delineation of the biological roles of protein activators will lead to a better understanding of the catal~olisiiiof glycosphingolipids, as well as of the pathogeneses of sphiiigolipidoses.

7. Concluding Remarks The accumulation of partially degruded, complex carbohydrates in a variety of storage diseases has druwii attention to the roles of glycosidases in the catabolism of glycocoii~ngates.However, very little is known about the specificities of various glycosidases towards different glycoconjugates. From the nature of accumulated glycoconjugates in various storage-diseases, it appears that some glycosidases may catabolize both glycoproteins and glycosphingolipids, whereas some may catabolize only glycosphingolipids, or only glycoproteins. For example, the deficiency of acidic p-D-galactosidase results in the accumulation of both GM 1-ganglioside and oligosaccharides derived from glycoproteiiis that contain p-linked teiiiiinal D-galactosyl On the other hand, patients with sialidosis accumulate only sialosyloligosaccharides derived from g l y c o p r o t e i n ~ . ~ ~ ~ The conventional nomenclatiire for exo-glycosidases is based on their specificities towards the glycoiis, but this kind of n o m e i d * d t llre is sometimes very misleading, Iwcause glycosidases of the sanie category (for example, a-D-galactosidnse or P-D-galactosidase), isolated froin different sources, frequently differ considerably in their specificity towards different substrates. The nature of the aglycon and the linkage can also affect the rate of hytlrolysis. It is also noteworthy that some glycosidases exhibit niultiple specificities. For example, P-Nacetylhexosaminidase hydrolyzes the p-linked 2-acetaniido-2-deoxyD-glucosides and -galactositles, and a-N-acetylgalactosaminidase hydrolyzes 2-acetamido-2-deox~-~-~-g~ilactosides and n-D-galactO(286) K. Komfeld and S. Komfeld, A t i i i u . He(;. Hiochem., 45 (1976) 217-237 (287) J. A. Lowden and J. S. O’Brien, Am /, llrini. Genet., 31 (1979) 1-18.

286

YU-TEH L1 A N D S U - C H E N LI

sides. Therefore, the results of i l l 2;itro degradation of sugar chains in glycosphingolipids should be interpreted with caution. So far, much attention has been focused on the catabolism of those glycospliiiigolipids iiitiniately related to varioiis lipid-storage diseases. The available evidence indicates that gangliositles having ganglio-series sugarchains are iiiainly catabolized through the scheme originally proposed by Leibovitz and Gutt (see Section 111,.2).It may he safe to assume that globotetraosylceraniide is catabolized through the stepwise removal ofthe glycosyl group froni the nonreducing eiid ofthe sugar chain b y a series of exo-glycosidases. Virtually nothing is known aliout the catabolism of such other glycosphingolipids a s glycosphingolipids of the lacto series, mannose-contaiiiiiig glycosphingolipids, or polyglycosylceramides. We know very little about the control and regulation of the catalm lism of glycosphingolipids. The discovery of protein activators that stimulate tlie hydrolysis of glycosphingolipids is one of the impoitant developments in this field. The fact that the hydrolysis of glycosphingolipids is fhcilitated b y the presence of protein activators suggests that the catabolism of glycosphingolipids is more complex than had been recognized. It is well known that, in many cases, the deficiency of one lysosomal glycosidase is accompanied liy the increase of' other glycosidase activities. For example, in the liver of patients with fucosiclosis, the activities of glycosidases, except a-L-fucosidase, are several times higher than that of the control.zxxDespite this increase, no excessive degradation of glycocoiijugates has been demonstrated; this strongly indicates that tlie catal)olism of glycocoiijugates is not controlled by glycosidases alone. Future research on the roles of activators will contribute to fundainental understanding of the catabolism of glycosphingolipids.

ACKNOWLEDGMENTS W e thank Dr. James E. Miildrey of T u l a n e University for his valuable advice a n d comments d u r i n g t h e preparation of this article. Thanks a r e also d u e Dr. William H. Baricos of T u l a n e University for reading t h e inairiiscript. W e express o u r appreciation to Mrs. Sharon Nastavi and Mrs. Mary Soike tor their assistance in typing a n d editing.

(288) F. Van Hoof, in Ref. 1.58, pp. 277-290.

THE LIPID PATHWAY OF PROTEIN GLYCOSYLATION AND ITS INHIBITORS: THE BIOLOGICAL SIGNIFICANCE OF PROTEIN-BOUND CARBOHYDRATES

B Y RALPH

T.

SCHWAHZ AND

ROELF DATEMA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 . . . . . . . . . . . . . . . . . . . 288 11. Biosynthesis of Lipid-linked 01igosaccharidcxs 1 . T h e Synthesis of Dolichol and 1)olichol-link onosaccharicles . . . . . . . 288 2. Assembly of the Lipid-linketl Oligosaccharide, 299 and its Transfer to Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cytological ;md Topological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . 314 111. Inhibitors of Protein Glycosylatioir . . . . . . . . ..................... 321 1 . General Coinnients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 2. Inhibitors of Fonnation of Ilolic,hol Phosphate . . . . . . . . . . . . . . . . . . . . . 322 . . . 326 3. Inhibition by Sugar Analogs . . . . . . . . . . . . 4. Inhibition b y Antibiotic Substatices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 5. Other Inhibitors of Protein GIyco\yl;itioii . . . . . . . . . . . . . . . . . . . . . . . . . 344 IV. Biological Effects of Inhibition of Glycosylation . . . . . I . General Comments . . . . . . . . . . . . . . . . . . . . . . . . . 2. Effects on Conformation of Proteiiis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 3. Effects 011 Limited Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 4. Effects on “Routing,” Secretioir. I-lccognition, and Uptake of Glycoproteiir\ . . . . . . . . .. 5. Effects 011 Collagen and Protcogl\~c~:tns. . . . . . . . . . . . . . . . . . . . . . . . . . . 364 ti. Effects 0 1 1 Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 7. Effects 011 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 8. Effects 011 Iriterferon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 9. Effects on Other Cellular P h e ~ i o i i r ~ .~.~. i.~. t. . . . . . . . . . . . . . . . . . . . . . . 374

I. INTRODUCTION In glycopepticles, the oligosuccharides are linked to peptides b y two types of covalent linkage: (1 )The glycosidic type, and (2)the glycosylN linkage. The latter linkage type always occurs between GlcNAc and ASII, to give 2-aceta1niclo-l-N-(L-aspart-4-o~l)-2-deoxy-~-D-gluco“7

CoprrtKl,t @ 1882 I,) Acadenric P r c \ \ , Inc A I Lright, <,Ireprt,durtlm 111 dny L m r r t w w e t l . 1SRN 0- 12-(X17240-8

288

RALPH T. SCIIWAHZ A N D HOELF D A T E M A

pyranosylarnine (or “asparaginyl-N-acetylglucos~ii~iiii~”). The biosynthesis of the oligosaccharide linked to protein by glycosyl-N linkages, the inhibitors of this biosynthesis, and the biological consequences of inhibiting glycosylation of proteins are the subjects of this article. In the first Section, the dolichol pathway of protein glycosylation is introduced, and the reader is made familiar with the various reactions in the formation of the lipid and carbohydrate moieties of lipid-linked saccharides. Three different classes of compound are known so far: ( a ) isoprenoid alcohol esters of monosaccharide nionophosphates, such as D-mannosyl and D-glucosyl (dolichol phosphate), ( h )such isoprenoid alcohol esters of saccharide diphosphates as dolichol diphosphate linked to 2-acetaniido-2-deoxy-D-glucose and to oligosaccharides, and (c) retinol (D-mannosyl phosphate). The clolichol-linked sugars occur in all eukaryotes. In Section I1 (on the biosynthesis of oligosaccharides), emphasis is placed on the biosynthesis of the dolichol-linked sugars, because the inhibitors mainly interfere with steps in the dolichol cycle of protein glycosylation. Modification of the oligosaccharide side-chain, once it has been transferred from the carrier lipid to the protein, is not discussed in any great detail. The mechanisms of inhibition of glycosylation of proteins, and the metabolism of the inhibitors, are outlined in Section 111, and, in Section IV, the effects of widely used inhibitors in a variety of biological systems is discussed. The contribution of the inhibitors to an understanding of the role of carbohydrates in, for example, the conformation of proteins, secretion of glycoproteins from cells, the processing of protein precursors, and a number of biological functions, is indicated. Although the article concentrates on a discussion of inhibitors of glycosylation of proteins acting at the level of forniation of lipid-linked saccharides, compounds that interfere with stages after transfer of the oligosaccharides to the protein are also discussed. 11. BIOSYNTHESISOF LIPID-LINKED OLIGOSACCHARIDES

1. The Synthesis of Dolichol and Dolichol-linked Monosaccharides Dolichols are long-chain poly(isopreno1)s found in eukaryotes only, and consist of 13-22 isoprene units. There are two internal, trans-isoprene units, the other internal units being cis-isoprene residues.’ (1) F. W. Hemming, i n T. W. Coodwin (Ed.), Biochemistry o f Lipids, M T P Int. Rea. Sci.: Biochetn. Ser. One, 4 (1974) 39-98.

Characteristically, the a-isoprctic. unit is saturated, u n l i k e 1)acterial undecaprenol, in which it is t i t tsatiirated. The dolichol tilono- and tliphosphoric esters function a s gl! )s>.latedinteriiiediates’ in the s y n thesis of glycoproteins, keratati sril fate, cellulose, and yeast iiiiinniin, and that is why they are c1iscitssc.d Iierc~. The formation de noco of do1ichol phosphate is considered to produce the diphosphoric ester of tlrr, polypreno1 b y a patliwa), in which the initial steps are shared w i t h thc 1)iosynthesis of cliolesterolL.:l(see Scheme 1). The 4(S)-hydrogc.n atonr of 3,5-tlili1\ldroxy-3-methyI pentano-1,5-lactone (“mevaloiiatt.”) is rctainetl i n the synthesis of tlolichols4 and, after injection of rats with 4(S)-[”H]ine\ialoiiate, [3€II]dolichol was found, for example, i t t the liver. Incubatiori of subcellular fi-actions froin these livers with (:111~-[’4C]Ma~i gave a doii1)ly lahelled mannolipid,5 probably D-niantiosyl (dolichol phosphate). Furtherinore, the labe 11in g in c iuo of ( 1( ) 1i chc)1 d ipho s p hate -1 inked o 1 i go saccharide b y radiolabellecl mevalotiate h a s been clenio~istratetl.”~ The addition of cis-isoprene to 3,7,1l-tritiiethyl-2,Fi,l~~-dodecatl-ien).l(farnesyl) diphosphate should give ~,3-delr~drodolic.hol diphosphate (see Scheme 1). This conipound has, indecd, been isolatedx from supernatant fractions of chick liver iticiil,ated with f:miesyl diphosphate and isopentenyl diphosphate. The t~ionophosphoricester had previoiisly 1)een identified after incril)ating a particulate fraction from hen oviduct with these substrates.“ A partially purified pren~ltr;itisti.rast. froin Ehrlich tumor cells yielded 2,3-deliydroclolichol phospliate contaiiring 17- 19 isoprene units.“’ It is very likely that this c,onipoutid is foritiecl by the action, on the corre s pon (1 in g dipho s phate ,I I ( ) f a co ri tam ii I ati 11g pho s phata se it11 (1, therefore, the probable reactiot t seqiience is that phosphatase action precedes saturation of the cY-isol)rt.ne unit, as s h o w i ~in Scheine 1. The initial demonstration t h a t isopentenyl diphosphate ma!. he used i n the biosynthesis of c l o l icliol phosphate i n animals and plants (2) 4 . J. Parodi and L. F. Leloir, N i o c l t i t t t . B i o p / i ! / . s .f i c . t n , 559 (1979) 1 4 7 . (3) F. W. Heiriining, Biocherit. S o c 7 ’ r . ( t i i , \ . , 5 (1977) 1223-1231. (4) D. P. Cough a i i t l F. W. Heiniiiiiig, H i r ~ c ~ h c , t t i . / 118 ., (1070) l(i3-166. . 138 (1974) 281-289. (5) H. G . Martin antl K. J. I. Thorirc-. H i o c . l i c ~ t t r /., ( 6 ) \ l . J . Spiro, K. G . Spiro, ;ind V. 1). H h o y r o o , / . H i d C / i ~ , r t t , . 251 (1976) 6400-6408. H i o l . ~ ~ h ~ ~ i253 t t . (1978) , 5270-5273. (7) J. T. Mills antl A. bl. Atlaiiiaii. B S I , c , t / . , 104 (1979) 379-38.3. (8) R. R. Wellner and J . J. Lucas, (9) 13. K. Grange and W. L. Adair, N i o c . / t c , i t t . H i o p h ; / . ~ . Re.\. C o t n t t i u r t . , 79 (1977) 734740. (10) W . L. Adair and D. Trepanier,E’c~tl.Z’roc. Fed. Am. Soc. E:x)J. R i o l . , 39 (1980) Ahstr. 1551. (11) H.H . Wellriel- and J . J. Lucas, F”/.I’t-oc.. 1;c.d ilm. Soc. E s p . B i o l . , 39 (1980) ..\bstr. 1553.

RALPH T. SCHWARZ A N D ROELF DATEMA

290

Acelvl-CoA

I

Acetoacetyl-CoA

1 I

3 - Hydroxv- 3 -methyl glutaryl-CoA

Mevalonate

Geraiivl diphosphate (C,")

Fariiesyl diphosphatc (Cl.,) - - - - - - -

Cholesterol

t

2.3-Dehydlodoli[.h[)l diphosphate (Ca,-C,lo) Acyl ester of dolichol

t

I I

2.3-Dehydrodolie hol phosphate

uD/P-Man(D*,pDp

Dolic hol

Dolichol phosphate

i

Dolichol diphosphatc

4

UDP-GlcNAc

G l c - P-Do1

GlcNAc - PP- Do1 Man - P- Do1

, t I I

+

Glc, - M a n - (GlcNAc),- P P - Do1 polypeptide

PP-Dol-

Polypeptide- (GlcNAc),-Man,-Glc,

Scheme l.-Pathway of the Biosynthesis of Dolichol Phosphate and of Dolichollinked Saccharides. (Reactions requiring more than one enzyme are indicated by dashed arrows. T h e Scheme is based 011 work cited in Refs. 1, 3 , 8-25, 35, and 50.)

T H E LIP1D PAT I1 W A Y ( 1 t' PI{( )'lE I N C LYCO b YL 4TIO N

29 1

came fro i n Pon t Lezica and cow ( r rs .I2- l4 Apparent 1y , the i r e 11z y i n e preparations contained the ap11ro p ri ate e nz ynie for reducing the d 0 1 1 ble bond of the a-isoprene unit. The high specific activity of polypreiiyl phosphate synthesized ( 1 t~w G o in the outer, iiiitocliontlrial ni e 111bran e s is cons i s te i i t w itlt t 11 e C Y ) I 1 ce pt that i i i i tocho IIdria are capable of a ~ i t o n o ~ i ~ o glycosylation us of proteins (see Section 11,3). As shown in Scheme 1, two ot1ic.r pathways lead to the formation of clolichol phosphate. These arc ( ( I ) phosphorylatiou of the free polyprenol, and ( h )dephosphoiylatiotr oftlolichol cliphosphate. A dolichol phosphate-cleaving phosphatwe hits heen described a s occurring in T et rc1h y 172eria 11 11rifomzis ,l5 h u i r 1 an 1 v 111phoc y te s,I 6 calf l) rai n , and ratnerve tissue.18The protozoal c i t z ~ m cwas ~ a soluble acid phosphatase, although it was clearly tliff&i-eiit from the bulk phosphatase activity, whereas the man~nialianenzyiiies had optima at iteiitrul pH antl were i n eni1)ran e- bo ti rid. A significant, if not major, part of the polyprenols is often present a s free alcohol or as fatty acid ester.'!' A liver-n~icrosomalfraction contained high specific activity of clolichol acyltransfer;~se.'OThe esterification of dolichol was not affected b y hexaclecanoyl-~oA(palniitylCoA), antl the a u t h o r P suggested that phospliatidylcholine m a y he the acyl donor. Dietary tlolichol tiray contribute to the pool of entlogenous dolichol,21and, indeed, the isolation from pig liver of a C,,-dolicliol having three, rather tliait t-wo, internal trci us-isoprene units points to the hydrogenation of' a polypreno1 (ficaprenol) of dietary origin .22 The demonstration that tlolichol added to hepatocytes can be kcb

(12) G. R. I>aleo and R. Pont Lezicii, I;f:'HS I , c t t . , 74 (1977) 247-250. (13) G. R. Daleo, H . E. Hopp, P. A. R o i i i c ~ r o ,and R. Pont Lczica, F E R S Letf., 81 (1977) 41 1-414. (14) H. E. Hopp, G . R. Dalco, P. '4. Hoinel-o, ant1 R. Pont I,t.zic,it, Plutit P k ! / s i o / . , 61 (1978) 248-251. (15) G. S. Adrian and R. W. Keenan, B i o ( , / i i t t i . HiophC/s. A c f a , 575 (lY7Y) 431-438. (16) J , F. Wetlgwood and J . I,. Stroiiiiiigvr, / . Hi()/. Chetn., 255 (1980) 1120- 1123. (17) W. A. Burton, Sf.G. Scher, and <:. 1 . \l'aechter,Fed. Proc.. Fed. .Atti, SOC.E x p . B i o / . , 39 (1980) Abstr. 1554. (18) V. Idoyaga-Vargas, E. Rt.locopito\v. .4.\1cntaberr>, a n d ki. C:arniinnatti, I:l:'BS Lett., 112 (1980) 63-66. (19) P. H. W. Butterworth mid F. \V. Iletnniiiig, Arch. Biochcttr B i o ? i h ! / . c . ,128 (1968) 503 -508. (20) R. W. Kcenan and M ,E. Kruc k , Hioc,~ic.ttiisfri/, 15 (1976) 1586-1591. (21) R. W. Keenan, J. B. Fischer, and S1. k:. Krnczek, A r c h Hiocltctti. Bioph!/s., 179 (1977) 634-642. (22) T. Mankowski, W. Jankowski, 1'.(:hojrracki, and P. Fraiike, Bioclteniistr!/, 15 (1976) 2125-2130.

292

RALPH T. SCHWARZ A N D ROELF DATEMA

phosphorylatedZ3iiiay suggest that the pool of free polyprenols can be mobilized in v i m to give “active” dolichol phosphate. The dolichol kinases needed for this activation have been described for several maininalian-cell type^'^.'^ and insect cells.26The phosphate donor is CTP in mammalian cells, arid may be ATP in insect cells. The newly synthesized dolicliol phosphate can be glycosylated by either GDP-Man or UDP-GlcNAc, to form the inonosaccharide lipid derivatives.25Although dolichol phosphate synthesized tle notio from isoit is neverpentenyl cliphosphate can a l s o accept glycosyl groups,12”:$ theless possible that part of the newly synthesized dolichol phosphate is first dephosphorylated before entering the glycosylation cycle after rephosphorylation. A pai-ticulate, enzyme preparation from human lymphocytes contains a phosphatase that converts dolichol diphosphate into dolichol phosphate and phosphate.I6 This alkaline phosphatase may play a role in the recycling of dolichol phosphate, if this conqmund is to act as a coenzyme. However, the specificity of the phosphatase for clolichol diphosphate, 2,3-dehydrodolicliol diphosphate, or dolichol diphosphate-linked oligosaccharidesZ7has not yet been detennined. The activities of dolichol phospliate-phosphatase and of dolichol phosphokinase may control the amount of dolichol phosphate;’ as suggested for the corresponding enzymes in the formation of undecaprenol phosphate in Sta),hylococczi.r. uiireus.2xThe fkwt that membrane preparations usually contain subsaturating amounts of dolichol phosphate may be indicative of a stringent regulation of the pool size of this compound. It would inean that the amount of‘dolichol phosphate is rate-limiting in protein glycosylation in oivo. Apart from the demonstration that dolichol phosphate added to slices of oviduct tissue only indirect evidence has so far speeds up glycosylation of been obtained to show that the level of dolichol phosphate may increase in differentiating tissues that require increased glycosylation of T. Chojnacki, T. Ekstriiin, and G. Dallner, F E B S Lett., 113 (1980)218-220. C. M. Allen, J . R. Kaliii, J . Sack, atid D. Verizzo, Biochettiistr!y, 17 (1978) 50205026. W. A. Burton, M. G. Schcr, a i i t l C. J . Waechter,] R i d . Clzeni., 254 (1979) 71297136. L. A. Quesada Allue, F E B S Lett., 97 (1979) 225-229. R. Cacau, B. H o f l a c k , and A. Verbrrt, Eur. J. B i o c h e m . , 106 (1980)473-479. J. L. Stroininger, Y. Higashi, H. Sanderinann, K. J. Stone, and E. Willoughby, in R. Piras arid H. G. Pontis (Etls.), Riochentistr!/ of the Gl!ycosidic Linkage, Acatleinic Press, New York, 1972, pp. 135-154. D. I>. Carson, F e d . Pro<. F e d . Atti. Soc. E x p . B i o l . , 39 (1980) Abstr. 1552.

THE LIPID PATHWAY OF PROTEIN GLYCOSYLATION

293

p r ~ t e i n . ~ OThe - ~ ~role ~ of 3-hydroxy-3-methylglutaryl-CoA reductase in controlling the rate of protein glycosylation by way of (EC 1.1.1.34) the rate of synthesis of dolichol phosphate has been suggested,' but may have no general ~ i g n i f i c a n c e , because ~ , ~ ~ . ~ the ~ rate of synthesis of dolichol phosphate in other cells was shown to be controlled in the first steps, specific for synthesis of dolichol phosphate, namely, the condensation of isopentenyl diphosphate with famesyl diphosHowever, a clear answer regarding the role of dolichol phosphate in the regulation of glycoprotein synthesis awaits an accurate method of chemical assay, and discovery of specific inhibitors of the various pathways leading to dolichol phosphate. The finding that dolichols differ in chain length has, of course, raised the question of the polyprenyl specificity of the various glycosyltransferases. The idea that different (that is, not miscible) pools of dolichol phosphate exist for different glycosyltransferases has also emerged, and was discussed in a review by Elbein.35Conflicting evidence has been and this point needs further investigation. Although it could be established that saturation of the a-isoprene unit of polyprenol phosphate is important for these compounds to act as effective acceptors of glycosyl moieties in eukaryotic cells, little chain-length specificity was observed in such s t u d i e ~ . ~This ~ - ~ obserI vation found support in the result that the same set of prenylogs occurs in total-yeast dolichol and in dolichol diphosphate-linked 2-acetamido-2-deoxy-~-glucose synthesized in oitro with yeast-cell h o m o g e n a t e ~As . ~ ~already noted by Chojnacki and dur(30) J. B. Harford, C. J. Waechter, and F. L. Earl, Biochem. Biophys. Res. Commun., 76 (1977) 1036-1043. (31) J. J. Lucas and E. Levin,J. B i d . C h e m . , 252 (1977) 4330-4336. (32) J. B. Harford and C. J. Waechter, Biochem. J., 188 (1980) 481-490. (32a) M . J. James and A. A. Kandutsch, Biochim. Biophys. Acta, 619 (1980) 432-435. (33) M . J. James and A. A. Kandutsch,J. Biol. Chem., 254 (1979) 8442-8446. (34) R. K. Keller, W. L. Adair, and C . C. Ness,]. Biol. Chem., 254 (1979) 9966-9969. (35) A. D. Elbein, Annu. Reo. Plant Ph!ysiol., 30 (1979) 239-272. (36) D. A. Vessey, N. Lysenko, and D. Zakim, Biochirn. Biophys. Acta, 428 (1976) 138-145. (37) D. Codelaine, H. Beaufay, and M . Wibo, Eur. J . Biochem., 96 (1979) 27-34. (38) M. C. Ericson, J . T. Gafford, and A. D. Elbein, Plant Physiol., 61 (1978) 274-277. (39) T. Mankowski, W. Sasak, and T. Chojnacki, Biochem. Biophys. Res. Commun., 65 (1975) 1292- 1297. (40) T. Mankowski, W. Sasak, E. Janczura, and T. Chojnacki,Arch. Biochem. Biophys., 181 (1977) 393-401. (41) D. D . Pless and C. Palarnarczyk, Biochim. Biophys. Acta, 529 (1978) 21-28. (42) F. Reuvers, P. Boer, and F. W. Hemming, Biochem. J . , 169 (1978) 505-508.

294

RALPH T. SCHWAKZ A N D HOELF DATEMA

ing a study of the polyprenol specificity of Salinonelln tgplzinauriuin enzymes, such results should be treated with caution, because unpurified, membrane-bound enzymes were used in these investigations. Thus, using solubilized enzymes froin the yeast Sacclzan,iriyces cerevisiae, and polypreriyl phosphates having 35 to 100 carhon atoms, a clearer picture of preferences could be obtained.4" A requirement for a-saturation of the polyprenol phosphate was shown for the fonnation of GlcNAc-PP-Do1 froin UDP-GlcNAc and Dol-P, but not for the formation of Man-P-Do1 from Dol-P and GDP-Man, but the a-saturated fonns were more effective in this reaction than the unsaturated polyprenyl phosphates tested (C3,, C,,, and Cleo). Both enzyme preparations had a requirement for a minimum chain length of 30-50 carbon atoms. Interestingly, in the formation of GlcNAc-PP-Dol, the chain length mainly affected the apparent K, (from 31 pM for C,,,-dolichol phosphate to 117 pM for C,,-dolichol phosphate), whereas, in the formation of Man-P-Dol, the maximal velocity of the reaction was affected, the apparent K, being 1-2 p M for the different dolichol phosphates. The variation of activity with chain length as found in yeast may be different in other species. The chain-length preference in the fommation of GlcNAc-PP-Do1 and (GlcNAc),-PP-Do1 (C,,, > C,, > C,,) was reversed when the transfer of (GlcNAc), from the lipid intermediate to a peptide was studied.43Although the significance ofthis finding in this artificial system (see Section II,2,11) is not y e t clear, it should challenge the implicit notion that the same dolichol diphosphate carrier serves in the glycosylation cycle shown in Scheme 2, with only the oligosaccharide moiety unclergoing inodific.at'1011s. The possibility that polyprenyl phosphates are also physical carriers of glycosyl groups in transmembrane translocation is doubtful. The observed rates of unassisted flip-flop of spin-labeled dolichol phosphoric esters in unilamellar phosphatidylcholine vesicles44is too low if such flip-flop should keep pace with the rate of protein glycosylation (measured as rate of protein s e c r e t i o ~ i ~Also, ~ ) . after its biosynthesis, (GlcNAc),-PP-Do1 does not change its transbilayer distribution in closed, membrane vesicles (having the carbohydrate inside).45 When incubated with the appropriate nucleotide esters of sugars, membranes from several sources2 were effective in catalyzing the forniatioii of Man-P-Dol, Glc-P-Dol, and GlcNAc-PP-Do1 (reactions I , 2 , (43) G . Palamarczyk, L. Lehle, T. Marrkowski, T. Chojnacki, and W. Taniier,Eirr.j. H i o chern., 105 (1980) 1517-1523. (44) M . A . McCloskey and F. A. Troy, Biochetttistr!y, 19 (1980) 2061-2066. (45) J. A. Hanover arid W. Lennarz,]. Riol. Cherri., 254 (1979) 9237-9246.

Dol-P UDP-GlrNAc +UMP

t

GleNAc-PP-Do1 UDP- GlcN A(, %UDP Dol-P

an,- ( G ~ ~ N A ~ ) , - P P - D ~ ~

Man,. ( G ~ c N A c ) , - P P - D o ~ @ Man-P Ikil UDP-Glc

Dol-P

Dol- P Man,. (GlcNAc),. PP-Do1

Dol- P

Dol-P

Glc,-Man,- (GlcNAc),. PI’- Do1

Glc,-Man,- (GI? N A c ) , - A s n

G k , - Man,- (GkNAc),. Asii

Ce Man

Man,

13

(GlrNAc),-

Scheme 2.-Proposal for Pathways i i i the Biosynthesis of the Lipid-linked Oligosaccharide Precursors ofthe L-Asparagirit.-liiihctl Glycans. [ I n the pathway in the celiter of the Scheme, Man for GDP-Man is r i d to give an endo-p-N-;icetylalii~[)s~~iriitla.;e-Hresistant heptasaccliaride-lipid (structiil-c c*j at the hottom), whereas Man from hlanP-Dol, subsequently elongates the hel~tasacchnride-lipid to give Man,-(GIcNAc),-PPDol. T h e alternative fate of the heptas;icc.Iiaride-lipid is u-gliicosylation to give GI<;,Man,-(GlcNAc),-PP-Dol. A different patliway (on the left-hand side of the Scheme) to Man,-(GlcNAc)2-PP-Do1 uses Man froni Iwth GDP-Man and Man-P-Do1 in the initial stapes of assembly; by this pathway, etid~~-~-N-acetylglucoaaminidase-H-se~isitive hesaand hepta-saccharide-lipids are formed (coinpiire structure @at the bottom). The Scheme is based on work cited in Refs. 2, 35,ri0, 75, 117, 117a, and 119-122.1

296

RALPH T. SCHWAHZ AND HOELF DATEMA

and 3 ) . Also, the formation of Xyl-P-Do1 and Gal-P-Do1 has been reported,’ but the biological significance of these compounds is not yet clear. GDP-hlan + Dol-P + Man-P-Do1 + C D P

(1)

UDP-GIC + Dol-P + Glc-P-Do1 + UDP

(2) (31

+ 1101-P+ GICNAC-PP-DOI+ UMP UDP-Glc + Dol-P + Clc-PP-Do1 + UhlP

UDP-GIcNAc

(4)

The dolichol-linked D-rnannosy!, D-glucosyl, and 2-acetamido-2deoxy-D-gliicosyl residues are used in the glycosylation of proteins and in the biosynthesis ofD-mannan.2’35GlcNAc-PP-Do1 may also play a role in the synthesis of glycosaminoglycans. Reaction 4 is the first step in the formation of lipid-linked precursors for The enzymes catalyzing these reactions are membrane-bound, ancl are stimulated by exogenous dolichol phosphate. It has been suggested, and now shown43(at least in the foilnation of GlcNAc-PP-Do1 b y enzymes from yeast), that Dol-P stimulates the incorporation of sugar residues into the lipid intermediates because it serves as a substrate. Reactions 1 and 2 proceed with inversion ofthe aiiorneric configuration of the glycosyl group, that is, in the dolichol phosphate-linked intermediates, the glycosyl group occurs in the p configuration. These derivatives are fonned by transfer of glycosyl groups, and these reactions are reversed by the nucleoside &phosphate, whereas, in the formation of lipid diphosphate derivatives, the glycosyl phosphates are transferred.‘ These reactions are inhibited by UMP, which is also a competitive i11hibitol-1~ of the formation of Glc-P-Do1 (reaction 2). Because several reviews have now appeared that cover the formation of monosaccharide derivatives of dolichol, the reader is referred to these for detailed information and original ita at ion^.**:^^,^^-^^ Solubilized, and partially purified, preparations have been obtained4”47,”-5“ for enzymes catalyzing reactions 1 , 2 , and 3 . Solubiliza(46) H. E. Hopp, P. A. Romero, G. R. Daleo, ant1 R. Pont Lezica, Eur. 1.Biochena., 84 (1978) 561-571. (47) C. L. Villemez and P. L. Carlo,]. R i d . Chern., 254 (1979) 4814-4819. (48) C. J. Waechter and W. J. Lennarz, Anrru. Hev. Biochem., 45 (1976) 95-112. (49) N. Sharon and H. Lis, Biochem. Soc. Trans., 7 (1979)783-799. ( S O ) D. K. Struck ancl W. J. Lennarz, in W. J. Lennarz (Ecl.) The Biochemistr!!ofGl!/coprotein7 atid Proteoglycciris, Plenum Press, New York, 1980, pp. 35-83. (51) P. Babczinski, A. Haselbeck, and W. Tanner, Eur. J . Biochem., 105 (1980) 509-515. (52) R. K. Keller, D. Y. Boon, ant1 F. C . Crum, Biochemistry, 18 (1979) 3946-3952. (53) A. Heifetz and A. D. Elbein,]. B i d . Chem., 252 (1977)3057-3063.

T H E 1,IPID P A T I I W A Y O F I'HOTEIN GLkCObYLA'l'lOh

"17

tion, usually with detergents, caiisctl an increase in the dependence of the enzyme activity on added dolichol phosphate. Most of the solu1,leenzyme preparations catalyzing reaction 3 fo~iii(GlcNAc),-PP-Dol, i n addition to GlcNAc-PP-Dol. From bisubstrate, kinetic aiialysis with a transferase froin hen oviduct that, under the conditions of the assay, fonned only GlcNAc-PPDol, it followed that both do1ichol phosphate and UDP-GlcNAc have to be bound to the enzyme I)txfore release of the product occurs."z However, the fact that only pirti;ilIy purified preparations have thus far lieen obtained (the preparations niay also still be coirtaniinutetl with substrates and product), to,syther with experimental difficulties in handling both the substrate tlolichol phosphate (which, furthermore, is not one compound, s e t ' tlie earlier discussion) and tlie unstable enzyme (enveloped in niicelles of detergent), make difficult a sensible interpretation and conrparison of the kinetic parameters determined for the different eiizvine-preparations. The solubilized enzymes catalyzing reactions I , 8,and 3 have i n coiniiion their alkaline pH optima and dependc>nct,on Mg2+or hclii2+ions. The latter fact makes (ethylenedinitri1o)tetraacetic acid (EDTA) a reversible inliibitor of enzyme activity and an iinpoitant experimental tool. M o s t of the so1u b il ized-e 11zy I I 1 e preparations i i n d er di s c u s s ion h ere are unstable (an exception being tlie polyprenyl phosphate: UDP-Glc, glucosyltransferase froin Acaiitlrti t r i o c h c i castcllani prepared h y sonic o ~ c i l l a t i o nand, ~ ~ ) of course, this may suggest loss of an essential coinporient during solubilization. Thus, specific lipids may n~odulatethe enzymic activity, either by serving as a cofactor o r substrate, o r b y influencing the membrane environnient. The observations that ( a ) inactivation by detergent of the yeast enzymes catalyzing reactions 1 and 3 could be counteracted b y those polyprenol phosphates that are substrates for the enzymes,s4 and ( h ) phosphatidylglycerol can enhance the synthesis of GlcNAc-PP-Do1 b y a microsoinal preparation from rat serve as examples. The participation of derivatives of vitamin A in glycosylation reactions seems indisputable.2Their role became evident when it was observed that a deficiency, or ail c~xcess,of the vitamin affects the glycosylation of at least some proteins (see Refs. 56 and 57, and the (54) G. Palaniarczyk, L. Lehle, and W. Taniler, F E B S Lett., 108 (1979) 111-115. (55) P. L. Plouhar and R. K. Bretthaiic.r, Hiochetn. B i o p h ! / s . R c s . Contmurt., 90 (1979) 1186-1193. (56) L. M. de L L I CP. ~ ,V. Bhat, W. S i i s a k , and S. .i\damo, Fed. Proc. F e d . Am. Soc. E x p . Biol., 38 (19791 2535-2539. (57) G . Wolf, T. C. Kiorpes, S. Masushige, $1. J . Smith, and R. S . Anderson, F e d . Proc. Fed. A m . Soc. E x p . B i o l . , 38 (197'3)2.540-2543.

298

RALPH T. SCHWARZ A N D ROELF DATEMA

literature cited therein). Especially decreased D-mannosylation is apparent under retinol deprivation. In one case,jx the fonnation of shorter-than-normal, protein-bound oligosaccharides has been observed in vitamin A deficiency. How, exactly, retinol affects protein glycosylation is not yet known, but the fonnation of retinol phosphate, the D-mannosylation of retinol phosphate by GDP-Man, and the transfer of Man from D-mannosyl (retinol phosphate) to pronase-sensitive glycoconjugates are well est a b l i ~ h e d . 5On ~ ~the ~ ~other * ~ ~ hand, the fonnation and role of other glyIt is very cosy1 esters of retinol phosphate are still probable that the D-mannosylation of retiiiol phosphate, and also the transfer of Man froin this lipid derivative to protein, are catalyzed by enzymes different from those involved in the dolichol p a t h ~ a y , ~ l - ~ ~ and this finding points to the different physiological roles of Man-PDo1 and D-mannosyl (retinol phosphate). Thus, whereas Man-P-Do1 can serve to D-mannosylate dolichol diphosphate-linked oligosaccharides (see Section 111),such a role has not been found for D-mannosyl (retinol phosphate)."-63 Although it is interesting to know the nature of the proteins D-maniiosylated by D-mannosyl (retinol phosphate), more-intriguing questions are the nature of the carbohydrate accepto?' and of the glycosidic linkage formed, because, in the retinol-mediated D-mannosylation of proteins, there may be involved a post-translational D-mannosy~ation, a reaction but little studied so far (compare Sections II,2,a and 11,3), It has been suggested that retinol phosphate may play a role in shuttling Man across the lipid bilayer [according to the subcellular localization of the enzyme, forming D-mannosyl (retinol phosphate)] of membranes from the rough, endoplasmic reticulum64 (dolichol phosphate does not play such a role, see earlier). Although the donor of the Man group for this possible shuttle-system may be GDP-Man, defined acceptors of the Man group from D-mannosyl (retinol phosphate) have not been indicated. As the results of studies in vitro indicated that these acceptors cannot be the intermediates of the dolichol pathway, (58) W. Sasak, L. M. de Luca, and J. G. Bieri, Fed. Proc. Fed. A n , . SOL.. E x p . Biol., 39 (1980) Abstr. 1558. (59) L. M . de Luca, Vitcini. H o r m . ( L e i p z i g ) ,35 (1977) 1-57. (60) S. Adarno, L. M. de Lwa, C. S. Silverinan-Jones, and S . H . Yuspa,]. B i o / . C:heln., 254 (1979) 3279-3287. (61) G. C. Rosso, S. Masusliige, H. Quill, and G. Wolf, Proc. Natl. Acud. Sci. U , S.A , , 74 (1977) 3762-3766. (62) J. Frot-Coutaz, R. Letoublon, and R. Got, F E B S Lptt., 107 (1979) 375-378. (63) W. Sasak and L. M. de Luca, F E B S Lett., 114 (1978) 313-318. (64) M . J. Smith, J . B. Schreilxr, and G. Wolf, Biochem. J,, 180 (1979) 449-453.

THE LIPID PATHWAY OF PROTEIN GLYCOSYLATION

299

it would seem interesting to study, with intact cells, the fate of D-mannosy1 (retinol phosphate) when the synthesis of Man-P-Do], but not of GDP-Man, is inhibited. 2. Assembly of the Lipid-linked Oligosaccharide, and its Transfer to Protein a. Studies in uiuo.-Slices of calf thyroid incorporate radiolabelled sugars, phosphate, and mevalonate into a lipid-linked oligosaccharide that can be extracted rather specifically with 10 : 10 : 3 (v/v) chloroform - m e t h a n ~ l - w a t e r . ~Following *~~ purification by chromatography on DEAE-cellulose, it could be shown that phosphorus occurs as a diphosphate bridge between the oligosaccharide and the lipid moiety (dolichol). The oligosaccharide was branched and consisted of mannose, glucose, and 2-acetamido-2-deoxy-~-glucose. Radiolabelled oligosaccharides of similar, if not identical, structure were isolated from slices of a number of other tissues incubated with D-['4C]glucOse or D-['4C]mannose.66 The biological function of this dolichol-linked oligosaccharide was revealed in pulse-chase experiments which showed that the intact oligosaccharide was transferred, as such, to protein. Subsequent studies, to be discussed, confirmed that the formation of the oligosaccharide linked to the Asn residues of glycoproteins is probably initiated by transfer of the oligosaccharide from the dolichol diphosphate intermediate to the asparagine residue of a nascent polypeptide (see Refs. 35, 49,50, and 67 for reviews). The structure of the dolichol diphosphate-linked oligosaccharide from cells infected with vesicular stomatitis virus68 was determined after differentially labelling glucose, mannose, and GlcNAc by feeding the cells isotopically labelled sugars. T h e analyses were consistent with formula 1. Apart from the D-glucosyl residues, the structure resembles the core structure of the Asn-linked chains of yeast mannan'j9 and the high-mannose oligosaccharides of g l y c o p r ~ t e i n sThe .~~ part of the molecule shown in formula 2 occurs in all Asn-linked oligosaccharides analyzed so far.70*71 In fact, studies made mainly with virus-infected cells have led to the proposal that the lipid-linked oli(65) R. G. Spiro, M. J . Spiro, and V. D. Bhoyroo,]. B i d . Chenz., 251 (1976) 6409-6419. (66) M. J. Spiro, R. C . Spiro, and V. D. Bhoyroo,J. B i d . Chenl., 251 (1976) 6420-6425. (67) H. Schachter and S. Roseman, in Ref: 50, pp. 85-160. (68) E. Li, I. Tabas, and S. Komfeld,]. B i d . Chem., 253 (1978) 7762-7770. (69) C. E . Ballau, Adu. Microbiol. P h y ~ i o l . 14 , (1976) 93-158. (70) J . Montreuil, Adu. Carbohydr. Chrtn. Biochem., 37 (1980) 157-223. (71) R. Komfeld and S. Komfeld, in Ref. 50, pp. 1-34.

RALPH T. S C H W A l U A N D R O E L F DATEMA

300 (y

- Man- (1- 6)- a - Man- (1 2

3

1

1

t

01 -Man

-

-

6)-0Man- (1- 4) - (3 - GlcNAc - (1 3

4) - N - Glc N Ac - P P- Do1

t

t

1

a - Man

N

- Man 2

2

t

t

1 01 - Man

1 CY - Man

t

1 N

- Man 3

t

1

01 - Glc

3

t

1 a-Glc

P1 a-Glc 1

@-Man- (1-

6)- (3- Man- (13

4) -$- Glc NAc - (1-

t

H O I I1 4) -13- GlcNAc -N-C-CH,

I

c=o

I -CH

I MI

I

1 a-Man 2

gosaccharide 1 is the only precursor for both the high-mannose and the complex oligosaccharides found linked to L-asparagine residues in g l y c o p r ~ t e i n s ~(for '-~~ reviews, see Refs. 50,67, and 71).This proposal has been extended to include the notion that this lipid-linked oligosaccharide plays this role in m a n y , if not all, eukaryotic cells. Although lipid-linked oligosaccharides having structures compatible with 1 have been isolated from n i a i i i ~ i i a l i a n avian,72 , ~ ~ ~ ~plant,7Y ~~~~~~~ (72) P. W. Robbins, S. C. Hubbard, S. J. Turco, and D. F. Wirth, Cell, 12 (1977) 893900. (73) I. T a b a s , S. Schlesinger, atid S. Kornfeld,]. B i o l . Chern., 253 (1978) 716-722. (74) P. W. Robbitis, Biochern. Soc. Trcins., 7 (1979) 320-322. (75) S. C. H u b b a r d and P. W. Rohbins,]. B i d . Cheni., 254 (1979) 4568-4576. (76) B. K. Speake a n d D. A. White, Biochem. I., 170 (1978) 273-283. (77) B. K. Speake and D. A. White, Biochern..\., 176 (1978) 993-1000. (78) D. S. Bailey, M. Diirr, J . Rut-ke, atid G . .hlaclachlan,]. Supruinol. Struct., 11 (1979) 123- 138. (79) S. K. Browder and L. Beevers, P l a t i t Ph!ysiol., 65 (1980) 924-930.

T H E LIPID PATHWAY O F PROTEIN C:I,YCOSY1,~2'1'1ON

301

and fungalH0.8ce 11s , a p recurs o I'- p rc)t 1tict re lat ic ) n s 1) i 1) 1)etw ee t i 1i p i tland protein-linked oligosaccharicles has been shown i n o n l k - a few instances. In 1977, SeftoiP showed that ii single, lipid-linked oligosaccharide i n sindbis virus-infected cells triitisfers its oligos ace1I'lllc .' 1t t 0 a llascellt (that is, polyri~osoine-bound)protein. In the saiiie year, Robhina and c ~ o w o r k e rfound ~ ~ ~ that the piilw-laI)elled high-mannose oligosaccharide released from viral gl>,cop,roteins,and the> oligosaccharide from the lipid-linked oligosaccharide, are similar, and later w o r k showed that they are, indeed, itlenticd.is I h r i n g a chase period, the oligosaccharides released froin the pulse-labelled, viral glycoprotclin at first lost D-glucosyl, and then l>-mannosyl,groups, and were, i n part, converted into complex oligosaccl~arides.iz~i3~~~~ requiring at least two The removal of the D-glucosyl D-g~ucosidases,8x-x9 can be followed I)y the clipping of ii variable niiniher of D-maniiosyl groups, giving rise to the various high-D-mannose oligosaccharides found in mature ~ l y e o ~ ~ r o t e i ~ iAfter s . ~ "the ~ " removal, from deglucosylated oligosaccharide, of four a-(1+2)-liiiked ~>-iwinnosy1 groups, for which one enzynic' may be responsible,!'"' GlcNAc can be added and then two iiiore I>-mannosyl residues will Iw excised,833Y1392 giving a substrate for ii second N-acetylglucosatiiiiiyltratisf e r a ~ eThis . ~ ~ oligosaccliaritle ciitt be extended with D-galactosyl and sialic acid residue^.^^*^" This seqiience of reactions, called "processing," whereby an original, high-D-mannose oligosaccl~aridecan be converted into a complex t y i ~ c , ~occurs :' not only with menl1)raneh i n d gl ycoproteins (for exannplc, viral g l y c o ~ r o t e i i ~ s i ~ ~ i ' l - i ~but ~x"~"), also with secreted glycoprotei~is.~' In the formation of yeast tnanno(80) A. J. Parotli,]. B i o l . C;hem., 254 (1979) 10,051-10,060. (81) L. Lehle, Eur. Biochenr., 109 (1980) 589-601. (82) B. b l . Sefton, ( / I , 10 (1977)659-6f58. (83) S. Konifeltl, E. Li, and I. Tabus./. H i d C / W J ~ 253 I , , (1978) 7771-7778. (84) S. J . Tiirco ant1 P. W.Rol>l>ins,].Hiol. C / I C , J I254 . , (1979) 4560-4567. , (1979) 2630-26.37. (85) hl. G . Scher a i i d C. J . Waechtrr,/. Biol. C h c ~ m .254 (86) R. G. Spiro, hl. J . Spiro, and 1'. 1). Bho! r o o , / , B i d . C\wm., 254 (1979) 7659-7667. (87) W. W.Chen and W. J. Lennarz,]. H i o l . C h e m , , 253 (1978) 5780-5783. (88) L. S . Grinna ant1 P. W. Hobbiiis.] B i i d C h e ~ n . 254 , (1979) 8814-8818. Biol. C h e ~ f 255 ~ . . (1980)2325-2331. (89) J. J. Elting, W. W. Chen, and W. J . Le-ii~~arz,/. (90) I. Tabas and S. Korliklt1,J. Rial. ( : / t c , f f i . , 254 (1979) 11.655- 11,663. (91) I. Tabas and S. Kornfeld,J. B i o l . C / w f f t , , 253 (1978) 777s)-7786. , (1980) 4894-4902. (92) N . Harpaz and H. Schachter,]. Biol. ( , ' h t ~ r ~ r .255 (93) N . H:~rpazand H. Schachter,/. H i d . C:/zc,,tc., 25.5 (1980) 4885-4893. (94) L. A. Hunt, J . K. Etchisoil, and 1). F. Siiiiimers, Proc. Nafl. Accitl. Sc.i. (1. S. A . . 75 (1978) 754-758. (95) A. Tartakoff m c l P. Vassalli,]. Cc4i H i o l . , 83 (1979) 284-299.

302

RALPH

-r. SCHWAHZ

A N D HOELF DATEMA

proteins, de-D-glucosylation has been s1iown,80sx1~96 aiid de-D-inaniiosylatiori may also occur,xn but reinoval of D-mannose is difficult to demonstrate, because the deglucosylated, high-inannose oligosaccharide can be extended with D-mannosy1 groups coming directly from G D P - b l a ~ i .The ~ ~ *latter ~ ~ reaction has not yet been shown to occur in animal cells. Definite proof for the excision of D-mannosyl residues in yeast will depend on the isolation of a specific, yeast a-Dinannosiclase. An interesting question is: what deterinines that one oligosaccharide is processed to a complex oligosaccharide, and another (on the same molecule) to a high-inannose oligosaccharide? A certain cell has to be equipped with a set of iiiaiiiiosidases, in order to trim the highiiiaiinose chains, and a set of glycosyltransferases, in order to add the terminal sugars GlcNAc, Gal, and Ne~iAc.Fungi appear to lack some of these enzymes, because they do not fo1-m complex-carbohydrat~ chain^.'^.'^ Mosquito cells have low, if not negligible, activities of N acetylglucosai~iinyl,galactosyl, and sialyl t r a n s f e r a s e ~and , ~ ~ in line with this finding is the observation that the L-asparagine-linked oligosaccharides from a mosquito, cell-membrane glycoprotein are of the high-mannose type Interestingly, this high-mannose oligosaccharide appears to contain three glucose residues, and it may be identical with the dolichol diphospliiite-linked tetradecasaccliaride. If this finding turns out to be true for several arthropod-cell types, it would mean that the forniation of coniplex, Asn-linked carbohydrate chairis is an evolutionarily late development. As shown by Kobata and coworkers,'"' bovine rhodopsin contains Asn-linked oligosaccharides identical with processing intermediates, suggesting that the rods of the tiovine retina do not have, or have lost, the glycosyltransferases that, in other cells, complete the processing to complex oligosaccharides. Studies on the glycosylation of one viral protein in different or of different viral proteins in one cell type,'04-10X or comparison of carbohydrate chains of a viral glycoprotein and an immuno(96) H. D. Kilker, A. Herscovics, J . R. Tkacz, aiid K.W. Jt.mloz, Fed. Proc. F d . Am. Soc. E x p . R i d . , 39 (1980) Abstr. 2078. (97) A. Parodi,]. Biol. Chetn., 254 (1979) 8343-8352. (98) V. FarkaS, Microhiol. Rec., 43 (1979) 117- 144. (99) T. D. Butters, R. C. Hughes, and P. Vischer, H i o c l i i t n . H i o p h y r . . Actci, 640 (1981) 672 - 686. (100) 1'.11. Butters and R. C. Hughes, Riochint. B i o p l i y s . Actn, 640 (1981) 655-671. (101) C.-J. Liang, K. Yamashita, C. G. Muellenberg, H. Shichi, and A. Kobata,]. B i o l . Chetti., 254 (1979) 6414-6418. (102) J. R. Etchison and J. J . Holland, Proc. N a t l . Acutl. Sci. (1. S. A., 71 (1974) 40114014.

THE LIPID PATHWAY ()P I'HOTEIN GLYCOSY1,ATIO.L

30 3

globnlin glycosylated in the saiiic' cell,")9 all led to the same concliision, namely, that the strticturtl of the protein to be glycosyluted determines the processing of tliv high-mannose chains iiiitially attached: the same protein is glycosylated similarly liy different cells (the cells all have the f d l set of processing enzymes), whereas different proteins are glycosylated tliffr,reiitly by the same cell. Consethe cellular glycoquently, as indicated by Keegstra and svlating system can produce ;I variety of different oligos~iccharides, the oligosaccharicles found in niattirca gl>.coproteinsIxing the restilt of the interactions of a (folding) polypeptide with the glycosylatiiig enzynies. It should lie noted that l)otli secreted"" aiitl meiiibraiie-l,otind glycoproteins" undergo chi3ngc.s i t i coiifommation during inattiration. As the glycosylation process is svclur.iitia1, that is, different steps occur in different, cellular conipaitnicnts (see Section 11,3), glycosvlation i n a pro xi inal coin pai-tnie n t can i 11N I ic' n c e the con form at ion o f ii po 1)'peptide and, hence, its glycosylation i n a distal conipartment. The assembly of the lipid-linkod oligosaccharide i r i c k o is fast.x' After 2.5 inin of labelling with [:'I-I]Man, the fiill-sized, lipid-linked oligosaccharide Glc,Man,(GlcN Ac),-PP-Do1 can already be detected.i5 In chick and hamster c.t~lls,it reaches ii steady state after 1O min. Intennediates in the iissetnlily of the tetradecasaccharide are labelled to only a small e x t e i i t , and, also, Glc-P-Do1 and Man-PDo1 are usually difficult to isolutc, froin intact cell^.^;.^^,^^ This indicates that the traiisfer froin the oligosac,c.li~~ride of this lipid-linked oligosaccharide to protein is a rate-liniitirig step in protein glycosylatioir. It also implies that, were any ot1ic.r lipid-linked oligosaccharide present that could function as a donor of oligosaccharitles, it might escape tietection. A heptasacchuride-lipid having 5 Man and 2 CIcNAc residues appears to be labelled rather cltiic.kly i n Chinese-hamster ovary-cells,

-

(103) K. Keegstra, €3. Sefton, and U. H i i r I , ( , , / . Viro/., 16 (1975) 613-620. (104) R. M . Sefton,/. Virol., 17 (1'376)85-93. (105) R. T. Schwarz, X I . F. G. Schiiiitlt, I].An\ver, and H.-I>. Klenk,]. Virol., 23 (1977) 217-226. (106) K. Nakamnra a i i d R. W. Conip:iii\, \'irO/Og!/, 95 (1979) 8-23. (107) H. T. Schwarz and H.-D. K l e i i k , I I I H . Schauer, P. Boer, E. Butltlecke, \1. F. Kramer, J . F. G. Vliegenthart, a i i t l H . Ll'iegaiidt (Eds.).Proc. I t i t . S ! y r i i p . G ~ ! / C Y J ~ O I I jugates, Sth, 'Thieme Verlag, Stuttcgart, 1979, pp. 678-679. ., (108) M. R. Rosner. L. S. Griniia, a i i t l 1'. LV. Rohliiiis, Pro(,. .%'tit/. Accitl. S c i . CI. S. .I77 (1980) 67-71. . , (1979)5377-5382. (109) S.Weitzmaii, M ,Grennoii, and I<. Kt~cgstla,].B i o l . C ~ h w t ~254 (110) E. W. Siitherland, D. H. Ziiiiiiic'i-iii;ciiii~ aiid M. Kcrn, Proc. S o t / . Accltl. Sci. U . S. A , , 69 (1972) 167-171. (111) G. Kaluza, R. Rott, and R. T. ScIi\v;iIr, i'iro/og!y, 102 (1980) 886-2519.

304

RALPH T. SCHWAHZ A N D ROELF DATEMA

reaching the steady state"' in 1.5 min. A lipid-linked heptasaccharide, Man,(GlcNAc)2-PP-Do1, together with the normal, lipid-linked tetradecasaccharide, was isolated from Chinese-hamster ovary-cells infected with vesicular stoinatitis virus, and its structure was determined to be aMan( 1+2)aMan( 1+2)aMan( 1+3)[aMan( 1+6)]PMan( 1+4 or3)PGlcNAc(lb4)aGlcNAc-PP-Dol.Itwas ~ u g g e s t e d ~ l ' , " ~ that the heptasaccharide lipid was an intermediate in the biosyiithesis of Glc,Man,(GlcNAc),-PP-Dol, but the evidence (decreased labelling after a chase) is not convincing. These experiments would only give conclusive results were the transfer of the oligosaccharide to protein to be inhibited; this could, in principle, be achieved b y inhibiting protein synthesis, but it has been shown that, at least in some cells, inhibition of protein synthesis leads to inhibition of oligosaccharide as~ e n i b l y , "or ~ even to degradation of lipid-linked oligosaccharides.lli A decreased rate of assembly of the oligosaccharide can be achieved b y using inhibitors of the assembly process. When prelabelling cells with radiolabelled Man or GlcN, and then treating them with the inhibitor 2-deoxy-2-fluoro-~-glucose (see Section 111,3,a), a series of lipid-linked oligosaccharides can be isolated that are biosynthetic intermediates.'I6 Also, the fact that more than one lipid-linked oligosaccharide [namely, Glc,Man,-(GlcNAc),-PP-Doll is detected when cells radiolabelled with for 10 min are harvested in cold buffers, instead of directly in organic solvents, may be related to the inhibitory effects on assembly of lipid-linked oligosaccharides caused liy withdrawal o f t h e carbon source (see Section 11,5). Still another approach to solving the problem of detecting interniediates in the assembly of the lipid-linked oligosaccharide comes from the use of cell mutants having defects in the synthesis of an essential intermediate. This approach has proved productive (see later). Despite the problems involved in analyzing small amounts of radiolabelled oligosaccharides, and the foregoing reservations made as to the interpretation of pulse-chase experiments, Kornfeld and coworke r ~ determined " ~ the structure of 9 oligosaccharides, presumptive intermediates in a sequence of orderly additions of glycose residues leading to Glc,Man,-(GlcNAc),-PP-Dol. The assenibly was called "ordered," because each precursor oligosaccharide consists of only a sin(112) S. C. Hubbard, Fed. Proc. Fed. A T T SOC. I . E x p . B i d . , 39 (1980) Ahstr. 350. (113) E. Li and S. Kornfeltl,]. B i d . Cherti., 254 (1979) 2754-2758. (114) J. W. Schmitt a n d A. D. Elbein,]. B i d . Cheni., 254 (1979) 12,291-12,294. (115) B. K. Speake and D. A. White, Biocheni. SOC.Trails., 7 (1979) 372-373. (116) R. Datema, H. T. Schwarz, ant1 A. W. Jankowski,Eur.J. Biochem., 109 (1980) 331341. (117) A. Chapman, E. Li, and S. Kornfeld,]. B i d . Chem., 254 (1979) 10,243-10,249.

gle isomer. Studies on thc asscinl)ly of the lipid-linked oligosaccharide i n uitro, however, contradicted the conclusion iis to the ordered sequence, because isomers of the hexasacclraride- and heptasaccharide-lipid were dett,cted.i'i;l The proposed reactions e q i ~ e i i c edoes ~ ~ ~not, of tours(', s o l v c ~the nature of, for exainple, tlic donors for the D-mannosy1 groiips. lising a mutant, u~iouse-l~~inphoma cell-lineiixthat is not capable of s1,irtlresizing Man-P-Dol,"" Kornf&ld and coworkers were able t o show that the lieptasaccharide lipid or 3)aMan( l+2)aMan( 1+2)-aMan( 1+3)[LuMan( 1+6)]PPvlan( 1-4 pGlcNAc( 1+4)aGlcNAc-PP-lh)l is still synthesized b y this cell Consequently, at least i i i iiioiise-lyinphot~i~i cells, D-rriannosy~ groups coming fi-om Man-P-Dol a p p c w to be involved oiily in the elongatio~i"~ of Man,(GlcN,4c),-PP-I)ol to Maii,(GlcNAc),-PP-nol. The heptasaccharide-lipitl can also be elongated with 3 D-glucosyl residues, to Glc,Man, (GlcNAc),-PP-Do1 (3),the lipid-linked decas accli ari de be in g an in1n r e ( 1i ate p rc~ci~rsor of the coiii111e x o 1i gosaccharides of vesicular stonratitis v i r u s G protein when the viriis is propagated in the mutant, nrouse-lyiiiplioii~acells."" ~

a(1- ~ 6).13 -- Man- (1-

4)-,4- C;l,,NAc- (1-

4 ) - I ? - GlcNAc- PP- Do1

3 I

1 N - Man 2

f

1 e- Man 2

t

1 u - Man 3

t

01-

1 Glc 3

t

1 a - Glc 2

t

1 a - Glc 3

(117a) I. K. Vijay a i d G . H. Perdew,./. Hiol. C h ( , f t i . , 255 (1980) 11,221-1 1,226. (118) I. S. Trowbridge and R. Hyniaii, (:c//, 17 (1979) 503-508. (119) A. Chapiiiaii, K. Fujimoto, and S. K o r i i t e l ~ I , / R , i d . Ch4,tn., 255 (1980)4441-4446. (120) A. Chapman, 1. S. Trowbridgc., R . I I ! i i i ; i i i , and S. Kornfeltl, Cell, I7 (1979) 509515. (121) S. Kornfeld, W. Gregory, and A . ( ; h a p i i u i i , / . B i o l . Chcnt., 254 (1979) 11,64911.654.

306

RALPH T. SCHWARZ A N D ROELF D A T E M A

The finding of an alternative pathway of lipid-dependent, protein glycosylation cast doubt on the proposal that a common intermediate [Glc,Man, (GlcNAc),-PP-Doll might be the precursor both of complex and high-mannose oligosaccharides, because both the lipid-linked tetradecasaccliaride and the lipid-linked decasaccharide [Glc,Man, (GlcNAc),] can serve as precursor for protein-linked oligosaccharides. A pathway using the intermediates leading to Glc,Man, (GlcNAc),-PP-Do1 can be readily detected, because the assembly intermediates resist the cleavage by endo-p-N-acetylglucosaminidase H (EC 3.2.1.96,H), of the linkage between the two, @-(1-+4)linked, GlcNAc residues at the reducing end of the oligosaccharide.lls That this alternative pathway is not restricted to the mouse-lymphoma cells was shown b y studies using 2-deoxy-2-fluoro-D-g~~1cose.~~~ This sugar analog inhibits the synthesis of Man-P-Dol, but not of GDPMan,116and, in its presence, glycosylation of influenza virus protein in chick-embryo cells still occurs, by way of a pathway of endo-p-N-acetylglucosaniinidase H-resistant oligosaccharides.12z Some important points concerning this alternative pathway require attention. When, and to what extent, do cells use this pathway, instead of the one involving Glc,Man, (GlcNAc),-PP-Do]? Are both oligosaccharides transferred to protein by the same enzyme? Do there exist two pathways leading to Man, (GlcNAc),-PP-Dol, one using Man-PDo1 as a D-mannosyl donor for the a-linked residues, and another using only GDP-Man; or is Man-P-Do1 used only in the elongation of Man,(GlcNAc),-PP-Do1 to Man,(GlcNAc),-PP-Dol? If the latter possibility turns out to be correct, does Man,(GlcNAc),-PP-Do1 serve as a branching point in the pathway, so that, when it accepts Glc groups, it does not receive additional Man groups, but is immediately transferred to protein? The fact that Glc,Man, (GlcNAc),-PP-Do1 is barely detectable in nonnal cells, or in cells treated with 2-deoxy-2-fluoro-~glucose,122points to a high rate of turnover, and supports this suggestion. Does Glc-P-Do1 serve as a D-glucosyl donor to both Man,(GlcNAc),-PP-Do1 and Man, (GlcNAc),-PP-Dol? Can the protein-linked oligosaccharide Glc,Man, (GlcNAc), also be processed to a high-mannose oligosaccharide? If so, does it then function as a protein-bound acceptor of Man groups? Whatever may be the answers to these questions, the regulation of the synthesis of ( a ) Man-P-Dol, arid ( b )Man,(GlcNAc),-PP-Do1 may each turn out to be an important, control point in protein glycosylation. Scheme 2 gives a possible sequence of reactions that may be involved in the dolichol pathway of protein glycosylation. (122) R. Datenla, R. T. Schwarz, and J . Winkler, Eur. /. Hioc.hc~ri.,110 (1980)355-361.

b. Studies in citro.-hlany cr.11-frec~systems are unable to synthesize the lipid-linked oligosacclr;it-ide foriiietl iii intact cells, tiatnely,

Glc,Mali,(GlcNAc),-PP-DoI,01' ~ v t ' l i : ' ~~atr,(GlcNAc),-PP-I)ol. ~ 111stead, in many, but not all, cell-1'rt.e systems, a series of lipid-linked oligosaccharides is fomied. Also, wlien incubated with GDP-hlaii, the se s y s te in s us i d l y accum i i 1at c M a t 1 - P-Do1, a c()mpot 111tl 1):ire 1j, tletectable iii u i u ~ " ~ - . this ' ~ " ;suggests that cither ( N ) the cell-free systems are ineffective or even inefficietit (tor c,xample, b y being iiicotirplete, or because of poor accessibility of substrates to the t i i e i i i l , r a t i e - l , o i i i i ~ ~ enzymes; compare Ref. 124), o r ( 1 ) ) the extraction a n d labelliiig procedures used with intact cells miss cisential intermediates, for exaiiiple, those having a high turnover r a t c ( s e e tlie prc,vious Section). This speculation is based on the fitrtlitig that transfer i l l citro of oligosaccharides from lipid interniediates to endogenous and exogeiioiis acceptors does not require coinpletion of the oligosaccharitle chain to Glc,M\/lan,(GlcNAc)2.In fact, thr. siiiallcst oligosaccharide trmsfet-red to protein in uitro is (GlcNAc), , c o t t i i ~ t from g ~ ~(GlcNAc),-PP-Dol. ~~~~~ Apparently, the di-N-acetylclritol,iosyl portion is an importiint deteriiiinaiit in the substrate-specifici ty of'tlie enzyme(s ) , transferring oligosaccharides to protein. However, tlie finding b y Kornfeld and coLvorkers that the structures of the ittteritietliates in the asseml,ly of the lipid-linked oligosaccharide"' tliffer f r o m those of those fomiecl d u r ing processing of protein-linked oligosaccharides"~ (see later, however) suggests that the asset I 1l) l ?'- i 1 I te riii e d iate s are no t traiis fe rre d direct 1y to pro te i t i . That some cell-free systenis we're iticomplete I)ecame evident when the role of UDP-Glc was apprcci;itc.tl (sce Refs. 127-13s). Only i i i the presence of UDP-Glc (in atltlitiotr to UDP-GlcNAc and GDP-hlan, when no endogenous acceptors arc. prc'sent) was a lipid-linked oligosaccharide o1)taiiied that had thcl siitiic cheinical properties a s the D(123) S. S . Krag,J. H i d . Cheljt., 254 (1979) 9167-9177. (124) T. Cooll)e.ar, S. Mookejea, an11I;.\V. Iieiiliniiig, B i o t A c , t i t . J . , 184 (1979) 3 9 - 3 9 7 . (125) F. Kcuvers, C. I-Iabcts-\.Z'ilIeiii~,A . Hciirking, and P. Boer, Bi(Jc/Lilii. H ~ I J / ) / I ! I . s . Actel, 486 (1977) 541-552. (126) \V. \V. Chen ; w t l W. J. Lenirarz,/. /(io/. ( : / i i , i l i . , 252 (1977) 3473-3479. (127) A. J . Parotli,FEBS Lett., 71 (1976) W - 2 8 6 . (128) A. J. Parotli, E u r . 1 . Biochen., 75 (ICJ77) 171-180. (129) P. W. Hobbiris, S. S. Krag, ant1 7 ' . I , i i i , , / H i d Chi~tir.,252 (1977) 1780-1785. (130) A. Hcrscovics, B. Buggc,, antl K. i f ' . J i ~ m i l o x , J .Hied. C / l c ~ i r t . 252 . (1977) 2271-2277. (131) C. J . Wnechter antl M .G. Sclrcr, , A t - ( , / t . Hioclicni. B I e ~ p h ! y ~188 . , (1978) 385-393. (132) C. Rotiin and S. Bouchilloux, H i i ~ i ~ / t i 13iei/di!/,s. t~t. Actel, 539 (1978) 481-488. (133) \Y. W' Chen and W. J . Lennarz,]. R ~ ( J (/ .: / I ~ I ~253 I . , (1978) 5774-5779. (134) hl. S. Kang and A. D. Elbein, Arc,/t H i o c , / i c J i i i .B~cJ)J/I!/.s., 198 (1979) 304-313. (135) hl. 1. Spiro, R. C . Spiro, a n d V. 11. B h o > r o o , J . B i d . C/wnt., 254 (1979) 5668-7674.

308

RALPH T. SCHWARZ A N D ROELF DATEMA

glucosylated, lipid-linked oligosaccharide obtained from intact cells; for example, that having the three D-glucosyl residues arranged linearly Glc-(1+2)-Glc-( 1+3)-Glc(Man . . . .) at one of the nonreducing ends.136Although the earlier work of Leloir and coworkers had indicated the presence of D-glucosylated derivatives of dolichol (see Ref. 2 for a review), the biological significance of the presence of the D-glucosyl residues remained to be assessed b y Robbins and when they determined that the D-glucosy~atedspecies was a better substrate for transfer to protein than the nonglucosylated. The removal of Glc, but not ofperipheral Man groups and residues, from the lipid-linked oligosaccharide also abolished its function as an oligosaccharide donor in a microsomal preparation from thyroid.':j5 In fact, only the oligosaccharide having three D-glucosyl residues is efficiently transferred to Hence, the presence of three D-glucosyl residues, in addition to two GlcNAc residues, seems to be a recognition signal in the transfer reaction. The inicrosomal D-glucosidase that, immediately after transfer, removes D-glucosyl residues from the protein-bound oligosaccharides was also found to be active towards the D-glucosylated, lipid-linked oligosaccharide.s6,H~ The significance of this finding may be that D-glucosylation is a control point in glycosylation of proteins, because the enzyme would detennine the availability of that lipid-linked oligosaccharide that can serve a s the donor. The implication of this, namely, that a deglucosylated, lipid-linked oligosaccharide should be detectable in vivo when there are no specific requirements for enhanced protein glycosylation, should find suppoi-t in the frequent detection of Glc,Man, (GlcNAc),-PP-Dol. It is tempting to speculate that the donor of the D-glucosyl groups and residues in the lipid-linked oligosaccharide is2,50Glc-P-Dol, and, because some evidence in favor of this has been obtained,138at least in the D-glucosylation of Man,(GlcNAc),-PP-Dol, the following reactions may be proposed as occurring in the later stages of assembly of the lipid-linked oligosaccharide, as in equations 5 - 7 . 2 Glc-P-Do1 + (~lan),(ClcNAc),-PP-Do1 + C;lc-( 1~.3)-Glc-(l-t3)-(Mari)~~(GlcNAc),-PP-I~ol + 2 Dol-P

(5)

(136) T. Liu, B. Stetson, S. J . Turco, S . C. Hubbard, and P. W. Robhins,J. B i d . Cheiii., 254 (1979) 4554-4559. (137) S. J. Turco, B. Stetson, and P. W. Rohhins, Proc. Nut/. Acad. Sci. U . S. A , , 74 (1977) 441 1-4414. (138) R. J . Staneloni, R. Ugalde, antl I,. F. Leloir, E u r . , / .Rioclzetn., 105 (1980)275-278. (139) L. A. M u r p h y antl R. C. Spiro, Fed. Proc. Fed. Am. Soc. E x p . B i o l . , 39 (1980) Abstr. 351.

-

C~lc-P-Dol+ ( ( : I ~ ) ~ ( , ~ ~ ~ I ~ ) , ( G ~ C N . ~ C ) ~ - I ’ P - I ) ~ ~ I

Glc-(l-*2)-((:lc),(\laii),(GlcN,4c),-PP-I)oIi- L>o-lJ ( 6 )

(Clc),(Mai~),(GIcNAcI,-PP-Dol+ polypr.ptitlc + ((:l~~):,(~laii),(C:lcNA~),-polypeptide + PP-l)ol

(7)

The enzyme that catalyzes reactioii 7 h a s been s ~ l i i h i l i % e d , la ~i i ”d ~ ~ ~ ~ purified1412000-fold. The enzynit. activity is depenclent on m a n g a n c ~ s e ions. The enzymes catalyzing wactioiis 5 and 6 have not yet ltee~isolubilized, purified, or separated, a l i t l , therefore, the substrate specificities indicated reiiiain specul.‘1 t IVC’. ’ The history ofbiochemistiy teaches 11s that a full rinclerstancling o f a pathway can be obtained only wlien tlefiiied siilistrates and purified enzymes are used. A proglaiii ot‘ cheiiiical synthesis of dolichollinked saccharides has been iindcitaken Ily C. D. W7aIren and coworkers,145-144 and the first attempts at purification of the enzyines have

I,een

pll~~iSlie~~~4i,S1-S3,14S,146

The pathway of assembly of’I>- I t i an iI (1 s y 1 residues in the 1i p i d-1inked oligosaccharides is not yet untlerstootl i n detail, b u t an overall picture of the assembly process, based on studies in cell-free systems, h a s emerged (see Refs. 2, 35, 49, and $50).The first step in the iisseiiil)l!. of the lipid-linked oligosaccharide is pro\)al)ly the formation of GlcNAcPP-Do1 (see Section II,l,b). It is rc.asonable to assume that the siicceeding two steps are, first, t h v atltlitioii of another GlcNAc group (from UDP-GlcNAc), and secoirtl the, xldition of a Man group (from

GDP-Man).2,:$5 a-GlcNAc-PP-Do1 + UDP-GlcNAc

+

(GlcNAc)2-PP-Dol + CUP-hlair

P-(:lc.NAc-( 1~4)-a-GlcNAc-l’P-l)(,l + LJDP

(8)

fl-Mail-( I ~ ? ) - ( G l c ~ A c ) , - P P - D+ o lGDP

(9)

-

However, these reactions are iiiisiippoited b y evidence from studies with intact cells. The eiizynic. that catalyzes reactioii 9 requires Mg2+ i o l l s 1 4 5 , I 4 i , aiid the enzyme. trolii nervous tissue,14iat least, is strongly inhibited b y EDTA.

(140) C. R o i i i n , F E B S Lett., 113 (1980):340-344. (141) R. C . Das and E. C. Heath, P n w .Ycit/.,4cwtl. Sci. U . S. A , , 70 (1980) :3811-3815. (142) C. D. Warren arid R. W. Jeanloz, , 2 / i ~ t / i o c F~ s; i ~ q m o / . 50 , (1970) 122-137. (143) C. D. Warren, C. Auge, M. L. L;ivr.r, S. Suzuki, D. Power, and R. W. Jranloz, Carhohydr. R ~ . Y82 . , (1980) 71-83, (144) C. A@, C. D. Warren, and R. W. Jr,miloz, (:urhoh!/dr.R w . , 82 (1980) 85-95 (145) A. Heifetz arid A. D. Elbein, B i o c ~ h r ~ i iBi i. o l i / i ~ / , sR. e s . C O I I I I I I 75 ~ L(1977) ~ I . , 20-28. (146) J. S. Schutzlxwh, J. D. SpringfitXl(1,a i i d J . W. Jenscn,j. B i o l . C : h c ’ m . , 235 (1980) 4170-4175. (147) C. J . Wawhter and J. B. Harfortl, , 4 n , / l . H i o c l i e i n . B i o p / i ! / , ~ I92 . , (1979) 380-:390.

310

RALPH T. SCHWAIIZ AND ROELF DATEMA

Incubation of (GlcNAc),-PP-Do1 or Man(GlcNAc),-PP-Do1 with GDP-Man and Man-P-Do1 results in the rapid elongation of the lipidlinked saccharides with a-linked Man groups, and, in several cell-free systems, transfer of the origiiial and elongated oligosaccharicles to enThis transfer was made en bloc to Asii residogenous protein s .* dues, as shown by using doubly-labelled oligosaccharides, and b y analysis of the protein-linked olig~sacch~irides.~n Elongation of the protein-linked di-N-acetylchitobiose or mannosyl-di-N-acetylchitobiose with GDP-Man could not be d e ~ n o n s t r a t e d , ~ ~ "exemplify,'~~,'~~ ing the artificial nature of the transfer to protein, because glycosy1-Nlinked di- and tri-saccharides have not been cleinonstrated.Tn Both GDP-Man and Man-P-Do1 play a role in the fonnation of (a- M an),-p- M an - (G 1c N Ac ), - P P- Do1 ( T I > 4). Man iio s y 1 transfer froin GDP-Man to endogenous acceptor-lipids or exogenous oligosaccharide-lipids can occur in the presence of EDTA. A heptasaccharidelipid is then f 0 n i 1 e [ l . ~ ~ This ~ , ~means ~ ~ , ~that ~ ~(1-) ~the ~ acceptor-lipids ~ must be p-Man-(GlcNAc),-PP-Do1 (or larger) if the addition of the plinked Man group is inhibited b y EDTA, as described for this reaction in nervous and (2) (when the possibility may be excluded that endogenous Man-P-Do1 is present in the enzyme preparations) Man-P-Do1 does not play a role in this reaction, because its foniiation is also inhibited by EDTA (see Section I,l,b). The heptasaccharidelipid is probably the saiiie a s that isolated froin the mutant inouse-lymphoma cells unable to synthesize Man-P-Do1 (see Section 11,.2,a). The work of Schutzbach and co-authors indicated that at least the fonnation of the a-mannosyl-(1+2)-mannose linkage occurs b y transfer d i r e ~ t l froin y ~ ~GDP-Man. ~ ~ ~ ~ ~They partially purified the enzyme that catalyzes the fonnation of thi s bond, aiid demonstrated146that it is not capable of forming Man-P-Dol. Thus, it is probable that, in the fonnatiori of' an a-Man-(1+2)-Man unit, not only in the oligosaccharides linked to L-serine or L-threonine in the yeast iiiariiioproteiiis,ls~-l~ti but 4y35n

(148) W. W. Cheii aiid W. J . Lennarz,J. B i o l . Cheni.,251 (1976) 7802-7809. (149) L. Lehle and W. T a i i n e r , Eur. J . Hioclieiii., 83 (1970) 563-570. (150) W.T. Forsee, J. A. Griffin, antl J . S. Schutzbach, Aiochem. Bioph!/s. R u . C o i i i rnuir., 75 (1977) 799-805. (151) J. Chainbers, W. T. Forsee and A. D. Elbein,J. Biol. C h e i i i . , 252 (1977) 24982506. (152) J. P. Spencer and A . 11. Elbein, Proc. Nntl. Accitl. Sci. U . S. A., 77 (1980) 2.5242527. (153) P. Babczinski and W. Tanner, Rioc41eiti. Biop/i~/s.Hex Coriiniuii., 54 (1973) 11191124. (154) L. Lehle and W. Tanner, B i o c / i i i i r . B i o p / q s . Acta, 350 (1974) 225-235. (155) C. B. Shartna, P. Bal)czinski, L. Lehle, antl W.Tanner, Eur. J . Bioclwm., 46 (1974) 35-41. (156) E. Barise and L. Lehle, Eur. J . Riochrnr., 101 (1979) 531-540.

THE LIPID PATHWAY O F PROTEIN GLYCOSYI,.ATI~N

31 1

also in those linked to L-asparagine residues, GDP-Man (not Man-PDol) is involved. As indicated i n Section II,2,a, hlan-P-Do1 plays a role in the elongation of Mati,(C:lcNAc),-PP-Dol to Man, (GlcNAc),PP-Dol. Although it has become feasible that Man(GlcNAc),-PP-Do1 can accept D-inannosy1 residues directlyis' froin GDP-Man, it has still to be demonstrated that incul)ation of the trisaccharide-lipid with Man-P-Do1 does not result in elongation. Thus, evidence for the existence of two pathways of assembly of D-mannosyl groups has become available.ii7aIn one pathway, already outlined, D-inannosy1 groups froin GDP-Man elongate (GlcNAc),-PP-Dol, to give an endo-pN-acetylglucosaininidase H-resistant heptasaccharide, namely, Man,(GlcNAc),-PP-Do1 (see also, Hef. 156a). In the other pathway,t17a endo-P-N-acetylglucosaniinidase H-sensitive hexasaccharides and heptasaccharides are formed (see Scheme 2), and Man-P-Do1 might be involved in the assembly of this Man,(GlcNAc),-PP-Dol. As already indicated, control points in the regulation of the rate of glycosylation of proteins may be ( ( I ) the availability of Dol-P (Section II,l,a), ( b )the D-glucosylation of the lipid-linked oligosaccharide, o r its transfer to protein, or both (Section I,2,a), and, as already discussed, (c) the metabolic fate of' GDP-Man. Furthennore, GDP-Man inhibits fonnation of Glc-P-Do1 i n cell-free preparations front liver,',' and activates formation of GlcNAc-PP-Do1 in cell-free preparations from tissues of chick embryo.i5H An interesting parallel, in the eiikaryotes, to the dolichol pathway of protein glycosylation was descrilwtl for the initial stages of cellulose synthesis in the alga Protothecu z011$i.46.159 Here, Glc-PP-Do1 serves as an acceptor of Glc groups froin UDP-Glc, to give (Glc),-PP-Dol; the trisaccharide-lipid can, in turn, accept further 1)-glucmyl groups, from Glc-P-Dol, to give a cello-oligosaccharide-lipi(~(Glc),-PP-Do1 (ri 10). The oligosaccharide inoietl. is then transferred to I I protein, where it serves as a primer for cellrilose synthetase (which requires GDP-Glc as the D-glucosyl donor). There is little further evidence that lipid intermediates play a rolc, i n the forniation of cell-wall poly1 i i e r s , 4 ~ with , ~ ~ " the notable exceptions of yeast ~ i i a i i ~ i a r i ~ant1 ~"~ a1' gal ~'~ t i i a i i n a i ~A, ~question ~~ arising in this context is whether, for lipid-de-

-

; c i i t l 1 . S. Schiitzbach,]. B i d . C l i c . r t r . . 2.55 (1980) 11,268-11,272. (157) A. K. A. Kerr a i d F. W. Heinming. l i u r . /. RioclarJiit.,83 (1978) 581-586. (158) E. L. Kean,/. R i o l . Cherii., 255 (1980) 1921-1927. (159) R. Poiit Lezica, Bioclzrmi. Soc. 'l'ruji.\., 7 (1979) 334-337. (160) P. Boer, tliocheitt. Soc. Truiis., 7 (1979) 331-333. (161) W. Taiiner, P. Babczinski, hl. hl;ii-iott, A. Hasilik, iiiid I,. Lehle, R i o c / i c , ~ t iSoc. . Trutts., 7 (1979) 329-331. (162) P. A. Romero, H. E. Hopp, ; u > t l 11. I'oiit Ixzica, Biochiiti. Riop/i!/.c..Acfri, 586 (1979) 545-559.

(156a) J. W. Jeiiseii, J. D. Springfield,

312

RALPH T. SCHWAHZ A N D ROELF DATEMA

pendent synthesis of a cell-wall polysaccharide, a protein-acceptor is a prerequisite and, if so, what the nature of this acceptor is. The nature of the acceptor for foiination of Asn-linked oligosaccharides has been studied i n some detail in cell-free systems from aninial tissues and from yeast.ig6-i63+16X These studies (where synthetic peptides, or protein-derived, small peptides of known sequence, were incubated with lipid-linked saccharides, and the rates of transfer of the saccharide to the peptide were measured) confirmed a proposal16Y that the occurrence of the sequence Asn-X-Ser/Thr in a peptide is a minimal requirement for glycosylation. A tripeptide, for example, Asn-Leu-Thr, is, however, a very poor acceptor,I6’ but the rate of glycosylation is increased when larger peptides, elongated at either the C-terminus or the N-terminus, or both, or peptides having otherwise blocked amino and carboxyl termini, are u ~ e d . ~A ~ ~ , for ~ ~ ~ , ~ reason these better acceptor properties may be the diminution of charge effects around L-asparagine. Glycosylation in zjiwo will, in addition, require exposure of the tripeptide sequence, that is, accessibility to the oligosaccharide transferases.50si6H Although studies with cell-free systems and synthetic peptides indicated that Pro and Asp in position X of the sequence Asn-X-Ser/Thr will give poor substrates,16g,166 only data from sequence analysis of glycoproteins can be used to contradict the supposition that some amino acids (Pro or Asp, for example) are “forbidden” in position X.

c. Cell Mutants Having Defects in the Synthesis of Lipid-linked 0ligosaccharides.-In the past, a number of cell mutants having defects in the glycosylation of glycoproteins and of glycolipids have been described. They have been selected mainly by using toxic, plant lectins as selection agents. Lack of a particular glycosylating enzyme may lead to altered, cell-surface carbohydrates, revealed b y virtue of an altered binding of lectins: the cells that do not interact with the lectins survive, and are cloned. Some cells have been shown to lack defined, enzymic activities, but, in other instances, the biochemical basis for carbohydrate alterations are not clear (for a review, see Ref. (163) D. K. Struck, W. J. Lennarz, aiid K. Brew,J. Biol. Chetn., 253 (1978) 5786-5794. (164) C. Ronin, C . Granier, J. van Rietschoten, and S. Bouchilloux, Biochetn. Biophys. Res. Cornmuti., 81 (1978) 772-778. (165) C. Ronin, S. Bouchilloux, C. Granier, and J . van Rietschoten, FEBS Lett., 96 (1978) 179- 182. (166) E. Bause, FEBS Lett., 103 (1979) 296-299. (167) E . Bause and H. Hettkamp, FEBS Lett., 108 (1979) 341-344. (168) G. W. Hart, K. Brew, G. A. Grant, R. A. Bradshaw, and W. J . Lennarz, J . B i d . Chem., 254 (1979) 9747-9753. (169) R. D. Marshal1,Annu. Reu. Biochem., 41 (1972) 673-702.

THE LIPID PATHWAY O F I’ROTEIN CLYCO\YI,ATION

313

170). Here, we shall discuss nititants having defects in the lipid pathway. Soiiie con A-resistant mutants of‘C:hinese-haiiister ovary-cells show a lesser synthesis of [‘4C]mannos);l-lipid-linked oligosaccharide if the cells are not co~ifluent.”~ One of‘ the niritants has been shown to be deficient iii the synthesis of [3H]glucosyl oligosiiccharide-lipid. In other words, the transfer o f D-glucose from Glc-P-Do1 to lipid-linked oligosaccharide was impaired; t h i s , i n tnrn, could lead to an accuniulation of Glc-P-Dol, thereby deplcting the pool of Dol-P. Decreased amounts of Dol-P are, indeed, sirggcsted b y the olxervation that only after addition of Uol-P to iiieiiibrancs from mutant cells was synthesis of lipid-linked oligosaccharicles ol,served in amoiints comparable to those formed b y the wild types. Thus, although it is not clear if this mutant has one, or two, genetic tlefect(s), deficient glucosylation of lipid-linked oligosaccharides ins!' also account for the decrease i n Dol-P. Other mutants of Chinese-humster ovary-cells having decreased am o tin t s of 1i pi d-1i ii ke d 01 igosacc*h ari tle s are kn own, a1tho ugh they are less well characterized. G-Protcin of vesicular stomatitis virus grown in one of these mutants appearcd to contain fewer, but f‘iill-sized rather than truncated, oligosacchiiricle side-chains. There may be iiisufficient amounts of oligosaccharide precursor available for transfer to nascent glycopi-otein~.’~~ Also, mutants having a defect i n the foimation ofnomia1 ainounts of lipid-linked oligosaccharides have been is01ated.I~:’However, in acldition, these cells show increased incorporation of [L4C]-labelfrom [’4C]inannose into glycoprotein. It h a s been demonstrated that these cells show an increased flux in the pathway leading from GDP-Man to G D P - F U ~ .In ” ~class E Thy-1 negative, niouse-lymphoma cells, which have been isolated b y iniiii~itros~,l~~ctioii froin mouse-lymphoiiia cells,’74an alternative pathway to complex oligosaccharides is used (see also Section 11,2,a). Instead of Glc3Man,(GlcNAc),-PP-Dol, Glc,Man,(GlcNAc),-PP-Do1 is forrried and transferred to G-protein of vesicular stomatitis virus, where the oligosaccharide moiety can be converted into the complex type.”’ Formation of Glc,Man,(GlcNAc),PP-Do1 does not require the participation of Man-P-Do1 (see Section 11,2,a),and, indeed, an inability of’these cells to form Man-P-Do1 has (170) P. Stanley, in Ref. 50, pp. 161-180. (171) S. S. Krag, hf. Cifone, P. W. Rohliins, m i d R. hl. Baker,/. H i o l . Chern., 252 (1977) 356 1 -3564. (172) E. B. Briles, S. Schlesinger, a i i t l S. K o I t i f e l d , J . Cell Biol.. 79 (1978) 405a. . Rc.r., 121 (1979) 1-8. (173) J. A. Wright, J. C. Jamieson, and 14.( k r i , E x ~ JCell (174) R. Hyman and I. Trowbridge, C O I I /(;y c / / Prolif., 5 (1978) 741-754.

314

RALPH T. SCHWAHZ AND ROELF DATEMA

been shown. Only after addition of exogenous Man-P-Do1 to homogenates of the mutant cells will the complete, lipid-linked oligosaccharide Glc,Man,(GlcNAc),-PP-Do1 be fornied.1i9 Another mutant cell-line synthesizing truncated rather than fullsized lipid-linked oligosaccharide was obtained by selection against both phytohemagglutinin and concanavalin The absence of a(1~6)-mannosyltransferasewould explain the synthesis of an oligosaccharide containing seven, rather than nine, D-niannosyl residues. As discussed in Sections II,2a and II,2b, lack of peripheral Man in the lipid-linked oligosaccharicle would not hamper the transfer to protein. A Chinese-hamster lung-cell having a temperature-sensitive lesion i n synthesis of glycoprotein synthesizes lipid-linked oligosaccharides normally; but here, a temperature-sensitive step in glycoprotein synthesis appears to be the transfer of the oligosaccharide core from the lipid-oligosaccharide intennediates to the nascent, polypeptide chain. However, further investigations are needed in order to decide whether the enzyme that transfers the oligosaccharide from the lipidlinked oligosaccharide to the protein is defective, o r whether its activity is impaired because of the environmental factors, such as the lipid composition of the ~ n e i n b r a n e s . ' ~ ~ It is clear that mutants of the lipid pathway are useful in elucidating the individual steps in the assembly (see Section II,2,a) and transfer of the lipid-linked oligosaccharides, and possibly will become important in future work on the synthesis of lipid-linked oligosaccharides. A.1i53175a

3. Cytological and Topological Aspects Soon after the discovery of dolicliol,lii the question as to its physiological function arose, and it was believed that a study of its distribution in the various cellular fractions would help to establish its role. The determination of the intracellular distribution of total dolichol in pig liver showed that it is fairly evenly distributed. The values obtained in the various fractions were in the range of 1 ing per g ofprotein. Microsoines contained 0.78, and ground plasm 0.30, whereas nuclei and mitochondria were richest, with respective values of 1.34 and 1.07 mg per g of ~ r 0 t e i n . IA~ re-examination ~ that was undertaken when it became clear that dolichol also occurs in esterified form re-

(175) L. A. Hunt,]. Virol., 35 (1980) 362-370. (175a) L. A. Hunt, Cell, 21 (1980) 407-415. (176) A. J. Tenner and I. E. Scheffler,]. Cell. Ph!lsiol., 98 (1979) 251-266. (177) F. W. Hemming, R. A. Morton, and J. F. Pennock, Biochem. ]., 74 (1960) 3 8 ~ (178) J. Burgos and R. A. Morton. Biochern. I , , 82 (1962) 454-456.

vealed that cell debris and nuc1r.i contain 53% of the estcrified dolichol, whereas 77% of the free f o r t i t o c c ~ i r sin t~ritoclroiidria.'" a. Subcellular Localization of Dolichol-dependent Glycosylation. -The localization of dolichol in siibcellular structures of' rat-liver cells has heen rnonitored after itrtritvthnoits injection of tritiatetl dolicliol of high specific activity. h l o s t of' tlie radioactivity w a s recovered free dolichol, although small proportions ofdoliclrol tatty acid ester were foiiried with increasing tittrc. Significant tnetalwlism was not found, and part of the unaltercxl tiraterial was excreted in the feces, presumably through the hile, I'hc striking resiilt of this stud!, was that most of the dolichol was found i t 1 tlte nritochondrial fraction, which was more highly radioactive tliaii a i r y other organelle, ancl that this radioactivity was concentrated witlriii the outer, mitocl?ondrial inc'iiibrane. Anrounts in the Golgi f r x t i o i i were fairl!. sniall." Also, aftcr injecting [4-(S)-3H]mevalonate into partially hepatectotirizecl rats, the dolichol isolated froirr the mitoc~lrontlriaa r c 1 the cell debris contaiiied t h e highest specific activity.5 From siiel-1stiidies, it is clear that no conclusions can be drawn concerniiig the sul)cellular t1istril)ution of 1101P, Dol-P-monosaccharides, and I)ol-PP-oligos~tccharictes,ant1 the localization of pathways that u s t ~tliest, siibstrates. Attc.tnpts have b e ~ t r made to estimate, for example, tlw amounts of Dol-P h y using the forniation of Glc-P-Do1 from UDP-Glc a s a method of a ~ s a ! - . ' ~ Extracts '" from the nuclear and Golgi tractions prodiiced thc greatest stitnulation, and, according to the aiithors, had the highest Itvels of 1101-P. Lipid-mediated glycosylatioii w a s found to occiir in yeast nuclear~ ~ ~ 'proves ~' the occurrence o f the membranes and rat n ~ c l e i , ~ *which corresponding enzymes and snl)stratc>sin this organelle. In the light of the aforementioned results, it is not surprising that, in liver iiiitochondria, glycosylation of proteins appt'ars, at least in part, to occur h y the lipid pathway. The outer meinl)rane, especially, contains e t i z y n e s that incorporate D-glucose from LJI)P-glucose, and D-nraniiost~from G1lP-mannose, into Glc-P-Dol, Mart-P-Dol, and lipid-linkecl oligosaccharides and p r o t e i i ~ . ' ~ ~ Interestingly, -~*~ initoclionclria from influenza v iru s-i11 fecte d ce 11s display a s t i ii t I 1at io 11 of a i i A!-acety I g l uco s at 11 i I I ylas

(179) G. Dallner, N . H. Behrens, A. J . l';irotli, and L. F. Leloir, F K H S L e t t . , 24 (1972) 315-317. (180) G. Palanrarczyk and E. Janczura. FI:'NS L r t t . , 77 (1977) 169-172. 6 0 (1978) 593-599. (181) M .Richard, F. Tytgat, arid P. L,ouisot, Hi(~chitnz~,, (182) 0. Gateau, R. Morelis, and P. Liiuirot, C R . Acud. Sci. Ser. 11. 290 (1980) 413416. (183) 0. Gateau, R. Morelis, arid P. Loriisot, C. R . Accitl. Sci. Ser. I ) , 285 (1977) 15231525. (184) 0. Gateau, R. hlorelis, a n d P. Lotii\i)t, Eirr. J . Hioclieni., 88 (1978) 613-622.

316

RALPH T. SCHWARZ AND ROELF DATEMA

transferase, which leads to increased formation of GlcNAc-PP-Do1 and (GlcNAc),-PP-Dol.'85The sigiiificance of the latter finding awaits further investigation. Mitochondria from yeast have also been found to possess enzymes of the lipid-linked pathway. Palainarczyk'*'j reported the formation of Man-P-Dol, GlcNAc-PP-Dol, and (GlcNAc),PP-Dol, and the incorporation of their sugars into water-insoluble polymers. The main sites of glycoprotein biosynthesis are, however, the endoplasrnic reticulum and the Golgi apparatus. Therefore, attempts have been made to assay dolichol-depeiidei~treactions in these organelles. Rough and smooth membranes from rat liver have been shown to synthesize Man-P-Dol, GlcNAc-PP-Do], Glc-P-Dol, and lipid-linked oligosaccharides, and to mediate glycosylation of protein. Golgi-derived nieinbranes were also found to be capable of performing these reactions, but to a lesser e ~ t e n t . ' ~The ~ , ~significance ~" ofthis finding might possibly be explained by a flow of intracellular membranes froni endoplasmic reticulum to Golgi and plasma membrane, and transferases of the lipid-linked pathway may be inactivated or degraded during this transport. Throughout the systems investigated, the endoplasmic reticulum has usually been found to be the main site for lipid-dependent glycosylation of protein. In yeast, all of the transfer reactions in which dolichol phosphate is glycosylated, or dolichol phosphate- and diphosphate-activated sugars serve as glycosyl donors, showed the highest specific activity, and had most of the total activity in the endoplasinic reticulum. PlasmaleInma- or Golgi-containing fractions contained significantly smaller amounts of these a c t i v i t i e ~ . ~However, ~ ~ . ' ~ ~ the lipiddependent glycosylatiori of protein in the plasma membrane of yeast has been well docunieiited.l9' D-Mannosylation of lipid-linked oligosaccharides and of proteins has been found to occur in rough, endoplasinic retictilum.18K*19z In hen-

(185) 0. Gateau, R. Morelis, and P. Louisot, Biochiniie, 62 (1980) 79-84. (186) G. Palamarczyk, Actu Biochim. Poloti., 14 (1976) 1290-1292. (187) A. Bergman, T. Mankowski, T. Chojnacki, L. M . tle Luca, E. Peterson, and G. Dallner, Biocheni. /., 172 (1978) 123-127. (188) 0. S. Wilson, M. E . Ile Thomas, E. Peterson, .4.Bergman, G. Dallner, and F. W. Hetnming, Eur. /. Biochem., 89 (1978) 619-628. (189) M. Mariott and W. Tanner,]. Bacteriol., 139 (1979) 565-572. (190) L. Lehle, F. Bauer, and W. Tanner, Arch. Microbiol., 114 (1977) 77-81. (191) G. W. Welten-Versteegen, P. Boer, and E. P. Steyn-Parvi.,/. Bucteriol., 141 (1980) 342-349. (192) V. Idoyaga-Vargas, M. Perelmuter, 0. Burrorre, and H. Carminatti, M o l . Cell. Biochem., 26 (1979) 123-130.

T H E LIPID PATHWAY 0 1 ; P'HOTEIN GLYCOSYL.4TION

317

oviduct membranes, enzymes iirvolvetl in the torination of glycoproteins by way of the lipid-linked pathway were localiztd almost exclusively in tlae rough, entloplasniic reticulum. I n contrast, a galactosyltrarisferase that catalyzed transfer of galactose to asialo-agalactoorosomucoid was localized i n tlie s nioo th-in em 1, r a n e fraction. The re was no evidence for the involvemelit of lipid intemiediates in the galactosyl transfer observed in this f r ~ t i o n The . ~ ~extent ~ of separation in this study1s3of the pathway involving lipid-linked oligosaccharides and single-step transfer of sacx*harides i n rough and smooth nit'iaibranes appears to be exceptionally complete. The role of glycosy1transft.rusr.s found in tlie plasma niemljraire of animals cells is not clear; for ;I rcAview, see Ref'. 194. Evidence f o r lipid-dependent glycosylation b y ectoglycosyltraiisferascs h a s BCCIIni d a t e d .2i,3gs- 9 i The p lasnaa n I t' 11I 11 riin e s from 11 tin1 an e i-yth rocy te s o r reticulocytes contain the enzynies that fomi Glc-P-Dol, Man-P-Dol, GlcNAc-PP-Dol, (GlcNAc),-PP-llol, and Dol-PP-lirikecl oligosaccharides consisting of GlcNAc, hlan, and Glc, litit the transfer to enclogenous acceptors has been observed only with reticulocyte plasmaiiieiiil>rane.T h e lack of glycosylation in erythrocytes is possibly due to the absence of either the enzyinc that transfers the oligosaccharide from tlae lipid-carrier to the protcliii, or of suitable t~ndogenousacc'eptors. i g ~ - z n n The s ubce 11u lar di s tri butio 11 of 1i 1, i c I-de pe nd e n t, gl yco s y 1,'I t 1011 reactions has also been investigated i i i a number of plant systems. In plant cells, the situation is, however, more coniplicatetl, a s their iiieniljriines often have the capability to transfer activated siigars, not o n l y to lipidbound saccliaricles201~203 and to ~irotei~is,"","~-""" but a l s o to cell-wall '

(193) U . Czichi antl W. J. Lei1narz.J. B I ( J /C. h c t i t . , 252 (1977) 7901-7904. (194) B. D. Schur a n d S. Roth, Biochiiti. / j i o ) j \ i ! / , Y , clctcr, 415 (l97Fi) 473-.512. (195) L. 11. Patt a n c l W. J. Grimes, Bioc,/iirti. H i o p / i ! / s . Actcr, 444 (1976) 97-107. (196) U . Arnold, E . Iloinmel, antl H . J . Ri\\e. .lfo/. C : c l l . BioclwiTi., 11 (1976) 137-145. (197) 11. K. Struck airtl W. J. Lern1arz.J H i o l . Clwiti., 251 (1976) 2511-2519. (198) A. J . Parodi and J . Maitiir-Baricnto\, ~ i ( J C h i 1 l iB. i o p l i ! / ~.4cfo, 500 (1977) 80-88. (199) J . Martin-Baririrtos antl .4. J. Pai-o(Ii, .\fo/, C e l l . B ~ o c ~ I ( ~16 I J (1977) I., 111-117. (200) J . J . L L I C ~ and S C . Nevar, B i o c h i t i ~ Bioph!/,T. . Actcl, 528 (1978) 475-482. (201) L. Lehle antl W. Taiiirer, Riochiiii, B i o ! i / i ! / , s .ilcfn, 399 (1975) 365-37.2. (202) R. Pont Lezica, P. A. Hoinero, ;uid 11. A. Uankert, P l n ~ i t/ ' / i ! / . $ i o / . , 58 (1976) 65;680. (203) C. T. Brett a r i d L. F. Leloir, Bioc,/rc,tlr./.. 161 (1977) 93-101. (204) L. Lehle, F. Fxtaczek, Mr. T m r i i ~ ~and i , H. Kauss, Arch. Hioc/ic,trr. Biop/i!/,y., 175 (1976) 419-426. (20s) 11.C . Ericsoir ant1 D. P. D e l i i i ~N~u, t i t P \ t ! / . ~ i o l , ,59 (1977) 341-347. (206) H. Pont Lezica, P. A. Ronrero, mid 11. E. Ilopp, P E U J I ~( R O c r l i r i ) , 140 (1978) 177183.

G 18

RALPH T. SCHWARZ A N D KOELF DATEMA

polysaccharides"'i'20X;for a review, see Ref'. 49. In addition, the different species niay show differences in the intracellular localization of glycosylating enzymes due to the different products they fonn. Encloplasmic-reticululn membranes from the endospenn of castor beans glycosylated sulfitolyzed ribonuclease A i t i uitro, starting from labelled lipid-linked oligos~~ccharides."O" In the green alga Protothecti zopfii, the enzymes reponsible for the fomiation of oligosaccharidelipids, and for the subsequent transfer from lipid to protein, were associated with the rough endoplasmic reticuluin.210These results are in agreement with those for protein glycosylation in Phaseolus mreus211 and Pisum .satiuuin.212 On the other hand, cellulose synthetase (for involvement ofdolichol derivatives, see Section II$,b) was consistently found to be associated with the Golgi-rich fractions.210Similar results were obtained in peas.210~212a Endoplasmic reticulum from peas contains most of the recovered capacity for glycosylation of endogenous polyprenol phosphate, but a direct involvement of Man-P-Do1 fonned in these meinbranes in the synthesis of other lipid-linked oligosaccharides could not be demonstrated. Polyprenol diphosphate and lipid-bound oligosaccharides occurred to a limited extent in the endoplasmic reticulum and in Golgi and, possibly, plasma iiieinbrmes.21R In soybean, UDP-g1ucose:dolichol phosphate glucosyltransferase had the highest specificity in fractions containing the plasma inembrane, but some activity was also fouiid in regions of Golgi meinbranes and endoplasinic Enzymes in the different membrane fractions may be used for different glycosylation processes. In conclusion, lipid-dependent glycosylation-reactions appear to occur throughout all subcellular compartments of various cells, especially those from plants. As inany investigations were conducted at a time when many details of the pathways for lipid-dependent glycosylation were still unknown, re-investigations appear necessary.

(207) P. M. Ray, T. L. Shininger, and M . M . Hay, Proc. Nntl. Acad. Sci. 71. S. A., 64 (1969) 605-612. (208) G. Shore arid G. Maclachlan,J. Cell Riol., 64 (1975) 557-571. (209) B. Mellor, L. M .Roherts and J . XI. Lord, A i o c h ~ iJ., . 182 (1979) 629-631. (210) H. E. Hopp, P. A. Romero, G . R. Daleo, airtl R. Pont Lezica, in L.-A. Applequist and C. Liljenberg (Ecls.),Adoa J I C C S i i i tlie Hiochemistr!y arid Ph!/siolog!yo . f P h t Lipicls, Elsevier/North-Hollair(~Bioniedical Press, Ainsterdam, 1979, pp. 313318. (211) L. Lehle, D. J . Bowles, and W . Ta11ner, P l n n t Sci. L r t f . , 11 (1978)27-34. (212) J. Nagahashi and L. Beevers, Pk17lt P h l s i o l . , 61 (1978) 451-459. (212a) J. P. F. G. Helsper, J. H. Veerkamp, and M . M . A. Sassen, Plaritu, 133 (1977) 303-308. (213) M. Diirr, D. S. Bailey, and G. Maclachlan, E u r . J . Biochem., 97 (1979) 445-453. (214) C. M. Chadwick and D. H . Northcote, Biochem.J., 186 (1980)411-421.

THE LIPID PATHWAY 01’ I’IiO‘L’EIN GLYCOSYLATION

31 9

b. Sidedness, Signal Theory, and the Theory of Membrane-triggered Folding.-Here, some aspects of protein glycosylation in the endoplasmic reticulum will be discussed. Some proteins are synthesizctl on ribosomes that are attached on the cytoplasmic side of the entloplasmic reticuluiti. They are either destined for secretion to the exterior of the cell, or, after proper iiisertion, become integral, membrane proteins. Some are destined for the lysosomes. In the past decade, viral glycoproteiiis have seived as models for studying the synthesis aircl insertion of the membrane proteins. Central questions are how these molecules are ( a ) synthesized, and ( h ) integrated into the cellular membranes. Two theories that share some features are currently used to answer them. (1 ) The signal theory suggests that the N-terminal, hydrophobic leader-peptide of membrane proteins binds to a receptor in the hilayer of the endoplasmic reticulum and is recognized b y a tiieinbrane-boiind, peptide-transport system. The polypeptide synthesizing ribosonie binds to this system, and “pushes” the nascent protein through a specific, transient pore. The leader seqiience is clipped off by a sigiialpeptidase while the protein is still being synthesized, but these sequences can also remain as a part of the end product. This means that removal of the hydrophobic, leader sequence from the protein is not in all cases ~ i e c e s s a r y . ” ~(2) - ~The ~ ~ hypothesis of nieinbrane-triggered folding postulates that the leader peptide interacts, in concert with other regions of the nascent or completed protein, with the hydrophobic regions ofthe membrane, and thus allows (in a process that resembles self-assembly processes) passage through, or insertion into, iiieinbranes. Neither a specific, peptitle-transport system nor active protein synthesis is required in this theory.’LXFuture studies should demonstrate whether both theories upply. It is, however, well established tliat inembrane glycoproteiris and secreted proteins are glycosylated during the synthesis of their peptide lxickbone,21“-22’ although post-translational glycosylatiori has also been r e p ~ r t e d . * The ~ ~ -questioii ~ ~ ~ ~ arises as to whether these proteins (215) G. Blobel and B. Dobberstein,/. (,’(,// H i d . , 67 (1975) 835-851. (216) G. Blobel and B. l h b b e r s t e i n , / . ( : P / / fjfol., 67 (1975) 852-862. (217) G. Blobel, Proc. N u t l . Acad. S c i . l / . S. A , , 77 (1980) 1496-1.500. T J(1979) I., 23-45. (218) W. Wickner, Annu. Reo. B ~ O C ~ C J48 (219) F. Katz, J. E. Rothman, V. Lingaplxi. (2. Blobel, and €1. Lodish, Proc. Y u t l . Acud. Sci. U . S. A , , 74 (1977) 3278-3282. (220) J. E. Rothman and H. F. Lodish, Vutiirc., 269 (1977) 775-780. (221) L. W. Bergman and W. M. Kiiehl, Hiocherllistry, 16 (1977) 4490-4497. (222) L. W. Bergman and W. M. K u e h l , f~ioc.lieiiiistr!/, 17 (1978) 5174-5180. (223) V. Idoyaga Vargas and H. Carminutti, Alol. Cell. BiochrrJi., 16 (1977) 171- 176. (223a) J. E. Strickler and C. L. Patton, Ft-oc.. N u t / . Accitl. S c i . U . S.A , , 77 (1980) 15291533.

320

RALPH T. SCHWARZ A N D ROELF DATEAMMA

are glycosylated before, or after, passage, or insertion, into the meinbranous system of the cells. In a series of elegant studies in oitro, it has been shown that glycosylation of nascent proteins requires the presence of membranes, and that, after synthesis of the glycoprotein, the glycoproteins are inaccessible to pro tease^,^^^.^^^ indicating that they are inside the vesicles. These experiments do not, however, indicate where the inernbrane-bound, oligosaccharide transferase is situated-on the cytoplasmic, or the luminal, side of the membrane vesicle. Location on the cytoplasmic aspect would imply that the protein has to cross the bilayer together with its carbohydrate side-chains, a process that appears unfivorable from the thermodynamic point of view. Using sealed, microsomal vesicles from hen oviduct and endoP-N-acetylglucosaininidase H as a topological probe, Hanover and LennarzZZ6 coiicluded that transfer of oligosaccharide from the lipid carrier occurs within the lumen of the endoplasinic reticulum. This assumption is supported by the results of studies on the location of (GlcNAc),-PP-Do1 i n natural and artificial membranes, using the formation of Gal(GlcNAc),-PP-Do1 by exogenously added galactosyltransferase and UDP-galactose as the indicator reaction. It was coricluded that N,N'-diacetylchitobiosyl (dolichol diphosphate) faces the lumen of the endoplasniic reticulum, and that no significant, unassisted flip-flop occurs.45The same conclusion has also been obtained from studies using spin-labelled, glycosyl-carrier lipids and phosphatidylcholine vesicles.44 In other words, it appears that the inolecule (GlcNAc),-PP-Do1 is already inside the lumen of the vesicles, and that its elongation to give the final product probably has also to occur in the same orientation; this suggests that trans-membrane movement of GlcNAc-1-P, GlcNAc, Man, and Glc could be an integral part of'the enzymic reactions leading to the formation of the final, lipid-linked oligosaccharide. Treatment of sealed vesicles from rat-liver microsomes with protease, and subsequent assay for the capacity to synthesize Glc-P-Dol, Man-P-Dol, and GlcNAc-PP-Dol, and to transfer a D-glucosyl group from Glc-P-Do1 to the oligosaccharide-lipid, showed that all of these activities of oligosaccharide-lipid synthesis were labile to pronase and trypsin digestion. As only the cytoplasmic face of the membrane was accessible to the protease, it is suggested that active centers of the corresponding enzymes reside on the cytoplasmic face.227Similar con(224) H. Garoff, K. Simons, and B. Dobberstein,J. M o l . B i d . , 124 (1978) 587-600. (225) F. Toneguzzo and H. P. Gosh, Proc. N a t l . Acod. Sci. U . S. A., 75 (1978) 715-719. (226) J . A. Hanover and W. J. Lennarz,]. B i d . Chern., 255 (1980) 3600-3604. (227) M. D. Snider, L. A. Sulzman, and P. W. Robbins, Cell, 21 (1980) 385-392.

T H E LlPID PATHW.41 OF PROTEIN CLYCObYL.ATION

32 1

clusions had been drawn earlic~r.i24-1’xx These authors, however, restricted the protease-sensitive activities to the synthesis of oligosaccharide side-chains of glycoproteins situated at the cytoplasmic f~iceof the endoplasmic reticulum. Further studies will be neetl(1tl i n order to resolve the iiitrigiiing question a s to how the glycosyl groiips of the activated sugars that are usually found in the cytoplasiii :ire' transferred to the growing chain of the lipid-bound oligosaccharitle inside the lumen of the endoplasmic reticul urn.

111. INHIBITORS OF h O T E I N GLYCOSYLATION

1. General Comments Inhibitors of lipid-dependeiit gl?,cosylation of proteins are also antiviral and antibacterial agents.228The multiplication of soine enveloped viruses requires proper glycosylation of their envelope glycoproteins,229and formation of 1)actc.rial peptidogl in is iiiitiated b y lipid-dependent transfer of a siigar phosphate c vative.‘”) In fact, some of the most-used inhibitors of protein glycosylation were cliscovered b y virtue of their antiviral propei-ties.”,22””:’ Also, several antibiotics known for some time to intcrfcre with bacterial growth were subsequently found to inhibit the glycosylation of eukaryotic proteins? Other sources of glycosylation inhibitors are sugar analogs, or analogs of nucleotide esters of sugars. A strate wards development of such inhibitors has been outlined i n r c v i e The best known inhibitors of gly 11 of proteins interfere with the lipid-dependent step 22H Substances that specifically block reactions taking place after the transfer of the oligosaccharide to the protein are little known. As several, incompletely (or differently) glycosylated, viral glycoproteins iwe still biologically active (see Section IV), these substances would escape the screening procedure based on

(228) R. T . Schwarz and R. Datema, ‘l’rt~ritl.~ R i o c h e m . Sci., 5 (1980)65-67. (229) R. Rott antl H.-D. Klenk, in G. Postc a n d C . L. Nicolaon (Etls.),Vir7c.r.Irifi,c.fiori urid tlw Cell Surface, Elsevier/N~)i-th-HollandBioinrtlical Preas, Amstertlam, 1977, pp. 47-81. (230) S. J . Tonri antl J. E. Gander, Anrirl. H ~ L Microbial., . 33 (1979) 169- 199. (231) A. Takatsuki and G . Tamura,]. .4ri/il1io/.,24 (1971) 224-231. (232) A. Takatsuki and G. Tamura,]. A t i f i b i o t . ,24 (1971) 232-238. (233) C. Scholtissek. Curr. T o p . M i t ~ ~ h [rrirtiunol., i ~ ~ l 70 (1975) 101- 119. ., (234) R. Berrracki, C:. Porter, W. Korytiiyk. m d E. Mihich, Atlr;. Erizynic R ~ g i ~ l 16 (1978) 217-237. (235) V. N . Shibaev, Pure Appl. Clicrri., 5 0 (1978) 1421-1430.

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antiviral properties. Thus, studies on the functional significance of carbohydrate side-chains of proteins have centered on the properties of fully glycosylated, compared to nonglycosylated, species that are fonned during inhibition of the dolichol cycle. The biological properties of differently glycosylated proteins have thus far mainly been studied by using cell mutants having a block in one of the many glycosyltransferases .I7'' Many of the reactions in the dolichol cycle may be perfonned i n vitro by using crude, microsomal preparations (compare Ref'. 236). It is, therefore, possible to locate the step inhibited by a certain drug. It should be borne in mind that some inhibitors of protein glycosyl,at'1011 have to be metabolized in order to exert their inhibitory effects.237 Thus, 2-deoxy-~-c~rr~hiiio-hexose ("cleoxyglucose") inhibits protein glycosylation in viuo, but, for inhibition in uitro, the nucleotide derivatives are necessary. Envelope glycoproteins of viruses have been used extensively as models for co- and post-translational modifications of glycoproteiIis.'"X Beca~isemany enveloped viruses effectively inhibit host-cell synthesis of protein, only the viral glycoproteins are glycosylated, and this situation niakes biochemical analysis easier. Thus, the effects of glycosylation inhibitors on intact cells may also be studied best with virus-infected cells. Before release of virus, the glycoproteins are detected in the water-insoluble, membranous fraction. Furthermore, the lipid-linked oligosaccharides may be rather specifically extracted from whole cells, and monosaccharide-lipids may also be d e t e ~ ~ n i n e d . It ~ .is" ~thus seen that the various tools of virology and of lipid and carbohydrate biochemistry have proved productive in establishing the mode of action of inhibitors oflipid-dependent glycosylation of proteins.

2. Inhibitors of Formation of Dolichol Phosphate Substances that interfere with the formation of polyprenyl phosphates are of 3 types: (1)those that interfere with the biosynthesis of polypreiiyl diphosphate; for example, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), (2) compounds that prevent the recycling of polyprenyl diphosphate (bacitracin), and ( 3 ) compounds that prevent the phosphorylatioii of (236) N. H . Behrens and T. Tibora, Methods Etinyniol., 50 (1978) 402-435. (237) R. T. Schwarz, M. F. G . Schmidt, and R . Dateina, Biochem. S O C . Trans., 7 (1979) 322 - 326. (238) L. KBariaiiren and 0. Renkonen, in G. Poste and G. L. Nicolson (Eds.),The Synthesis, A.sseinhl!~arid l'tiriiocer of Crll Siirfalcc Conilmiieiits, Elsevier/NorthHolland Biomedical Press, Amsterdam, 1977, pp. 741-801.

polyprenols. The preseiit author\ we not aware of an!. sulistaiicc that specifically blocks the CTP-delx~ncletitphosphorylutioii of dolichol, b u t agents that inhibit fonnatioir of CTP from LJTP (for exainple, 3[ l e ~ i z a ~ i r i ( l i t i eiiiay ~ : ~ do ~ ) so. Inhibitors of HMG-CoA retlrictaae activity (for example coinpactinz4"),o r coiiipoui-lds that l o w e r tlie levels of tlie enzyme (iiicludiiig ii number of oxygenated cholesterol tlerivatives,24124:1a such a s 25-1qdroxycliolesterol), not o n l y r l c ~ c r c a s c the fortnatioii of polk-l,rciiyl diphosphate, but also affect tlre foriiiation of cholesterol atid the polyprenyl side-chains of coenzynrcb 0. Consequeiitly, prolongetl trvatment with such compounds i i r a y - caiisc sick. cffccts, for caxainple, changes in meri-lbrane fluidity ( s w also, Sectioii 111,,5), clecreascd activity of membrane eiizyiiic~s,2'~'~215 ant1 inactivation of iiieiiil)rmr(~ transport s ys tern s ,24G and, tlir,rc,1;) r o , i Ii tli rectl y preve 11t gl\'cosyl at ioi i of protcins. As indicated in Section II,1 ,a, s o i n e cells ciiii iriclependetitly rc'giilate the forination of cholcstc~rola i i c l dolichol tliphosphate. Tliiis, the, effectiveness of inhibitors of HAI(;-CoA reductase i n iii1iiI)itiiig the fonnation of lipid-linked oligosaccliaritles not o i l l l - depcnds oii t h e pool of'dolichol phosphate (that ins! lie sinall:'), hut also (see Sclicine 1) on the pools of substrates f o r tlolichol phospliate synthetase (tliat can he 1argeP3).In other worcls, i i l soiiie cells, HIIG-CoArediictase may not be actively rate-controllitig i t r the synthesis of dolichol.".' 111 h c t , in L cells and MOPC 104 E c c ~ l l sa, strong t l ( ~ ~ e : i in s ethe synthesis of sterols h y 25-hydroxycholcstt.1-ol was accompai-lied b y oiily siiiall alterations in the rates of dolicliol synthesis.:':' €€owever, it is not known whether these small altc,ratioris were sufficient to decrease the amouirts of clolichol phosphate-liirlit.tl saccharides. The inhibitioii of the conversion of HMG-CoA itito nre\donic acid b y 3-hydroxy-3metl-lylglutaric acid in slices froiii tliyroid6 did iiot leud to inhihitioil of fotination of lipid-linked oligosacclr~iricles.

(239) H. P. McPortlaird, M . C . LVairfi, A . tilo(,li, i i i i c l H , Wciiit;ltl, C ' a f i w r - RK\., :34 (1974) 3 107-3 1 11. (240) A. Eiitlo, Cl. Kurotla, and K. T ~ i i i ~ a u iI;E:BS i, Lctt., 72 (1976) 323-326. Browii aiid J. L. Goldstt,iii,/. H i ( ) / . C/fcjm,, 249 (1CJ73) 7806-7814. (242) A. A. Kaiidutsclr and H. W.C l r c i i , / . B i t ) / . C;/icni., 2-15) (1974) 6507-MiOI. (243) J . L. B r e s l o w , D. A. Lothrop, I > . H. Spaiil(liiig, a i i t l A. A . Kairtlutsch, H i o c . / i i r i i . B i o l h ! / , y .Actci, 398 (1975) 10- 17. (243a) J. J. Bell, T. E . Sargeaiit, ant1 1. A . LV.itaoir,/. B i d . C / w i t f , . 251 (1976) 1745- 1758. (244) H. K. K i m e l b e r g and D. Papali;icl;o~jiilt~s, / . H i d . ( ; / w i i f . . 249 (1974) 1071- 1080. (245) A. K. Siirha, S. J. Shattil, aiitl 13. LV. ~ ~ ~ ~ o l i i,/.i i Bu i d, . C ' / t c > r r i . , 252 (1977) X3103.314. (246) J . J . Baltlassare, Y. Saito, and I). I.Silljc.l-t,/. H i o l . C l i t J r , i . , 254 (1979) 1108- 1 113.

324

RALPH T. SCHWARZ AND ROELF DATEMA

In contrast to these results, 25-hydroxycholesterol (and also, 20-hydroxycholesterol, 7-ketocholesterol, and diosgenin) in aortic, smoothmuscle cells effectively blocks the incorporation of acetate into lipidlinked oligosaccharides (and, also, into cholesterol7). Thus, less of the lipid-linked oligosaccharides were available for glycosylation of proteins. In harmony with the presumed, inhibitory mechanism was the observation that incorporation of mevalonate into lipid-linked oligosaccharides was not inhibited, and that mevalonate itself (the product formed by HMG-CoA reductase from HMG-CoA and NADPH) could reverse the inhibition of glycosylation of protein (see Scheme 1). Compactin is a metabolite isolated from Penicillium strainsz4”and the molecule contains a structure resembling that of the lactone named “mevalonic acid.” It could, indeed, be shown that compactin is a competitive inhibitor of the enzyme, with respect to HMG-CoA (and noncompetitive with respect to NADPH).247In the sea urchin (Strongylocerztrotus purpurutus), compactin inhibits not only cholesterol biosynthesis but also the formation of lipid-linked oligosaccharides and g l y c o p r o t e i n ~although ,~~~ the synthesis of RNA and protein was not affected. Exogenous dolichol, but not cholesterol or coenzyme Q, could overconie this inhibition of glycosylation of proteins, because it could be used by the sea-urchin embryos to form lipid-linked oligosaccharides.”* Mevinolinic acid,249another strong competitive inhibitor of HMG-CoA reductase, may have effects on protein glycosylation similar to those of compactin. Cerulenin inhibits formation of polyisoprenol, probably b y uncoinpetitively inhibiting HMG-CoA synthetase.2s”It strongly inhibited production of Rous-sarcoma virus by infected, chick-embryo cells, but an effect on the viral glycoproteins was not observed.251Other effects of cerulenin, such as its inhibition of fatty acid synthesis, may have caused inhibition of virus production. The inhibition, by cerulenin, of secretion of proteins by bacilli has been noted for some time, but no satisfactory explanation has as yet been offered (see Ref. 252, and ref-

(247) K. Tanzawa and A. Entfo, Eur. ]. Biochetti., 98 (1979) 195-201. (248) D. D. Carson and W. J. Lenuarz, Proc. N a t l . Acuci. Sci. U . S. A., 76 (1979)57095713. (249) A. W. Alberts, J. Chen, G . Kuro, V. Hunt, J. Huff, C. Hoffiiian, I. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchet, R. hlonaghan, S. Currie, E . Stapley, G. Albers-Schonberg, 0. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch, and J . Springer, Proc. N a t l . Acad. Sci. U . S. A , , 77 (1980) 3957-3961. (250) S. Omura, Bacteriol. Rezj., 40 (1976) 681-697. (251) H. Goldfine, J. B. Marley, and J. A. Wyke, Biochim. Biophys. Acta, 512 (1978) 229-240. (252) J. C. Paton, B. K. May, and W. H. Elliot,]. Gem Microhiol., 118 (1980) 179-187.

ereiices cited therein). Cleurl>, it would be interesting to have polyprenyl analogs that more spc,cifically block the pathway of dolichol synthesis beyond the part that is shared with sterol synthesis. Bacitracin, a cyclic polypeptide antil)iotic isolated from Bacillro.y lichenifi)rniis, forms a complex with polyprenyl dipliosphates a i i d a tlivalent cation, thus inhibiting tlic tlcpliosphorylatioii of, for example, undecaprenyl diphosphate (sec, Refs. 253 and 254). I n addition, lxtcitraciii can inhibit the biosyiithesis ofpolyprenols (antl sterols) h y comple xirig with the i sopre n y 1 d i ph ( ) s pha t e in te n ne d i a t e s in polyp re n y l &phosphate synthesi -'"Thc synthesis of peptidoglycan i n bacteria can be blocked b y bacitracin, I)ecause (regenerated) undecaprenyl phosphate becc )mes 1imit in g . € I (1 lo h i c't e ri 14 711 .xi1i I I u riu 711 11se s 11n tl ecaprenyl phosphate-linked Mair a i r t l Glc, antl undecaprenyl diphosphate-linked GlcNAc, to glycosylate its major s i i r ~ ~ c e - g l y c o p r o t e i ~ ~ s . ~ ~ ~ Although bacitracin does not t,iiter these cells, it inhibits the glycosylation of the membrane proteiii, pro1)ably because the polyprenyl dipho s p hate i s gene rated 011 t s i ( ICJ t lit. I 11emb ran e s . This re s1 11t s ugge s t s that the lipid pathway here, a s i i i peptidoglycair syiithesis, is cyclic.25g Consequently, it is not surprising t o learn that the dephosphoryl at'I o n of dolichol diphosphate is a l s o iii1~il)itetlb y lxicitracin.'6 The antibiotic substance forms a complcx with dolichol &phosphate, antl coinplexation, as well as inhibition, is prevented when bacitracin is preincubated with EDTA. This s i i g g e s t s a need for a clivalent cation in the formation ofbacitracin-tlolicliol cliphosphate coniplexes also. The dolichol diphosphate phospliatase investigated in this study"' was present in membranes froin h i i i i i i t i i lyinphocytes, and vet bacitracin does not inhibit the synthesis of glycoproteins i n the intact cells, because dolichol cliphosphate is located in the endoplasmic reticulum (see Section II,3) and the cells are, probably, impermeable to lxicitracin. Lymphocytes from rat splectn synthcsize cell-surface-bouiicl, lipidlinked oligo~accliarides,~~ and, here, the presumed first step in the ca(253) G. Siewart and J. L. Stromingr~r,Z'roc. ,\'a//. Accid. Sc,i. C ' . S. A . , 37 (1967)767-773. (254) K. J. Stone and J. L. Stromingctr, P ~ ( J cN. .a t l . Acutl. Sci. U . S. A , , 68 (1971) 3227. (255) K. J. Stone antl J. L. Stromiiigvr. Z'r-oc,. N n t l . Acatl. Sri. U . S. A , , 69 (1972) 12871289. (256) N.Schechter, K. Momose, a i i t l H . H i i t l i i c y , Rioclw,ti. R i ~ p h ! l .H~c .s . CottittiiLri., 48 (1972) 833-839. (257) N. Schechter, T. Nishino, ant1 H. Hutlney, Arch. Hioclreni. Bioph!/s., 158 (1973) 282 -287. (258) M. F. hlescher, U.Hanseir, a i i c l 1. I , . Stroiiiiiiger,J. Biol. Chem., 251 (1976)72897294. (259) M. F. Mescher arid J. L. Stroiiiiiigear, FF;.:HS Lett., 89 (1978)37-41.

326

RALPH T. SCHWAIG AS11 KOELF IIATEMA

tabolism of the dolichol dipliosphate-liiiketl oligosaccharide, namely, cleavage of the phosphoric diester l ~ o n d is , inhibited b y bacitracin.2i In a plant system, namely, carrot slices, bacitracin inhibited the fonnation of Man-P-Dol.26G" Also, in yeast protoplasts, the incorporation of maunose into lipid-linked sugars was inhibited b y bacitracin.261It has not been determined whether bacitracin enters these cells (for example, by measuring its effects on prenol synthesis). Thus, in such cellwall-synthesizing organisms a s plants and fungi, a substantial part of the lipid-dependent, glycosylation reactions may occur at the plasma ineinbraiie,191,2fi2 and therefore be accessible to bacitracin. Bacitracin has also l x e n used to study lipicl-dependent glycosylation with cell-free systems. Thus, Elbein and coworkersX6lnoted, in aortal microsoines, inhibition of the transfer of GlcNAc and Man from the nucleotide derivatives to lipid, but they also showed that the effect ofbacitracin on incorporation of GlcNAc into GlcNAc-PP-Do1 and (GlcNAc),-PP-Do1 was different froin its effect on the formation of Man-P-Dol: only in the latter case could the inhibition be overcome b y exogenous Dol-P. In calf-pancreas microsoines, inhibition of formation of GlcNAc-PP-Dol, but not of (GlcNAc),-PP-Dol, was observed.26"In yeast-membrane preparations, the situation was the other the trisaccharide-lipid way around264;and, in oviduct Man-(GlcNAc),-PP-Dol accumulated to sollie extent in the presence of bacitracin. These multiple 01- diverse effects, o r both, and also the significance of finding inhibition in uitro, are not yet understood, but may be related to the extent to which recycling of Dol-PP contributes to glycosylation in ~itro.'"~*

3. Inhibition by Sugar Analogs

a. Inhibitors of Formation of Lipid-linked Oligosaccharides .-The carbohydrates 2-amino-2-deoxy-u-glucose (GlcN), ~ - ~ ~ o x ~ - D - u T - u bino-hexose ("2-deoxy-D-glucose," dGlc), 2-deoxy-2-fluoro-D-glucose (FGlc), and 2-deoxy-2-fluoro-D-mlannose (FMan) have for several years been known to be inhibitors of' the multiplication of enveloped viruses.22R,233,22"i Crucial to the elucidation ofthe inhibitory mechanism (260) M. C. Ericson, J. Gaf'fortl, and A . D. Elbein, Plant Ph!/siol., 62 (1978) 373-376. (261) J. C. Spencer, M.S. Kang, and A. D. Elbeiti,Arch. Biochem. Biophys., 190 (1978) 829-837. (262) D. H. Northcote, i n Ref: 238, pp. 717-739. (263) A. Herscovics, B. Bugge, and R. W. Jeanloz,F E R S Lett., 82 (1977) 215-218. (264) F. Reuvers, P. Boer, and E. Steyn-Parvt, Riochetii. Bioph!/s. Res. Conwnun., 82 (1978) 800-804. (264a) S. Kato, M . Tsuji, Y. Nakanishi, and S. Suzuki, Biocheni. Biophys. Res. Coint n t m . , 95 (1980) 770-776.

T H E LIPID PATHWAY 01' PROTEIN (;LYCX)SYL24TIOU

327

was the finding that they iiihil)it t h e glycosylation ofthe viral glycoproteins.265Conditions were established under which these sugar analogs specifically inhibit glycosylation of proteins, and do not significantly affect glycolysis, the ener gy charge, or the pool size of n iicl eot ide esters of sugars . U 11t l c>r s uch con cl i t io n s , they interfere with the assembly o f the lipid-linked oligosaccharitle. However, the rate of inhibition of incorporation of siigaIs into the lipid-linked oligosaccharide is differently affected 1)y different c ~ ~ r l ~ o l ~ ~ [ l Thus, r a t e sin .~~~ order to understand how thesr, siigal-s inhibit, a detailed analysis of their metabolism is a prereclitisitc,.

(i) 2-Deoxy-~~irabino-hexose (dGlc).-Dcrivatives of dGlc are extremely sensitive to acid, antl are hydrolyzed within a few minutes at 0" when cells are extracted with 0.9 A4 perchloric acid. Therefore, ethanolic extractions were developtd, niaking possihle the detection o f dGlc 1-P, dGlc 6-P, 2-deoxy-IMt-ubirio-hexoiiic acid 6-P, and UDPand G D P - ~ G ~InCthe . ~ yeast ~ ~ Sn(.(.1i(/rorii!Ict's corecisiae, wherein the metabolism of clGlc was first studied, 2-deoxy-~-cir-crE?irlo-hexouic acid, and not its 6-phosphate, was detectect.'""~2~"8"" The fotmation of both nucleotide derivatives had I~eriimticipated, because 2-deoxy-~-cirabino-hexose is related to both u-g1ucose and D-mannose. It had been known for sotiw tiiiie that D-iiiantiose, inore effectively than D-glucose, prevented the iiihi1,ition of virus formation bv dGlc."' An analysis of the nucleotitle esters of deoxy sugars from cells treated with both D-mannose and dC;lc, showed that the arnount of iiitracellular GDP-dGlc, but not of UDP-dGlc, was strongly lessened, compared with cells treated only with dGle.2i2Therefore, inhibitions caused b y GDP-dGlc inore strongly contribute to inhibition of glycosylation than those caused by UDP-tlGlc. The fict that cells maintained in media containing both dGlc and ma glucose have lowered amounts of UDP-dGlc, but not of GDP-tlGlc, arid do riot allow virus multiplication,"' substantiates this concliision.

(26s) R. T. Schwarz and H.-D. K1eiik.J. \ ' i r d . , 14 (1974) 102.3-1034. 184 (1979) 11.3- 123. (266) R. Datema and R. T. Schwarz, R i o c , / w r r i . I., (267) M.F. G. Schmidt, R. T. Schwarz, aiitl C . Scholtissek, Kur, / . Rioclic~tri.,49 (1974) 237-247. (268) W. Fischer and G. Weideniaiiii, f/rili),c.-Seyler's Z. P h ! / s i o / . C l w r ~ t .3.36 , (1964) 206-218. (269) P. Biely arid $. Bauer, Biochir~i.N i o p k ! / s . Actu, 121 (1966) 213-214. (270) P. Biely antl 5. Bauer, B ~ O C ~ ~H iJo pJ hI! /,s . Actu, IS6 (1968) 432-434. (271) G. Kaluza, \4. F. G. Schmidt, a i i t l <:. Scholtissek, Virology, 54 (1973) 179-189. (272) 1%.F. G . Schinidt, R. T. Schwarz, ; u i d C. Scholtissek, E u r . /. Rioclwrti., 70 (1976) 55-62.

328

RALPH T. SCHWARZ A N D ROELF DATEMA

dGlc does not inhibit the uptake of sugars by fibroblasts, and the pool sizes of GDP-Man, UDP-GLCNA~,’~’ and, at least in baby-hamster-kidney cells,277“ UDP-Glc, are actually increased. This demonstrates that, under the conditions used in these experiments, dGlc inhibits glycosylation of proteins without a significant contribution of the inhibitory effects of dGlc 6-P on phosplioglucoisomerase (EC 5.3.1.9),because a decreased foimation of Man 6-P would lead to a decreased pool size of GDP-Man. Rather, “nucleotide deoxy sugars” were shown to inhibit the synthesis o f the dolichol-linked sugars.22X Nucleotide esters ofdeoxy sugars interfere i n four different ways with the formation of the Glc,Man,(GlcNAc), lipid-linked oligosaccharide, as shown by in citro studies using synthetic UDP- and GDP-dGlc. Firstly, dGlc-P-Do1 is formed from the GDP derivative.274Thus, the amount of Dol-P available for formation of M ~ ~ - P - DGO~ c~- ,P ~- D~ o~~ , * ~ ~ and G ~ ~ N A ~ - P P -isDlessened, O ~ ~ ~ ~and the forniation of these substances is, indeed, inhibited. Exogenous Dol-P relieves these inhibitions in uitro, indicating that GDP-dGlc and the physiological nucleotide esters of sugars compete for limiting amounts of Dol-P. By this mechanism, the formation of the essential intermediates of the lipid pathway is prevented, and, if pools of Dol-P are rather small, as appears to be the case,:
sylation I)y dGlc could, therefore, l)e ol)tained 11>, iiii increased pool o f GDP-Man relative to GDP-dGlc. This has, indeed, 1)eenobservecl i n 21 dGlc-resistant, baby hamster-kitlncxy cell-line.27"Thus, under partially inhibitory concentrations of dC:lc, viral glycoproteins are fomied that contain lessened amounts of c.arlx)hydrate, but the oligosaccharide side-chains are of the same s i n , ;IS those found on fiilly glycosylated proteins.",ln".'fi,i Apparently, only tlre fiill-sized, lipid-linked oligosaccharides can serve as donors for tlie glycosylation of proteins. Thirdly, GDP-dGlc interfc.~-t,s~'~ with the I~-gliicosylatioii of Man,(GlcNAc),PP-Dol to forni Glc,~lan,(GlcNAc),-PP-Dol; t h i s is caused, in part, b y the fict that the forimtion of Glc-P-Do1 is inhibited, and this inhibition can be relicvetl, in part, b y exogenoiis Dol-P. However, in addition, dGlc resicliit,s, possibly coming from dGlc-P-1101, are incorporated instead of ~ g l u c o ~ but e , ~it ~is~ not yet k n o w n whether these oligosaccharides c ~ n be i transferrecl to protein. Fourthly, UDP-dGlc inhibits thc synthesis of Glc-P-1101and, p r o h a bly therefore, also the D-gliicosylntioii of Man, ( G I ~ N A ~ ) , - P P - D o ~ . ~ ~ ~ The formation of Man-P-Do], GlcNAc-PP-Do], and (GlcNAc),-PP-l1ol was not inhibited by UDP-dGlc. 7'he inhibitions caused b y UDPdGlc are not reversed by Dol-P, and tlGlc-P-Do1 is not foiiiied from this derivative; thus, the inhibitory effects of UDP-dC:lc i n intact cells will be limited if pools of Glc-P-Do1 are high. The effects of tlGlc on the formation of lipid-linked oligosaccharides are siiiiiiiiarizc~d in Scheme 3 . It shows that dGlc iiiterferes with inany of' tlie steps involved in lipid-dependent glycosylation of proteins. The last step i n the reaction sequence, the traiisfc>rof the oligosaccharide to the protein, is not inhibited by 2-tleos),-1,-circibi,i(~-hexose.""~~~ 2-Deoxy-D-urabino-hexose a l s o interferes with the formation of Dglucan and D-niannan in, for e x a m p l e , Saccliaro?,i!Iccs c c ~ r c . u i s i c ~ c . ~ ~ Knitkg and coworker^'^' reported preferential inhibition b y tlGlc of formation of cell-wall D-glucan whcii D-inannose was used as the carbon source, and of foniiation o f ceIl-wall D-mannan when D-glucose was used as the carbon source. I n addition, preferential incoi-pol-ation of dGlc occurred into that gl\~caii whose synthesis was the most strongly inhibited. The incorporatioii of dGlc into I)-iiiaiiiian alters its structure, and causes chain tern~ination."~ Apparently, UDP-dGlc and GDP-dGlc can serve as sugar cloiiors in the formation of yeast glycan. Thus, although UDP-dGlc is not converted into dGlc-P-Dol, it may not be a metabolic dead-end for the dcoxy sugar in yeast. The incorporation of dGlc into components of the yeast cell-w7all is, however, (277) Z. KrAtkv, P. Biely, and 5 . Baiirr, E~cr..,/. Hioclzeiri., 54 (1975) 459-467. (278) P. Biely, Z . Kratk?, and 5 . Bailer, Biocliiir1. B i 0 p h / , ~Actci, . 352 (1974) 268-274.

GlcNAc-PP-Do1

z

~~

UI3 P - G l c N A c dGlc-P-Do1 ,/

UDP- GlcNAc (GICNAC),-PP-D~I

t

dGlc- (GIcNAc),-PP-DoI cannot b e extended by addition of Man; not t r a n s f e r r e d t o protein

/'

~-M~~-(G~cNAc),-PP-D~ UDP-Glc ,,,'

Man- P- Do1 dGlc- P-Do1

UDP-dGlc

cudGlc -6Man- (GlcNAc),- PP- Do1 cannot b e extended by addition of Man; not t r a n s f e r r e d to protein

v

dGlc, -Many-(GlcNAc),-PP-Dol

+

x ~ = 9 . 8 , 7. . . not t r a n s f e r r e d to p r o t e i n

'

dGlc-P-Dol,

Glc - P- Do1

/' (Glc, dGlc),-aMan,-fl Man-(GlcNAc),-PP-Dol

aGlc3-aMan,-/3Man- (GIcNAc),-PP-Do~

f a t e unknown Scheme 3.- Interference b y GDP-dClc and UDP-dClc with the Assembly of the Lipid-linked Oligosaccharide, Glc,-Man,-(GlcNAc),-PP-Dol. [The pathway of the assembly of the normal, lipid-linked oligosaccharide is shown b y formulas connected b y heavy arrows. The incorporation of 2-deoxy-D-arnbino-hexosyl (dClc) groups from GDP-dClc into lipid-linked saccharides is shown by dashed arrows. The block in the biosynthesis of Clc-P-Dol, caused b y UDP-dClc, is shown by an arrow head. The Scheme is based on work cited in Refs. 228,237, arid 274-276.1

riot the primary cause of in1iil)itioti of'tiiosynthcsis of glycoproteiiis in this organism. Iiuiz-Herrent and Seirtandreu2i9found that dGlc inhiliits the glycosylation of pol?,soiiie-l,o~iti[lpolypeptides, kvitliout incorporation ofdGlc into these protliicts. Evidently, this result i s in accord with inhibition Iiy clGlc of thc. asst.ml)ly of thc, ligitl-linkc-d oligosaccharide. The interference b y dGlc \vith t h c clolichol pathwa>.of fonnation of glycans could be shown i n tlrc C'MC'S of oligoinaniros>rl sitlcA-chains linked glycosidically to Ser o r Thr i n yeast D-tn~tiiti~in,z80 and the lipidliiiked cellulosc~precursors o~'rr-olotliccc,a zo/if;i.'x' In the fot-nrer, tlGlc residues from tlGlc-P-Do1 w c~ t-egl\,cosidically linked to the hydroxyl group of Ser or Thr, antl fiirt1ic.r c,xtensiori with the (1+2)-linked Uman iio sy l re s i d tie s was, th c rc' ti)rcb , 1) locked . I I i i n enibran cl p re p ~ - a tions from the alga P. zopfii, Imtli UDP-dGlc aiitl GDP-dGlc inhibited the fonnation of Glc-P-Do1 ant1 Glc-PP-Dol. The inhibitory inc.chanisins in the a l g a were the s a i n t ' a s those in chick cells: inhibitions caused b y GDP-dGk were rvversc~tlI)!, Dol-P, and dGlc-P-1101 w a s foiiiied from this nucleotitle c.Ater, whereas inhiliitioiis caiisetl b y UDP-dGlc were riot reversc.tl I)\, 1101-P, antl tlGlc-P-11ol w a s not fonnetl from the UDP c1eriv:itivc~.Tlius, in yeast,28x" algal,"' chick,274 and hainstei2x2cells, UDP-dC:lc. c*;innotlie convertecl into dGlc-P-Dol, whereas GDP-dGlc can; this i i i ; t > . suggest that the OH-2 group of Glc in UDP-Glc is crucial for the actiiity ofpolyprenyl phosphate: UDP-Dglucose D-glucc,syltransfe~ises tliroughout the eiikaryotes, wliercas the OH-2 group of D-mannose i i i CDP-hlan is iiot essential for thc. Dinaniiosyltransferase. The inhibitory effects of dGlc o i i the fomiation of celliilosc~clearly demonstrate that dGlc not o n l k . inhiliits glycos?.latioii because it is a D-inannose analog but that it also inhibits liDid-deDencleiit D-gliicosylation reactions. When using S-tlcosy-D-nrcibirto-hexose a s itii i i i l i i h itor of glycosylation, it shoiiltl I)cx borne in mind that sonic' cells may be able to catabolize dGlc, iis \v;ts ol,servecl in leukocytes treated with a tunnor-pron7otiiig, phorbol e s t e r , namely, 12~>-tetradecaiioyIphorbol 1 3 - ; ~ e t a t eT . ~h e~ ~general sigiiificwice of this catabolic pathway is, 1iowe\w, not yet clear.

(279) J. Ruiz-Herrcmra arid R. Sentantlwii, ./. U o c f e r i o l . , 124 (1O7S) 127- 133. (280) L. Lehle arid H. T. Schwarz, Etii.. / H i o c 4 1 c , m . , 67 (1976) 238-245. (281) K . Datema, I<. T. Schwarz, I , . A . Hi\ ;is. antl R. Pont 1 ica, P / n i i / P l i y ~ i o l . ,in press (1982). (282) H. Ilatema a i r t l H.T. Schwarz, uirpiil)lished observutioir, (283) P. Zabos, I). Kyiier, N . h l e i i t l ~ ~ l ~ o l(:. i i i Schreibcr, , S. W:ixiii:iiiii, J . C:hristnitw, and C. Acs, Proc. h'citl. Acotl. Sci. C'. S .+I.,75 (1978) 5422-5426.

332

RALPH T. SCHWARZ AND ROELF DATEMA

(ii) 2-Deoxy-2-fluoro-~-glucose. -2-Deoxy-2-fluoro-D-glucose

(FGlc)

is an inhibitor of the synthesis of yeast glycan,2X4 and of virus mul-

tiplication,'"j I)ut inhibits less efficiently than 2-deoxy-2-fluoro-Dinannose or 2-deoxy-~-circil?iiio-hexose.Interestingly, D-mannose, more effectively than D-glucose, prevents FGlc-inhil~ition,"~ indicating that the cell recognizes FGlc not exclusively as an analog of D - ~ ~ L I cose. Indeed, subsequent studies on the metabolism of FGlc showed that, in yeast and in chick-embryo cells, both GDP-FGlc and UDPFGlc are fornied.2H6However, FGlc residues are very poorly, if at all, incoi-porated"7 into oligosaccharides used for protein glycosylation, and this indicates that the mechanism of inhibition of protein glycosylation by FGlc differs from that of' dGlc. FGlc inhibits glycosylation of protein by preventing the assembly of the lipid-linked oligosaccharide.266If cells are prelabelled with tritiated sugars and then treated with FGlc, lipid-linked oligosaccharides containing decreased proportions of Glc and Man are detected."'j They do not arise by breakdown of the full-sized, lipid-linked oligosaccharide, nor by incorporation of FGlc residues. Instead, it could be shown that they were intermediates in the biosynthesis of the Glc,Man,(GlcNAc), , lipid-linked oligosaccharide. Subsequent analysis"6 showed that FGlc inhibits the formation of GlcP-Do1 (and UDP-Glc) and Man-P-Do], but not of GDP-Man, UDPGlcNAc, and (GlcNAc),-PP-Dol. Rather, the last three compounds accumulate in FGlc-treated cells. The transfer of Glc and Man from their Dol-P derivatives is not prevented by FGlc, and, consequently, meinbranes from FGlc-treated cells contain decreased amounts of Man-PDo1 and Glc-P-Dol.116 The secretion of D-inannoprotein by protoplasts from S. cerevisiae was inhibited by FGlc, but to a much smaller extent than the forniation of the D-glucan Thus, in yeast, FGlc may be mainly a D-glucose antagonist. Possibly, although this is not yet proved, FGlc in yeast cells also depletes the pools of UDP-Glc, thereby inhibiting forniation of D-glucan. This inhibition of the structural, D-glucan components shifts towards degradation the balance between synthesis and degradation of D-glucan during the growth of yeast, and this causes lysis of the cells.'** (284) P. Biely, J. Kovarik, and 5. Bauer,J. Bacteriol., 115 (1973) 1108-1120. (285) M. F. G. Schmidt, R. T. Schwarz, and H. Ludwig,]. Virol., 18 (1976) 819-823. (286) M. F. G. Schmidt, P. Biely, 2. Krritkq, and R. T. Schwarz, k ; w . J . Biochein., 87 (1978) 55-68. (287) R. Datema, R. T. Schwarz, and R. Pont Lezica, in Ref. 107, pp. 218-219. (288) S. Bartnicki-Garcia, in J. M. Ashworth and J. E. Smith (Eds.),Microbiological Difjerentiation, University Press, Camb~idge,England, 1973, pp. 245-267.

On prolonged treatiiient of virus-iiifected, chick-embi-yx)cells with FGlc, and also in cells pretreated with FGlc, the fonnation of lipidlinked oligosaccharides and the glycosylatioii of viral protein are strongly, but not completelj,, ii~liil)ited.122 Residual glycosylatioii of iiifliienza-virus proteins was dependent on the formation of GlcNAcPP-Dol; that is, it could lie iiiliil)ited h y tunicamycin. However, tlie oligosaccharides transferred to 1)roteiii i r i cico in tlie presence of FGlc were snialler than the nornral, ciido-p-N-acetylglucosaminiclase Hsensitive, high-iiiannose oligosacc.haride and, ftirtliemmre, resistant towards digestion with eiitlo-p-:V-~icetylglucosaiiiitiidaseH. It has 1)een that glycosbTlation i i i the presence of FGlc occurs by way of a pathway similar to that clescri1)etl in tlz!y-l-iiegative, iiioiiselyiiiphoma cell^^'*^^^' (see Section II,2,a).

(iii) 2-Deoxy-2-fluoro-~-maiiiiose. - ~-~eoxy-~-fluoro-D-lnanuose (FMan) is converted in yeast a i i t l chick-enibryo cells into h t h GDPFh4an and UDP-FMan.'X6It also inhihits the assembly of lipid-linked oligosaccharide in chick-eml,r~.ocells,266and is poorly, if at all, iiicorporated into glycoproteiiis."X" It should be noted that both FGlc and Fhlan were incorporated significantly, but to a very low extent, into y e as t D-man 11 an .286 It i s po s s i 1) I however, that t hi s i nco iporat io n occurs in the tenninal parts of thy side chains of the D-inaiinan (which do not require lipid intermediates for their synthesis),ant1 may not be related to inhibition of glycosylatioii of proteins. Bauer and cowork61,

e r P 4 observed lysis of yeast c c ~ l l sb y FMan, although this would not

be expected if FMair were to iiiterfere with ~ - m a n n a r synthesis i only. A speculative explanation ma?. that UDP-FMan is an inhibitor of D-g 1ucan : U D P-Glc D-gl uco s y1t ran s fe rase , where b y F M an thus in h i 11its the fonnatioii of the structural L>-glucan fibrils. The difference in electroiir'gativity between OH and fluorine apparently does not prevent iiic,t~il)olizatioiiof fluoro sugars. It may, however, be sufficient to explain tlie observation that the niicleotitle I ) c b

esters of 2-deoxy-2-fluoro sugars are poor sugar-donors, 1)ecause of the stabilization of tlie glycosyl-O I)ontl: this would also explain why these iiucleotide esters of fluoro sugars are more stable towards acid than their normal counteiyai-ts. If the fluorine atom is farther away from the anonieric center (for exainple at C-6), it will have less &ariiatic effects on the biological properties of the analog (see Section 111,3,b).It should, furthemiore. lie noted that 2-fluoro sugars call assiiine an unusual confonnatioii, :is shown for a nucleositk of a fluoro sugaPH";this may also affect its hiological properties. (289) J. Kiburis, A. B. Foster, and J. 11. h'estwood, J . Chetn. Soc. Chcrri. Corrimun., (1975) 44-45.

334

RALPH T. SCHWAHZ AN11 HOELF DATEMA

The mechanism of inhibition of glycosylation of protein b y FMan has not yet been investigated i n detail; however, the result that residual glycosylation of protein, noted in the presence of FGlc, does not occur in the presence of FMari indicates a different inhibitoiy mechaiiisni.2x2Nucleotide esters of fluoro sugars have not yet been synthesized in sufficient amount to permit testing this idea.

(iv) 2-Amino-2-deoxy-~-glucose.-At those concentrations of 2amino-2-deoxy-D-glucose (GlcN) that inhibit virus multiplication, id of the amino sugar (GlcNAc, GlcNAc only the the ~ i s ~ metabolites 6-P, and UDP-GlcNAc) were detected.2Y0 On washing these cells with GlcN-free medium, GlcN diffuses out of the cells, and protein glycosylation is reinitiated within 15 min. During this time, the concentrations ofthe metabolites of GlcN and of the nucleotide esters of sugars (GDP-Man, UDP-Glc, and UDP-Gal) do not change significantly, indicating that GlcN itself is the inhibitor of protein glycosylation.2Y0 A kinetic analysis2(j6 of the inhihition of glycosylation of GlcN showed that, first, the assembly of the lipid-linked oligosaccharide was inhibited, and then the glycosylation of proteins (after a lag period). Thus, at subinhibitory concentrations of GlcN, the uiiderglycosylated glycoproteins lack complete oligosaccharide chains. 105m Depending on the riiiiiiber of side chains eliminated, different subspecies of a given glycoprotein will be present. Using high resolution, poly(acry1amide)-gel electrophoresis, iinderglycosylated, influenza virus heinagglutinin could be resolved into 8 bands, suggesting 7 glycosylation sites on this glycoprotein.z9' Consequently, inhibition b y GlcN can be u s e d for estimating the number ofcarbohydrate side-chains of a glycoprotein. The inhibition of incorporation of D-inannose into lipid-linked oligosaccharide extracted with cliloroforin-methanol-water occurred veiy rapidly, showing that an early step in the biosynthesis was inhiliited,z66but which step this is, is not yet clear. The inability to inhibit the foniiation of lipid-linked oligosaccharide in citro by GlcN indicates that an intact men-lbraiie-system might be nece tion by GlcN to be effective. In f x t , to date, no direct evideiice is available that indicates that the prime target of GlcN-inhibition is the assembly of lipid-linked oligosaccharides. GlcN modifies the ultrastructure of cellular ineiiibranes; for example, it causes fragmentation of the rough, endoplasmic reticulum, and proliferation of the Golgi (290) H. U. Koch, R. T. Schwarz, a n t l C . Scholtissek, EIW./ . Biochern., 94 (1979) 512522. (291) M. A. Horisberger, C . tle Staritzky, antl J . Content, Arch. Virol., 64 (1980) 9-16

THE LIPID PATHWAY O F PROTEIN GLYCOSYLATION

335

system.29z*293 The rapid effect that GlcN has on the formation of dolichol saccharides266is parallelled by similar, rapid changes in cellular ultrastructure.z9zInterestingly, local anesthetics that are known to affect membrane fluidityzg4potentiate the cytotoxic effects of the amino and the authors suggestedzg3that the specific cytotoxicity of GlcN towards tumor cells results from inhibition of the synthesis of membrane constituents. It would, therefore, be interesting to correlate the membrane effects of GlcN with its inhibition of the glycosylation of proteins. Also, because of the known effects of primary amines on the pH of intracellular compartments, for instance, lysosomes (see Refs. 295 and 296 for a discussion), it may be speculated that GlcN affects a step, early in the assembly of the lipid-linked oligosaccharide, that is sensitive to changes in the concentration of hydrogen ions. Thus, inhibition by GlcN has the advantage that it is readily reversible. Inhibition by tunicamycin is poorly reversible, because of the hydrophobic nature of the antibiotic. Inhibitions by dGlc, FGlc, or FMan are only slowly reversible, because the inhibitory, phosphorylated derivatives do not readily leave the cells. Although the reversibility of inhibition b y GlcN is a useful property, it should be noted that, in media containing pyruvate, instead of D-ghCOSe as the carbon, or energy, source, GlcN causes depletion of UTP and ATP pools, because excessive amounts of UDP-GlcNAc are formed.z33Hence, the synthesis of RNA is inhibited. Furthermore, the nature and extent of inhibition by GlcN can differ with the cell type. For example, Nakamura and corn pan^"^ still found incorporation of L-fucose into the hemagglutinin of influenza virus WSN grown in MDBK cells maintained in a medium containing 40 mA4 GlcN. Also, the incorporation of sulfate into the carbohydrate chains of this hemagglutinin (another late event in post-translational modification) was not completely inhibited by GlcN. In BHK cells infected with vesicular-stomatitis virus, GlcN did not cause uridylate trapping, but synthesis of viral protein and RNA was inhibited before decrease of incorporation of Dmannose into the viral G-glycoprotein was observed.z98As host-pro(292) Z. Molnar and J . G. Bekesi, Cancer Res., 32 (1972) 384-389. (293) S. J . Friedman and P. Skehan, Proc. Natl. Acad. Sci. U . S. A , , 77 (1980) 11721176. (294) S. H. Roth, Annu. Reu. Pharnaucol. Toxicol., 19 (1979) 159-178. (295) D. J. Reijngoud and J. M. Tager, Biochim. Biophys. Acta, 492 (1977) 419-499. (296) P. D. Stahl and P. H. Schlesinger, Trends Biochem. Sci., 5 (1980) 194-196. (297) K. Nakamura and R. W. Coinpans, Virology, 84 (1978) 303-319. (298) L. L. Marnell and G . W. Wertz, Virology, 98 (1979) 88-98.

336

RALPH T. SCHWAKZ A N D ROELF DATEMA

tein synthesis was oiily minimally inhibited, it might again be speculated that GlcN affects cellular membranes, the integrity of which is necessary for production of virus.

b. Other Sugar Analogs.-Here will be briefly discussed some of the sugar analogs that are known to interfere with the glycosylation of proteins, or, at least, the synthesis of glycoconjugates, but of which an effect on the lipid pathway has not been demonstrated. Reviews by Decker and Kepplerzss and Bernacki and coworkers234give inore details. Naturally, the effects of sugar analogs on cultured cells depend on the inediuin in which these cells are maintained, or grown, as shown for the uridylate trapping b y GlcN in virus-infected, chick-embryo ce11s.233*299 Furthennore, the effects of the sugar analogs depend on the cell type; that is, on the specificities arid abundance of the enzymes of the Leloir pathway and the salvage pathways.zss Thus, 2-aniino-2deoxy-D-galactose (GalN) depletes rat liver,300but not chick-embryo cells,301of UTP. The most extensively studied sugar analogs are simple, fluorinated carbohydrates302(2-, 3-, and 6-FGlc, 2-FMan, 2- and 6-F-Fuc, 6-FGal, and FGlcNAc), deoxy sugars (dGlc, dGal), arid amino sugars (GlcN, GalN). It has been shown that the metabolic or antineoplastic effects, or both, of some analogs are enhanced if they are 0-acetylated, possibly because cellular permeability is increased.300"-300" Pe nta-0-acetyl-p-GlcNAc was shown to be O-deacetylated intracellularly, arid converted into UDP-GlcNAc, thereby depleting300"the cells of UTP and CTP. Several fluoro sugars are incorporated into glycoconjugates, and therefore probably compete with the nonfluorinated sugars. Thus, 6FGal preferentially inhibits Gal incorporatio~i':~~,"~; 6-F-Fuc and 2-F~ * 2-deoxy-2-(fluoroaceta~~~; Fuc, the incorporation of ~ - f u c o s e ~ "and (299) K. Decker a n d D. Keppler, Reo. P h i p i o l . Biochem. Phurttiucol., 71 (1974) 77-106. (300) D. 0. R. Keppler, J. F. M. Rudigier, E . Bischoff, and K. F. A. Decker, E w . 1.B i o chem., 17 (1970) 246-253. (301) C. Scholtissek, Eur. /. Biocliem., 24 (1971) 358-365. (302) A. A. E. Penglis, A&. Carbohyclr. Chmn. Biochem., 38 (1981) 195-285. (303) R. J. Bemacki, M. Sharrna, N. K. Porter, Y. Riistum, B. Paul, and W. Korytnyk, /. Supmmol. Strucf.,7 (1977) 235-250. (304) J. R. Sufrin, R. J . Bernacki, M. J. Morin, a n d W. Korytnyk,]. M e d . Chem.,23 (1980) 143-149. (305) P. Simon, W. J. Burlingham, R. Conklik, and T. P. Fondy, Cancer Res., 39 (1979) 3897-3902. (306) M. J. Morin, R. J. Bernacki, C . W. Porter, and W. Korytnyk, Fed. Proc. Fed. A m . Soc. E x p . Biol., 39 (1980) Abstr. 2094. (307) D. J. Winterbourne, C . C:. Butchard, a n d P. W. Kent, Biochem. Bioph!ls. Res. Commun., 87 (1979) 989-992.

T H E LIPID PATHWAY O F PROTEIN GlJYCOSYL4T1OK

.3.37

mido)-D-glucose and 2-deoxy-~-(trifli1oroacetamido)-D-g~ucose, the incorporation of GlcNAc (see Refs. 303, 308, aiid 309). Although these results suggest that some glycos!ltransferases are still active when h y clrogen atoms or hydroxyl groups i n their substrates are replaced by fluorine atoms [see, however, Section 111,3,a,(iii)], this has not y e t heen directly tested with the fliioro sugar esters of'niicleotitles. However, in the light of the results ol)tained by Shilxiev and coworkers2'j5 with gl y co sy 1trans fe rase s in vo 1vc t l i n the bio s y I i the s i s of S (1 1m o t 1 e 1la polysaccharides, this is not s o siirprising (for a review, see Ref'. 235). They found that, for UDP-Glc, o n l y the NH group oftlie iiracil residue and OH-3 of the D-glucosyl grorip are critically essential for enzynie activity. The importance of OH-3 of the D-glucosyl group in UDP-glucose or CX-D-G~C-P was also noted i n the synthesis of glycogen, starch, and a , a - t r e h a l o ~ e . ~ ~The. O ~ " activity ~ of the enzymes was not cornpletely abolished, but was stroiigly decreased. In the case of GDPMan, the 2-, 3- and 6-hydroxyl groups of the D-niannosyl group do not participate in the interaction with the Salmonella transferase.235Thus, it is fairly possible, a s suggested b y Korytnyk antl that the glycosyl group ofGDP-6-fliioro-L-fiicose can be transferred to glycoprotein acceptors in maniiiidian cells. However, although their GDP and UDP derivatives arv formed, 2-deoxy-2-fliioro-D-~liic(~se aiid ~-deoxy-2-fluoro-D-i1lannoseare poorly incorporated into glycoproteins.116.286 These sugar aiialogs primarily inhibit synthesis of dolichol diphosphate-linked oligos~~ccharides (see Sections III,3a,ii and iii), possibly because their iiiicleotide esters are inhibitors of the Dglucosyl- and D-nianiiosyl-transft.I.ases.116 The 6-fluoro derivatives of Gal antl FUCdo not decrease the pools of ribonucleotides, nor do they inhibit protein ~ynthesis."j~-'~"~ Interestingly, when present in low concentrations, most fluoro sugars do not give cytotoxic effect^."^.^^^ When the size of the halogen atom is increased (Ci- Br + I derivativt,s), incorporation of L-fucose is lessened, and, for as-yet-unknown re'asons, a concomitant increase in cytotoxicity was noted."04 The conversion of dGal iirto dGal l-P causes trapping of phosphate."I4,"'j Furthermore, especially in liver, UDP-dGal is fonned, and (308) P. W. Kent and D. J. Winterbourirc., Biochem. SOC. T r a i i . q . , 5 (1977) 439-440. (309) K. Barrett-Bee and W. Hendrrsoii. 111 Ref. 107, pp. 312-313. KuGr, antl Btiuer, b;iit-. J . Biochem., 40 (1973) 195-199. (310) J . Zemek, (311) J . Zemek, J . Stmiefi, KuEBr, and Bauer, E u r . ] . Biochetri., 64 (1976) 283-286. (312) J. Zernek, 5 . KuEBr, and J. ZBinochj, E u r . ] . Biocherti., 89 (1978) 291-295. (313) E. M . Bessel, V. D. Courtenay, A . H. Foster, M. Jones, and J. H. Westwood, E u r . ] . Cancer, 9 (1973) 463-470. (314) D. F. Smith and D. Keppler, Eur, J . Biochem., 73 (1977) 83-92. (315) J . J. Starling antl D. Keppler, b:itr, J . Biochetn., 8 0 (1977) 373-379.

s.

s.

s.

s.

338

RALPH T. SCHWARZ A N D ROELF DATEMA

this leads to UTP and UDP-glucose deficiencies.299However, in addition, dGal is incorporated into glycoproteins, and this lowers the incorporation of L-fucose into g l y c o p r ~ t e i n s This . ~ ~ ~decreased incorporation is not the consequence of inhibition of synthesis of protein, or of trapping of UTP. The additional administration of uridine restored the levels of UTP, but did not overcome the inhibition of incorporation of L-fucose. The authors316proposed that the lack of the OH-2 group of Gal specifically blocks the incorporation of the (1-+2)-linked, L-fucosyl residues. In accordance with this proposal was their finding that incorporation of NeuAc (mainly to 04 or 0-6 of Gal) was not affected. dGlc, also, can be incorporated into g l y ~ o p r o t e i n sbut , ~ ~this ~ does not seem to be the reason for its inhibitory effect on the glycosylation of proteins [see Section 111,3,a(i)].In this respect, the metabolic fate and inhibitory properties of 4-deoxy-~-xylo-hexose:~~~ “4deoxy-D-galactose”) may be interesting to study. In liver, GalN, like dGal, is metabolized on the galactose pathway, GalN 1-Pbeing uridylated by UDP-Glc, thus giving rise to UDP-GalN that, in contrast to UDP-dGal, can be rapidly epimerized, and further metabolized.2YY The content of UDP-Glc, UDP-Gal, and uridine phosphates thus become deficient, and the administration of GalN results in liver injury (“galactosamine hepatitis”). Also, addition of GalN gives a transient decrease in intracellular, inorganic phosphate, thereby lowering the rate of breakdown of glycogen.318The altered pattern of UDP-sugars in galactosamine hepatitis probably gives rise to the different pattern of glycosylation of the a p o l i p o p r o t e i n ~ . ~ ~ ~ Nucleotide analogs and polyprenyl phosphate analogs are potential inhibitors of glycosyltransferases. Methods for their synthesis are available, and, as the elegant, chemical approach described in Shibaev’s indicates, such compounds may readily be designed, because only certain functional groups are essential for the enzymic activity of glycosyltransferases. Realizing the possible impact of such an approach, Korytnyk and coworkers320tested cytidine nucleotides and some analogs thereof on ectosialyl- and serum sialyl-transferases. The inhibitory properties observed may be exploited to learn more about the role of sialic acid residues in glycocoiijugates.

(316) R. Buchsel and W. Reutter, Eur. J. Biocheni., 111 (1980) 445-453. (317) S. K . Sinha and K. Brew, Curhohytlr. Res., 81 (1980) 239-247. (318) R. Steman, S. R. Wagle, and K. Decker, Eur. J. Biochem., 88 (1978) 79-85. (319) P. Kiss and R . Kattennan, in Ref. 107, pp. 316-317. (320) W. D. Klohs, R. J . Beriiacki, and W. Korytnyk, Cancer Res., 39 (1979) 1231-1238.

4. Inhibition b y Antibiotic Substances Some coinpounds known to hc. inhibitors of peptidoglycan h r m a tion in txicteria have proved also to inhibit t h e lipid pathway of glycosylation of proteins. Moreovclr, thc~sec.ompouiids show aiitiviral activity, because t h e maturation o f c~iivc~lopetl virus in erika impaired if glycosylation of \,irtil glycoproteiiis is inhihited (see Section IV). a. Tunicamycin-like Substances.-T~inic~~tii\.ciii,Strepto\.iriidiii, Antibiotic 24010, and Mycospocicliiis l ~ e l o n gto a class of S f r c p t o t , i y ct' t e an till i o t ic s that in h ill it e i r z\r I I I c' s , rc, ve rs i b 1y trans loca t ir i g G lc N Ac 1-P (or derivatives of GlcNAc 1-I], siicli as phosphono-MiirNAc-pt,iitapeptide) to polyprenol llhosl,lltrte,22X .2:ll.2:12,921-:{:j2;1 a s shown in reaction 10. UIIP-GIcbNAc

+

polypreii\ 1-1'

(:lcNAc-PP-pcil\~)rc.nol + V h l P

(10)

Thus, transfer of GalNAc to afford lipid dil>hosphate-linked Gal NAc is riot inhibited by tunicamyciii.::" The polyprenol can b e either tlolichol or nndecaprenol, and I ) o t l i t l i v forward a i i d lxrckwartl reactions As tunicamycin is irot t i i e t ~ ~ ~ ~ j l ithe z ~ iininoclified ~~l,'~2~ are antibiotic is probably the itiliil>itor.Tuiiicariiyciii-setisitive e n z ~ . m e s thus occur in t h e first steps of((/)the tlolichol cycle in eukaiyotes, and ( h )the synthesis of peptidoglycaii, a i i t l of the linkage uiiits attaching teichoic acids to the peptidogl!.c,~iii.Iwth in prokaryotes (see Ref. 321 for a suminary). The cleinonstrat ioii that glycophorin A froni tunicairiycin-treated leukemia cells lacks oiily the oligosacch~~ride-i[ie linked to L(321) J. B. Ward, A. W. Wyke, a r d <:. A . \I Ciirti\, B ~ O C ~ W Soc. I J I . ' l ' r ( i t ~ , ~8 .(1980) , 164166. (322) A . Takatsuki, K. Ariina, ;ind G . ' l ' a i i i i i r a , / . Atitihiot., 24 (1971) 215-223. (323) A. Tnkatsuki, K. Shiinizii, and C . T < t i i i i i i - : i , / , Aiitiljiot., 25 (1972) 75-85. (324) .4.Takatsuki, K. Kawaiiiiira, h l . O k i i i a , Y.Kotlama, T. I t o , ant1 G.Tanini-a..4grtc. B i d . Ckeni., 41 (1977) 2307-2308. (325) S. C . Kuo antl J. 0. Laiiipen. 1 2 ~ 1 1R. i o c h c J i i i . B i o p h y $ . , 172 (1976) 574-581. (326) J . B. Lliard, F E B S Lett., 78 (1977) 151 - 159. (327) .4.W.Wyke a i d J. B. W d , / . Roc./c.riol., 130 (1977) 305,5-1063. (328) K. Eckarclt, H. Thruni, G. BrmllfAi, E. 'I'oiie\v, antl \ I ' l ' o t i c ~ v , ,/. Atitihic>t., 28 (1975) 274-279. (329) A. 11. Elbeiri, J. Gafford, a n t l J1. S. Kaiig, Arch. H i o c , l w j i t . B ~ o ) J ~ ! /196 . s . ,(1979) 311-318. (330) S . Yamamori, N. Slurazuini, 1'. .Araki, m i t l E. Ito,]. H i ( ) / .( ~ / i c t t i . . L53 (1978) 65lfj6522. (331) S.Nakaniura, \I.Arai, K . Karas;t\\a~i i i i d 11. Youehara./. Aiitihiot , 10 (19.57)2482.53. (332) J . S. Tkacz and A. Wang, F e d . Pt-oc Fctl. A m . Soc. E X ~ Biol., J. 37 (1978) 1766. (332~1) G . Tamura (Ed.), Tunicumy/cin. Jiip;iii Scientific S o c i e t i e s 1'1-ess, T o k y o . 1982.

340

RALPH T. SCHWAKZ A N D HOELF DATEMA

asparagine residues elegantly shows the specific inhibition b y tunicamycin, because the biosynthesis ofoligosaccharides linked to L-serine or L-threonine residues was not inhibited.33:3 Tunicainycin (4), originally isolated from cultures of Streptoni!/ces

1

wo

HO

HO

Ac HN

C-

HN-C-C=

11

0

(CH,),-CHMe,

H

Tunicamycin'*'

0) =

8.9,lO. or 11)

4

Zysosuperi$cus by Tamiira and coworkers,322has been extensively studied. It is a iiucleosicle antibiotic containing residues of uracil, GlcNAc, an unsaturated fatty acid, and a n uriusual, C,, aminoclideoxylO-ainino-6,10-dideoxy-~-gd~icto-~dialdose, namely, tunicaiiiine~iz4 allo-u1ideca1iodialdo-l,4-furanose-ll,7-pyraiiose.~~~ The chair]-length of the fatty acid linked to the tunicamine moiety is variable. The homologous members making up tunicamycin differ in the fatty acid group, and ten different species have been isolated and their structures d e t e i i i i i i i e ~ l .Mycospocidins :~~~ constitute a complex of antibiotics having virtually the same components a s those present in tunicamycin, whereas the streptoviruclins are niore polar, because the fatty acid chain is shorter than that in tiinicamyciii.:j3sAll of these antibiotics are potent, and rather specific, inhibitors of the fonnation of GlcNAc-PP-Dol, and, hence, of lipid-linked oligosaccharides in both (333) C. G. Gahmberg, M. Johinen, K. K. Karlii, and L. C. Antlersson,J. B i d . Chetn., 255 (1980) 2169-2175. (334) T. Ito, A. Takatsuki, K. Kawainura, K. Sato, aiid G. Tamura,Agric. B i d . Chem., 44 (1980)695-698; see also, T. Ito, Y. Kotlani;~,K. Suzuki, A. Takatsuki, and G. Tamura, ibitl., 4 3 (1979) 1187-1 195. (335) J. S. Tkacz, Fed. Proc. Fed. A m . SOC. Err). H i o l . , 39 (1980) 1166.

THE LIPID PATHWAY OF PHOTEIN GLYCOSYLATION

34 1

animal and plant systems, 329,336 but data on the effectiveness of inhibition of the isolated, purified components, on a molar basis, are not yet available. The specificity of the antibiotics tunicamycin, streptovirudin, and antibiotic 24010 towards inhibiting enzymes catalyzing transfer of phosphorylated residues is not absolute, because high concentrations of these antibiotics also inhibit the formation of G ~ C - P - D O ~ ~ ~ ~ 11). and, competitively, chitin ~ y n t h e t a s e(see ~ ~ reaction ~ (GlcNAc),

+ n UDP-GlcNAc + (GlcNAc),., + n UDP

(11)

Two major, homologous components isolated from a tunicamycin preparation were to differ slightly in their ability to inhibit the synthesis of GlcNAc-PP-Dol, but, and this may be of practical importance, the homologs (and, therefore, possibly different batches of tunicamycin) differed in the extent to which they affected protein synthesis. For example, on studying the effects of tunicamycin on the formation of proteoglycans, Hart and L e n n a r found ~ ~ ~ ~that the inhibitory effects that occur at high concentrations of the drug may be indirectly caused by inhibition of the synthesis of proteins. By using solubilized UDP-GlcNAc :dolichol phosphate GlcNAc 1-P transferase from pig or hen o v i d ~ c t , 3 the ~ ~mechanism of inhibition b y tunicamycin was studied in some detail. It has been proposed from these studies that tunicamycin is a bisubstrate analog mimicking the substrate-product transition-state formed during catalysis (see Section II,l,b), the fatty acid chain of tunicamycin occupying a hydrophobic “cleft” on the enzyme reserved for dolichol phosphate. This proposal explains the observation that the transfer of GlcNAc from UDP-GlcNAc in reactions 12,13, a n d 8 is not, or is only slightly, inhibited by t u n i ~ a m y c i n . ~ ~ ~ , ~ ~ ~ . ~ ~ ‘ MurNAc-PP-undecaprenol

+ UDP-GlcNAc +

pehapeptide GlcNAc- MurNAc-undecaprenol

+ UDP ( 1 2 )

peitapept ide Undecaprenol-P + UDP-GlcNAc + GlcNAc-P-undecaprenol + UDP GlcNAc-PP-Do1

+ UDP-GlcNAc -+

(GlcNAc),-PP-Dol

+ UDP

(13)

(8)

(336) D . W. James and A. D . Elbein, Plunt Physiol., 65 (1980) 460-464. (337) C. P. Selitrennikoff, Arch. Biocheni. Biophys., 195 (1979) 243-244. (338) W. C . Mahoney and D. Duksin,J. Biol. Chem., 254 (1979) 6572-6576. (339) G. W. Hart and W. J . Lennarz,]. B i o l . Chem., 253 (1978) 5795-5801. (340) A. Heifetz, R. W. Keenan, and A. D. Elbein, Biochemistry, 18 (1979) 2186-2192. (341) R. K. Keller, D. Y. Boon, and C. F. Crun, Biochemistry, 18 (1979) 3946-3952. (342) L. Lehle and W. Tanner, F E E S Lett., 71 (1976) 167-170.

342

RALPH T. SCHWAKZ .4ND ROELF DATEMA

Keller and coworkers"41proposed that tuiiicainycin is a reversible, tight-binding, and, therefore, competitive inhibitor of the GlcNAc 1-P transferase. The association rate-constant was 7 x lo4M-'.s-' (at 23"). Inhibition can be overcome by increasing the proportion of enzyme, and, because preincubation of the enzyme with UDP-GlcNAc prevented inhibition by t u n i c ~ i i i i y c i ~ isoiiie , : ~ ~ ~experimental support for competitive inhibition was obtained. The known affinity of the antibiotic for p h o s p h o i i o l i p i ~ l smay ~ ~ ~facilitate its access to the inembrane1)ouiid enzyme, but the lipids do not prevent inhibition of the enzyme by tiinicai~iyciii.~~"

b. Amphomycin.-Amphoiiiyciii ( 5 ) belongs to a group of aiitibiotics consisting of a fatty acid and a peptide chain. Other antibiotics belonging to this group are tsushimycin, asparticin, and laspartomy.34:3-:34ti Amphomycin inhibits the synthesis of peptidoglycan in Bacillus niegateriurn b y blocking the phosphono-MurNAc-pentapeptide translocase,:346 the same eiizyiiie found to be inhibited by tunicamycin (see Section 111,4,a). (MeCH,CH(CH2)5CH=CHCH2C0 -Asp- MeAsp- Asp- Gly- Asp- G1y-Dab'- Val-Pro I Me P i p - Aabt

Dab'

:

Dab'

=

1

n - entlri-o -2.3-diarninobutanoic acid ~ - t / i r e -2,3-diaminobutanoic o acid

P i p = pipecolic a c id Amphomyc i n ' 4 i

5

In eukaryotic cells, amphomycin inhibits the formation of GlcNAcPP-Dol, Man-P-Dol, and Glc-P-Do1 froin their respective nucleotide esters of sugars aiid Do1-P.134.1s8,347,348 These reactions have in coninioii the fact that they require manganese ions and Dol-P, but high concentrations of neither the ion nor the lipid phosphate were able to overcome these blocks. The transfer of preformed M a ~ i - P - D o l (and, " ~ ~ pos(343) H. Tanaka, Y . Iwai, H. Oiwa, S. Shinohara, S. Shoji, T. Oka, and S. Oinnra, Bioc h i i t i . Bioph!/s.A c f a , 497 (1977) 633-640. (344) H. Bodansky, G. F. Sigler, and A. Bodailsky,]. Aiii, C l i c . , , i . Soc., 95 (1973) 23522357. (345) J. Shoji, S. Kozaki, S. Okamoto, R. Sakazaki, and H. Otsiika,]. A t i t i b i o t . , 21 (1968) 439-443. (346) H . Tanaka, H.Oiwa. S. Matsukura, ant1 S. Oinura, B i ~ ~ h e i iBiophys. i. Rcs. Coininuri., 86 (1979) 902-908. (347) M. S. Kang, J. P. Spencer, and A. D. Elbein,]. B i d . Chon., 253 (1978) 8860-8866. (348) M. C . Ericson, J. T. Gafford, and A. D. Elhein, Arch. Biochem. Biophys., 191 (1978) 698-704.

THE LIPID PATHWAY 0 1 : PROTEIN GLYCOSYLATION

343

~ i b l y , ' "Glc-P-Dol) ~ to the growing, lipid-linked oligosaccharide, and

also the transfer of the lipid-liiiked oligosaccharide to the protein, were not prevented by amphoniycin. At concentrations of ainphomyciii that completely block the fonnation of Man-P-Dol, the incoi-poration ofa-linked D-mannose into lipidlinked oligosaccharides is n o t coinpletely inhibited."' As was mentioned in Sections II,2,a and II,2,b, EDTA in uitro and 2-deoxy-2fluoro-D-glucose in uiuo have a similar effect, namely, the formation of a heptasaccharide-lipid, Man,(GlcNAc),-PP-l)ol, is still possihle in the absence of Man-P-Dol. Thc, question as to whether these mannosy1 residues come directly froin GDP-Man is discussed in Section II,B,b. c . Showdomycin and Diumycin.-Showdomycin (6, 3-p-D-ril)ofuran osy 1male iin ide) is a n 11 c 1ec ) si ( le antit) iot icJ4!' that preferential 1y inhibits formation of Glc-P-Uol, its shown with a solubilized enzymepreparation from pig aorta.:'"' The membrane-bound, particulatc enzyme is less strongly inhibited, probably because it is poorly accessible to the drug. Much larger proportions of the antibiotic were needed in order to inhibit the formation of Man-P-Do1 and GlcNAcPP-Dol. On the other hand, diiiniycin (a 2-amin0-2-deoxy-D-glucoseand phosphate-containing anti1)iotic) in solubilizecl enzyine-preparations"j' ,352 inhibits the formation of Man-P-Dol, GlcNAc-PP-Uol, and (GlcNAc),-PP-Dol, and only slightly affects the biosynthesis of Glc-PDol. The formation of (GlcNAc.),-PP-Dol from UDP-GlcNAc and GlcNAc-PP-Do1 is most strongly iiihit)ited.351

0

OH

130

Showdomycin''9

6

(349) H. Nishimura, M. Mayaina, Y. Koitiatsu, H. Kato, N. Shiinaoka, ;ind Y. Taii;tka, J . Arrtihiot., 177 (1964) 148-1%. (350) M. S. Kang, J. P. Spencer, and A . I). EIliein,J. B i d . C,'hc,m., 254 (1979) 10,03710,043. (351) C. L. Villeriiez and P. L. CaIlo,J 8i0l. Clwrn., 255 (1980) 8174-8178. (352) P. Babczinski, Eur. J . Biochrrri., 112 (1980)53-58.

344

RALPH T. SCHWARZ A N D ROELF DATEMA

The results with showdomycin are interesting, in that they show that the antibiotic inhibits formation of Glc-P-Do1 from UDP-Glc, but not the incorporation of Glc into lipid-linked oligosaccharides. This noncompetitive i n h i b i t o P may, therefore, be used in attempts to learn more about the origins of D-glucosyl residues in the lipid-linked oligosaccharides. Because of its inhibition of the formation of Man-P-Do1 and GlcNAc-PP-Dol, diumycin may be useful in attempts to discover possible roles of Glc-P-Do1 in D-glucan formation, because it would block concomitant formation of D-mannan and glycosylation of proteins. 5. Other Inhibitors of Protein Glycosylation A few other compounds have been found to interfere with the glycosylation of proteins. For example, 6-diazo-5-oxo-~-norleucine(DON) is an antagonist of ~ - g l u t a n ~ i n eand, , 3 ~ ~therefore, may interfere with the conversion of D-fructose 6-P into 2-amino-2-deoxy-D-glucose 6-P. Indeed, in epithelial cells from rat palate, the inhibition of synthesis of glycoproteins and glycosaminoglycans by DON was prevented when 2-amino-2-deoxy-D-glucose or L-glutamine was simultaneously added.354Thus, inhibition by DON in these cells is probably caused b y lowered levels of GlcN 6-P, and not by its interference with other cellular processes, such as diminution of NAD pools or of biosynthesis of cytidine. It is not y e t known whether the proportion of Man-P-Do1 or Glc-P-Do1 increases when the analog is added to lessen the formation of UDP-GlcNAc (and probably, therefore, also of GlcNAc-PPDol). Coumarin (2H-1-benzopyran-2-one) is a known inhibitor of cellulose f o ~ - m a t i o n , and ~ ~ ~Hopp - ~ ~ ~and coworkers358found that, in membranes from the alga Prototheca xop$i, it inhibits the transfer in vitro of the lipid-linked cello-oligosaccharide to its protein acceptor (see Section 11,2,b). Warfarin is a coumarin derivative, namely, 3-(c~-acetonylbenzyl)-4hydroxycoumarin, known to be an antagonist of vitamin K, 2-methyl-3phytyl-1,4-naphthoquinone.Some reports (for a review, see Ref. 359) (353) A. Telser, H . C. Robinson, and A. Dorfman, Proc. N a t l . Acad. Sci. U . S. A., 54 (1965) 912-919. (354) R. M . Greene and R. M. Pratt, E x p . Cell Res., 105 (1977) 27-37. (355) M. Hara, N . Umetsu, C. Miyamoto, and K. Tamari, Plant Cell Physiol., 14 (1973) 11-15. (356) D. Montezinos and D. P. Delmer, Planta, 148 (1980) 305-311. (357) J . R. Colvin and D. E. Witter, Plant Sci. Lett., 10 (1980) 33-38. (358) H. E. Hopp, P. A. Romero, and R. Pont Lezica, FEBS Lett., 86 (1978) 259-262. (359) J . Stenflo and J. W. Snttie, Annu. Rev. Biochem., 46 (1977) 157-172.

T H E LIPID PATHWAY OF PHOTEIN GLYC:OSYLATION

345

indicate that warfarin blocks the vitamin K-mediated carboxylation of the L-glutaniinyl residues of tlie glycoprotein prothrombin. However, in another coinmiinication,""~wnrfirin was shown to inhibit the glycosylation i n vivo, and not the car\)oxylatioii,of protlirombin. If warfar in, like coumarin, inhibits lipid-tlepeiident, glycosylation reactions, it should also inhibit the glycosylntioii of proteins other than prothronibin. Although this has not yet b c ~ shown, n inhibition ofthe incorporainto lipid-linked saccharide intertion of 2-aiiiino-2-deoxy-D-g~iieosc~ mediates (possibly, the dolichol-linked saccharides) h a s heen described.:<61 Interestingly, the inhibitions caused b y warfiirin are reversed by vitainin K. Members of a group of chetiiically unrelated compounds, for example, dibucaine [2-butoxy-N-(2-diethylaininoethyl)ci1iclio1iinaniicle], ethanol, phenobarbital (5-ethyl-5-phenylbarbitiiric acid), or lidocaine (2-diethylamin0-2',6'-acetoxylitlitle)~~~~~~~ (see the literature cited in Ref. 364) interfere with the secrc,tion of collagen, glycoproteins, and glycosaiiiiiioglycaiis, or with the forination of viruses; this niay be caused by the disruption of the niicrotubule system, but not nece ily so.364In fact, some of these conipoiinds, for example, ethanol,:'w2inhibit glycosylation of secretory proteins. From the pharmacological point of view, however, these coiiiponents are all known as anesthetics, and they exert their effects b y altering iiieiribrane properties.2"* 366p36R As most of the glycosyltraiisferases are membrane-bound enzymes, the anesthetics may possibly affect the enzyme activities, and hence, influence the carbohytlratc composition36s of the secreted products, or even their secretion:164J69 (see also, Section IV). Another proposal is that several anesthetics, for example, the tertiary amines, may detach microtubules from membranes, or, at least, affect the linkage between the cytoskeleton and the membrane proteins, and,

(360) R. G. Meeks and D. Couri, Biodtit)i. B i o p h ! l . ~Actci, . 544 (1978) 634-637. (361) R. G. Meeks and D. Conri, B i d t i t i t . Hiolh(/,s. Actu, 630 (1980) 238-245. (362) G. Nanni, M .-4. Pronzato, M. M . Avrfiiinc, G . R. Garnbella, D. Cottalaaso, and U . M . Marinari, F E B S Lett., 93 (1978) 242-246. (363) D. J. Tuma, R. B. Jennet, and 51. F. Sorrell, Biochitri. B i o p h y s . Actn, 544 (1978) 144-152. (364) J. H. Eichhorn and B. Peterkofsk>,,/.CcIl B i o l . , 81 (1979) 26-42. (365) C. D. Richardson and D. E. Vaiic.e,j. Hiol. Chern., 253 (1978) 4584-4.589. (366) G. F r e u d , Cancer Res., 39 (1979) 2899-2901. (367) H. Kutchai, L. H. Chandler, ant1 I,. hl. Geddis, R i o c h i r n . Bioph!ls. Actcr, 600 (1980) 870-881. (368) L. M. Gordon. R. D. Sauerheber, J . A. Esgate, I. Dipple, R. J. Xlarchmont, and M. D. Houslay, J . Biol. Cherri., 255 (1980) 4519-4527. (369) G. A. Read and C. J. Flickingrr, E x ~ JCell . R E S . ,127 (1980) 115-126.

346

RALPH T. SCHWAlU A N D ROELF DATEMA

hence, interfere with the secretion of p r o t e i ~ i s The . ~ ~hypotheses ~~~~~ on the mechanism of action of anesthetics (see Ref. 294 for a review) all center on their direct interaction with the membrane, and explain the rapid effects on membrane phenomena. However, these compounds also have long-term, biochemical effects: several local anesthetics inhibit the synthesis of c h o l e s t e r 0 1 , ~ and ~ ~may ~ ~ ~thus ~ alter membrane structure. Impairment of glycosylation of glycoproteins occurs b y omission of sugars from the surrounding medium of cultured cells (“starvation”). The viral glycoproteins, pulse-labelled under such conditions, have a decreased molecular weight, but are, still, partially g l y c ~ s y l a t e d . ~ ~ ~ The inhibition caused by starvation is reversible, because, after addition of D-glucose, normal glycoproteins are again The appearance of under- or non-glycosylated, membrane proteins during Dglucose starvation also occurs in non-infected, chick-embryo 37s and in the mouse-myeloma t u m o P MOPC-46. The deprivation of D-glucose can, for example, occur when the chick-embryo fibroblasts are transformed with Rous sarcoma virus, because the transfomied cells have an increased rate of utilization of D - g l u c o ~ e . ~ ~ ~ Reversal of the inhibition of glycosylation, by D-glucose starvation, through addition of D-glucose to MOPC-46 cells was prevented when tunicamycin was present.376This indicates that the dolichol pathway of glycosylation of proteins is involved in this glycosylation process regulated by D-glucose. Other evidence supports this notion.378Thus, the lipid-linked and protein-linked oligosaccharides from virus-infected, D-glucose-starved cells contain species that are smaller than the normal oligosaccharides, and, furthermore, r e s i s t a n P towards endo-@-N-acetylglucosaminidase H. These results resemble those obtained with cells treated with 2-deoxy-2-fluoro-~-glucose*~~ or with cells lacking Man-P-Dol, and suggest that D-glucose starvation decreases the levels of Man-P-Dol, and, hence, glycosylation of proteins, b y way of the Glc,Man, (GlcNAc), , lipid-linked oligosaccharide. (370) B. H. Woda, J. Yguerabide, and J . D. Feldman, Ex?). Cell Res., 126 (1980) 327331. (371) S. Friedman and P. Skehan, FEBS Lett., 102 (1979) 235-240. (372) F. B. Bell and E. V. Hubert, Biochirn. Biophys. Actu, 619 (1980) 302-307. (373) G. Kaluza,/. Virol., 16 (1975) 602-612. (374) R. P. C. Shiu, J . PouyssCgur, and I. Pastan, Proc. Nutl. Acud. Sci. U . S. A , , 74 (1977) 3840-3844. (375) J. Pouyssegur and K. M. Yamada, Cell, 13 (1978) 139-150. (376) N. J. Stark and E. C. Heath, Arch. Biochem. Biophys., 192 (1979) 599-609. (377) 0. Warburg, The Metabolism ofTumors, Smith, New York, 1931. (378) S. J. Turco. Arch. Biochern. Biophys., 205 (1980) 330-339.

THE LIPID PATklWAY O F I'KOI'EIN GL,Y(:OSYL,ATION

347

It is apparent that interference. with protein glycosylation can also be achieved by iising cell mutants that have a block in the synthesis of an essential intermediate. Siic*lr cell iriutants were discussed in Section II,2,c. In acldition, virus nriitunts have been clescribed that have defects in the migration of the viral glycoproteins from one ineml,rane system to a n ~ t h e r . " ~ Thus, , ~ ~ t~h y Ireinagglutinin of the influenza mutant ts 227, at the nonpennissivr, teinperaturc, does not reach the Golgi Consequently, the protein does not become equipped with the complex, carhohydrate chains. Although mutants have proved to be invaluable tools in glycoprotciii research, the), are noti0 so generally applicable a s tlriigs are, and y e t , these results point to some future trends: (1 ) inhibitors o f protein glycosylation that, like dGal,3l 6 interfere with steps aftc r the transfer of the o 1i gomann o s i d ic side-chains from the lipid to thc protein, and (2) inhibitors of intracellular transport. It is now known, l o r example, that the ionophore inon(for a ensin prevents exit of glycoprotcins from Golgi cisternae:3XL-:3X5 review, see Ref. 383).The atltlition ofthe sugars Gal, Fuc, and NeuAc to the immunoglobulins takes place in a compartment distal to the inonensin-sensitive cistemae.!':' I Irnce, in the presence of monensin, these sugars cannot be incorporated. Other ionophores (of which A23187 is an example) may, i n S ~ I I I Csystems, block transport of proteins at a site between the endoplnsmic reticiiliim and the Golgi sysand also indirectly 1)loc.k the maturation of high-mannose chains to afford complex, carl)ohyclrate chains. Also, modification of membranes by incorporation of different phosphonolipids hampers migration to the plasma i n e ~ n t ) r m i eand " ~ ~may, therefore, indirectly affect glycosylation. Table I siiiiiiiiiirizes the effects on protein glycosylation of some of the inhibitors discussed here.

The enkephalins, a group of pcptitle neurotransinitters, are examples of physiological substancc,s that l , l o ~ k " ~glycosylation ~" (of glyco(379) J . Lohineyer and H.-D. Klenk, Vzro/og!y, 93 (1979) 134- 145. (380) J. Saraste, C.-H. von Bonsdorff; K . Hashiinoto, L. KLidriiiinen, and S. Keriinen, Virology, 100 (1980) 229-245. (381) A. M. Tartakoff and P. Vassalli,J. K q , . A 4 r d . , 146 (1977) 1332-1345. (382) A. Tartakoff and P. Vassalli,J. Cv!l W i o l . , 79 (1978) 694-707. Pnfhol., 22 (1980) 227-251. (383) A. M. Tartakoff, Int. Rea. EXJJ. (384) N . Uchida, H. Smilowitz, P. W. Lxciger, aiid M . L. Tanzer,J. R i o l . Chrrn., 255 (1980) 8638-8644. (385) D. C. Johnson and M . J. Schlesinger, Virology, 103 (1980) 407-424. (386) E. Fries and J. E. Rothman, Proc. N a t l . Accitl. Sci. U . S. A., 77 (1980) 3870-3874. (387) M .Maeda, 0. Doi, and Y. Akainatsa, Hiochini. Rio)~h!ys.Actci, 597 (1980)552-563. (387a) G . Dawson, H. McLawhon, and H . J . Miller, Proc. N u t l . Acad. S c i . U . S . A . , 76 (1979) 605-609.

TABLEI Inhibitors of Glycosylation of Proteins

Category, and inhibitor

Inhibitors of Formation of Dolichol Phosphate Bacitracin 25-H ydroxycholesterol

Reaction inhibited

Dol-PP

+

Dol-P

Remarks

+ Pi

inhibitor may not enter the cell inhibition of this reaction need not necessarily lead to inhibition of glycosylation (see Section III,2)

HMG-CoA + 2 NADPH + 2 H+ + nievalonate + 2 NADP+ + Co-A

w

e

a2

References

16 6,7,248

7-Ketocholesterol 3-Hydroxy-3-methylglutaric acid

Compactin Inhibitors of Assembly of Lipidlinked Oligosaccharides I. Antibiotics Tunicamycin-like compounds (mycospocidins, streptovirudins, antibiotic 24010, tunicamycin) Amphomycin ( 5 )

Showdomycin (6)

GlcNAc-PP-Do1

+ UMP

32 1 332a

UDP-GIcNAc + D d - P + GlcNAc-PP-Do1 GDP-Man + Dol-P + Man-P-Do1 + G D P UDP-Glc + Dol-P + Glc-P-Do1 + UDP UDP-Glc + Do1 P 4 Glc-P-Do1 + UDP

+ UMP

35

UDP-GlcNAc

+ Dol-P

4

high concentrations also inhibit the formation of ClcNAc-PP-Do1 and Man-P-Do1

350

Dirt i i i ycin

11. Sugar Analogs 2-Deoxy-D-nmbiiio-hexos~. (dGlc)

GDP-Xlan + Dol-P + hlaii-P-Do1 + GDP UDP-GlcNAc + 1101-P 4 GlcNAc-PP-Do1 + UMP GlcNAc-PP-I>ol + UDP-GlcNAc 4 (GlcNAc),-PP-Dol + UDP the forrnation of Man-P-Dol, Glc-P-Dol, and GlcNAc-PPDol, b y trapping of Dol-P as dGlc-P-Do1 the tnannosylation of(GlcNAc),-PP-Do1 and .2.1an-(GlcNAc)2caused b y PI'-Dol, because of formation of dGle(GlcNAc)z-PP-Dol and dClc-Man(GlcNAc)l-PP-Dol the D-glucosylation of Man,-(GlcNAc),-PP-Do1 Uol-P + UDP-Glc + Glc-P-Do1 UDP inhibition is cawed by

+

-

351,352

274-276

275 UDP-dGlc 2-Deoxy-2-fluoro-D-g~ucose(FGlc) in intact cells, the formation of Man-P-Do1 and Glc-P-Do1 is inhibitions are probably 116 inhibited; the pool size of UDP-Glc is lessened iiietliated l)y CDP-FGlc. or LTIIP-FGlc,o r both 286 2-1~eo*)-8-fluoro-~-iiiatiiiost, ullkno\Vll inhibition i s pro1)ably b y 266,286 (FSlati) way of GI)P-Fk.lan. o r C'DP-FMan, or both 2-.~mino-2-deox!--D-fil~ieo~e (GlcN) unknown inhibition is caused b y 266,290 GlcN, and not b y one of its metaholites Inhihitors of the As.sc~mlh/of Prof riti-bou rid Oli~oscrc,c.ltciritles I. Sugar analogs 2-Deoxy-D-l!ixo-hexose (dGal) tioii at OH-2 of Gal, due to incorporatioii of dGal itthibitiott is caused 1,) 316 UDP-tlG>d 11. Inhibitors of Intracellular Transport Slonerisin inhibits incoi-poration of Gal ; ~ n t lNeriAc into irihil)its transport of 95 immunofilobulins glycoproteins to a distal, Colgi coinpartintrnt

350

RALPH T. SCHWARZ AND HOELF DATEMA

lipids and glycoproteins). It is important that these effects are only seen in cells that possess receptors for these neurotransmitters, These results raise the question as to whether there are more substances having the biological fiinction of inhibiting the glycosylation of proteins. Such substances should exert their function in tissues devoted to synthesis or secretion, or both, of a few special polypeptides, because the structures of carbohydrate side-chains of glycoproteins do not vary a great deal.'" In the following Sections, the biological consequences of inhibition of glycosylation of proteins will be outlined. The consequences niay be dramatic, as proper processing through proteolytic cleavage can be impaired. Thus, it should be no cause for wonder if inhibitors of protein glycosylation affect the formation of an unglycosylated end-product, the carbohydrate-bearing portion of its precursor taking care of proper processing."x7h I v . BIOLOGICAL EFFECTSOF INHIBITION OF GLYCOSYLATION

1. General Comments Glycoproteins are widely distributed in Nature, being found in the cytoplasm of cells, and in plasma iiienibranes, cell walls, secretions, mucins, and body fluids. In fact, proteins that had been considered to be pure proteins have since been found to contain sugars as an integral part of the molecule, and are thus now recognized as being glycoproteins. Such niolecules as enzymes, hormones, membrane carriers, and receptors may contain oligosaccharides, and a question arises concerning the functional contribution of the sugar side-chains. Certain monographs are recoinmended for further information on glycoproteills .3xx-:39:3 In some instances, the fiinctioii has been recognized. Carbohydrates have been found to be essential in recognition or binding, (387b) J. W. Jacobs, P. K. Luntl, J. T. Potts, N . H. Bell, and J . F. HalxnerJ. B i o l . Chem., 256 (1981) 2803-2807. (388) A. Gottschalk, GI!ycoprofeiiis,Elsevier, Anisterdam, 1972. (389) R. C. Hughes, Membrutie Glycoproteins, Butterworth, London, 1976. Z. (390) J. G. Sniellie, The bfetubo~ismuticl Futictiori of Gl!ycoproteiiis,B ~ O C ~ W JSoc. S!ymp., London, 1974. (391) W. Piginan and D. Horton, The Carhohytlrutes: Cheniistr!/ utitl Biochemistry, Vol. IA, Academic Press, New York, 1972. (392) M. I . Horowitz and W. Pigman, The Gl!icocorijugutes,Acadelnic Press, New York, Vol. I, 1977; Vol. 11, 1978. (393) N . Sharon, Cornplex Curhoh!/drutr:s,Addison-Wesley, London, 1975.

T H E LIPID PATHWAY OF PHC)?'EIN GLYCOSYLATION

351

or both, of l e c t i n ~ , 3 ~ ~ arid viruses""; they protect proteins against changes in temperature and pH. E y W g 9postulated that the carbohydrate i s necessary for export of proteins from cells, in, for instance, secretion. However, a n i i i n b e r of proteins having no carliohyclrate attached are also known to he secreted. For example, bovine RNase [ribonucleate pyriinic~iiie-iiucleotido-2'-traIlsferase (cyclizing), EC 2.7.7.161exists in glycosylated and nonglycosylated fonns that are both secreted, and that exhibit siinilar catalytic properties.38xY Also, the enzyniic activity of RNase is not affected after removal of carbohydrate from the enzyme by g l y c o ~ i t l a s e s . ~ ~ ~ ~ ~ ~ ~ Also, variations in the carbohydrate contents of lipase (glycerolester hydrolase, EC 3.1.1.3),4'y2 glucoamylase [( 1+4)-a-D-glucan glucohydrolase, EC 3.2.1.3],40"and chloroperoxidase (3-oxoadipate: hydrogen peroxide oxidoreductase, EC l.11.1.a)404 are known to have no influence on their catalytic activity. However, in some cases, the carbohydrate was found to protect enzymes from proteolytic degradation, as, for instance, RNase, P-L)-glUcosiduror1ase (~-D-glucosiduronate glucosiduronatehydrolase, EC 3.2.1 31),and the secretory coinponent405-407 of iinmunoglobuliir A. Glycoproteins are needed a s lubricants, and also function as protective agents of surfaces. The viscosity ofmucins is due to the carbohydrate moiety.3y3Some species of arctic fish contain an antifreeze glycob y preventing protein that lowers the freezing point of their the lattice fonnation of water-ice clusters, a step that is a prerequisite (394) N. Sharon atid H. Lis, Science, 177 (1972) 949-959. (395) R. W. Jeanloz and J. F. Codingtoii, i n A Rosenberg and C. L. Schenigrund (Eds.), Bio/ogicuZ Roles of Siulic A c i d , Plc.nnin, New York, 1976. pp. 201-238. (396) W. E. van Heyningen, Nature, 248 (1974) 415-417. (397) V. Bennet, I<. O'Keefe, and P. Cri;itrecas;is, Proc. Natl. Accitl. Sci. C7. S. A . , 7 2 (1975) 33-37. (398) S . E. Luria, J. E. Darnell, Jr., 11.Haltiinore, and A. Campbell, Geiiercil Virology, 3rd edn., Wiley, New York, 1978. (399) E. H. Eylar,]. Theor. Biol., 10 (1966) 89-113. (400) A. L. Tarentino, T. H. Planiiner, and F. Maley,]. B i d . Clzetti., 249 (1974) 818824. (401) R. B. Trimble and F. Maley, H i o c / i c , r n . Biophys. Rrs. Coiniriuii., 78 (1977) 935944. (402) J. H. Pazur, K. R. Knull, and D. I , . Siiiipson, Biocheni.Hiop/i!/s. Re.r. C o m n m u . , 40 (1970) 110-116. (403) I. Sijiiholm, Actu Phurni. Surc., 4 (1967) 81-86. (404) T. Lee and L. P. Hager, Fed. P ~ Y J cF. , c d . A m . Soc. Lxp. B i o l . , 29 (1970) 599. (405) F. F. C. Wang atid C. H. W. Hirs,]. R i o l . Chem., 252 (1977) 8358-8364. (406) S. Hadulescu and C . Motas, Rcti. R(~itiii. Riochivi., 13 (1976) 219-223. (407) L. Buzila atid C. Motas, Reo. R o u i i i , Riochini., 14 (1977) 155-160. (408) H. E. Feeiiey and D. T . O s n g a , 7 ' r v i i d s Hioc/wni. Sci., 2 (1977) 269-271.

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to freezing. Sugars play an important role in the binding of glycoproteins to the receptors of cells. For example, the uptake of certain serum glycoproteins by liver cells is prevented by the tenninal sialic acid residues. When these residues are removed, a galactosyl group is unmasked, and this is recognized by the hepatic receptor. This receptor is also a glycoprotein and it needs its terminal, sialic acid residues so as not to bind to itself.409It has been shown for some hormones that, after removal of sugars, the target cells were not reactive in uiuo, but that the hormones were still active in uitro. The specific case of the removal of sialic acid makes them more accessible to proteolytic digestion in uiuo.410+412 Antigenicity of blood-group substances, such as those responsible for A, B, H (Ref. 390), and M N (Ref. 413) activities have been found to be dependent on sugars, especially the terminal ones. Antibodies raised to a foreign glycoprotein are largely directed against the polypeptide part of the molecule.38xNo obvious role for the carbohydrates of histocompatibility antigens is Although not required for antibody specificity, the carbohydrates of immunoglobulin are needed for complement-induced c y t o t o x i ~ i t y . ~ ' ~ Two approaches have been used in studying the role of the carbohydrate on the protein: ( a ) elimination, using glycosidases, of sugars from a molecule that has already been synthesized, and ( b )prevention of the attachment of sugars during synthesis of protein b y using inhibitors of glycosylation. The latter approach offers the advantage that it allows, in addition, conclusions about the role of sugars during the synthesis and maturation of a glycoprotein. The first method has been widely applied. In some instances, removal of terminal sialic acid by using sialidases modified the function of the glycoprotein, but, in others, the function was not changed. The results of such investigations are listed in a review by L. Warren and coworkers.416 After removal of the sugars, increased sensitivity against proteolytic (409) G. Ashwell and A. G. Morell, Trends Biochern. Sci., 2 (1977) 76-78. (410) E. van Hall, J. L. Vaitukaitis, G. T. Ross, J. W. Hickman, and G. Ashwel1,Etdm-inology, 88 (1971) 456-464. (411) A. G. Morell, G. Gregoriades, I. H. Schneider, J. W. Hickman, and G. Ashwell, J . Biol. Chem., 246 (1971) 1461-1467. (412) V. Bocci, Experientia, 32 (1976) 135- 140. (413) G. F. Springer and N. J. Ansell, Proc. N a t l . Acad. Sci. U . S. A , , 44 (1958) 182-189. (414) S. G. Nathenson and S. E. Cullen, Biochim. Bio7hys. Acta, 344 (1974) 1-25. (415) N . Koide, N. Nose, and T. Muramatsu, Biochenl. Biopkys. Res. Cornmuti., 75 (1977) 838-844. (416) L. Warren, C. A. Buck, and G. P. Tuszynski, Biochim. B i o p h y s . Acta, 516 (1978) 97- 127.

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degradation is frequently encountered, possibly by alteration of the conformation, thus exposing susceptible sites of the protein backbone. In addition, a s carbohydrate chairis have now been reniovetl, they cannot protect sites that are srisceptible to protease. However, removal of carbohydrates from glycoproteins b y means of glycositlases was not complete in all instances r e p o ~ t e d , l ' " ~ ~and ~ , " ~this may weaken the conclusions drawn from this approach. Therefore, inhibitors of protein glycosylation niay be inore useful for producing glycoproteins that are free froin carbohydrate. The first part of this Section will deal with the biological effects of the inhibition of protein glycosylation, and the coilsequences for conformation, limited proteolysis, roitting, secretion, recognition, and uptake of glycoproteins. The more comprehensive topics, such as the effects on differentiation, the inultiplication of' enveloped viruses, and cellular phenomena, will also lw described.

2. Effects on Conformation of Proteins The attachment of oligosaccharide side-chains constitutes a substantial modification of a protein. The sugar side-chains not only alter the molecular weight of thc, niolecule but also change its shape, that is, its confonnation-a factor that is responsible, among other roles, for different physicocheinical properties, such a s solubility. At present, few data are available concerning the primary sequences or the conformations of glycoproteins; however, evidciice from the proteins so far studied iiidicates that 0 -and N-glycosylically linked sugar sidechains are mainly situated i n the p-turns of the protein For instance , of 3 1 L-as parag i n e I-es id ue s, su 1) st itu ted with o 1igo saccharides, that have been investigated, 30 were found to occur i n sequences favoring turn or loop structures. Of the glycosylated asparagine residues, 22 occuP' in tetrapeptides predicted to have the p-turn conformation. The carbohydrates of ovomucoid4" are also found to be attached on loops of the protein. In cow K-casein, L-threonine residue 133, and, in sheep casein, L-threoniiw 135, carry an 0-glycosylic sidechain, and, in both, this amino acid is situated4" in a &turn. Interest-

(417) 11. McCaithy and S. C. Harriso~r,,/. \ T i n ) / . , 23 (1977) 61-73. (418) S. I. T. Kennedy,]. Geii. Virol.. 23 (1974) 129-143. (419) J . P. Aribeit, G. Biserte, and hl. 11. Loucheux-Lefel,vl-e,Ar(,/~. BiochiJI. Bioph!/.T., 175 (1976) 410-418. (420) J. G. Beelcy, Riochein. i?ioph!/.(.. H m . C O I J I J J ~76U (1977) ~ I . , 1051- 1055. (421) J. G. Beeley, Biocheni. I., 159 (1976) 335-345. (422) M . H. Louclit.~ix-Lefehvre,J. P. .411l)ett, aiid P. Jolli..;, i?ioph!/.s./., 23 (1978)323336.

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ingly, the authors422also found that the sites of phosphorylation are parts of loop structures of the molecules as well. The crystallographic structures of an IgG molecule and its Fc fragment have been published, and the interplay between the carbohydrate moiety and the protein part has been discussed in functional terms.423,424 The antibody is a very large glycoprotein (molecular weight, 170,000) which consists of two light and two heavy chains linked together by disulfide bonds. The N-glycosylically linked oligosaccharide turned out to be attached to a p-turn of the heavy chain. It was proposed that binding of the antigen converts the antibody from a “flexible Y” into a “rigid T” shape, a change that is, at least in part, facilitated by its carbohydrate moiety. Stabilization of the T shape seems to be essential for binding of complement after the antibody has bound its antigen. This assumption is supported by findings of Koide and which showed that removal of most of the carbohydrate by treatment of IgG with a glycosidase results in losses of cytotoxicity, rosette formation, and coniplement-dependent heniolysis. Also, in a number of other instances, carbohydrate side-chains may act as conformational modifiers of proteins. For example, certain enzymes, such as yeast invertase, N-acetylglucosaminidase, and a-D-galactosidase, remain active after removal of the sugar part, but become much more prone to denaturation, suggesting that a source of stability has been removed with the carbohydrate.388This is in accord with findings of Maley and removal of 90% of the carbohydrate of yeast extemal-invertase with endo-P-N-acetylglucosaminidase H had no significant effect on its catalytic properties; however, it became much less stable to multiple freeze-thaw treatment, acidic conditions, heat, and trypsin digestion. From de- and re-naturation experiments, it appeared that the carbohydrate of this enzyme promotes the folding of the enzyme protein to a more stable and more resistant conformation. This conclusion is in harmony with observations that , ~ ~release ~ of N-acetylneuraminic deglycosylation of r i b o n u c l e a ~ eand acid from p - D - g l u c o s i d u r ~ n a s eand ~ ~ ~the secretory componenPo7 of immunoglobulin A, rendered these proteins more susceptible to proteolytic degradation in uitro.Thus, lessening the amount, or the lack, of carbohydrate seems to lead to an exposure of turn or loop structures that may be readily attacked b y proteases. Lack of the oligosac-

-

(423) J. Huber, J. Deisenhofer, P. M . Colnian, M. Matushima, and W. Palni,Nature, 264 (1976) 415-420. (424) D. A. Rees, Polysaccharide Shapes, Chapman and Hall, London, 1977. (425) F. K. Chu, R. B. Trimble, and F. Maley,J. B i d . Chem., 253 (1978) 8691-8693.

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charides on a glycoprotein will tkilitate assumption of a different orie t i tat io n of the protein bac k b o t I ts that i n ay favor read ie r den at ti ration of carbohydrate-free proteins, Flirther examples are indicated i n Ref. 393. However, in addition, reiiioval of the hulky carbohydrate chains will expose additional sites on tlic. protein that m a y be cleaved b y proteases. 111 a series of elegant experinic,iits, the properties of different glycosylated, or nonglycosylated, G protein (synthesized in the presence of tunicamycin) of a variety of different strains of vesicular stonlatitis virus (VSV) have been tested. It1 contrast to the glycosylated viral glycoproteins, the nonglycosylated G molecules of VSV Indiana were in~ o l i i b l in e ~buffers ~~ containing such non-ionic detergents as Triton X100. G protein lacking the c a r l d i ydrate was precipitated when guanidinium hydrochloride, used to solubilize the protein i n this buffer, was removed b y dialysis. The iiiterpretation of this phenomenon is that lack of carbohydrate alters the ~)liysicocliemicaIproperties of glycoproteins, including their so1ul)ility. This assumption is further substantiated b y the following findings. After extraction of inenibranes from infected cells with 0.2% 'lriton X-100, G proteins from several strains of VSV were studied. The nonglycosylated G protein of VSV Orsay, synthesized at 38", pelletrtl to the bottoni of a sucrose gradient; in contrast, the nonglycosylated G protein of VSV Orsay, synthesized at 30", stayed at the top of the gradient, as did the glycosylated protein.4z7j42x This means that lowc>riiigof the temperature can substitute for the carbohydrate attachiment, and that, at lower temperatures, nonglycosylated G protein has tlie conformation of the glycosylated species. Glycosylated G protein, dialyzed either at 4 or 38", remained in solution at both temperatures. Nouglycosylated G protein that had been dissolved in buffers containing guanidinium hydrochloride remained in solution when dialyzed at 4", but aggregated, and was precipitated, at elevated temperatures. In the Sections dealing with the transport between intracellular iiieinbranes (Section IV,4) and with formation of virus particles (Section IV,7), it will be seen that the physicocheniical properties tlisciissed here have a direct influence and significance for biological properties of the glycoproteins. The functional role of the oligosaccharide side-chains in maintaining conformation and antigenicity has also been studied by use of an immunological approach. Antibodies directed against nonglycosylated, envelope proteins of Semliki Forest virus did not react with the (426) R. Leavitt, S. Schlesinger, and S. Koriifeld,]. B i d . C h e m . , 252 (1977) 9018-9023. (427) R. Gibson, R. Leavitt, S. Kornfeltl, ancl S. Schlesinger, Cell, 13 (1978) 671-679. (428) R. Gibson, S. Schlesinger, and S . Kornfeld,]. Bid. Chem., 254 (1979)3600-3607.

356

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rr.

SCHWARZ A N D ROELF DATEMA

fully glycosylated glycoproteins of the virus particles, but precipitated viral glycoproteins labelled during a short pulse with ["H]mannose. After a 20-min chase, these iminature foiiiis only reacted with antibodies raised against glycosylated glycoproteins. From this result, it was concluded that confoiinational changes of the protein are correlated with modifications ofthe carbohydrate side-chains, and that antigenic sites are exposed on nonmatiire, o r lion-glycosylated, glycoproteins which become hidden, whereas those found on mature fonns become exposed, during confomiational rearraiigeinent.lll Our understanding of sugar-protein interactions in glycoproteins is still far froin complete. Removal of oligosaccharides from glycoproteins that have already been synthesized may have quite different effects, as compared to preventing glycosylation of a glycoprotein that is being synthesized and has still to fold into the final conformation. In other words, the final conformation of the protein need not be identical in these two different situations, and may depend on the method and extent of sugar deprivation. Even a difference of a few sugars in an oligosaccharidic side-chain may have drastic effects. Experiments have been undertaken with vesicular stomatitis virusG-glycoprotein equipped with oligosaccharide side-chains having either the formula ManJGlcNAc), or Man,(GlcNAc), . Both glycoproteins were denatured by guanidiniuni chloride and thus unfolded, but, after renaturation of these glycoproteins, correct refolding of the glycoprotein having the shorter carbohydrate side-chain was prevented.429

3. Effects on Limited Proteolysis Many proteins and glycoproteins are first synthesized in the form of a protein that is larger than the final product. Here, the primary translation-product is subject to one or more steps of controlled proteol y ~ i s . Good ~ ~ " model-systems for study of the conversion of a precursor protein into the final product are cells infected with enveloped viruse s,229,431-435 such as influenza, Semliki Forest, Sindbis, and avian sarcoma viruses (and others). Some of these viruses stop synthesis of

(429) R. Gibson, S. Komfeld, ancl S. Schlesinger,J. Biol. Chern., 256 (1981)456-462. (430) H. Holzer ancl H. Tschesche, Colloq. Ces. Biol. Chem., 30 (1979). (431) H.-D. Klenk and R. Rott, Curr. Top. Microbid. Zmniunol., 90 (1980) 19-48. (432) R. W. Cornpans and H.-D. Klenk, Conipr. Virol., 13 (1979)293-407. (433) I. T. Schulze, Adu. Virus Res., 18 (1973) 1-55. (434) W. G. Laver, Adu. Virus Res., 18 (1973) 57-103. (435) L. Kaariainen and H. Sorlerlund, C u m . Top. Microbiol. Irnmunol., 82 (1978) 1569.

THE LIPID PATHW \ Y 0 1 ; PROTEIN C,I,YCOSfLrlTIOh

<3s7

protein by the host cell sooii aftcbr iiifection, and, after labelling of the cells with radioactive precursors, viral proteins clearly emerge over a low background; for example, cliiring poly(acrylamic1e)-gel electrophoresis of the cell lysates. Sonic, viruses, for instance, Rous sarcoma virus, do not stop cell-coded, protein synthesis, and, under these circumstances, iminunoprecipitalcs from cell lysates 1,y using specific anti bod ie s s ho ti 1cl lie prepared pri o r to anal y s i s b y pol y ( acry lam i de )gel electrophoresis. For Semliki Forest virus, the gl>.coproteinsare designated E 1, E2, and E3, and these are made i i i clclriilnolar amounts from a large, precursor, protein molecule that h a s a niolecular weight of 130,000. The first proteolytic cleavage liberates the core protein C, and gives rise to a precursor (ofmol. wt. 97,000) which is cleaved to produce E l and a precursor of mol. wt. 62,000 (P62) which, in turn, after a third cleavage, is ~ o n v e r t e d into ~ ” ~ E2 and E3. Another example of processing of glycoproteins is found in the synthesis of pituitary hormones. A<:T€I,PLPH, a-MSH, and ,&endorphin are synthesized from a coinnioii precursor in the neurointennedi~~te lobe. Controlled, proteolytic cleavage liberates the filial products, which are then secreted. Nuiii(,roiis other examples could be nienti on ed.3xx-3y3 A common trait in the synthesis of membrane glycoproteins is that they are fornied on the intraineiiil)raiit,us systenn of the cell. The first amino acids at the N-temiiiiiis constitute ii so-called “leader sequence” which, possibly ducx to its hydrophobic character, mediates the tunnelling of the nascent peptide chain to the lumen of the intrameinbranous system215,216 (see Scction 11,3). During the transfer of the nascent peptide into the lumen of the cisteriiae of the endoplasmic reticnliinr, the leader sequence is cleaved off’. The glycoproteins remaiii aiichored into the membrane b y either their N- or carboxyl-terminus. The attachment of the oligosaccharide occurs concomitantly with the synthesis of the protein. As discussed in Section 11,2, this is a high-miinnose structure that can be modified during transport on in trace 1111 1i i r 11i e m b rane s , and then y i e 1d s a coniplex type. The glycoproteins are trailsported froin the endoplasmic reticulum to the Golgi apparatus and the plasma membrane. Details of the phenomena of membrane transport a r e to be foiiiicl in References 383, 386, and 436. It was of interest to ascertain whether such inhibitors of glycosyla(436) D. J. MorrC, J . Kartenbeck, and W. W. Franke, Biochim. Biophys. Actci, 559 (1979) 71-152.

358

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tion as 2-deoxy-~-urubino-hexose, 2-arnino-2-deoxy-D-glucose, 2deoxy-2-fluoro-~-glucose,and tunicamycin would interfere with controlled proteolysis of the precursor molecule. Different effects of glycosylation inhibitors on the biochemical fate of glycoproteins have been recognized in these studies. For instance, the nonglycosylated fonns of enveloped glycoproteins from Seinliki Forest and Sindbis viruses are metabolically stable i i i t r a c e l l u l a ~ - l y ,and ~ ~ ~are * ~properly ~~ processed. Treatment of chick-embryo fibroblasts that had been infected with fowl-plague virus (an influenza A virus) with 2-arnino-2deoxy-D-glucose or 2-deoxy-D-c~rahi?zo-hexoseled to the formation of nonglycosylated, hemagglutinin protein HA,,, and its cleavage products HA,,, and HA,,z. (The glycosylated counterparts are designated HA, HA,, and HA, .) They were of heterogeneous size, most probably due to a lack of specificity of the proteolytic cleavage of the protein precursor HA,, that was devoid of protecting carbohydrates.265Proteolytic degradation in tunicainycin-treated, chick-embryo fibroblasts infected with fowl-plague virus prevented the detection of HA,,, unless a protease inhibitor was added.437This difference in the effect of the inhibitors is not yet understood. However, using a different strain of influenza virus (A/WSN) i n MDBK-cells, Nakainura and Compans reportedzg7that HA, was found associated with smooth meinbranes in the absence of a protease inhibitor. This means that results obtained for closely related biological systems inay differ. Lack of the glycosylation of the common precursor of ACTH-PLPH resulted in its rapid degradation, and formation of atypically processed peptides which were, however, still secreted. The decreased stability against proteases in vivo was also reflected by its lowered proteolytic stability in uitro: the isolated, nonglycosylated precursor was degraded much more readily by protease than the glycosylated counterpa~-t.~~~-~~ Firestone and Heath44' found that the alkaline phosphatase (orthophosphoric rnonoester phosphate hydrolase, E C 3.1.3.1) that is induced in cultured cells b y dibutanoyl-CAMP is not detectable in the presence of inhibitors of glycosylation, although messenger RNA is found in proportions that are comparable to those of noninhibited (437) R. T. Schwarz, J . M.Rohrschneider, and M . F. G. Schmidt,]. Virol.,19 (1976)782791. (438) E. Duda and M. Schlesinger,]. Virol., 15 (1975) 416-419. (439) Y. P. Loh and H. Gainer, Endocrinology, 105 (1979) 474-487. (440) Y. P. Loh and H. Gainer, FEBS Lett., 96 (1978) 269-272. (441) P. Crine, F. Gossard, N. G. Seidah, L. B. Blanchette, M. Lis, and M. Chrktien, Proc. Natl. Acad. Sci. U . S. A , , 76 (1979) 5085-5089. (442) G. L. Firestone and E. C. Heath,J. B i o l . Chem., 256 (1981) 1404-1411.

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cells. This may mean that, before having acquired a stahle confonnation, the nascent protein has already been proteolytically degraded during its synthesis. In yeast, however, the formation of enzyrnically active, nonglycosylated, alkaline phosphatase was not prevented by t ~ i n i c a r n y c i n These . ~ ~ ~ two examples show that we are far from predicting the effects of sugar deprivation on a protein; this will need a coinparison of the tertiary structures of the glycosylated and nonglycosylated forms. One possibility, namely, that the protein is proteolytically degraded during synthesis when lacking sugars, but shows resistance to protease treatment once it possesses its correct, three-dimensional structure, has not yet been found. However, the ohervations reported by Braatz and Heath444are interesting in this connection. These authors showed that cosecretion of polysaccharide is essential in order to stabilize newly fonned, alkaline phosphatase from Micrococcus sotfonensis. Inhibition of saccharide formation by bacitracin, 2-deoxy-D-uraresulted in degradation of hino-hexose, or 2-amino-2-deoxv-~-glucose the enzyme. The authors concluded that the polypeptide chain of the alkaline phosphatase is only vulnerable to proteolysis for a short period of time during the secretion process, and that, after the protein has been secreted, it complexes with calcium ions arid assumes a conformation that renders the polypeptide chain resistant to proteolysis. Removal of calcium ions froin the alkaline phosphatase indeed coil~ e r t the s ~protein ~ ~ into a form that is highly susceptible to proteolytic digestion.

4. Effects on “Routing,” Secretion, Recognition, and Uptake of Glycoproteins

Intriguing questions in niolt~cularbiology are as follows: how do proteins penetrate cellular meiirliranes, and how do membrane proteins become inserted into cellular membranes. Although some features that govern such processes have been recognized, much remains to be done before a fuller understanding becomes possible. Participation of carbohydrate has not been recognized in the process of insei-ting proteins into membranes, but it has been suggested that the ciirbohydrate does act as a “lock” to keep a protein inserted in the

(443) H. R. Onishi, J. S. Tkacz, and J . 0 . Lampen,J. Biol. C huit., 254 (1979) 11,94311,952. (444) J . A. Braatz and E. C. Heath,J. B i o l . Chrm., 249 (1974) 2536-2547. (445) R. H. Glew and E. C. Heath,]. H i d . CIiem., 246 (1971) 1566-1574.

360

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membrane after its synthesis, a possibility that has been widely disAlthough sugars appear not to be involved in the first step o f s e questration of a glycoprotein froin the cytoplasm to the lumen of the rough membranes during its synthesis, they might play a role in stabilizing the conformation for correct anchoring of the protein into the membrane. This assumption has, however, not yet received experimental support. Membrane vesicles prepared from cells that had been infected with Sindbis, Semliki Forest, or vesicular stomatitis virus contain the viral glycoproteins in membrane-inserted fonn. On treatment of such vesicles with trypsin, the glycoproteins are protected, regardless of whether they originated from tunicamycin-treated or from control cells: this means that the orientation to the luminal side of the vesicles of the glycoproteins has not ~ h a n g e d . ~ ~ ~ - ~ ~ ~ The results not only hold true for viral glycoproteins but may be generalized for cellular glycoproteins as well. For instance, the glycosylation of rhodopsin is not required for its insertion into the disk membrane of the rod outer segment or the rod cell of bovine retina.45" Similarly, rneinbranes from tunicamycin-treated cells failed to glycosylate newly synthesized, placental-protein hormones in a cell-fr. 2, translation system, but allowed sequestration into their lipid Lilayer.45' Conclusions concerning correct insertion into membranes could also be drawn from earlier reports in which it had been slrown that the nonglycosylated glycoproteins were able to move from r m g h However, . ~ ~ ~ ~ ~the ~ ~ failure of some to smooth e n d o p l a s m i c - r e t i c u l ~ ~ m nonglycosylated proteins to reach the plasma membranes showed that restrictions may exist in translocation. Although G protein of VSV was correctly inserted into membranes, movement from rough to smooth endoplasniic-reticulum was restricted.454Treatment of IgG1-synthesizing cells with 2-deoxy-~-clrabino-hexoseprevented the migration of this glycoprotein from rough to smooth membranes, but did not inhibit transfer of IgGl molecules from smooth membranes to the outside of the cells.455 (446) (447) (448) (449) (450) (451) (452) (453) (454) (455)

M. S. Bretscher and M. C. Raff, Nature, 258 (1975) 43-49. J. E . Rothman, F. N. Katz, and €3. F. Lodish, Cell, 15 (1978) 1447-1454. H. Garoff a n d R. T. Schwarz, Nature, 274 (1978) 487-490. D. F. Wirth, H. F. Lodish, and P. W. Robbins,]. Cell B i o l . , 81 (1979) 154-162. J. J. Plantner, L. Poncz, and E . L. Kean,Arch. Biochetn. Biophys., 201 (1980) 527532. M. Bielinska, G. A. Grant, and I . Boime,]. B i d . Chern., 253 (1978) 7117-7119. H.-D. Klenk, W. Wiillert, R. T. Schwarz, and R. Rott, I t a t . Congr. Virol., Madrid, S p a i n , 3rd, Abstr. W35. H.-D. Klenk, W. Wiillert, R. Rott, and C . Scholtissek, Virology, 57 (1974) 28-41. T. G. Morrison, C. 0. McQuain, and D. Simpson,]. Virol., 28 (1978) 368-374. F. Melchers, Biocheniistrtj, 12 (1973) 1471-1476.

These examples indicate that sugars also act as necessary modiilators, and help in establishing a conformation that is needed for meinbrane transport. Indeed, the abilit). of nonglycosylated G proteins of VSV to be transported intracellularly under certain conditions, and to fonn virus particles (as mentioned i n Section IV,2, o n confoilnation), is correlated with their physicocheniical properties. The influence of 2-deoxy-~-crrcihiiio-hexose or tunicamycin 011 the secretion of immunoglobulins has I,een widely studied. The reports are quite conflicting, but the discrepancies might be due to differences in the systems employed. For instance, a conformation that is required for secretion in one type of cell might not be suitable for secretion in another. The secretion of the nonglycosylated, light chain formed b y a mouse-myeloma tumor (46-B) in the presence of 2-deoxyD-umbitzo-hexose was not prevented.456The presence of residual, short sugar-chains on the protein appears unlikely, as these moleciiles fiinction as acceptors of oligosaccharides in an in citro system that requires nonglycosylated ~ - a s p a r a g i r i e .Tunicamycin ~~~ inhibited the secretion of IgM and IgA by 81 and 6470,respectively, whereas the inhibition of secretion of IgG w;1s45x,459 28%. The finding that secretion ofnonglycosylated, IgA alpha-chains from MOPC 315 cells was inhibited by tunicamycin, but that, after treatment of the cell surface with trypsin, nonglycosylated IgA receptors rea?peared, appears interesti t ~ g . ~In ~O other words, secretion of IgA is ,-revented,but not its insertion, and correct exposure, 011 the p l a s n ~membrane. These results are, however, challenged b y a report h y Williamson and c o w o r k e r F that concluded that secretion of the tionglycosylated forms of IgA and IgC was not significantly altered. If. is surprising that no inhibition of the secretion of nonglycosylated IgA relative to glycosylated IgA was observed, although, in this a cell line (315.40) was studied that is derived from the tumor examined by Hickniari and K ~ r n f e l dFurther . ~ ~ ~ investigations will be needed in order that the reported differences may be understood. At the present time, the resrilts of an investigation on the expression of IgM receptors, and disappearance of IgC receptors, of cultured human lymphocytes are also d cult to interpret. Under conditions (456) P. K. Eagon and E. C. Heath,J. Hiol. Chcin., 252 (1977) 2372-2383. (457) P. K. Eagon, A. F. Hsu, and E. C . Hrath, F e d . Proc., F c d Am. SOC. E x ) ) . B i o l . , 34 (1975) 678. (458) S. Hickman, A. Kulczycki, R. G . Lyiich, antl S. Kornfeld,J. B i d C h e m , 252 (1977) 4402-4408. (459) S. Hickman antl S. Komfeld,J. Zmniunol., 121 (1978) 990-996. (460) S. Hickman and Y. P. Wong, J . Z i r i t n t i i i o / . , 123 (1979) 389-395. (461) A. R. Williamson, H. H. Singer, P. A. Singer, a i d T. R. Mosman, Biochetri. SOC. Truns., 8 (1980) 168-170.

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that allow glycosylation of proteins, expression of IgM receptors is parallelled by a decrease of IgG receptor, due, in part, to shedding of receptor molecules from the cell. Inhibition of glycosylation by tunicaniycin inhibited the IgM receptor expression, and blocked the decrease of IgG receptor. The underlying mechanisms for regulation of both receptor types in opposite directions is unknown; glycosylation of protein appears, however, to be a common factor.462 Secretion of nonglycosylated macromolecules in the presence of tunicamycin has also been investigated in a number of other cells. Rat-liver cell-secretion of albumin (a carbohydrate-free protein), transferrin, and a-acid glycoprotein was not inhibited, and, in chickliver cells, only a decrease by 10-25% in the secretion of transferrin and the apoprotein B chain of very-low-density lipoprotein was noted.463,464 The secretion of ovalbumin (a glycoprotein) from hen oviduct was not blocked by t u n i c a m y ~ i n . ~ ~ ~ Glycosylation seems to be necessary for the secretion of the fourth component of complement (sS protein) from murine m a c r ~ p h a g e s . ~ ~ ~ T r e a t m e n F with gibberellic acid increased the secretion of alpha amylase [( 1-4)-a-glucan 4-glucanohydrolase, EC 3.2.1.11from the secretory tissue of barley grains, and led to an increased capacity ofthe crude membrane-fractions to transfer GlcNAc-P, GlcNAc, and Man from the iiucleotide esters of sugars to dolichol monophosphate, which may be needed for the increased rate of production of alpha amylase. Tunicamycin inhibited the secretion of the enzyme, but did not lead to an accumulation thereof'. Although it is possible that nonglycosylated alpha amylase is degraded more rapidly than the native enzyme, it is probable that prevention of glycosylation inhibits the de nooo synthesis of the glycoproteins, as had previously been demonstrated468*469 for synthesis of carboxypeptidase Y. These few examples support the notion that glycosylation is not, intrinsically, a mandatory requirement for secretion of glycoprotein, but that it may, in some instances, be an auxiliary modification. In this connection, a study of de (462) K. Itoh and K. Kumagai,]. I7nmutiol., 124 (1980) 1830-1836. (463) K. Edwards, M. Nagashima, H. Dryburgh, A. Wykes, and G. Schreiber, FEBS Lett., 100 (1979) 269-272. (464) D. K. Struck, P. B. Siuta, M.D. Lane, a n d W. J. LennarzJ. Biol. Cherri., 253 (1978) 5332-5337. (465) R. K. Keller and G. D. Swank, Biochem. Biophys. Res. Corrimun., 85 (1978) 762768. (466) M. H. Roos, D. C. Shreffler, a n d S. Kornfeld,J. Immunol., 125 (1980) 1869-1871. (467) H. Schwaiger and W. Tanner, Eur. J . Biochem., 102 (1979) 375-381. (468) A. Hasilik and W. Tanner, Antiniicrob. Agents Chernother., 10 (1976) 402-410. (469) A. Hasilik and W. Tanner, Eur. J. Biochem., 91 (1978) 567-575.

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o bio s y n the s i s of thy ro i (I- s t i I iII 11at i 11 g ho 1111o 11e (TS H ) is i i i te re s ting; it showed that preventioii ot'glycosylatioii of the a- ant1 the p-subunit led to the secretion of iiii a-sut,uiiit having decreased molecular weight, the coinhination of which with the p-subunit was inhibited. It had been suggested that the p-siil)tiiiit is limiting i i i TSH hiosynthesis, and that glycosylation may l i r l revluired for the combinatioii of the a - and p-subunit, but not for secwtion.""' ,oiic.ct - - , . routing is likely to pla!- a significant pait i n directing l!.so~' aclvanced b y Hicksoma1 enzymes into the l y s o s o i i ~ c ~A. ~hypothesis man and N e ~ f e l dsuggested ~~~ tliat the lysosoiiial e n z y m e s are secreted to the exterior of the coll, and bind to receptors iii the cell stirface b y way of their specifics, rt.cvgiiition m a r k e r ( a carl)ohytlrate structure), an event that ini t i;itc' s t' 11 docy tos i s, an (1 fo 1 x 1 atio I I of pi no cytotic vacuoles containing tlic- I\.sosomal enzymes. Although the phenoiiieiioii of secretion and uptakcx of' Iysosomal enzymes is freqiieiitlv obseivecl, it has been proposed that packaging o f lysoiiial enzymc~sis an intracellular process, which iiiiplicxs that this so-called "secretion re capture 11 y p othe s i s 111u s t lie I 11o t l i fie(1. How e vc>r , ;I iiio s t i inpo rtan t characteristic is not changed: t h y Ijw)somal enzynics are synthesized as precursors, having oligosaccli~iridechains that contain D-inaniiose 6-phosphate masked by a 2-~~cctairiitlo-2-tleoxIv-D-glucosyl After removal of the 2-acetamido-2-~leoxy-~-~lucosyl group, the 1111masked hexose phosphate scrv('s ;is it recognitioir marker, and biiiding ofthe recognition marker is r c y r i i r e t l for the packaging of the enz!mies in primary l y s o s o ~ n e s . ~Thus, " ~ ~ ~it~has ~ *been ~ ~ ~proposecl tliat a hexose phosphate acts as a signal for iiitracellular segregation of acid hydrolases from secretory glycoproteiiis destined for export.4i5 It is clear, then, that lack of the carbohvdrate-recognitioii niarker may lead to inislocations of lysosomal enz).iiies, and this has, for instance, been suggested as being the reasoii for the stop in programnied cell-death in secondary palate-foniiation (see Section IV,6). I 1 GO

c

"

(470) B. D. Weintraub, B. S. S t a n l i d , I ) . L,innekin, arid 11. l\larchall, / . R i o l . ( : ~ ~ I J I , 255 (1980)5715-5723. Tnger, I. P. G . de Groot, \l, U. II;uners, M.Holleinans, li. K ~ i l s l w e k A. , Strij(471) J. & land, antl F. P. W. Tegclaers, i n W. 1'.Daems, E. H. Burger, and B. A . Afelirl\ (Etls.), Cell Biological A . s p t ~ ~ tosf Ilisc,cisc: The P/tr.sinci .\l(~i~ihraiic,~ L I L!/soI ~ y o i i i e . ~ Vol. , 19, Leiden Univel-sit? PI-C.SS, The Hague, 1980, pp. 235-250. (472) S. Hickman and E. F. Neufeltl, H i o c , / t c , i t i . B i o p h / s . Rra. C ~ J ~ J I J 49 J I (1972) I ~ I I . ,992999. (473) I. Tahas and S. Kornfeld,J. Hiill. ( ; / i o i i i . , 255 (1980)6633-6639. (474) A. Hasilik, U.Kleiii, A. Waheed, <;. Stl-eckcr, and K. von Figur't, Pro(.. Ricitl. Actid. Sci. I/. S.A , , 77 (1980)7074-7078. (475) W. S. Sly, i n L. Svennerholnr, P. \ l , i l ~ ( l t ~ l lH. , Drt,yfilss. arid P.-F. Urban (Ed\.), Structure arid Function of f h c ( h ~ i g / i o . s i d ePlenum, , New York, 1980.

,

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RALPH T. SCHWARZ AND HOELF UATEMA

In cultures of confluent, human fibroblasts, 2-deoxy-~-arubino-hexose prevents the increase in the activity of lysosoinal enzymes that norinally occurs when the culture becomes confluent. This effect is probably caused b y inhibition of glycosylatioii, leading to a feedback inhibition of protein synthesis, or to a rapid breakdown of unglycosylated protein^."^ On the other hand, tunicamycin has been shown to have a biphasic effect on the secretion of lysosonial 0-N-acetylglucosaminidase, which was, at first, almost completely inhibited, and thereafter, increased more than fivefold over controls. Treatment with tunicamycin led to the foi-niation and secretion of lysosoinal enzymes deficient in the recognition marker, and the inhibition of expression of receptors for lysosoinal-enzyiiie uptake. This, in turn, resulted in a failure to internalize lysosonial enzyme. It was a s s ~ i i i i e dthat ~ ~ the ~ increased secretion is due to interference with the formation of the recognition marker on the enzyme. Uptake of low-density lipoprotein has been a s a prototype of a receptor-mediated pathway for internalization of external inacromolecules. It is a coupled process b y which selected, extracelliilar proteins or peptides are first bound to specific, cell-surface receptors, and then rapidly iriteriialized h y the cell. Internalization follows clustering of receptors in specialized regions of the cell surface, called “coated pits,” that invaginate, to form coated vesicles. Incubation of fibroblasts with tunicamycin resulted in a dose-dependent inhibition of binding, and internalization of low-density lipoprotein. Upon removal of the inhibiting medium, this effect is reversible within 3 days. It was hypothesized that the expression of LDL receptors on the surfice of fibroblasts is dependent on intact N-glycosylation,4x0which iiiay be necessary for proper orientation and function of LDL-receptors on the fibroblast cell-surfiace.

5. Effects on Collagen and Proteoglycans Collagen, the principal protein of connective tissue, is synthesized in the form of a precursor (procollagen) having amino- and carboxylterminal regions that are removed during conversion of procollagen into collagen. It is probable that more than one enzymic activity is involved in processing. Procollagen contains carbohydrate inoieties, (476) P. G. dr Groot, A. Strijlantl, H. Kalslwek, P. lleera Khan, A. Westerveld, 41. Hmiiers, and J. W . Tager, E x p . Ccll R r s . , 126 (1980) 207-216. (477) K. voti Figura, hl. H e y , H. Pritrz, B . Voss, a t r d K. Ullrich, E u r . / . B i [ ~ c h e i r ~101 ., (1979) 103-109. (479) J. L. Coldstein, R . C . W. Anderson, and k l . S . Browir,Notnrc,, 279 (1979)679-685. (480)I. Filipovic and K. v o Figiira, ~ Biochmti. /,, 186 (1980) 373-375.

some, at least, of which have I)c-en shown to be situated in the carImxyl-terminal, non-triple-helical tlomain. The fiinction of the carlmhydrate in this domain is not yet understood, hut it has been suggested that it could be important for secretion and for the regulation of the conversion of procollagen into collagen. These procollagen a p pendages appear to resemble most glycoproteins, because they contain D-niannose, 2-amino-2-deox\i.-1>-glllcose,a r i d 2-aiiiiiio-2-deox~-~~galactose, whereas these inonosaccharic~esare not present in the collagenous regions. It was claimed that tunicaiiiycin tiiarkedly impaired secretion of all macromolecules of chickeii-einl)r!.o cells, but did not exert any specific effect on the secretion of procollagen; the iiitracellular content of procollagen polypeptides was iiiic*hanged in the presence of tunica~iiycin.~ However, ~' reinvestigation of the effects of tunicamycin on the biosynthesis of procollagen also showed a retardation of its seci-etion from huinan fibroblasts.'l"21 1 1 another study, however, procollagen synthesized in the presence of tunicainycin was found to be secreted nonnally, but the incorporation of 1)-[2-:'H]mannose into procollagen was inhibited b y over 90%. Although the immiinological properties, as detected by an antiserum to t h e intact protein, were unchanged, a biosynthetic intennediate cont:kining c~isulfide-bontled,carboxyl-terin inal e xte n s ion s was foun (1; c 1c,avage of am i n o-te rni i n a1 e x te n s ions was not detectably impaired. The possible explanation for t h v inhibition of processing of procollagen b y tunicaniycin is that the carboxyl-tenninal, procollagen pcptidase is a glycoprotein, and that inctlia from cells cultured with tuiiicamycin lack this procollagen - protease activity. This hypothesis receives support from the finding that u~ic~erglycosylated procollagen fomied in the presence of tunicamycin was nornially processed b y protease from the culture niediiim of chick-tendon Because concanavalin A also inhibited the conversion of procollagen into collagen by carboxyl-tc~rmiliralprotease, it was suggested that this protease contains oligosaccharide side-chains that are recognized b y concanavalin A, and that tiinicaniycin affects the secretion, activity, or activation of this enzyme?

(481) bf. L. Tanzer, F.N . Rowlaird, L. M'. bfrrrray, and J . I
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RALPH T . SCHWAHZ A N D ROELF DATEMA

Turiicamycin can affect the synthesis of acid glycosaiiiinoglycans. Incorporation into total glycosaminoglycans of 2-amino-2-deoxy-~['4C]glucose and H,"5SS04was inhibited, but individual classes of glycosaminoglycans were not analyzed in this i n v e ~ t i g a t i o nThe .~~~ biosynthesis of corneal keratan sulfate, which, in contrast to skeletal keratan sulfites, contains N-glycosylic linkages to protein, is inhibited by tunicamycin, but the biosynthesis of hyaluronic acid is not affected. Also, the synthesis of sulfated proteoglycaris in chondrocytes was not impaired, and the partial inhibition of the foimation of chondroitin sulfiate and heparan sulfate is possibly due to secondary effects of tunicamycin on protein

6. Effects on Differentiation Inhibitors of glycosylatiori have been used as tools in the study of the development of a variety of organisms and organs, such as cornea, palatal epithelial-cells, mouse embryos, sea urchin, Tetrahymena, and yeast, in order to investigate the possible involvement of glycoproteins in differentiation. Although only a part of the reports that have been published on this matter deal with experiments at the molecular level, they provided supplementary evidence that glycoproteins do, indeed, participate in certain steps of development. The morphogenesis of the sea-urchin egg has been studied b y embryologists for a long time. In an early study, it was reported that, when applied in high concentrations ( 10-2-0.5 M ) , 2-deoxy-D-urubino-hexose interfered with cleavage of the egg and with oxygen consumption. Low concentrations ( 1 0 ~ ~ - M ) prevented eggs from gastrulating, without interfering with the early steps of development. at high concentraWhereas the effects of 2-deoxy-~-urabino-hexose tions are due to competition with the metabolism of D-glucose and interference with the consumption of oxygen, the effect of low doses can, in the light of present knowledge, be explained b y an inhibition of g l y ~ o s y l a t i o nThis . ~ ~ ~is in agreement with the data obtained with tunicamycin, which show that it specifically inhibits gastrulation, but does not prevent f e r t i l i ~ a t i o n . ~ ~ " Also, it has been found489that the presence of the drug has no detectable effect on the morphogenetic changes in the development of the embryo from the 16-cell stage to the early gastrula. It is at the gas(485) A. Takatsuki, Y. Fukui, and G. Tamura, Agric. Biol. Chem., 41 (1977) 425-427. (486) R. M. Pratt, K. M. Yamada, K. Olden, S. H. Ohanian, and V. C. Hascall, Exp. Cell Res., 118 (1979) 245-252. (487) G. S. Bernstein and R. E. Black, Proc. SOC.E r p . B i d . Med., 102 (1959) 531-534. (488) R. Lallier, C. R. Acad. Sci., Ser. D , 287 (1978) 543-546. (489) E. G. Schneider, H . T. Nguyen, and W. J . Lennarz,]. B i d . Chern., 253 (1978) 2348-2355.

THE LIPID PATHWAY OF PHOTEIN GLYCOSYLATION

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trula stage, when extensive, morphogenetic movements of mesenchyma1 cells begin, that an effect of prior treatment with tunicamycin becomes apparent. Treatment of embryos after gastrulation, or later, results in the arrest (or retardation) of spicule development and arm growth. Thus, it was suggested4H9 that the synthesis of N-glycosylically linked oligosaccharides of glycoproteins relevant for morphogenesis does not occur in embryos prior to the initiation of gastrulation. Similar, but reversible, effects were o l ) s e r ~ e dwhen ~ ~ ~ compactin (see Section II1,B) was used instead of tunicarnycin. New surface-glycoproteins that may be essential for nornial cell-movements and interactions at gastrulation contain unsulfated, and sulfated, N-glycosylically linked oligosaccharides, and t h i s is in keeping with a report that gastrulation does not occur i n sulfate-free ~ea-water.~"' Early cleavage divisions, aggregation of mouse embryos, and the initial phase of compaction are riot affected b y tunicaniycin, and this bears some resemblance to the action of this drug on early steps of the development ofthe sea urchin. 'The later stages of compaction ofblastomers, as well as the adhesion a n d outgrowth of trophectodenn cells of blastocysts, are, however, prevented by tunicamycin, and this is p a ~ - a l l e l l e by d ~a~decrease ~ ~ ~ ~ ~in the binding of radiolabelled concanavalin A. Therefore, membrane-1)ound glycoproteins having N-glycosylically linked oligosaccharides evidently play a role during compaction and trophoblast adhesion. Cell interactions leading to kidney-tubule detenination are tunica' mycin-sensitive. When tunicamycin was applied at concentrat ions that prevent induction of differentiation, cells did not contain laminin, a glycoprotein that is detected i n early stages of differentiation. As tunicamycin did not prevent differentiation when applied later during the inorphogenetic period, the authors considered it unlikely that the drug interferes with tubule formation by inhibiting the secretion of laminin.49"494 Tunicamycin specifically inhibits cell division and pairing between the mating types of Tetrahymotia pyrilfomnis. The glycoproteins involved in this mating process have not y e t been characterized, but may coincide with new, concanavalin A receptor-sites that appear during conjugation.49s (490) A. Heifetz and W. J . Lennarz,J. B i d Chern., 254 (1979) 6119-6127. (491) M . A. H. Suratii, Cell, 18 (1979) 217-227. (492) M . I. Sherman, Annu. Reu. B i o c l w m . , 48 (1979) 443-470. (493) P. Ekblom, S.Nordling, L. Saxbn, M . L. Rasilo, and 0.Reukoneii, Cell Iliffer., 8 (1979) 347-352. (494) P. Ekblom, K. Alitalo, A. Vaheri, R. Timpl, and L. Saxbn, P m c . N a t l . Acnrl. Sci. U . S.A., 77 (1980) 485-489. (495) A. Frisch, A. Levkowitz, a n d A. Loyter, Riochem. B i o p h y s . Res. Comn~zrn.,72 (1977) 138- 145.

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RALPH T. SCHWAHZ A N D ROELF DATEMA

Sacchuromyces cerevisiae, mating type, a-cells enlarge and elongate when exposed to a-factor, a sex pheromone produced by mating a-cells. The extensive synthesis of the cell wall is accompanied by alterations in its gross and fine structure. The wall becomes more susceptible to digestion b y glucanases, and the mannan contained an increased proportion of shorter side-chains and unsubstituted, backbone D-mannOSyl residues. Morphogenesis was blocked by such inhibitors of cell-wall biosynthesis as 2-deoxy-~-arabino-hexose, 2deoxy-2-fluoro-D-g~ucose,and 2-deoxy-2-fluoro-D-mannose, but noVs6 by polyoxin D, and an inhibitor of synthesis of chitin. The mechanism of inhibition has not yet been studied in detail, but it is probably similar to the mechanisms proposed in Sections 111,3,a,(ii) and (iii). Cellular death is a common occiirrence throughout embryonic development, despite pronounced proliferation and rapid, embryonic growth. This phenomenon norinally affects specific cell-populations, leaving adjacent, cellular areas intact. The tenn “programmed” implies that those cells are influenced at a precise, embryonic stage, well before death. Programmed cell-death has not yet been extensively studied in mammalian, embryonic development, with the exception of epithelial cell-death occurring during secondary-palate fonnation. Apart from inhibiting adhesion of medial-edge, epithelial cells, 6diazo-5-oxo-~-norleucine(DON) (see Section III,5) prevented programmed cell-death and autolysis of midline epithelium. This effect is reverted b y addition of 2-amino-2-deoxy-D-glucose, the synthesis of which is prevented by this L-glutamine analog. Degradation in programmed cell-death is most probably mediated by lysosomal enzymes. No significant differences in the activity of lysosomal enzymes in DON-treated and control cells appeared, but their intracellular 10calization was different. In DON-treated epithelia, lysosomal acid phosphatase (orthophosphoric monoester phosphate hydrolase, EC 3.1.3.2), for instance, is restricted to lysosomes, whereas, in the control, activity is found throughout the cytoplasm. Thus, DON appears to inhibit the availability of lysosoinal enzymes to intracellular coinponents. Inhibition of glycos ylation of the lysosomal enzymes, or membranes, may prevent release from the lysosomes. Defective glycosylation is also likely to interfere with the correct “routing” of the lysosomal e n ~ y m e s ~(see ~ ~Section , ~ ~ IV,4). ~ , ~ ~ ~ (496) P. N. Lipke, A. Taylor, and C. E. Ballou, ]. Bacteriol., 127 (1976) 610-618. (497) R. M . Pratt and R. M. Greene, in D. Neuhert and J. Merker (Eds.), New Approaches to the Evaluation of Abnormal Embryonic Developrnents, Thietne Verlag, Stuttgart, 1975. (498) R . M. Greene and R. M . Pratt,]. Histochern. Cytochem., 26 (1978) 1109-1114.

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Inhibition of glycosylation was shown to interfere with certain stages of development, as shown i n the previous examples. However, in cultured, human and myeloid leiikeinia cells, tunicamycin treatment induced differentiation. Froin this observation, it has been concluded that glycosylation of cellular proteins plays a role in maintaining these cells in a transformed state."""

7. Effects on Viruses In preceding Sections, nonglycosylated glycoproteins of viruses have frequently been mentioned i n a variety of contexts. Although no fundamental differences have thus far been found between viral and nonviral glycoproteins regarding their biosynthesis and intracellular transport, viral glycoproteins participate in the formation of complex structures, such as viral envelopes, and possess functions that are relevant in the spreading ofthe disease caused by the virus. For instance, heinagglutination activity displayed b y the influenza viral hemagglutinin mirrors the potential of this glycoprotein to interact with cell surfaces and to mediate absorption and penetration of virus. Other viruscoded proteins are involved in virus-induced, cell -cell fusion, a s , for instance, the fusion factor F of parainfluenza viruses, which is also a constituent of the viral envelope. Because of these special roles, viral glycoproteins merit special discussion. Inhibition of glycosylation i n virus-infected cells usually has dramatic effects on virus multiplication. This discovery prompted promulgation of a new concept in the experimental therapy of virus-induced diseases. Local treatment of the affected regions with 2-deoxy-D-urubino-hexose1ed50"-502 to significant improvements in human-genital herpes infections, or Herpes simplex virus infection of the eye. The diminution in infectivity could be caused either by the production of virus particles having a lessened or abolished infectivity, or a decreased formation of infectious virions. The envelope glycoproteins of Semliki Forest virus and Sindbis virus synthesized in the presence of tunicamycin (and, thus, devoid of carbohydrate) are metabolically stable. They do not participate i n the assembly of virus particles, although nucleocapsids are still fonned under these conditions and are (499) M. Nakayasu, M. Terada, G . Tairrnra, and T. Sugimura, Proc. N u t / . Acutl. Sci. U . S. A,, 77 (1980) 409-413. (500) J. Bettner, Bu.Pine.ss Week, (July 30, 1979) 79-80. (501) E. K. Ray, D. B. Levitan, B. L. Halpern, and H. A. Blough, Lancet, (Sept.21, 1974) 680-683. (502) H. A. Blough and R. L. Giuntoli,j. A I I I M . ed . Assoc., 241 (1979) 2798-2801.

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visible, under the electron microscope, as pseudo-microcrystalline arrays.503On the other hand, proteolytic degradation, in the presence of tunicamycin, of hemagglutinin in cells infected with fowl-plague virus is possibly the reason for the lack of virion formation; in contrast, MDBK cells infected with another strain of influenza virus (A/WSN) can produce virus particles devoid of hemagglutinin. It is conceivable that HA,, the nonglycosylated hemagglutinin protein, which is associated with smooth membranes and the plasma membrane, may be essential for the budding process, although it may subsequently be degraded after release of virus from the infected cells. These virions do not hemagglutinate, as is also true for 2-deoxy-D-urubino-hexosetreated cells infected with influenza virus. In the presence of tunicaniycin, cells infected with ROW sarcoma virus produced virus particles that lacked infectivity, and were devoid of the envelope glycoproteins gp85 and gp35. Virus particles lacking envelope glycoproteins had previously been found in a deletion niutant of Rous sarcoma virus and in a temperature-sensitive mutant of VSV.504-507 Herpes virus particles having a decreased infectivity were produced from cells fed with 2-deoxy-Darubino-hexose, and were shown508to contain altered glycoproteins. A similar result has been obtained b y using tunicaniycin. The morphology of the noninfectious virions is similar to that of intact virus particles, as judged from electron-microscope studies.509 In another study, viral polypeptides having a diminished molecular weight have also been detected.51" Lack of particle formation in the latter investigation may be due to the use of a different host-cell. Sub-inhibitory doses of 2-deoxy-D-urubino-hexose ledIo5to the formation of influenza virus particles that had a decreased stability. The viral glycoproteins lacked complete, oligosaccharide side-chains and did not possess shorter ones. The implications for the mechanisms of inhibition of glycoprotein formation mediated b y 2-deoxy-~-uruhino-hexosewas discussed in Section III,3. Vesicular-stomatitis virus (VSV) is synthesized in the presence of tuni(503) H. Ogura, M. F. G. Schmidt, and R. T. Schwarz,Arch. Virol., 55 (1977) 155-159. (504) C. M. Scheele and H. Hanafusa, Virology, 45 (1971) 401-410. (505) H. Ogura and R. R. Friis,]. Vim/., 16 (1975) 443-446. (506) M. S. Halpern, D. P. Bolognesi, and R. R. Friis,]. Viro/., 18 (1976) 504-510. (507) T. J. Schnitzer and H. F. Lodish,]. Virol., 29 (1979) 443-447. (508) R. J . Courtney, S. M. Steiner, aiid M. Benyesh-Melnick. Virology, 52 (1973) 447455. (509) E. Katz, E. Margalith, and D. Duksin, Antiinicrob. Agents Chemother., 17 (1980) 1014-1022. (510) L. I. Pizer, G . H. Cohen, and R. J . Eisenberg,]. Virol.,34 (1980) 142-153.

THE LIPID PATHWAY OF P R O T E I N G1,YCOSYLATION

:37 1

camycin ifthe temperature is lowered to 30"; this is due to the fact that the lowering in temperature is ii siil,stitute4" in niaintaining a conformation that allows transport of' the C: protein into the plasma i i i e i i r brane (see Section IV,2), and thiis ena1)les prodiiction of irrfectious virus. Tunicaniycin and other in1ril)itors of glycosylation will unt1oul)ttdly be increasingly used a s adtlitioiial tools i n investigations on precursor -product relationships of viral proteins, i n o r d e r to distingiiish between glycosylated and nonglycos!.lated forins; thus, iuhibition of glycosylation m a y become part of tlre standard procedures i n virus research. Using tunicamycin, siippleinentary evidence h a s been obtained that a protein of niol. wt. r)O,OOO that is found i n cells infected with iiiurine leukemia virus is iiot ii precursor of 1165 (\vliich is not a glycoprotein), froin which, b y lititlier processing, internal, striictural proteins of the virion are dcri\ic.tl. After being fiirther gly-cosylattd, this molecule, now having a i r ~ o l c c ~ i lweight ~ir of95,000, is cleaved instead, to yield glycoproteins haL-iiig iirolecular weights of 55,000 and 40,000 that are re1eased"l into tlic. cell-culture fluid. Cells infected with ROLISsarcoiii;i virus that were niaintained in medium containing 2-ainino-2-cleox>-I+gliicose produced a irovel protein"I2 that has a molecular weight of 70,000; it may be related to the envelope glycoprotein gp85. Botli envelope glycoproteins gp8Fj and gp37 are derived b y proteolytic c*leavagefroin a glycoprotein precut-sor of mol. wt. 92,000. In the presence of tunicaniyciti, this precursor has not been fbund, but, instead, novel proteins having inolccular weights of 62,000 (p62) and 57,000 (pS7), respclctively, are forined; these are nonglycosylated foriirs of the glycoprotein. Discrepancies regarding the molecular weight mid the metabolic stability ofp62 and p57 may be due to the different srtl)groups of Hous sarcoiira virus used in the investigatioiis."~,~l~ Iiitcrestirigly, material having a inolcnilar weight of58,000 (in analogy to tliat of inolecular weight 57,000 formed in the presence oftunicamycin), fLrnretl in the prcsence of 2 - d e 0 ~ y - ~ a ra bi I I o -he x o se , shows an in c r c w e i t i ni ol e c u1iir we i glit up to 75,000 during a 2-h chase in the al)setic.c of the inhibitor, prol)al)l>.b y suhsequent addition of glycosyl grorips to tlie peptitle. The fact that earlier investigations of the effect of tuiiicam~.cinon the replication of ROW sarcotiia \ririis fiiiled to show the unglycosyl-

(511) S. A. Etlwartls aird H . F'an,,/. \'i)-ol,, 30 (1979) 551-,563. (512) L. J. Levandowski, R. E. Snlitlt, 1). 1'. Hologtresi, and \1. S. Halpc'rii. V i r o l o g ! / , 66 (1975)347-355. (513) H . IXggelmaiiir,]. Virol., 30 (1879) 799-804. (514) R. Stohrer atid E. Hunter,/. \'in)/., 32 (1979) 412-419.

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‘r. SCHWARZ

AND ROELF DATEMA

ated precursors is possibly due to the inability of the employed antiserum to recognize antigens lacking the ~ a r b o h y d r a t e . ~ ~ ’ The use of inhibitors of glycosylation also allows conclusions to be drawn as to whether glycoproteins or nonglycosylated proteins are involved in virus-induced, cell-cell fusion. This might become particularly helpful if the fusion proteins have not been characterized by other means. For example, 2-deoxy-~-nrabino-hexose blocks cell-cell fusion induced by Herpes virus, which is in accordance with a glycoprotein nature for the fusion f a ~ t o r . ~ However, ~ ~ - ” ~ cell-cell fusion of monolayers proceeded (to an extent indistinguishable from that of control cultures) in the presence of concentrations of 2-deoxy-u-umbino-hexose and 2-amino-2-deoxy-D-glucose that abolish vaccinia virus (a pox virus) specific hemagglutination linked to a vaccinia-specific glycoprotein. Hence, the factor involved in fusion in this example may not be a glycoprotein.5’RNewcastle disease virus is another virus that induces cell fusion. Administration of tunicarnycin or 2-deoxy-Duruhino-hexose to infected cells block^^^^-"^ glycosylation of the hemagglutinin-neurainirlidase coinplex HN aiid fusion factor F, and this delays the fusion. A reinarkable diminution in the protein biosynthesis of viral proteins has been observed with Newcastle disease virus in the presence of the inhibitor, an effect so far not seen with other v i r u ~ e s . A ~~ reg~~“~ ulatory link between glycosylation arid synthesis of protein may thered ~ ~other ~ ~ ~systems. ~~ fore exist, as has been s i ~ g g e s t e with An important observation is that certain viruses preferentially bud at different poles of their host cells. In MDCK-cell monolayers, VSV buds exclusively from the basal, or lateral, plasma membranes, and contains sialylated glycoproteins, whereas influenza virus buds exclus ive 1y from the apical plasm a-memb rane , an cl lacks 11euram inic acid . The question arises as to whether glycosylation of viral glycoproteins is needed in order to determine the site of budding. An electron-microscope study revealed that polarity in the maturation sites of these viruses was maintained under conditions of inhibition of glycosyla(515) H. Ludwig, €I. Becht, and R. Rott,]. V i d . , 14 (1974) 307-314. (516) H. Ludwig and R. Rott,]. Virol., 16 (1975) 217-221. (517) R. W. Knowles and S. Persoi1,J. Virol., 18 (1976) 644-651. (518) S. Weintrauh, W. Stem, and S. Dales, Virology, 78 (1977) 315-322. (519) K. Bortfeltl, Ph. D. Dissertation, Giesscn, 1974. (520) H.-D. Klenk, K. Bortfeld, M. F. G . Schlnitlt, aiicl R. T. Schwarz, unpublished ohstxrvation. (521) T. G. Morrison and 11. Siiiipsoii,Al,str-. I t i f , Corigr. Virol.,4 t h , The Hague, (1980) Ahti-. 204.

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tion by tunicamycin. Thus, proper glycosylation is not needed for the determination of the cellular-maturation site of these v i r u s e ~ . ~ ~ ~ , ~ ~ ~ Side effects of inhibitors of glycosylation have also been recognized as contributing to interference with virus multiplication. As already discussed (see Section 111,3), the mechanism of action of the inhibition of glycosylation brought about b y 2-amino-2-deoxy-D-glucose is not yet fully understood, and it may probably be caused by alterations of the status of intracellular membranes. It is, therefore, not surprising that membrane-associated events (apart from glycosylation) might also be affected. In the presence of 2-amino-2-deoxy-~-glucose,cells infected with avian sarcoma virus produce virus particles that lack gp37 and gp85, due to defective glycosylation. The dominant effect of the amino sugar, which accounts for the 50-fold diminution in number of particles is, however, due to a combination of inhibition of protein biosynthesis and prevention o f t h e cleavage of the polypeptide precursor p76 to yield p19, p27, p12, and p15, which presumably occurs in close connection with the plasma ~ e m b r a n e .Agents ~ ~ ~ that , ~ ~dis~ rupt membranes are known to inhibit this ~ l e a v a g e . ~ *Also, " ~ ~ 2~ amino-2-deoxy-D-glucose inhibits the multiplication of poliomyelitis virus (a non-enveloped virus) in Vero cells. The impaired conversion detected in vitro of 15 S polio particles into 70 S particles (one of a series of steps in virus maturation that is facilitated by addition of membranes398)may be one of' the causes for formation of defective virus in vivo. It is not unreasonable to assume that 2-amino-2-deoxy-~glucose impairs the assembly of mature virions due to its membrane on mRNA synthesis activity.528Effects of 2-amino-2-t~eoxy-D-g~ucose were discussed in Section III,3.

8. Effects on Interferon Information on the significance of carbohydrate chains of interferon, the protective agent against a number of virus diseases, has been ob(522) M . C . Roth, J . P. Fitzpatrick, and R. W. Compans, Proc. N u t l . Acud. Sci. U . S. A , , 76 (1979) 6430-6434. (523) E. R. Boulan and M. Pendergast, Cell, 20 (1980) 45-54. (524) E. Hunter, R. R. Friis, and P. K. Vogt, Virology, 58 (1974) 449-456. (525) M. J. Hayman, E. Hunter, and P. K. Vogt, Virolog!y, 71 (1976)402-411. (526) M. J. Hayman, Virology, 85 (1978) 475-486. (527) V. M. Vogt, R. Eisenmann, and H. Diggelmann,]. R l o l . B i d . , 96 (1975) 471-493. (528) R. A. Delgadillo, D. A. van Den Berghe, and S. R. Pattyn, Abstr. I n t . Corigr. Virol., 4th, The Hague, (1978) Abstr. 321.

074

KALPH T. SCHWARZ .4NU I-IOELF DATEMA

taiiied b y using inhibitors of glycosylation during its s y ~ i t h e s i s . ~ ~ ~ ~ ~

The results thus far obtained are in good harmony with those repoited for gly cop ro te i ii s froni ot he r source s . Non g1y co s y 1ate d i i i t e rferoii retained its antiviral activity, but showed a decreased thermal stability and decreased affinity for antibodies directed against its fiilly glycosylated These effects niay be explained in terms of alte red coil form.d t 1011. There also seeiued to be no requirelnent for glycosylation of interferon for secretion, although a partial involvement cannot be strictly excluded. Interferon fomied i n thc presence of glycosylatioii inhibitors showed 1e s s charge -het e ro ge 11 e it y , w 11ich is cause (1 b y N-ace t y 1neuraininic acid in the iiative molecule I n earlier iiivestigations, it had been r e c ~ g i i i z e d ~ : ' ~ that , " ~interferon retains full biological activity after reiiioval of sialic acid, or after removal of 50% of the total carbohydrate."i2 However, its apparent hydrophobicity, and affiiiit). for poly(riboniicleotides), are conferred only when glycosylation is ~ n i i i i p a i r e d . " ~ ~ '

9. EfYects on Other Cellular Phenomena Many aspects of the social behavior of cells are detemiined b y the coni po si t i or i , arrange 111 e 11t , and i 11 t e rac t i oI i of ce 11-surface 111 o 1ecul e s . Therefore, changes in the composition ant1 structure of plasma m e m branes appear to contribute to differences in such characteristics as cell adhesion, contact inhibition, antl tumorogenicity of cells. Cellsurface glycoproteins, in particular, participate in a n u m l ~ e rof membrane-modulated phenomena, including responsiveness to honnones, agglutination b y lectins, recognition b y antibotlies, or uptake of iiutri-

(529) K. C. Chatla, P. M .Grab, H. L. Haiiiill, a t i d E. Siilkowski,Arch. V i r o l . , 64 (1980) 109- 117. , (1977) (530) v. G. Ed?., 1. Llesmyter, A. Billian, at1d P. 1k S O I I I I~J ~l f C, J C f . ~ J t t J ~ t U J I .16 445-448. (531) J. Fiijisawa, Y. Iwakura, and Y. Kawatlc,J. Riol. Chetn., 253 (1978) 8677-8679. (532) E. A. Havell, J . V i l h k , E . Falcoff, and 8.Bermarur, Virolog!/, 63 (1975)475-483. (533) E. A. Havell, S. Yamazaki, and J . VilFek,J. Biol. Chettt., 252 (1977) 4425-4427. (534) W.E. Stewartl, M.Wiranowska-Stewartl, V. Koistiireri, aittl K. Cantell, Virology, 97 (1979) 473-476. (535) F. Domer, M . Scrilia. antl R. Weil, Proc. Not/. Accid. Sci. IT. S. A , , 70 (197.3)19811985. (536) E. Schoiine, A. Billiati, and P. De Soiner, i r i F. T. Perkins and K. H. Regatnay (Eds.), S!yni)i. Stciiitl~irrlizntioJi I i i f c r f i w i , i I?ifrv-feroit Int/ucer.v, Kii-ger, Basel, 1970, pp. 61-68. (537) A. Mizrahi, J. A. O'Malley, W. A. Carter, A. Takatsiiki, G . Tamura, and E. Sulk0wski.J. B i d . Chertz., 253 (1978) 7612-7615.

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ents. Extensive studies have beeii iiiade of cell-surface glycoproteins and of alterations that occur aftvr transformation. A prominent finding in traiistormecl cells has been the absence, or marked lessening of the content, of' a high-molecular-weiglit, cell-surface glycoproteiii known as LETS protein, or fibronectin, that is involved in the attachment of tlie cells to the sulxtratuni (for a review7, see Hynes and coworkers5"). Cultured BHK cells that had Iwen inaintainetl in a niediuin containing tunicainycin still shed varioiis niembrane gl>wq)roteins into tlie inedium,539and this is in keeping with observations mentioned in Section IV,4. However, the proportioii of fibronectin found in the medium appeared to be lessencd.54-"'~5~*~ Cells kept i n the presence of tunicamycin showed profound 1-iiorpliologiealchanges, from epitheloid to elongated, spindle-shaped niorpliology, and lowered adhesion to the s u h stratum. Although both nomial, and SV-40- o r polyorna-\.irus-trarisforiiie(l, 3T3 cells are inhibited in growth, pronounced cytotoxicity of tunicaniycin was demontrated only with the transformed cell sensitivity to tiinicamycin, foiintl i n another study o formed, C3H-2K mouse-cells, slipported this observation."2 The selective cytotoxicity of tunicaniyciii for transfornietl cells may indicate that tunicamycin interferes with some cellular processes critical for the s urvi val o f man y t ran s form et 1 ce 11s 1)ut I 10 t of 11011 -t ran s forme d cells. It has been speculated that the cytotoxicity- of this drug towards transfornied cells may result froiii iinpairetl rates of nutrient trans(see also, later). 1nhil)itioii of agglutination of transfoniied cells with concanavalin A, but not with wheat-gem or soybean agglutinin, was detected as a consequence‘ o f the action of tiinicarnyciii on the cell c u l t ~ r eThe . ~ ~3T3 ~ cells respoiiclecl to incubation with a mecliuin containing tunicamycin b y extt,iisive ruffling, which is usually o n l y restricted to the leading edge of actively migrating cells. Electroii-iiiicroscope exainiiiiitioii did not, however, reveal changes i n the organization of the rnicrofilaineiits o r microtubules, but the. endoplasmic reticulum was dilatcvl i n a sac-like Decreased proportions of fibronectin were ol)served, both for control and virally transfomied cells. In cells that I i a d been exposed to tunicamycin, (538) R. 0. Hynes, A. T. Destree, h l . E . Perkins, and 11. 0. Wagner, / . Stcprclmol. Struct., 11 (1979) 95-104. (539) C . H. Damsky, A. Levy-Benshiiiii)l, (:. A Buck, ant1 L. n'arren, E1.p. C c , / / . Hcs., 119 (1979) 1-13. (540) D. Duksin and P. Bom\trin, Proc.. .Vtit/. Acutl. Sci. C i . S. A , , 74 (1977) 3433-3437. (541) D. Duksin, K. Holbrook, K. Williaiii\, a i i d P. Bornstein, E r j i . ( ; P I / R m . , 116 (1978) 153-165.

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RALPH T. SCHWARZ A N D ROELF DATEMA

reappearance of fibronectin on the plasma inem1)rane after mild proteolysis of s u r f k e glycoproteins with trypsin was prevente~l,"~ or may not have been detected (because fibronectin is not re-exposed). This observation m a y be related to the finding that filironectin that is devoid of carbohydrate side-chains is still exposed nonnally on the cellular nieinbrane, hut has half the half-life, due to increased susceptibility to cellular pro tease^."^ Interestingly, a BHK cell mutant having decreased adhesion to the sulistratum displayed the loss of a glycoprotein of high molecular weight, presumably identical to f i b r o n e c t i ~ i . ~ ~ ~ An elegant i n v e s t i g a t i o ~ ishowed ~~~ that, when applied to SV-40transfonned cells, preparations of nonglycosylateci fibronectin were as effective in promoting a inore-fil)rol,lastic phenotype (flattening and elongation) as were the glycosylated fomis. Furthennore, the nonglycosylated fibronectin showed the same extent of hemagglutination as its glycosylated countei-part.54fi Other fiinctions of fibronectin also reside in the protein part. For cei-tain aspects of work with cell cultures, a useful property of tunicamycin is that it can be used to synchronize cell division. After release o f the block imposed b y the drug, the cloning efficiency was also higher, and the cloning size more regular, than in the control c i d ture .347 A notable finding was the defective transport o f D-glucose, uritline, and two amino acid analogs (2-aiiiinobutanoate and cycloleucine) in chick-embryo cells in which glycosylation of proteins was prevented b y tunicamycin. Other mernbrane-associated processes, such as the enzymic activities of N a / K ATPase and adenylate cyclase, or the stiinulation of adenylate cyclase I,? a prostaglandin and cholerae toxin, were not a f f e ~ t e d . " ~ A report on the inhibitory effect of wheat-genn agglutinin on cellular transport may be relevant to this study, as this lectiii binds to 2-acetainiclo-2-deoxy-~-glilcosepresent in the same, L-asparafiiiie-liiiketl class of oligosaccharide that is affected by ti~nicarnyciii."~ (542) A. Takatsuki, X1. hluirekatn, 11. Nishimura, K. K o h n o , K.Onodera, antl G . Tainiii-a, Agric. R i d Chenr.. 41 (1977) 1831-1834. (543) K. Olden, R. 41. Pratt, and K. 51. Yaniatla, Z t i t . J. Caticer, 24 (1979) 60-66. (544) K. Oltlen, H.M . Pratt, a i r t l K. X I . Yairiatla, Crll, 13 (1978) 461-473. (545) A. Me~ger,R. Nairn, ant1 H. C. Hughe.\, Eirr. J . Riochcnt., 72 (1977) 275-281. (546) K. Olden, R. M . Pratt, ant1 K . 51. Yamatla, Pro(,. N n t l . Accicl. Sci. L7. S. A , , 76 (1979) 3343-3347. (547) K. Watanabe, G. Tamura, and H. 5tit.;iii, C P / /Struct. Furictiori, 4 (1979) 127-134. (548) K. Olden, R. M. Pratt, C . Jaworski, and K. M. Yainatia, Pro(,. N u t / . Aced. S(.i. U . S. A . , 76 (1979) 791-795. (549) E. Li m d S. Kornfeld, Hiockim. Bio/i/r!/.c..Actu, 469 (1977) 202-210.

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Inhibition of glycosylation by tunicamycin elicited a rapid depletion of insulin-binding activity at the surface of 3T3-Ll adipocytes. The disappearance of insulin receptors was accompanied by a diminution in the sensitivity of the cells to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism. These results suggest, in D-glucose uptake and insulin binding, a specific role for the glycosylation by modulating the survival times of the receptors, or influencing the exposure and orientation of the receptors needed for function.550 ST 13 fibroblasts maintained in a medium containing insulin differentiate into adipose-like cells. This conversion is characterized by the appearance of lipid droplets in the cytoplasm and by an increase in synthesis and accumulation of cellular triglyceride. The insulin binding increases about 10-fold during differentiation. Tunicamycin inhibits the differentiation and suppresses insulin-binding Further uses of tunicamycin were shown by the following studies. Treatment of cultured, calf-aorta, smooth-muscle cells with tunicamycin resulted in progressive loss of receptors for epidermal growth-factor. From this, it may be concluded that this factor may probably be a glycoprotein, or is closely associated with one. The t,,, determined for epidermal growth factor using tunicamycin (or cycloheximide) was 6 h. The epidermal growth-factor thus appears to be turning over more rapidly than, for instance, the receptor for growth hormone (t,,, = 10 h) or the insulin receptor552(tl,2 = 30-40 h). The proliferation in vitro of granulocyte macrophages is dependent on the presence of a sufficient concentration of a protein called colony-stimulating factor. When the protein is synthesized in the presence of tunicamycin, the heterogeneity of its molecular size disappears, but it retains its biological activity. The authors553inferred that the carbohydrate moiety is not essential for the production and action of the factor, and that the heterogeneity in molecular size is caused by tunicamycin-sensitive glycosylation. Tunicamycin and 2-deoxy-~-iirabino-hexoseinterfere with the expression of lipase (glycerol-ester hydrolase, EC 3.1.1.3)in cultured, mesenchymal rat-heart cells. The causes of inhibition were not inves-

(550) 0. M. Rosen, G . H. Chia, C. Fung, and C. S. Rubin,]. Cell. Phlsiol., 99 (1979) 37-42. (551) K. Kohno, A. Hiragun, A. Takatsuki, G. Tamura, and H. Mitsui, Biochem. Biophys. Res. Commun., 93 (1980) 842-849. (552) G. Bhargava and M. H. Makman, Biochim. Biophys. Acta, 629 (1980) 107-112. (553) D. Ayusawa, K. Isaka, T. Sano, M. Tomida, Y. Yamamoto, M . Hozumi, A. Takatsuki, and G . Tamura, Biochem. Biophys. Res. Commun., 90 (1979) 783-787.

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RALPH T. SCHWAIU AND ROELF DATEMA

tigated in this study, but formation of an inactive enzyme lacking carbohydrate may be the reason for the behavior.5s4 Human-leukemic antigen (HLA-DR) has been found in remarkably low proportions on the plasma membrane of human, lymphoblastoid cells. In the presence of tunicamycin, both subunits of this cell-surface antigen showed lower apparent molecular weights. One of them completely lost its [3H]-labelled 2-acetamido-2-deoxy-~-glucoseportion, implying that HLA-DR antigens possess oligosaccharides whose synthesis is lipid carrier-depenclent.sss A special example of cell-cell interaction is the adherence of group B streptococci to canine, epithelial cells that are infected with influenza A virus. However, this capacity was blocked in the presence of tunicamycin, and this result supports the concept that adherence of streptococci to mammalian cells involves recognition of viral hemagglutinin, or its carbohydrate complement, the synthesis of which is blocked by tunicainycin.”j6 Incubation of mouse macrophages with a medium containing 2deoxy-D-urahino-hexose leads to inhibition of Fc and complement C3-receptor-mediated, opsonin-dependent phagocytosis by these cells, but phagocytosis of latex and zyinosan particles was not affected. It was thus concluded by the a ~ t h o r sthat ~ ~energy ~ , ~ depletion ~ ~ is not the primary cause for the inhibitory effect of the sugar analog in this system. Possibly, the alteration of glycosylation of a rnacrophage glycoprotein is the cause. Although 2-c~eoxy-~-urubino-hexose was shown mainly to affect glycosylation (in the systems studied so far), its mode of action in morecomplex, biological systems may not always depend on this well known property. Additional effects, not yet understood, which are, however, separate from an alteration in the energy content of the cells that may be induced b y the sugar analog, may play a role. Thus, the of T-cell-mediated selective inhibition, by 2-deoxy-~-uruhino-hexose, cytolysis seems not to be related to protein glycosylatioii.~5~-s6* Pre-

(554) G. Friedman, 0. Stein, and Y. Stein, Atherosclerosis, 36 (1980) 289-298. (555) Y. Nishikawa, Y. Yanianioto, K. Onodera, G. Tamura, and H. Mitsui, Biocherrt. Biophys. Res. Co?wnun.,87 (1979) 1235-1242. (556) Y. T. Pail, J. W. Schmidt, B. A. Sanford, antl A. D.Elbein,]. Racteriol., 139 (1979) 507-514. (557) J. Michl, D. J. Ohlbaum, antl C. Silverstein,]. E x p . Med., 144 (1976) 1465-1483. (558) J. Michl, D. J . Ohlbaum, and C. Silverstein,]. E x p . Med., 144 (1976) 1484-1493. (559) H. R. MacDonald antl C. J. Koch,]. E x p . Illed., 146 (1977) 698-709. (560) H. R. MacDonald,]. E x p . Med., 146 (1977) 710-719. (561) H. R. MacDonald and J . C. Cerottini,]. Intitiuitol., 122 (1979) 1067-1072. (562) H. R. MacDonald and J. C. Cerottini, Eur. J . Zittrnunol., 9 (1979) 466-470.

iiiciilxition of cells with tuiiicwiiyciu, in order to I)lock glycosyl.‘1t‘1011, did not prevent cytolysis, which, however, occiirrd after suppleiireutation of the antibiotic-contaitiii~gi i i t d i i i i r i w i t h 2-cleo..;y-u-trr-nhi~iohexose. The precise mechanism of action of this sugar in t h i s c;isca remains to be e s t al)1i shed. In ;i n y c‘ v t’ I it , i live s t i gat o rs s h o 111tl co 11s id e r that, occasionally, it does not act 1)). interfering with glycosylation, Imt b y other ineans that have yet to IKX tiilly elucitlatcd.

ACKNOWLEDGEMENTS Work of the present authors that is citcd in tliis articlc was suppoited h y Deiitsclie Forscliuiigsgemeiiiscliaft ( S o n d e r f o r ~ c l r i i i i ~ ~ 47). l~~~ We ~ i thank ~li h a . 1-I. Rott arid C . Sclioltissek for their encoiiragriririit. M i - s . h l . Seitz l o r secretarial work, ;inti MIS. C . Heitz for art woi-k.

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ADVANCES IN CARBOHYDRATE CIiE\fISTRY AND BIOCHE.Z.1ISTRY, VOL. 40

BIBLIOGRAPHY OF CRYSTAL STRUCTURES OF POLYSACCHARIDES 1977- 1979

R. SUNDARARAJAN AND ROBERT H. MARCHESSAULT

B Y PUDUPADI

Xerox Research Centre of Canada, 2480 Dunwin Drive, Mississauga, Ontario L5L 119, Canada I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 11. Amylose and Other a-D-Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 111. Cellulose and Other P-D-Glycans . . . . . . . . . . . . . . . . ., 3 8 6 . . . . . . . . 392 IV. Glycosaininoglycans (Amino Polysacc V. Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 VI. Peptidoglycan . . . . .................... 399

I. INTRODUCTION This article, which is the fourth in the current series,' presents a bibliographic account of the crystal structures of polysaccharides that have been published during 1977- 1979. Several comprehensive reviews on the structures of glycosaminoglycans,'-" bacterial capsular poly~accharides,"*~.~ ~tarch,~ and - ~ cell-wall polysaccharides,1°and on (1) P. K. Sundararajan and R. H. Marchessault, Adu. Carhohydr. Chem. Biochetn., 33 (1976) 387-404; 35 (1978) 377-385; 36 (1979) 315-332. (2) E. D. T. Atkins and I. A. Neiduszytiski, Fed. Proc., Fed. A m . Soc. E s p . Biol., 36 (1977) 78-83. (3) E. D. T. Atkins, Proc. Cleveland Syttip. Macromol., l s t , (1977) 61-85, (4)E. D. T. Atkins, Pure A p p l . Cheni.,49 (1977) 1135-1149. (5) S. Artiott and W. T. Winter, Fed. Proc., Fed. Am. Soc. E s p . Biol., 36 (1977) 73-78; J . D. Gregory and R. W. Jeanloz (Etls.), Glycoconjugute Res., Proc. Znt. Symp., Academic Press, New York, 1979, pp. 321-323. (6) R. D. Preston, Nature (London), 266 (1977) 302-303. (7) A. D. French and V. G. Murphy, Cereal Food World, 22 (1977) 61-70. (8) A. D. French, Baker's Dig., (1979) 39-46, 54. (9) A. D. French, Brew. Dig., 54 (1979) 32-43. (10) R. D. Preston,Annu. Rev. Plant Ph!/siol., 30 (1979) 55-78. 38 1

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382

P. R. SUNDARARAJAN AND R. H. MARCHESSAULT

the crystallography of mono-, oligo-, and poly-saccharides,” have been published during this period. The methodology of model building for structure analysis of polysaccharides seems to have reached a plateau, at least for the time being. However, the availability of a multitude of crystal-structure data on mono- and oligo-saccharides, and the unsurpassed accuracy that has been made possible in computer-aided model-building, have opened a “Pandora’s box,” and several structures are being revised. Since its proposal by Kainuma and D. French,12in 1972, the possibility of double-helical structures in starch has been investigated by Wu and Sarko,l3-I6who concluded that the structures of A- and B-starches are double-helical. This followed an era during which flat, sixfold helices had been widely advocated in the solution and crystal structure studies on starch. The question of chain packing in cellulose is still far from resolved. Blackwell and coworker^^^-^^ and Sarko and MuggliZ0proposed that the chains are packed parallel in cellulose I, and antiparallel in cellulose 11. This demands an ingenious scheme to envisage the conversion of cellulose I into 11, which requires chain reversal. However, A. D. Frenchz1suggested that antiparallel packing in the native celluloses of ramie and cotton cannot be excluded. Hunter and DweltzZ2 claimed that the X-ray data can be accounted for by the parallel packing of the chains in cellulose 11. If the polarity of the chains is the same in cellulose I and 11, the conversion I into I1 is readily visualized. However, whether both of them are parallel, or antiparallel, is still a matter of debate. Such geometrical features as bond lengths, bond angles, and torsion angles, derived from crystal structure analyses of mono- and oligo-saccharides, are being used extensively in the analysis of the crystal structure of polysaccharides. However, a few special features that are beginning to be realized from studies on simple sugars have not as yet (11) G. A. Jeffrey and A. D . French, Mol. Struct. Dzffr. Methods, 6 (1978) 183-223. (12) K. Kainuma and 1). French, Biopolymers, 11 (1972) 2241-2250. (13) H.-C. H. Wu and A. Sarko, Carhohydr. Res., 54 (1977) ~ 3 - ~ 6 . (14) A. Sarko and H.-C. H. Wu, Staerke, 30 (1978) 73-78. (15) H.-C. H. Wu and A. Sarko, Carbohydr. Res., 61 (1978) 7-25. (16) H.-C. H. Wu and A. Sarko, Carbohydr. Res., 61 (1978) 27-40. (17) W. C . Claffey and J. Blackwell, Biopol!jmers, 15 (1976) 1903-1915. (18) F. J . Kolpak and J. Blackwell, Macromolecules, 9 (1976) 273-278. (19) J . Blackwell, F. J . Kolpack, and K. H. Gardner, in J . C . Arthur, Jr., (Ed.) Cellulose Chemistry and Technology, A m . Chem. Soc. Symp. Ser., 48 (1977) 42-55. (20) A. Sarko and R. Muggli, Macromolecules, 7 (1974) 486-494. (21) A. D. French, Curbohydr. Res., 61 (1978) 67-80. (22) R. E. Hunter and N. E. Dweltz,J. A p p l . Polym. Sci., 23 (1979) 249-259.

BIBLIOGRAPHY OF CHYSTAL STRUCTURES

383

been included in model-builcling efforts for polysaccharides. The present procedures depend priiriarily on the minimization of Van der Waals repulsions, and maximization of the number of hydrogen I>onds.A trend in the donor-acceptor properties of hydroxyl groups in simple sugars was pointed out b y Jeffrey and Lewis.z3Perez and Marchessaultz4 considered the exo-aiioineric effect and the restriction it imposes on the orientations of the C-1-0 bond, and suggested that a potential function to simulate this force should be added to the functions in use at present. T h e y also called attention to the "peri" interactions and intramolecular hyclrogen-bonding involving the hydroxyniethyl gro1qxZ5It is pro1)d)le that, when included in the stereochemical constraints, such features may lead to a better understanding of the interactions in the crystal structures of polysaccharides. As before, significant features of the structures are given i n this article, in addition to the unit-cell diinensions. In the title to each abstract, H common name o r descriptive title for the polysaccharide described is given at the left, and the formula at the right. The other details follow the nomenclatiire set forth previously.'

11. AMYLOSE AND OTHERa-D-GLYCANS 1. A-Ainylose13,14,'6 c

The unit cell is orthorhomliic, with (1 = 1.19 nin, b = 1.77 nin, and 1.052 nm. The favored co~ifonnationis a parallel-stranded, double

=

helix. Each strand is a fi(O.351) helix. Equally good refinement was achieved with the OH-6 group in the g + [ x ( S )= 61'1 or t [ x ( S )= 144'1 states. The R factors are 37 arid 360/0,respectively, for these two positions. It was suggested that the true structure is a mixture of both. The double helices pack in an antiparallel array, with eight water molecules distributed along the (1 ant1 11 axes of the unit cell in the interstices between the helices. Thcl structural features of A- and B-amylose were compared.

2. B - A ~ n y l o s e ' ~ ~ ~ ~ Two parallel-stranded, c-loul)lehelices pack antiparallel i n a hexagonal unit-cell, with u = b = 1.8.511111, c = 1.04 11111,and y = 120". The individual chain is a 6(0.347) helix. There are 12 Glcp residues in the u n i t cell, with 36 water nioleciiles. The water is columnar, packed in ( 2 3 ) G. A. Jeffre!, and L. Lewis, Ccl?holtI/tlr-. RcF.,60 (1978) 179-182. (24) S. Perez and R. H. M~archessault,(~~ir/wlt!yc/r, R m . , 65 (1978) 114-120 (25) H. H. Marcheshault and S. Pi.i-cz, / ~ i ~ ) / ~ ( ~ / ~ / r t t18 [ , r(1979) .~, 2369-2374.

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P. R. SUNDAHARAJAN A N D R. H . M A R C H E S S A U L T

the corners of the unit cell, in the channel fonned by the hexagonally packed helices. The OH-6 group is in the g+[x(5) = 67”] position. The hydrogen bonds are of the types OH-3---0-6 (0.268 nm) and OH-2--0-6 (0.295 nm) l~etweenthe strands in the double helix, OH-2---0-5 (0.259) between equivalent chains (chain 1)in the two double-helices, and OH-6---0-2 (0.283 nm) between the other pair of chains (chain 2) in the two double-helices. The R factor is 22%.

3. NigeraiP

Poly[ ( 14)-a-D-Glcp-(1-4)-a-D-Glcp]

X-Ray and electron-diffraction studies showed that anhydrous nigeran crystallizes in an orthorhomhic unit-cell, with IL = 1.776 nin, h = 0.6 nm, and c = 1.462 nm. The space group is P2,2,2,. It was found that base-plane, a s well a s c-axis, dimensions change upon hydration. Two 2(0.73) chains pass through the unit cell. Both OH-6 groups are in the g- position. The conformation of the chain resembles a corrugated ribbon. An OH-2---0-3’ (0.28 nm), intrachain hydrogen-bond exists between the residues participating in the (1+4) linkage. Two interchain hydrogen-bonds, of the types OHi-4---Oi-6 (0.27 n m ) and OHi+,-6---Oi-2(0.264 nm),are between the corner chains related by the translation along the 11 axis. In addition, an OHi-4---Oi+,-3 (0.288nm) hydrogen-bond is possible between the central chain and its antiparallel countelpart. The transfomiation between the “dry” and hydrated” structures was discussed in terms of folded-chain, lainellar morphology. The R fkctor was 25% according to electron-diffraction data, and 30% from X-ray data. “

4. Ainylose - 1-butanol complex“

Poly[( 1+4)-a-D-Glcp]

Us in g e 1ectro n diffraction and que iich -froze n samples of amy 1os e 1-butanol complex, the base plane was shown to be rectangular, with u = 2.7 inn and 17 = 1.32 nin. The plane group is Pmg. On drying, a hexagonal symmetry was observed, with u = b = 1.35 n m . Rehydration restored the rectangular unit-cell, although the b diinension changed to 2.34 nni. This would indicate that the hexagonal syiimietry observed by earlier author^^^,'^ was due to drying of the sample in the vacuiini of the electron microscopc.

(26) S. Perez, M .Rorix, J. F. Kevol, a n d H. H. hfLlar-chessault,J. M o l . Biol., 129 (1979) 113-133. (27) F. P. Booy, H. Chanzy, and A. Sarko, B i o p o l y i r i c m , 18 (1979) 2261-2266. (28) R. S . J . Manley,J. PoZ!ym. Sci., Part A , 2 (1964) 4503-4515. (29) Y. Yamashita,J. P o h / i ~Sci., . Pnrt A , 3 (196s) 3251-3260.

5. T ri-0 -ethy1an?y lose:1°

Fol?.[(l~4)-cr-~-Glci)-Et,]

The unit cell is orthorhonil)ic, with u = 1.613 11111, b = 1.166 nni, and c = 1.548 n m . Left-handed, four-fold helices pack in the space group P2,2,2,. The 6-0-ethyl group is in the t position [x(5) = 148.2'1. The R factor is 33%. (This crystal fomi was denoted a s TEA1 111. the authors .) 6. Tri-O-ethyliiniylose31

Pol>.[( 1+4)-a-~-Glc~1-EtJ

Complexes of tii-O-ethyl~irn~~lo~c with nitromethane, chloroform (TEA1-CB),and dichloroii~ethallt.~ii~~ (TEA1-DCRIB)crystallize i n ;i pseuclotetragonal unit-cell, with N = I ) = 1.47 nni, and c = 1.548 nm.1 1 1 all cases, the chain is a 4( - 0.387) h c l i x . The eight giiest molccules occ u p y the grooves of the lieliccs, with a 2, axis and a statistically averaged orientation of their dipoles. The 6-0-ethyl group is in the t position [x(5) = 145"l. The R factor is 35% for the nitromethane and ch 1o r o fomi comp 1exes , i i i i d 32%' t;)r the di ch 1o ron 1e th m e c ( )I 11pl e x . 7. Tri-O-ethylamylose32

Poly[( 1-+4)-a-~-Glc/?-Et3]

Both the chloroform and dichloroinethaiie complexes of tri-0-ethylaiiiylose (TEA1-C1 and T E A I - l X M 1 , respectively) crystallize i n i i n orthorhombic unit-cell, with f o i i r small molecules per unit cell. The unit-cell dimensions are ( I = 1.676 nni, b = 1.428 nni, and c = 1.602 nni for the chloroform complex; a n d (1 = 1.652 nm, 12 = 1.395 nin, a n d c = 1.602 n m for t h e TEA1-DCM1 complex. The space group is P2,2,2, in both complexc~s.A 4( - 0.4) helix is the fiavored conformation. The R factor is 36%) f o r TEA1-C1, and 3<5% for TEA1DCM1. It was proposed that, cliiriiig the transfoimations TEAI-C1 + TEAl-C2 and TEA1-DCM1 +TEA1-IlCM2, the space group remains the same, the number of coiiiplcxing inolecules in the unit-cell increases to eight, the oi-thorhoinl)ic cell is converted into the pseudotetragonal, and the fiber repeat decreases to 1.548 11111. The small inolecules a r e well oriented, antl iiitc,rstitially located, in TEA1-C1 and TEA1-DCM1. They are situated i n the grooves of the helices, with statistical orientation of their clipoles in TEAl-C2 and TEA1-DCM2.

(30) T. I,. Bluhm, G. Rappeneckcr, antl 1'. Zrlgenmaier,C t / ~ - h h ! / t / r -H. r s . , 60 i 1978) 241250. (31) T. L. Bluhm aiid P. Zugenmaier, P r o g C o / / o i t l Z'o/~ym, S ( , i . 64 (1978) 132-138. ( 3 2 ) T. L. Blithm and F'. Zugeiinlaier, Z ' O / ! / J J I P ~ . 20 (1979) 23-30,

386

P. R. SUNDARARAJAN ,4ND R. H . MARCHESSAULT

8. Tri-O-ethylamylose:i3

POly[(1+4)-~i-D-Glcp-Et,]

Removal of the solvent from the tri-O-ethylamylose-chloroform complex (TEA1-C2) results in a new polymorph (TEA3) of tri-Oethylainylose. The unit cell is orthorhomhic, with II = 1.536 nin, h = 1.218 nm, and c = 1.548 nm. The space group is P2,2,2,. The chain confornution is a 4( - 0.387) helix. The C-6-0-6 bond is in the t position, with x(5) = 143.3'. The striictiiral relationship between the various polymorphs was discussed.

9. (1+3)-a-~-Glucan"~

Poly[( 1+3)-a-D-Glcp]

The unit cell is pseudo-orthorhombic, with a = 0.823 nm, b = 0.955 nm, and c = 0.844 nm. The space group is P2,. The chain conformation is close to a two-fold helix. 10. (1+3)-a-D-Gl~ican~~

PoIy[( 1+3)-a-D-Gkp]

Three polymorphic forms (I, 11, and 111) of (1+3)-a-D-glucan have been identified. The native tissues o f t h e fungi L. sulphzireus and P. betulinus show the presence of I. On precipitation from alkali solution, I1 was obtained. This is converted into I11 on moderate drying. In L. szdphureus, the d spacings of the D-glucan in the trama are slightly larger than those of the D-glucan in the context. A detailed comparison of the d spacings for the different polymorphs was given. 111. CELLULOSEAND OTHERp-D-GLYCANS 1. Native cellulosez1

Poly[( 1+4)-/3-D-Glcp]

Using the "two-chain" unit-cell,:'6 with Q = 0.817 nm, b = 0.785 nni, c = 1.034 nm, and y = 96.38", the modified intensity-data of Mann and coworker^,"^ and several residue-geometries, the structure of native ramie cellulose was refined. The resulting R factors were 15.8%, 18.5%, and 17.5% for, the antiparallel, parallel-up, and parallel-down models, respectively. A temperature factor of 0.23 nm2 was necessary in order to obtain a good fit with the obseived data. It was suggested that the antiparallel packing of the chains cannot be cliscounted for cotton and ramie celluloses. (33) T. L. Bluhrn and P. Zugentnaier, Carbohydr. Res., 68 (1979) 15-21. (34) K. Ogawa, A. Misaki, S. Oka, antl K. Okarnnra, Curhohyclr. Res., 75 (1979)c 1 3 - c l 6 . (35) J. Jelsma antl D. R. Kreger, Corboh!/tlr.Res., 71 (1979) 51-64. (36) H. J . Wellart1,J. Polyrn. Sci., 13 (1956) 471-476. (37) J . Mann, L. Roltlaii-Gonzalez, and H . 1. Wellard,/. P o l ! / m . Sci., 42 (1960) 165-171.

2.

ce11ulo

se”8

PoIy[( 1+4)-p-D-Glcp]

Electron diffraction patterns f r o n i the primal?; wall of lt5-da).-old cotton fiber showed sharp, meritlional reflections, with ti spacings of 0.517 iiin and 0.258 mu. Broad iiiiixiiiia on the equator, with spacings of 0.416 nm and 0.570 n m were also olxerved. On this basis, it w a s suggested that the primary widl of cotton contains the cellulose IV, polymorph, which is simply a laterally disordered structure of cellulose I. A discussion of the morphology of cellulose i n the priinary wall was given.

3. C e l l ~ l o s e “ ~

Polv[( 1+4)-p-D-Glcp]

The swelling of native, aiid iwrcerized, cellulose fibers in ethylenediamine (EDA), with sill, liicnt washing i n methanol, changes the cellulose I and I1 lattices to cellulose 11, and III,, respectively. The presence of EDA, in excess of that which is necessaiy f o r the cellulose-E DA complex to form, is essential for the conversion to occur. Treatment with boiling water o r li).tlrochloric acid converts the I11 lattices into the respective, parent types. Washing i n alkali converts both forins into cellulose II.

4. Cellulose I1 (Ref. 22)

Pol y[( 1+4)-p-~-Glcp]

It was possible to index d l of the observed reflectioiis b y iising a reduced, “one-chain,” inonoclinic iinit-cell, with u = 0.446 nni, h (fiber axis) = 1.034 nm, c = 0.725 nni, and /3 = 98.6’. The space group is P2,, with one chain per unit cell. Thus, the chains have palallel polarity. There are two intrachain hydrogen-bonds of the type OH-3--0-5’ and OH-2---0-6’, and an 0€1-6---0-3,interchain h y d r o g e n - h i d .

5. Cellulose I1 (Ref. 40)

Poly[( 1+4)-fl-~-Glc~1]

Cellulose 11, derived by mercerization of cotton, crystallizes in a monoclinic unit-cell, with a = 0.802 nm,19 = 0.899 nni, c = 1.036 nm, and y = 116.6’. The space groiip is P2,. Two chains, each with a 2(0.518) conformation, are packed in the uiiit-cell, with antiparallel polarity. In addition to the OH-3---0-5’(0.269 nm), intrachain hydrogen-bond, an OH-2’---0-6 (0.272 nm), intrachairr hydrogen-bond is possible onlyfor the center ‘“rlowri” chain. This is due to the g+ orientation of the C-6-0-6 bonds in the corner chains and t orientation i n (38) H. Chanzy, K. Imada, and R. Vuong, P r o f o ~ i l u s m u94 , (1978) 299-306. (39) P. K. Chidainl,areswarair, S. Sreenivasan, N. B. Patil, H. T. Lokhantie, and S. R. Shukla,]. A p p l . Pohlnl. Sci., 22 (1978) 3089-3099. (40) F. J. Kolpack, hl. Weih, and J. Blackwtall, Polymer, 19 (1978) 12.3-131.

.388

P. H. SU.UDAKAH.-\JAN A N D R. H. MARCMESSAULT

the center “down” chain. The interchain hydrogen-l)onds are OH“, (0.272 I ~ I I I ) , and OH.‘-2---0’’-2’ 6---0.‘-2 (0.276 I I ~ ~ I )OHf3-6---0.‘-3 (0.273 nin), where A = atom on the next chain along the (1 axis; = center “down” chain; (~ = corner chain. The R factor is 26.3%. 6. D-G1ucan4’ fro111 A cc to h~c te t- x!/ 1i t 1 u 171

Poly[( 1+4)-p-D-Glc)1]

Single crystals of the 13-glucan from A c e t o h c t e r myZinutn were prepared in water - n i e thano 1 solutions at rooi n te i n pe ra ture. U sing el ectron and X-ray diffraction, a lwxagonal unit-cell, with (1 = 11 = 0.518 nin and c = 2.0 mn, was derived. It was concliicted that the chain axes lie parallel to the surf& of the lamellar crystals, that there are 2.5 water molecules per p - ~ - G l c presidue, and that the structure is similar to that of cellulose hydrate I1 (Ref’. 42). 7. Alkali-cellulo~e~~

,

Poly[( 1+4)-p-D-GIcp]

Froin the variation of the tl,,, spacing in alkali-celluloses I, 11, and V, the number of water molecules h a s been calculated to be 3, 1, and 5, respectively. A value of dlol= 1.51 mi, which is the largest reported so far for alkali-celluloses, was obtained by mercerization of sulfite cellulose with 11% sodium hydroxide solution for one hour at 20”. Between six and eight water molecules are included in this structure. 8. Cellulose-diamine

Poly[(1+4)-p-D-GIcr1]

The N,N’-di1nethyl-1,3-propanediamine complex of cellulose, prepared froin ramie, requires a large unit-cell, with a = 3.364 nm, h (chain axis) = 1.026 nin, c = 3.040 nm, and p = 32.74’. One diainine inolecule per two P-D-Glcp residues exists in the complex.

9. Tri-0-acetylcellulose I (Ref. 45)

PoIy[( 1+4)-P-D-Glcp-AcJ

The unit cell is orthorhoinbic, with (1 =2.363 n m , b = 0.627 nm, and c = 1.043 nm. The space group is P2,, with two parallel, polarity chains in the unit cell. The helical parameters are 2(0.52). All of the C-6-0-6 bonds are in the g - position. The R factor is 24.2%.

(41) M .Takai and J . R. Colviii, A4rn~.Fac. E n g . Hokkuido Uiiiu., 15 (1979) 6:3-73. (42) P. H. Hermans and A. Weitlinger,]. Colloid Sci., 1 (1946) 185-193. (43) A. Sh. Goikhnian, A. L. Iialler, G. V. Polyakova, and N . P. Matsibora, Polyni. Sci. USSR, 19 (1977) 3000-3005. (44) J. J . Creely, R. H . Wade, a n d A. D. French, Text Rev. /., (1978)37-43. (45) A. J. Stipanovic and A. Sarko, Polt/rner, 19 (1978) 3-8.

10. ‘rri-O-acetylcellLilose I1 (Rct‘. 46)

Poly[( I - - + ~ ) - / ~ - I > - G ~ C ) I - A C J

C:onil,ined X-ray and elt’cti-oil clilYraction anal!.sis led to an ortl~orhonibicunit-cell, with (I = 2.468 nm, 11 = 1.152 11111, antl c = 1.054 nm.The space group is P212121.‘l’wo p:irallel chains are related, p i rw i se , b y a tw o-fo I d screw -axi s p a ral 1e 1 to t 11e ch iii n a x is , and pairs of chains pack in iin antiparallcxl Lirrity. The (I 1 0 ) growth planes of the tal are parallel to the clirec.tioii of highest atomic densities. The transfoiiiiatioii CTA I1 s celliilose I1 was discussed. The H fiictor is 30% with the X-ray diffractioii tlata, antl 26% \vith the electroil tliffi-action data.

Pol>.[(1+4)-p-D-c;lcj>(NO,),]

11. Cellulose Trinitrate4‘

(I = 0.9 n m . 1) = 1.46 11111, and The unit-cell cliniensioris c =2.54 nni. A h e l i x with fivr rc-sitliic-s i n two turns is the favored conforniation. The helices pack i i r m i approxiimitely hexagonal a r r a y . m

12.

s

Pol?.[(I+4)-/3-D-hlklnp]

D-MZtIII1~iIl4’

Laniellar, single crystals of i\.or>.-riiitinatiiian wert~stiiclied h y electron diffraction. The base-plauv cliinensions of the unit cell a r e rc = 0.722 nni and b = 0.892 niri. systematic iil)seiices confirinecl the space group P2,2,2, . The clitfrx~tioiipattern did not chaiige with the crystallization temperature. Oriclntc>dcrystallization of ~ - i i i a i i n a nwith its chain axis parallel to thc 111 i cr( fil ) ri 1 substrates , Vnlo t 1 i a oc t I t t-icosci and hacte rial ce 11111o se , was cl i s C Y )\.e re (1 (“hete ro- s h i sh-ke1MI, s ” ) . ‘l’lrcb

13. ( ~ + ~ ) - P - D - G ~ U C ; ~ I I ~ ’

Pol y[( 1+3)-p-D-Glc)j]

Th ree d ifferei i t , X-ray (1if Yract ioI 1 pattern s \Y e re 01) s e ilre(1, depending on the condition o f tht. sainple. The base plane is hexagonal in all cases. For the as-spiiii pol\mier (A), (1 = 1) = 1.7 11111 a i d c = 2.2 n i n . After annealing in watcsr, the hydrated form (B) is olituinetl, with = h = 1.571 ntn ant1 c = 1.882 nm. LJpoir “clehytlration” (form C), the unit-cell dimensions six' (I = 11 = 1.438 mi and c = 0.579 nm. A triple helix, with each strantl corresponding to a 6(0.289)helix, was proposed for the C form. Upoil h>.tlration,the triple-helical symmetry (46) E. Hoche, 13. Chanzy, h l . B o u t l t ~ i i l l t ~H., 11. 1Iarchca\arilt, i d P. R . Srrirtlal.nrajai1, ,\lmcromolccules, 11 (1978) 86-94. (47) D. Meader, E. D. T. Atkins, and F,k I c ~ p p e yPol!/rrtcar, , 19 (1978) 1371-1374. l l t , J. F. H e \ . ( ~ lBiolx)/cynter,s, , 18 (1979) (48) H. Chanzy, 1 4 . Dull@,R. H. M a r c ~ h t ~ s s ~ r aiid 887-898. (49)R. H. Marchessault, Y . Deslaiidc.s, K.Ogawa, and P. R. Suntlararajalr, C o r i . / . C h r . r i i . , 55 (1977) 300-303.

390

P. R. SUNDARARAJAN A N D R. H. M A R C H E S S A U 1 , T

is perturbed, and the c dimension corresponds to a full helix turn: 1.882 nm. The transition from B to C is reversible.

14. Lentinan"'

Poly[( 1+3)-/3-D-Glcp]

The (1+3)-P-D-glucan from Lentinus edodes crystallizes in a hexagonal unit-cell, with a = b = 1.58 nin and c = 0.6 nm. Comparison with the studies on (1+3)-p-~-glucans from other sources49J'.52 showed that, irrespective of the source, this polysaccharide crystallizes in almost the same, hexagonal unit-cell. The favored confomiation is a six-fold helix, with a repeat of 1.8 nin. A triple-helical structure was proposed, with the right- antl left-handed chiralities giving equally good fit with X-ray diffraction and stereochemical criteria. 15. 0 -Acety1pachyinans3

Poly[(1+3)-P-~-Glcr~,-A~3]

Two polymorphic forms were observed. On stretching b y 25-50% at 125", polymorph I was obtained; this crystallizes in an orthorhombic unit-cell with a = 1.10 nin, b = 1.90 nm, and c = 2.238 nm. The space group is P2,2,2,. Further stretching of the film to 300% at 215" gave polymorph 11, with rinit-cell dimensions a = 1.149 nm, b = 2.013 11111, and c = 1.86 nin. In both, the chain conformation is a right-handed, six-fold helix. The chains are packed antiparallel in I. A 1 : 1 statistical mixture of parallel and antiparallel polarities was derived for 11. The acetate groups attached to C-2 and C-4 are so positioned that the carbonyl bond is within 10" from the eclipsed orientation with respect to the corresponding C-H bond. The C - 6 - 0 4 bond is in the t orientation [x(5) = - 153'1. Refinement of the structures led to an R factor of 22.1% for I, and 23.4% for 11. 16. ( 1+3)-/3-D-Xyla1154

Pol)'[( 1+3)-p-D-xylp]

The triple-helical model previously proposedg5 for (1+3)-p-D-xylan was refined in the hexagonal unit-cell, with u = b = 1.54 nm, c = 0.612 rim, and y = 12W, by using least-squares methods. A procedure that obviated the use of Bessel functions was described. The R factor was 41%. (50) T. L. B l u h m antl A. Sarko, Can. J. Chem., 55 (1977) 293-299. (51) W. H e r t h , W. W. Franke, H. Bittiger, A. K u p p e l , and G. Keilich, Cytohiologie Z. E x p . Zellforsch., 9 (1974) 344-367. (52) J. Jelsma and U . R. Kreger, Curbohytlr. R e s . , 43 (1975) 200-203. (53)T. L. B l u h m and A. Sarko, Biopol!ymrrs, 16 (1977) 2067-2089. ( 5 4 ) M. A. Haleern a n d K. D. Parker, Z. Wnturforsch., Teil C, 32 (1977) 665-668. (55)E. D. T. Atkins and K. D. Parker,J. P o l y i t ~ .Sci., Part C, 28 (1969) 69-81.

BIBLIOGRAPHY O F CRYSTAL STRUCTUHES

39 1

17. Wutsoniu Xylan56*57

The gummy polysaccharidc froin the corni sacs of Watsotiin p!yruiniclutu crystallizes in a trigonal unit-cell, the base-plane dimensions of which depend on the relative liriniidity (r. h.). At 7670 r.h., the diinensions are Q = b = 1.40 nin, and, for the dry form, (I = h = 1.34 nni. A 3( - 0.495) helical conformation was proposed. The richness of the X-ray pattern led to the conclusion that the sulxtitution is highly regular in the crystalline regions.

18. PustulanSR

Po~Y[( 1+6)-P-D-Glcp]

Electron-diffraction patterns were recorded for the d r y and “frozenhydrate” fonns of pustulan froiii Pzistulun papullosu. The frozen-hydrate foriii crystallizes in a rectangular unit-cell, with u = 2.44 nni and b = 1.77 nm. The chain-axis repeat was not detemiinetl. Systematic absences led to the two-diitieiisional space-group Pgg. IIehydr~ ‘1 t ’1011 results in a reversible, partial collapse of the crystals.

19. C a l a c t o ~ n a n n a n ~ ~

n

X- Ray cli ffract ion pattern s for t 1ie gal actom an n an s from guar , locustbean, and tara gums show, with relative huiiiidity, continiious variation ofonly the (1 dimension oftlie Ixise plane. For guar g!um, the u axis varies from 1.35 nm at 0% r.11. to 3.32 nm at 78% r h . , whereas, for lo(56) C. Lelliott, E. I). T. Atkins, ]. \V. I . Jriritz. and A. \I.Stcphen, Polytric,r, 19 (1978) 363 - 367. (57) H. Chanzy, F. P. Booy, and E. 11. ‘I. Atkiirs, Polymrr, 19 (1978) 368-369. (58) H. Chanzy, C. Guizarcl, and H. Vuoiig,J. Microsc. (OufiJrtl), 111 (1977) 143- 150. (59) R. H . Marchessault, A . Buleon, Y,I > r h ~ a n c ~ eand s , T. Goto,,/,C [ ~ l l o i tIiilcrf’clcz / Sci., 71 (1979) 375-382.

392

P. R. SUNDARARAJAN 4ND H. H . MAHCHESSAULT

ciist-bean gum, it varies from 1.16 to 3.06 nm in the same range of r.h. A sheet-like structure is thus implied, and the sheets run parallel to the b axis, with water inserted between the backbone chains. I n all cases, the degree of crystallinity improved with increase in r.h. For the packing of the chain, a model was proposed that consists of galactoniannan chains at the corner, and nianiian chains at the center, ofthe a edge of the unit cell, and this accounts for the continuous variation of the largest ti spacing from that for the pure D-mannan to that of a galactomannaii chain having a ManplGalp ratio of 1: 1. IV. GLYCOSAMINOGLYCANS (AMINO POLYSACCHAFUDES) 1. a-Chitin60,61

Poly[( 1+4)-P-~-GlcpNAc]

Chitin from the mandibular tendon of the lobster H o m a r u s a m e r i canus crystallizes in an orthorhombic unit-cell, with a = 0.474 nm, b = 1.886 nm, and c = 1.032 m i . The space group is P2,2,2,, with one chain in the corner and another in the center of the a h projection. Two intrachain hydrogen-bonds, OH-3'---0-5 (0.272 nin) and OH-6'--0 - 7 (0.285 nm),the latter occurring only in the corner chain, were proposed. The chains along the a axis are bridged by NH---0-7 interchain hydrogen-bonds (0.27 nm), antl those along the ci b diagonal, by an OH-6---0-6' hydrogen-lmnd (0.283 nm). The hvo types of hydrogen bonds involving OH-6 groups are rendered possible b y their different rotational positions. A statistical mixture was proposed in order to maximize the number of hydrogen bonds. The weighted R Factor is 20.5%. 2. a-Chitino2

Poly[( 1+4)-fl-~-Gk71 NAc]

Electron-diffraction patterns for a-chitin from the grasping spines of the marine woim Sagittci led to an orthorhombic unit-cell, with a = 0.474 nni, b = 1.886 nm, and c = 1.032 nni. The appearance of the 001 (1, odd) and 0k0 (k,ocld) reflections cast doubt on the P2,2,2, space group proposed by Blackwell and coworkers.fi"*61 3. @-Chitinfi3

Poly[( l+d)-p-~-GlcpNAc]

Intensity calculations were performed for antiparallel and parallel layers, and the statistical layer-shifts were examined. It was con(60) R. Miiike and J. Blackwell,]. Mol. B i o l . , 120 (1978) 167-181. (61) J. Blackwell, R. Minke, antl K. H. Gartlner, MI?' Seu Grulit Rep.. 78-7 (1978) 108123. (62) E. D. T. Atkins, J . Dlugosz, a i d S. Foord, I u t . ] . Biol. hlacromol., 1 (1979) 29-32. (63) M. A. Haleeiii a n d K. D. Parker, Z. Nuturfor.vch., T e d C, 32 (1977) 669-671.

cluded that there might be

a i l airtiparallel, layer sequc'nce, with each layer randomly displaced froiri the position (i/2.This allo\\zs for strong h y droge 11 bonds , betw ee 11 n e i g111)o r i I Ig lay e r s , t h rough 0I 1-6 g ro u 11s.

4. P-Chitin6' Chitin from the pogoiiophortx t i i l ) e a (0ligohrricliiu i w t 1 o t - i )cr\,stallizes in a nionoclinic unit-cell, with (I = 0.485 i i i t i , 11 = 0.926 trm, c = 1.038 m i , and y = 97.5'. The syxicc group is PZl, with one chaiii per unit cell. In addition to the OH-:3'---0-5, intrachain liyclrogeii-l)oiid, there are intrasheet OH-6---0-7' (0.289) a nd NII---O=C: (0.276) liydrogen-bonds. The weighted H fiictor is 38%. The gelling properties of @-chitin were attributed to t h t > al)seuce of iiitersheet hydrogen-

bo I1CIS.

5. Regenerated Chitine4

P ~ l y [~( + ~ ) - @ - D - G I c ~ I N A ~ ]

0rie nte d, re ge 11e rate d chit i 11 13 r e p a red h y s p i 11 ti i t i g froin a fo ri 11i c ac i tl so 111tiori showed reflect i () I is co rre s pon (1 i ir g to s pac i I i gs of 0.25, 0.34, 0.51, and 1.01 iitn on tht, iiit~ridi;iti,a nd 0.46 and 1.12 i i i i i on the equator. It was suggested that rc-generated chititi iiia!. havcb the same strncturc a s native chitin. t 1 i c k

6. Hyaluronic

Poly[( 1-+4)-P-D-Glcr)i\-(l+3)-P-~-GlcpNAc]

The X-ray cliffract ion pa tte 1-1 1s 01) t a i t i e d froI 11 pot a s s i iiin h ~ \ . a l r( i i ) 11iit e films prepared from solutions of p H 3.0-4.0 were represent~~ti\.e of the patterns from Rb+, Cs+,a i i t l hH4* forms. At 90-98% r.li., the unit cell is tetragorial, with (1 = 1) = 1.714 iini, a i i t l ( ' = 3.28 ntii. Each chaiii is a 4(- 0.82) helix, and two such chaiiis foiin an antiparallelstranded clouhle-helix. There. a r e two double helices i n the unit cell, a r i d the space group is I4,22. ' l l t t ~ r eare no intratnoleciilal- hydrogeii1,onds. 7. Hyaluronic acid"6

Poly[(l--.4)-@-~-Glc))i\-( 1+3)-~-1>-G]q1 NAc]

Calciiiin and strontium salts of' Iryaluronic acid, at relativta huiriidities of 66-92%, crystallize i t i :i trigonal iinit-cell, witli (1 = h = 2.093 nin and (I = 2.83 mi. 0 1 1 drliirg, the Ijase-plane dimensions reduce to a = b = 1.832 nm, with ( - = 2.847 i i i i i . Srveii n7att.r iiioleciiles per disaccharide residiie c:xist i n the wct forin, and two i n the clry form. The adjacent chains arc^ antiparallel, ant1 tlic space groiip is P3,12. The three disaccharide> inlit.; i n the 3( - 0.94) I~elixare noiiequi(64) S. Tokura, N. Nishi, and J. Nogwlii, P o / ! / ~ t i .J . , 11 (1979) 781-786. (65)J . K. Sheehan,K. H. Cartlner, and 1.: I > . T. Atkiiis,J. .!f(~/. H i o l . , I17 (1977) 113-13.5. (66) W. 1'.Winter and S. Ariiott,J. ,\fo/.H i ( ) / . . 117 (1977) 761 -784.

394

P. R. SUNDARARAJAN AND R. H. MARCHESSAULT

valent. It was suggested that each hydroxymethyl group is disordered over at least two rotational positions. The lengths of the intramolecular, OH-3---0-5’ and OH-4’---0-5 hydrogen-bonds vary between 0.269 and 0.273 nm, and between 0.261 and 0.295 nm, respectively. Interinolecular hydrogen-bonds of the type OH-6---0-6, and several hydrogen bonds involving OH-6 groups and water molecules, were proposed. The antiparallel chains are bridged by the COO----Ca2+--- 0 O C interactions. The weighted R factor is 28.4%. It was suggested that the compressed forins of hyaluronate occur with monovalent cations, and that the divalent cations lead to extended chains. 8. Chondroitin 4 - s ~ l f a t e ~ ~ Poly[( ~ + ~ ) - P - D - G ~ ~ N A c - ~l+4)-p-D-GlcpA]CiY2+ SO~--(

The unit cell is trigonal, with a = b = 1.28 iim and c = 2.74 nm. Two chains, each with a 3(- 0.913) helix, pass through the unit cell. All three axes ofthe unit cell are shorter than those observed for the sodium salt fomPHaE9 ( u = h = 1.45 nm, and c = 2.88 nm). This was attributed to the greater degree of binding of calcium ion to the polysaccharide chain, compared to that of the sodium ions.

9. Choiidroitin 4 - s ~ l f a t e ~ ~ 1+4)-P-D-GlcpA]Nat Poly[( 1~3)-P-D-GalpNAc-4SO,--( The unit cell is hexagonal, with n = b = 1.452 nni and c = 2.832 nm. Two 3(- 0.944) helices pack in an antiparallel array, in the space group P3221, with up to 16 water molecules per disaccharide residue. An intramolecular OHA-3---OB-5hydrogen-bond (0.277 nm) is possible (where A = p - ~ - G l c p Aand = p-~-GalpNAc-4SO,-).No hydrogen bond exists between the residues participating in the (1-+3)-linkage. A statistical disordering of the hydroxyrnethyl group over the g+ and g- orientations was suggested. Each disaccharide participates, once as a donor and once as an acceptor, in two intennolecular hydrogen-bonds involving OHA-2 and 0’-7 (0.264 nm). Successive, hydrated cations are tied together by 0 - - - 0 contacts, affording an infinite helix that runs parallel to the polyanion helices, and fills the large, open channel at the corner of the unit cell. The weighted R factor is 29.8%.

(67) K. Tanaka,]. Bioclzem ( T o k ! / o ) 83 , (1978) 325-327. (68) D. H. Isaac and E. D. T. Atkins, Riattirc~(London)New Biol., 244 (1973) 252-253. (69) W. T.Winter, S. Arnott, D. H. Isaac, arid E. 1). T. Atkins,]. ~$401.B i o l . , 125 (1978) 1 - 19.

10. C hoiidro i t in 4 -s u 1f h ei 0 Poly[ (1~3)-P-D-Gall,NAc-1SO:,~ -( 1 ~ 4 ) - p - I ~ - G l c / , A ]' Cand a~ Nai The sodium salt form, at Y2?4 r . h . , ci tallizcs i n a trigonal unit-cell, with ci = b = 1.45 niii and c = 2.88 I I I I I , with a 3 ( - 0.96) conforiiiation of the chain. 0 1 1 conversion of'tlic. sodiuiii form into the calcium form, the unit cell converts to the orthorlioml,ic, with (1 = O.74Fj rim, 11 = 1.781 rim, and c' = 1.964 i i i i i . 'Tlrt~ chain is a 2(0.982) helix, witli the d i saccha r i tle re peat-tin it . Tw ( ) ;I I I t ipara11e 1 ch ai 11 s are con tai 11e tl i 11 the unit cell, along with- 30 water riiolc.cules. Tlic space group is P2,212,. When the sodium form is (IOIKYI with calcium to t h e extent of N a : Ca 3 20: 1, the unit-cell tlirirt~nsionsare close to that ot'the pure calcium salt form, with el = 0.776 nni, 1, = 1.756 iim, and c = 1.953 nm. The space group, I i o \ v c . ~ ~rednces ~r, to P2,. 'The calcium h r i i i was stutlietl in detail. 7'here are two iiitrachain hydrogen-l~oiidsofthe type 0H.'-3---Oi3--5(0.264 i i i n ) and OHA-2---OH7 (0.299 nm),one direct, OH"-6---0:\-2 (0.277 i i m ) , interchain hydrogen-ljond (* = P - D - G I ~ ~and A '$ = p-~-Gall,NAc-4SO;-), and extensive hydrogen-~,ondiIig bridgcvl l)y water niolecules. Acljacent, parallel chains interact through COO ---CiPi ----OOC bridges. The hydroxyiiiethyl group is i n tht. g + orielitation. The weighted R factor is 21.6%.

V. BACTERIAL POLYSACCHA~UDES

1. Esclterichici coli Mutant h14 1 (Ref'.71)

r

(1--2)-a - u - M m p - ( l -

3)-,i -r,-Glcp -(1-3)-,1-n-GlcpA-(l-3)-a-n-Galp

t

, ' -I I -GIrp - ( 1

6,

? c

-

4 1 2 ) - a -n -Ma!@

1

/ \

-

H , C CO,H

The capsular polysacchuriclc~l i o i i i E . coli native serotype K29 and its mutants 31113 and M41 has the smie molt~ciilarstnicture. At 92% r.h., the unit cell is orthorliotiil)ic, with ( I = 2.03 11111, 11 = 1.178 nm, and c = 3.044 11111. The space g r o i i p is P2,2,2,. It was proposed that the chain confoiiiiation is a 2( 1.Fi22) helix, with hexasaccharide repeat(70) J. J. Cnel, W. T. Winter, and S. .4riiott.J. ,\lo/. B i o / . . 1% (1978) 21-42. (71) R. Moorhouse, W. 'I. Witrtcr, S. h i - t i o t t , a i i t l hl. E. Hayvi-~,/.Mo/. H i o l . , 109 (1977) 373-391.

P. I<. SUNDARAKAJAN A N D R. H. MAHCHESSAULI'

396

units. On drying, the u and h dimensions reversibly reduce to 1.73aiid 1.02 nni, respectively. Whereas the polyanions are ordered, the cations and water molecules fill the available interstices. The three intrachain and two interchain hydrogen-bonds are: OH*-3---OF-4 (0.283 mn), OHB-4---0"-6 (0.26 i i i i i ) , OHE-3---O'-5 (0,305 r i m ) , OHD4(coriier)---OF-3(ceiiter)(0.284 nin), and OHK-4(corner)---OD-4(corner) (0.273 n i i i ) , where .' = a-D-h4anp, = p-D-GIcp, ') = a-D-Galp, " = aD-Man),, = p-D-Glcp, and and x e side-chain residues. The R fhctor is 26%.

'"

''

' '

2. Klebsiellri K57 polysaccl-laritlei2 r-

I

o-n-Manp 1

1

(1-3)-0

I 4 -11

-GalpA-(l-2)-a-1,

-Manp-(l-3)-;3-n

-Gal0 ll

A 3( - 1.143) helical conformation was proposed. A complete determination of the unit-cell dimensions ant1 the lateral arrangement of the chains was not possible, due to the paucity of the X-ray data. Two intrachain hydlogerr-boiitls, of the type O H - ~ - ( ~ - D - G ~ ~ ) - - - O - S - ( ~ - D M a n p ) and OH-2-(a-~-Gal?)A)---O-3-(a-1~-Man),), were proposed. N o intracliain hydrogen-bond is possible with the a - ~ - M a nside-groiip. p

4 ) - ,i-1' -. M a n p

I1

Limited, X-ray diffraction evidence suggested that the chain is a 2( 1.52) helix, with a n uncoortlinated variation al)oiit this average conforni at ion . As the project e d he i g 11 t of the t 1-isaccharide r e pe at-mi it is 1.52 nm, the average advance p e r saccharide is 0.507 11111, siinilar to that i n cellulose and D-iixinnaii.

4. Klcbsirlln K 9 polysacch~iritlt,;'

-

3 ) - a - i - R h a p - (1-

3)-a - I - Rh.I/) - (1

4

-

-

2)- a - L -Rha p - (1

1

3) -(I- ~ - G a l p

A layer line-spaciiig of 4.13 I I I I I \V:IS ol)served. Occurrence of iiieridional reflections only on l a y e r liiicxs with 1 = 3ri, ant1 stc-1-eocheiiiical analysis, led to a 3(- 1.377) coil foimation. The intracliain 1iytlrogc.nbonds are: OH-4---0-5 in the. R h q - ( 1+3)-Rhii/1 segment, OH-4---0-5 in the Rhap-(143)-Gal/i segnieiit, ant1 OH-2---0-2 in the Galp-(1+3)Hhap segment. The iiifliieiic*t, of' HIi:tl~ oil the coiiforiiiatioii o f the chain was disciissed.

5 . Klebsielln K16 polysacclraritl(~"'

X-Ray diffraction showed n i e r i c l i o i i a l reflectioiis on the third and the sixth layer-lines. Model-l)iiiltliiig calculations led to ii :3( - 1.291) helical confoimatioii, with the tijllowiiig, iiitrachain hytlrogen-\,onds: ( i ) OH-3-(Fucp)---O-5-(GlcpA),( i i ) OH-%( Fuci))---0-2-(Clc),),ant1 ( i i i ) OH-3-(GlcrI )---0-5-(G:dp>). 6. K 1e bs i el 1(I K25 po 1y sacch a ri ( Ic.j-' (1- 3 ) - , j

-1r-C31P-(1-*4)-;3 -n-Glcp 4

t

,> - 1 )

1

- G l ~A p 2

t

1 ,,-I,

Glcp

-4 3(- 0.97) helical confomiatioii w a s proposed from X-ray data arid s te re oc hem ical analysis . T 11 c 1jiic kl I ( )11 e , intrachai n hydro ge n-bo 11d s are OH-2---0-5 in the ~ - G d p1+4)-p-11-Glcg) ( seginent and OH-2---02 in the p-D-Glcp-( 1+3)-@-11-
398

P. R. SUNDAR.4RAJAN A N D R. H. MARCHESSAUL'I

bond between the back1)one and side-chain residues. A detailed comparison of this conformation with those proposed earlier for hyaluronate and chondroitin sulfate polysaccharides was given.

7. Klehsiellu K54 polysaccharide'"

p-

6)+ -r,-Glcp -(1-

4)-a-r,-GkpA-(l-

3 ) - u- I - F u c p

4

t

1

ii -ri-Glcp

The favored conformation is a 3(1.236) helix. Left-handed helices were excluded, because of unrelievable, short contacts. In the righthanded helix, intrachain hydrogen-bonds are of the type OH-3(GlcpA)---O-5-(Glcp),OH-2-(GlcpA)---0-2-(F ~ i c p )and , OH-3-(Glcp)--0-5-(Glcp).No intrachain hydrogen-bond could be fonned to stabilize the (1+6)-linked residues.

8. Klebsiella K 6 3 polysacchari~le~~ Poly[ 1+3)-a-D-Gd11-( 1+3)-a-D-GaIl1A-(~ + ~ ) - ~ - L - F U L ~ I ] The unit cell is monoclinic, with u = 1.025 nm, b = 1.210 nm, c = 2.37 nm, and y = 108.82". A e(l.185) helical conformation was derived. Intrachain hydrogen-bonds of the type OH-2-(Fucp)---O-2(Galp) and OH-2-(GalpA)---0-5-(Galp) were proposed. 9. Klebsiellu K 5 polysacchari~le~~ (1-3)-p

[

-u-Manp-(l6,

H,C

4)- p-n-GlcpA-(l-4)-fi

-n-Glcp

P C

/ \

CO,H

1.

OAc

?I

The sodium salt foim of the Klehsiella K 5 polysaccharide crystallizes in a 2(1.35) helical conformation. The intrachain hydrogen-bonds are: (i) OH-3-(GlcpA)---0-5-(Manp), ( i i ) OH-3-(Glcp)---O-5-(GlcpA), and ( i i i ) OH-2-(Manp)---0-2-(Glcp). On deacetylation, the conformation of the chain is unchanged, although the intensity and radial spacing of the reflections differ. This indicates that deacetylation causes changes only in the packing of the chains.

(75) L). H. Isaac, K. H . Gardner, C . Wolf-Ullish, E. 11. T. Atkins, and G. G. S. Untton, Znt. J . Biol. Macromol., 1 (1979) 107-110.

BIBLIOGRAPHY O F CRYS'1'4L STRUCTURES

10. Xunthonionas poly~accharide'"~~'

1

(1-4)-,i-u-Glcp-(1-4)-~~-1i-Gl~P

13 -D-Manp-(l-

6

H,C

\ I

4 ) - ii - 1 , -Glc p A - ( l

4

-

2 ) - a -n-Manp

C

6 I

3 99

I,

OAc

I \

C02H

Po 1y s accharide s fro in bo t 11 X (I ii t 11 o i i i o n(is p hu seo 1i and X u I I thoinonus c a m p e s t r i s crystallize with a repeat of4.7 nm.A S(0.94) helix is the favored confomiation, and a hexagonal unit-cell with u = h = 2.13 nm and c = 4.7 nm was chosen. The intrachain hydrogen-bonds are: OHA-3---OR-5, OH*-2---(jE-8, ()H*-6---OD-5, OHR-2---0'-7, OH'3---OA-6(or OH'-3---OD-5), and OH"-2---OD-6, where * = P-D-Glcp, = p - ~ - G l q iwith branching, = a - ~ - M a i i p ,I) = P - D - G ~ ~ ~ and A, = P-D-Manp. ('

VI PEPTIDOCLYCAN 1. Bacillus liclieniforrnis Peptitloglyca~i'~ 71 Poly[ (1+4)-P-~-Glq,NAc-(1 - + 4 ) - P - ~ - M ~Nr Ac] The polysaccharide from the cell walls arid extracted peptidoglycans froin the organism Bacillus licheiiiformis show a meridional reflection with a spacing of 0.98 nni at 1" and 100% r.h. Three lionoriented rings with spacings of 0.977 n m , 1.308 mi, and 1.892 m i , observed at 100% r.h., changed to 0.95 mn, 1.28 m i , and 1.82 nin, respectively, after drying. Coiifori~iationalanalysis and optical clif'fi-action led to a 4(0.98) helix, where chitoliiose is the asyminetrical unit.

(76) R. Moorhouse, h l . 1).Walkinsliaw, m l t l S. Arnott, i l l P . A . Santlfortl antl A. Laskin (Eds.),Ewfrcicellular Buctericil Pol!/.ccic.c.licirir/es,A J I LC. / I V J ISOC. I . S ! / i f t j i . Sc,r., 45 (1977) 90-102. (77) R. Moorhouse, M.D. Walkinshaw, W. l'. Winter, antl S. Arnott, i n J. C:. Arthur, Jr., (Ed.),Cellulose Chefitisfryuirt/ ' l ' w / i f i o / o g ! /A, m . Chcffi. Soc. S ! / f J l / i , S e r . , 48 (1977) 133- 152. (78) H. E. Burge, A. G. Fowler, atid 1). A. Rravrlley,J. Alol. B i ~ l . 117 , (1977) 927-5153,

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AUTHOR INDEX FOR VOLUME 40 Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred to although his name is not cited in the text.

A Abe, Y., 87 Achmatowicz, Jr., O., 38, 39, 41, 42, 44(138), 51(138), 60(138), 65, 66, 67, 68, 69(217), 70, 71, 72, 113(227) Acree, S. F., 72 Acs, C., 331 Ada, G . L., 198 Adachi, M., 284 Adair, W. L., 289, 290(9, lo), 293, 323(34) Adamany, A. M., 289, 293(7), 324(7), 348(7) Adamo, S., 297, 298(56) Adamowicz, H., 44 Adams, E. P., 249 Addor, R. W., 84 Adrian, G. S., 290(15), 291 Agneray, J., 153, 157(121), 158, 159(121, 134), 180,232 Ahmad, F., 218 Air, G. M., 198 Akamatsa, Y., 347 Akiyoshi, T., 228 Albers-Schonberg, G., 324 Albert, A. W., 324 Alhadeff, J . A., 193 Alitalo, K., 367 Allan, A., 218 Allen, C. M., 290(24), 292 Allingham, R. P., 65 Amano, R., 276 Ambrose, E. J., 228 Aminoff, D., 153, 154(122), 155(122) Anderson, R. G. W., 364 Anderson, R. S., 297, 298(57) Anderson, L. C., 174(201), 175, 340 Ando, S., 200, 240 Ando, T., 26 Andrade, A. F. B., 136, 176(26)

Andreae, M., 277 Andres, K. H., 171, 172(182), 173(182), 174(182),222, 223(484) Andrewes, C. H., 229 Angelino, N., 194 Angerbauer, R., 49 Ankel, H., 231 Anker, D., 44 Anker, M., 31 Ansell, N. J., 352 Antonova, N. D., 37 Anwer, U., 302(105), 303, 370(105) Aplin, J. D., 170, 171 Appel, M., 180 Appel, S. H., 194 Aquino, D., 219 Araki, Y., 2, 121, 339,340(330) Aranda, G., 122 Aranha, G. V., 227, 228(513) Arce, A., 255,257,258,263,264, 265 Ariga, T., 279 Arikawa, J., 210 Arima, K., 339, 340(322) Amold, D., 317 Amon, R., 270, 277(168) Amott, S., 381, 393, 394, 395, 399 Arshova, R. M., 12 Asai, M., 10 Ascari, E., 222. 225 Ashwell, G., 190, 220, 270, 352 Atkins, E. D. T., 381, 389, 390, 391, 392, 393,394,396,397,398(74) Aubert, J. P., 353,354(422) Auge, C., 309 A d a , P., 138, 147(38) Auria, M. D., 72 Austen, K. F., 224 Autio, S., 138, 147(38) Au Young, Y.-K., 112 Averame, M. M., 345 401

402

AUTHOR INDEX, VOLUME 40

Awasthi, Y. C., 270, 274(169), 277(167), 278(167) Aymard, M . , 203 Ayoub, E. M . , 196 Ayusawa, D., 377 Azliar, S., 230

B Babezinski, P., 296, 309(51), 310, 311, 343, 349(352) Bach, G., 207, 273, 278 Bachhawat, B. K., 177, 269 Bachrnayer, H., 198 Baer, H. H., 107 Baerlocher, K . , 206 Bagshawe, K . D., 226 Bahl, 0. P., 194 Baig, M. M., 196 Bailey, D. S., 300, 301(78), 318 Bain, A. D., 274 Baker, C. J., 206 Baker, R. M., 313 Baker, S. R., 266 Baldassare, J. J , , 323 Balduini, C., 222, 225 Balduini, C. L., 222, 225 Baldwin, M. J., 31, 40 Ballou, C . E., 299, 368 Balsley, R. B., 12 Baltimore, D., 351, 373(398) Banaszek, A,, 41, 42(123), 44(123), 46(l23), 47, 48(146), 49( l46), 50(146), 51(146), 5 4 , 5 6 , 57, 58(180, 184), 72 Bang, F. B., 229 Bardos, P., I94 Barenholz, Y., 203 Bargigli, V., 221 Barnett, J. E. G., 104 Barrett-Bee, K., 337 Barry, G. T., 142 Barszczak, B., 66 Bartnichi-Garcia, S., 332 Barton, H., 71 Bascliang, R., 133 Bassin, R. H., 266 Basu, M., 247, 248, 250, 252, 253

Basu, S., 192, 246, 247, 248, 249, 250(46, 83), 252, 253, 254, 255(108), 256, 257(46), 258(111), 259(108), 260(108), 264(108),265(108, 112), 266( 108) Bauer, C., 215, 227(419), 231(419) Railer, F., 316 Bauer, s., 327, 329, 332, 337 Baumann, N., 139, 144(50) Bauineister, L., 134, 153(23), 154(23), 155(23) Baumgarten, W., 115 Bause, E., 310, 312(156) Banx, G., 217 Baxter, A,, 193, 226 Bayer, E. A,, 171 Bayer, M. E., 395 Bazarian, E. R., 210, 211(405) Beau, J.-M., 146, 149, 170, 180(176), 185(238), 186(252), 187(252), 202(260), 209, 210(113), 211(113) Beaufay, H . , 293 Becher, P., 84 Becht, H., 372 Becker, B., 62 Beeley, J. G., 226, 353 Beevers, L., 300, 301(79),303(79), 3 18 Behrendt, H., 227 Behrens, N. H., 245, 315, 322 Bekesi, J. G., 228, 335 Bt.lisle, M., 208 Beljanskaja, G . K., 149 Beljean, M., 153, 157(121), 1 5 9 ( l 2 l ) , 210(114) Bell, F. B., 346 Bell, J. J., 323 Bell, N. H., 350 Belleau, B., 112 Bellelli, L., 227 Belniak, K., 4 1 Belocopitow, E., 290(18), 291 Bendlin, H., 24, 25, 115(52) Bennet, V., 351 Bentley, R., 164 Ben-Yoceph, Y., 276 Beranek, W. E., 189, 193(267) Berent, S., 283 Bergman, A., 316, 321(1883 Bergman, L. W., 319 Berman, B., 374

403 Benracki, R. J., 193. 194, 321, 336(2:34), 337(234, 3041,338 Benistein, G. S., 366 Uertistein, XI. A , , 171 Aeri-y, L. R., 221

Bcrti, G., 33, 43, 571141) Bewtrpoii, F., 231 B r u r l , E. X I . , 337 Bc.\ttn;cnn, €1. J., 115 Brttclheim, F. A , , 151 Rc~ttncr,J., 369 13cntler, E., 270, 277(167),278(1671, 279,

28O( 170) Heii\er>, E. C., 167 Avpc,r, T. A,, 189 HIlar,qa\.a, A. S., 141. 142166) Bll?tlg?c\a , G., 377 Allat. 1’. I.,, 297, 298156) Ahrtt:icIi;irjee, A . K., 139, 166. 169, 170(166, 1671

Uli;tttucll:try?;a, B. K., 181 Bll~tttncharyya,s. P., 181 Blr>c\a n a n d a n , 1’. P., 175, 205, 208 Rlroyroo, 1’. U., 289, 293(6), 299(6),301, 003(6),307, 308(86, 135), 348(6) Bial, XI., 154 B i e d l , A., 170 Bielinska, M., 360 Biclski, H., 71, 72, 113(227) Riel?;, €’,,327, 329, 337(286), 349(286) Bieri. J. G., 298 Bighouse, K. J., 187, 211(262) Billian, A , , 374 Bilow, A , , 46, 47(155j, 57(155) Biirette, J. P., 169 Binklep, S. B., 212 Bischoff, E., 336 Bisette, G., 138, 142(37), 353 Bislrayee, S., 175, 269 Bittigel-, H., 390 Black, 11. E., 366 Blacklow, H. S., 177 Blackwell, J., 382, 387, 392, 393(61) B l d i e r , D., 222, 226 Blanchette, L. B., 3.58 Blau, E. B., 227 H l i s , G., 132 Blot)el, G., 319, 3.57(215, 216) Bloch, A,, 101, 323 Hloch. H., 269

M.,199, 202 H l o k , J., 198 l 3 l l ~ o n l ,B. hl.. 62 13lotigh, H . A , , :I69 Rliihm, T. L., 3x5. 386, :390 Ihhek, XI., 101 Hocci, 1’.,221, 352 Blollnr,

I3otlansky, A , . 3 4 2 Hotl;insk>,, [ I . . 0.42 Bodo, G., 209 H o l i n i . P., 13.1. 153(231, 1.9, 155(2:3I Ihiei-, P., 293, ,307, 311, :316, :326(19l) Hog;itko\, S. L V . , .37 k3ognBr, K., 7:3. 74 lill~golnolevil.L’, I.. 63 13oiine, I., 360 Rolognesi. D. P.,370, 371 ~ o o n D. , Y., 296. 297(52),30Y(ij2), 34 1,

3Gq3-11) B o o n e k a m p . 1’. M,,167 Booy, F. P., 384, 391 Bornstein, l’.. 365, :375. 376(,541) Borst, P., 280 Bortfeld, K., :372 Boss, E., 62 Horichillotiu. S., 307, 312 13orldeulle, X l . , 389 Borllan, E. H., 373 Horirillon, H . , 112 Uotirsnell, J . (:.. 218 Bowen, D. \I., 274 Howles, D. J., 3 1 8

HI-adley, R . M . , 26.5, 274 Hradshaw, 11. A., 312 13ratly, R. O., 197, 203, 2.30, 231(54 11, 256, 257(113), 258, 265, 266(1.13), 269, 270. 272( 1601, 274, 277, 280, 28 1 Brairlorsley. C.- 266 Rrandsiitn;i, L., 3 0 Braun, G . , 4, 5 l3l->lLln,P.. 248 Breer, H., 138 Brcnkert, A , 248 Breslow, J . I , . , :32:3 Bretscher, hl. s., 360 Brrtt, C. T., :317 Bretthaner, H . K., 297 Brew, K., ,312. ,‘33S

404

AUTIlOR INDEX, \'OLUME 40

Brey, W.S., Jr., 157, 167(132) Briggs, P. A , , 218 Briles, E. B., 228, 313 Brimacotnbe, J. S., 35 Brodbeck, U., 219 Brooks, D. E., 170 Brooks, S. E., 253 Brossrner, R., 133, 167(15),170, 178, 195(15), 198, 199, 205, 209, 210 Brouwer-Kelder, B., 280 Brovelli, A , , 222 Bi-owder, S. K., 300, 301(79), 303(79) Brown, A . E., 181 Brown, D. M., 216 Brown, E. B., 157, 167(132) Brown, 1.1. C . , 56 Brown, M . S., 323, 364 Brown, R. K., 31(75),32, 33, 35, 36(65, 77),40(84), 41, 42, (65, 76, 84, 8.51, 44(65, 76, 126, leu), 45, 49(151), 50(151), 51(151), 55, 57(126, 160, 1611, 58(160, 161) Brunetti, P., 212 B w h a n a n , J. G., 45 Brick, C. A,, 352, 375 Budtfecke, E., 221 Briddrus, J., 83, 109 Biichsel, R., 221, 338, 347(316),349(316) B ~ c k i n g ,H. W., 199, 204(337) Bugge, B., 307, 326 Bukowski, P., 65, 66, 68, 71, 113(227) Huleon, A,, 391 Bundle, D. H., 139 Burge, R. E., 399 Burger, M . M., 269 Burgoe, J., 314 Burke, D., 302(10:3),303 Burke, J., 300, 301(78) Burlinglianr, W. J., 336 Brirnet, F. .M., 229 Biirrone, O., 316 Burton, R. M., 237, 244, 259, 26.3 Burton, W. A , , 290( 17, 251, 291, 292 Biirzynska, M . H., 67, 71 Riisclier, H.-P., 142, 143(74), 145(74), 146(74), 151(74), 156(74), 158(74), 162(74),163(74), 164, 165(146), 179(74), 181, 186(249), 192(249),205, 208(368) Bush, C . A., 170 Buss, D. H., 44

Britclrard, C . G . , 336 Britlerov, A., 2 Butterfield, D. A , , 171 Butters, T . D., 302 Britterwortli. I., 274 Butterwoi-th, P. 1-1. W., 290(19), 291, 3 15(19) Brizila, L.. 351, 354(407)

C C d ~ e z a sJ. , A , , 141, 142(70),143(70) Cwuii, R., 292, 317(27),325(27) Cacl, J . J., 395 Caesar, R., 207 Calran, L. D., 229 Calrri, M., 40, 41(107), 42(107), 44(107) Calldrarr, J . W,, 276 C;inrpl)ell, A,, 351, 373(398) C;tntell, K., 374 Cantz, M., 207 Capeari,J., 231 &q~ek,K., 107, 120 C;:ipkov2i, J., 120 Caplan, A. I., 138 Caputto, H . , 247, 255, 257, 258(118), 263(118),264, 265, 267 Carey, D. J . , 186 Carllmrg, L., 180 Carlo, P. L., 296, 297(47), 309(17),343, 349(35I ) Cnrminatti, H., 316, 319 Caroii, M., 231 Carret, G., 31 Carson, D. D., 292, 324, 348(248), 367(248) Caitci-, 1I . E., 236 Carter-, w. A . , 374 Cariilxlli, R . , 205, 206, 208 Carver, J. l'., 176 Casals-Stenzel, J., 142, 143(74), 145(74), 146(74),l,51(74),156(74), 158(74), 162(74),163(74),164, 165(146), 179(74),1x1, 183, 186(249), 192(219, 2.50) Castellricci, N. T., 3 0 Catelairi, C . , 33 cellllel-, W. u., 50 Ceri, H., 313 cernp, hl., 3 , 42

AUTHOR INIIEX, VOLUME 40 Cerottini, J. C., 378 Cestaro, B., 203, 205, 217 Chada, K. C., 374 Chadwick, C. M., 318 Chambers, J., 310 Chambers, J. P., 282 Chandler, L. H., 345 Chang, N.-C., 178, 181(227) Chantler, E., 194 Chanzy, H., 384,387,389,391 Chapman, A,, 295(117, 119-El), 304, 305(117),307(117),313(121), 314(119), 333(119, 120, 121) Chareire, M., 31, 44 Chargaff, E. J., 202 Chautemps, P., 27 Cheetham, 1’. S. J., 274 Chekareva, N. V., 146 Chen, J., 324 Chen, H. W., 323 Chen, W. W., 301, 307, 308(89), 310(126),326(148) Cherry, R., 105 Chester, M. A., 138, 147(38) Chia, G. H., 377 Chiao, Y. B., 282 Chidambareswaran, P. K., 387 Chien, J.-L., 240, 242(26),243, 247, 249, 250(46, 83), 252, 253, 254, 257 Chindemi, P. A., 221 Chiu, J., 227 Chizhov, 0. S., 146, 165(98) Chmielewski, M., 17, 18(37, 38), 39, 40, 41, 42, 44(139), 48(157), 49, 51(139). 54, 56, 57(179), 58(182) Choi, S., 222 Chojnacki, T., 290(22, 23), 291, 292, 293, 294,316 Choppin, P. W., 229 Chretien, M., 358 Christman, J., 331 Chn, F. K., 354 Cifone, M., 313 Claffey, W. C., 382 Clamp, J. R . , 164, 200(144), 208(144) Clark, C., 275 Clarke, J. T. R., 279 CI~USOII-K&IS, N., 61, 62, 72, 73 Clanssen, U., 59, 60(185) Clav. hl. G.. 172 Cleiand, W. W., 247

405

Clewe, T. H., 215, 219(425) Coates, S. W., 187 Cotlington, J. F., 140, 215, 229(416), 230(416), 351 Coffee, C. J., 282 Cohen, G . H., 370 Colien, M. M., 273 Cole, L., 249 Collins, J. M., 194 Colrnan, P. M., 35.2 Colvin, J. R., 344, 388 Comb, D. G., 133, 177, 186 Coinpans, R. W., 302(106), 303, 335, 356, 358,373 Conklik, R., 336 Content, J., 334 Conzelmann, E., 271, 277(178), 278(178), 284 Cook, G . M. W..216 Cook, T. M., 195 Cooke, K. B., 145, 173(92) Coolbear, T., 307, 321(124) Coolman, R. W., 323 Corfield, A. P., 138, 140, 142, 144(77), 145(77), 146(33), 147, 148(106), 149(55), 150(55), 155(141), 161, 162(77), 163(141),164(141), 166(141),171, 172(141, 182), 173(92, 182), 174(182),179, 180(233), 183(33), 184(33), 186(33), 192(250, 257), 196(55),197(55),198(320,324), 199(55),200(338),ZOl(55, 115), 202( 115), 203(77), 204(359), 205(55, 364), 206, 207(55),208(55, 1411, 209(115, ,324).210(112, 113), 211(113),212, 213(233),215(106), 222, 223(483), 225(483) Cornl)lath, M., 265, 266( 143) Cornforth, J . W., 132 Cornillot, P., 222, 226 Cortesi, S . , 222 Costantino-Ceccarini, E., 248, 265(63) <:ottalasso, D., 345 C o r i r i , D., 345 Chtttenay, V . l l . , 337 Coiiitney, R. J., 370 Cortrtois, J. E., 2116 Coyle, P., 282 Crmnipen, M., 195, 196(311) Creely, J. J., 388 Crtegee, R., 84

406

AUTHOR INDEX, VOLUME 40

Crine, P., 358 Crum, F. C., 296, 297(52), 309(52) Crun, C. F., 341, 342(341) Cuatrecasas, P., 198, 269, 351 Cuccione, M. A., 222 Cullen, S. E., 352 Culling, C. F. A,, 145, 170, 171(91), 172, 173(91) Cumar, F. A., 256,257,258,266 Current, S., 54 Currie, G. A., 226 Currie, S., 324 Curtino, J. A., 247 Curtis, C. A. M., 339, 348(321) Czarniecki. M. F.. 169, 202 Czichi, U., 317

D Da’Aboul, I., 35 Dain, J. A., 134, 136(19), 137(19), I38(19), 139(19), 140(19), 142(19), 143(19),203(19), 256,257(115), 258, 259 Daleo, G. R., 290(12-14), 291, 292(12, 13), 196, 311(46), 317(46), 318 Dales, S., 372 Dallaire, L., 208 Dallner, G., 290(23), 292, 315, 316, 32 1( 188) Damsky, C. H., 375 Dance, N. E., 274 Dankert, M. A., 317 Danon, D., 147, 148(106), 171, 215(106) Damell, J. E., Jr., 351, 373(398) Das, N. D., 219 Das, R. C., 509 Datema, R., 295(122),304, 306, 307(116), 310(116), 321, 322(116), 326(228, 237), 327, 328(228, 266), 329(273, 275), 330(228,237,275,276),331, 332(116, 266), 333(122),334(266), 335(266), 337(116), 339(228), 346(122),249(116, 266, 275, 276) Daub, J., 84 Dauber, S., 134, 153(23),154(23), 155(23) David, J. R., 232 David, S., 37(350), 52(355, 356). 53(356), 117, 122, 124, 125(95,352, 353), 126(353), 127

Davidson, J. M., 365 Davies, M . A,, 217 Davis, L., 196 Davy, J., 180 Dawson, G., 164, 192, 194(282), 200(144), 203, 205(355), 208(144), 259, 347 d’Azzo, A,, 207 De Adhikari, H., 181 Dean, K. J., 280 Dean, L., 154, 154(127) de Belder, A. N., 167 Debruyne, E., 194 Decker, K. F. A,, 336,338(299) de Groot, P. G., 363, 364 Deisenhofer, J., 354 Dejter-Juszynski, M., 240 Delcroix, C., 221 Delgadillo, R. A., 373 Delmer, D. P., 317, 344 Delmotte, F., 175, 176(206) De Luca, L. M., 297, 297(56), 316 De Luca, S., 138 Den, H., 250 DePierre, J. W., 215 Desai, P. R., 228 DeSandre, G., 222 Descotes, G., 39,40,41(107),42(107), 44(107) Descours, D., 31, 44 Deslandes, Y., 389, 390(49), 391 Desmyter, J., 374 Desnick, R. J., 279 DeSomer, P., 374 de Staritzky, C., 334 Destree, A. T., 375 DeThomas, M. E., 316,321(188) Deuel, H. J., Jr., 238 Deutsch, E., 193 DiCesare, J. L., 256, 257(115),270 Dickerson, J. W. T., 265 Dickinson, H. R., 170 Dickson, J. J., 208 Diggelmann, H., 371,373 DiMatteo, G., 280 Dipple, I., 345 Dittmer, J., 247 Dixon, J. F. P., 232 Dlugosz, J., 392 do Amaral-Corfield, C., 197, 198(320) Dohherstein, B., 319, 320, 357(215, 216)

A U T H O R I N D E X , VOLUME 40

Ddbereiner, J. W., 60 Doerr, N., 21 Doi, O., 347 Dijrrscheidt-Kiifer, M., 216 Donath, E., 215 Donker-Koopman, W. E., 280 Dorai, D. T., 175 Dorfman, A., 158,259,344 Dorland, L., 133, 139(18), 144, 145(17), 146(17), 157(17), 158(90), 167(16, 17), 168, 169(17), 170(18), 179, 180, 183(90), 187(18), 195(16, 17), 202, 204, 205(364), 2 l l ( l 8 ) , 268 Dorner, F., 374 Downs, F., 174 Draper, P., 177 Dreyfus, H., 208, 255, 265 Drurnmond, K. N., 227 Dryburgh, II., 362 Drzenick, R., 195, 196, 197(307), 199(307, 313),202, 271 Dube, M., 389 Duda, E., 358 DufTkird, R . O., 258, 267 Durr, M., 300, 301(78), 318 Dukes, P. P., 221 Duksin, D., 341, 365, 370, 375, 376(541j Diimont, J. P., 180 Dunn, W. L., 172, 173 Duran, G., 153, 157(121), 159(121) Durand, D., 180 Durand, G., 158, 159(134), 180, 232 Durand, P., 138, 207(40) Durham, J. P., 193 D’Urso, M., 280 Dutcher, J. D., 46 Dutton, G . G. S.,398 Dweltz, N. E., 382, 387(22) Dyong, I., 15, 17, 21, 23, 24, 25, 114(46), 115(52)

E Eagon, P. K., 361 Earl, F. L., 293 Ebert, W., 232 Ebisu, S., 175 Eckardt, K., 339 Edel-Harth, S., 265 Edo, H., 115, 117(331)

407

Edwards, K., 362 Edwards, S. A . , 371 Edy, V. G., 374 Egami, F., 252 Eg;in, W., 139, 144(47j, 233(47) Ehrlich, K., 141, 149, 187(72),211, 212(408),213(408) Elrrlich, P., 153 Eichberg, J., Jr., 177 Eichhorn, J. H., 345, 346(364) Eisenberg, R. J., 370 Eisenmann, R., 373 Ekblom, P., 367 Ekstrom, T., 290(23),292 Elbein, A. D., 290(35), 293, 295(35), 296(35), 299(35), 304, 307(35), 309(35, S ) ,310(35), 311(152), 321(35), 326, 339, 341(329), 342(134, 340), 343(134, 347), 344(350), 348(35, 350), 378 Elliot, W. H . , 324 Elloway, H. F., 396, 397, 398(74) Elming, N., 61 Elsasser-Beile, U., 396 Eltiiig, J. J., 301, 308(89) Elwing, €I., 230 Einmelot, P., 227 Endo, A , , 323, 324(240) Eng, L. F., 253 Engen, R. L., 181 Eppler, C . M., 192, 194 Erhardt, U., 84 Erickson, J. S., 282, 283(269) Ericson, M. C . , 293, 317, 326, 342 Eschenfelder, V., 170, 205, 206 Esgate, J. A , , 345 Etchison, J. R., 301, 302 Errstache, J., 37(350), 124, 125(95, 352, 353), 126(353) Evnngelopoulos. A. E., 212 Evstigneeva, N. A , , 202 Eylar, E. H., 351 Ezepchuk, Y u . V., 149, 210(114), 230

F Faillard, H., 132, 141, 142(69), 144, 145(89), 148(69), 155(89, log), 156(89),160(89),172(89), 178, 179(226), 182(226), 199, 200(89),

408

AUTHOR INDEX, VOLUME 40

202(89), 215, 217(415), 218(415), 219(415),227(415), 229(415) Fakstorp, J., 72 Falcoff, E., 374 Fan, H., 371 Farkas, V., 302, 329(98) Farquhar, M. G., 171, 215 Farriaux, J.-P., 138, 142(37), 143(34), 146(34), 147(34), 163(34), 165(34), 179, 185(34), 207(40), 209(34) Fartaczek, F., 317 Fearon, D. T., 224 Feather, M. S., 61 Feeney, R. E., 351 Feger, J., 153, 157(121), 158, 159(121), 134, 180, 232 Fehr, J., 226 Feix, J. B., 171 Feldman, J. D., 346 Felsenfeld, H., 177 Fenger, C., 172 Ferreira do Amaral, C., 138, 146(33), 158, 183(33), 184(33), 186(33), 187, 191(258), 199 Fiat, A.-M., 168 Filipe, M. I., 145, 172, 173(92) Filipovic, I., 221, 364 Finne, E., 221 Finne, J., 166, 189 Fiorilli, A., 308 Firestone, G. L., 358 Firth, M. E., 132 Fischer, E., 2, 115, 232 Fischer, G., 281, 282 Fischer, J. B., 290(21), 291, 315(21) Fischer, K., 206 Fischer, W., 327 Fishman, P. H., 230, 231(541), 256, 257(113), 258, 265,266( 143), 270 Fitzpatrick, J. P., 373 Fitzpatrick, P., 230 Flashner, M., 175, 206, 209, 210 Fletcher, P., 228 Fletcher, T. C., 176 Flickinger, C. J., 345 Flippen, J. L., 170 Flowers, H. M., 140,240,269, 277 Floyd, W. C., 79 Folch, J., 247, 271 Fondy, T. P., 336 Fontaine, G., 138, 142(37)

Fontaine, M., 221 F o o d , S., 392 Forsee, W. T., 310 Foster, A. B., 333, 337 Fournet, B., 138, 168, 169, 207(40) Fowler, A. G., 399 Franco, M., 222,223(483), 224, 225(483) Franke, P., 290(22), 291 Franke, W. W., 357, 390 Fraser, I. H., 194 Fraser, M. M., 27 Fraser-Reid, B., 31 Frednian, P., 230 Fredrickson, D. S., 275 Frelinger, J. A,, 232 French, A. D., 381, 382, 386(21),388 French, E. L., 198 French, W. C., 194 Freund, G., 345 Friebolin, H., 133,167(15), 195(15) Friedman, G., 378 Friedman, S. J,, 335, 346 Friege, €I., 15 Fries, E., 347, 357(386) Friis, R. R., 370, 373 Frisch, A,, 367 Frohwein, Y. Z., 272, 277(188) Fronza, G., 117 Frost, R. G., 276 Frot-Coutaz, J., 298 Fuganti, C., 117, 128 Fiijimoto, K., 295(119), 305, 314(119), 333(119) Fiijino, Y., 248 Fujisawa, J., 374 Fukami, €1., 108(312), 109 Fukui, Y., 366 Fung, C., 377

C Gabrielyan, N . D., 191, 193(278) Ggrtner, U., 220 Gafford, J. T., 293, 326, 339, 340(329), 342 Gahmberg, C. G., 174(201), 175, 340 Gainer, H., 358 Gal, A. E., 277 Galjaard, H., 207

409

AUTHOH INDEX, VOLUME 40 Galli, G., 240 Ganibella, G. R., 34.5 Gander, J. E., 321 Ganzinger, U., 193 Garcia, J. H., 265 Gardas, A,, 240, 242(26) Gardner, D. A., 249, 250(83), 253 Gardner, K. H., 382, 392, 393(61),396, 398 Gamer, A., 218 Garnier, M., 226 Garoff, H., 320, 360 Gateau, O., 192, 315, 316 Gatt, S., 203, 272, 273, 274(186), 277(188), 281 Gattegno, L., 222,226 Geddis, L. M., 345 Gee, S., 229 Gentinetta, R., 219 Geoghegan, P., 56 Georgias, L., 253 Germain, M. J., 232 Gerok, W., 215, 227(419), 231(419) Gerrie, J., 276 Gesner, B. M., 221 Geyer, G., 174 Ghalambor, M. A , , 186 Ghidoni, R., 139, 144(50), 217, 240 Ghiotto, G., 222 Gibson, R., 220, 355, 356(429),371(427) Gieger, B., 270, 277(168) Gielen, W., 141, 217 Gies, D., 202 Gillies, D., 193 Ginsburg, D., 62 Ginsburg, V., 176 Githens, S., 216 Giuntoli, R . L., 369 Glant, S., 168 Glaser, G. H., 178, 181(227) Glasgow, L. R., 230 Glattfeld, J. W. E., 5, 6(17), 7 Clew, R. H., 282,359 Glick, J . L., 216 Glick, M . C., 140 Glickman, I., 209 Glittenberg, D., 21, 114(46) Gluzinski, P., 67 Godelaine, D., 293 Goebel, W. F., 142 Gohring, K., 59, 60(185)

Goikhman, A. Sh., 388 Goldfine, H., 324 Goldman, J. E., 284 Goldstein, I . J., 175 Goldstein, J. L., 323, 364 Chrdon, I. L., 232 Cordon, L. M., 345 Gosh, H. P., 320 Gossard, F., 358 Cosstrau, R., 205 Got, R., 142, 298 Chto, T., 391 Chttschalk, A,, 132, 134, 137(21),138, 139(42), 141(21),142(21, 66), 143(21), 153(21), 154(21), 157(21), 179(42), 195, 196, 1991313). 202(313). , 218, 350, 352(388),354(388), 357(388) Chugh, D. P., 289 Gowland, G., 227 Grage, T. B., 227, 228(513) Graham, E. R. B., 141 Grange, D. K., 289, 290(9) Granier, C., 312 Grant, G. A . . 312, 360 Grant, P. T., 176 Grasmuk, H., 178 Grasselli, P., 117, 128 Graircob, E., 207 Graves, M., 248, 249(65) Gray, G . M., 249 Grazi, E., 212 Greenberg, J. P., 222 Greene, B. B., 66 Greene, R. M., 344, 368(354) Gregoriades, G., 352 Gregory, W., 228, 295(121), 305, 313(121),333(121) c.ieineder, . D. K.. 232 Grennon, M., 303 Griffin, J. A,, 310 Grimes, W. I., 317 Grinna, L. S., 301, 302(108),303 Grisebach, I-I., 21 Grob, E., 7 Grob, P. M., 374 Grozinger, K., 75, 77(249) Grynkiewicz, G., 65, 66, 69, 70, 71, 72, 128(215) Guenounou, M., 232 Giiizard, C., 391 I /

'

AUTHOR INDEX, VOLUME 40

410

Gunnarsson, A., 227, 228(513) Gurney, T., Jr., 187

H Haas, J. E., 227 Habener, J. F., 350 Habets-Willems, C., 307 Hadfield, A. F., 181 Haegert, D. G., 228 Hager, L. P., 351 Hahn, H., 142,221(80) Hajra, A. K., 274 Hakomori, S.-I., 138, 199, 238, 240, 243, 249, 252, 253, 260(28), 266, 279(76) Haksar, A., 230 Haleem, M. A,, 390,392 Hall, L. D., 44, 170, 171 Halliday, J., 216 Halliday, N., 238 Halpern, B. L., 369 Halpem, M. S., 370, 371 Hamanaka, S., 139 Hamers, M. N., 280,364,563 Hamill, R. L., 374 Hammarstrom S., 247, 248 Hammond, K. S., 156 Han, T., 229,232 Hanada, E., 270,274(169), 283 Hanafusa, H., 305 Hancock, K. W., 227 Handa, S., 139, 141, 143(71), 176(71), 259,263,279 Hanover, J. A., 2%,320(45) Hansen, U., 325 Hanson, V. A,, Jr., 201 Happey, F., 389 Haq, M. Z., 84 Hara, M., 344 Harding, S. E., 216 Hardy, S. W., 176 Harford, J. B., 293, 309, 310(147) Harpaz, N., 240, 269, 301 Harris, E., 324 Harris, J. F., 61 Harris, P. L., 204 Harrison, F. W., 171 Harrison, S. C., 353 Hart, G. W., 312, 341, 366(339)

Harth, S., 208, 255 Hartree, E. F., 218 Hascall, V. C., 138, 366 Hasegawa, A., 139, 141, 143(71), 176(71) Haselbeck, A,, 296, 309(5I) Hashimoto, K., 347 Hasilik, A., 277, 278, 311, 362, 363, 372(468, 469) Hatton, M. W. C., 221 Hauser, G., 247, 248(51), 249(66), 258(51) Havell, E. A., 374 Haverkamp, J., 133, 138, 139(18), 141, 143(34), 144, 145(17), 146(17, 34), 147(34), 156(93), 157(17, 93), 159, 160(93, 97), 163(34, 97), 164, 165(34, 97, 139, 147), 166(139, 147), 167(16, 17, 139), 169(17, 93), 170(18), 179, 180, 185(34,238), 186(252), 187(18, 72, 252), 195(16, 17),209(34), 211(18) Hay, G. W., 38,42 Hay, J. B., 249 Hayakawa, Y., 80 Hayashi, K., 106 Hayman, M. J., 373 Heath, E. C., 186, 309, 346, 358, 359, 361 Hechtman, P., 283, 284 Hegde, U. C., 227 Heifetz, A., 296, 309(53), 341, 342(340), 367 Heimer, R., 177 Heinz, R., 46,47(155), 57(155) Hellerqvist, C. G., 249, 279(76) Helsper, J. P. F. G., 318 Helting, T. B., 230 Hemming, F. W., 288, 289(1), 290(1, 3, 19), 291, 292(3), 293, 307, 311, 314, 315(19), 316, 321(124, 188),322(3), 328(3) Ilemminiki, K., 189 Henderson, W., 337 Henneberry, R. C., 270 Hensens, O., 324 Hentschel, H., 216 Herbertz, L., 223 Herczegh, P., 73, 74 Hermans, P. H., 388 Herp, A., 174, 218 Hers, H. G., 269

AUTHOR INDEX. VOLUME 40 Herscovics, A., 302, 307, 326 Herth, W., 390 Herzog, H., 83 Hestrin, S., 159 Hettkanip, H., 312 Heusner, A., 61 Hickman, J. W., 352 Hickman, S., 269, 361, 363 Higashi, Y., 292 Hildebrand, J., 247, 248(51), 249(66), 258 Hill, R. L., 189, 190(270), 193(267), 230, 267 Hinzen, D. H., 217 Hipp, F. X., 217 Hirabayashi, Y., 243, 260(27), 284 Hiragun,A., 377 Hiraoka, T., 10 Hirs, C. H. W., 351, 354(405) Hirschberg, C . B., 186, 187 Hirshfield, J., 324 Hitzig, W. H., 206 Ho, M. W., 274,282,283(269) Hoffler, U., 196 Hof, L., 171,274 Hoff, G . E., 139, 144(47), 233(47) Hoffman, C., 324 H o f h a n , L. M., 253 Hoflieinz, W., 21 Hoflack, B., 292, 317(27),325(27) Hogan, E. L., 243 Holbrook, K., 375, 376(541) Holcomb, A. G . , 84 Holland, J. F., 228 Holland, J. J., 302 Hollemans, M., 363 Holm, M., 265 Holnigren, J., 230 Holmquist, L., 170, 197, 198 Holzer, H., 356 Homniel, E., 317 Hoogeveen, A. T., 207 Hoogsteen, K., 324 Hopp, H. E., 290(13,14), 291, 292(13), 296, 311(46), 317(46),318, 344 Horecker, B. L., 212 Hori, T., 240 Horisberger, M. A,, 334 Horn, R. C . , 171 Horowitz, M. I., 138, 139(41), 140(41), 350, 357(392) Horton, D., 121,350, 357(391)

411

IIorvBth, T., 26, 29(53) Hosada, K., 85, 87(280),89(280) Ilotta, K., 141, 142(62),144(62), 165(62), 216(62) Hough, L., 44, 120, 164, 200(144), 208( 144) IIoiislay, M. D., 219, 345 Iforislay, T. J., 365 Iioward, R. J., 144, 165(85),215(85) flozunii, M . , 377 Iisu, A. F., 361 IIiilhard, S. C., 295(75), 300, 301(72, 75), 303(75), 304, 308 Hiiber, J., 354 I I i i l ) c r t , E. V., 346 IIiichzernieyer, R., 210 Huff, J., 324 rlllgiles,E. E., 72 Ilughes, J. B., 121 Iiiighes, R. C., 302, 328, 329(273),3.50, 351(389),376 1 1 1 1 1 1 , W. E., 168 Ilultherg, B., 277 Irllrlg,H.-K., 111 Iiiingerford, M., 276 Hunt, L. A,, 301, 314 Iiunt, V., 324 Iirinter, E., 371, 373 IIiinter, R. E., 382, 387(22) Hiird, C. D., 31 Ilynian, R., 295(120). 305, 306(118),313, 333(118, 120) Hyncs, H. o.,375

I Ichikawa, T., 121 Ide, J., 10 Idoyaga-Vargas,V.. 290(18),291, 316, 319 Iijiiiia, Y., 252 Illiano, G., 198 Imada, K., 387 Ingraham, H. A,, 193 Inokawa, S., 71, 109(232), 110(232) Inoue, J,, 139, 141, 142(45), 143(71), 176(71) Iiioue, S., 139 I p , C., 194 Iqbal, K., 254

AUTHOR INDEX, VOLUME 40

412

Isaka, K., 377 Isaka, S . , 216 Isemura, S., 19, 114(44) Ishibashi, T., 250 Ishida, N., 210 Ishido, Y., 2, 121 Ishiwata, Y., 260 Ishizuka, I., 144 Issac, D. H., 394, 396, 397, 398(74) ltasaka, O., 240 Ito, E., 339, 341(330) Ito, R., 80 Ito, T., 339, 340(324) Itoli, K., 229, 232(526), 362 Iwai, I., 8, 9, 10(24) Iwai, Y . , 342 Iwakiira, Y., 374 Iwamori, M., 139, 230,257 iwasaki, M., 139, 142(45) Iwashige, T., 8, 9(24), 10 Iwataki, I., 27, 35(323), 114 Iwatsaka, H., 231

J Jablonovskaya, S . D., 57 Jacobs, J. W., 350 Jacobson, C. B., 275 Jacques, L. W., 168 James, D. W., 341 James, M. J., 293, 323(33) Jameson, P., 199 Jamieson, J. C., 313 Jancik, J. M., 206, 209(371), 222, 223(484) Janczura, E., 293, 315 Janda, M., 62, 63, 64(205) Jankowski, A. W., 304, 307(116), 310(116), 322(116), 332(116), 337(116), 349( 116) Jankowski, K., 39 Jankowski, W., 290(22), 291 Janotovski, M. C., 42 J a e , J., 19, 110, 119, 120 Jatzkewitz, H., 277, 281, 282, 283 Jaworski, C., 376 Jeanloz, R. W., 140, 141, 215, 229(416), 230(416), 302, 307, 309, 326, 351 Jeffrey, G. A., 382, 383

Jeffrey, P. L., I94 Jelsma, J., 386, 390 Jennet, R. B., 345 Jennings, H. J., 139, 166, 169, 170(166, 167) Jensen, J. E., 23 Jensen, J. W., 309, 310(146), 311 Jersch, N . , 23, 24, 114 Jeserich, G., 208 Johannsen, R., 225 Johinen, M., 340 Johnson, D. C., 347 Johnson, L. W., 226 Johnson, P., 236 Johnson, W. G., 280 Joller, P., 206 Joll&s,P., 168, 353, 354(422) Jones, J. K. N., 3 Jones, M., 337 Jones, P. H., 51 Joseph, R., 177 Joshua, € I . , 324 Jourdian, G. W., 154, 155(127), 178, 212 Journey, L. J,, 222 Joziasse, D. H., 168, 268 Jucker, E., 62 Jurczak, J., 18,36, 37(88), 38, 39(88, 92), 40,41, 113 Juritz, J. W. F., 391 Just, G., 75, 77(249), 78

K

Kiiriiinen, L., 322, 347, 356 Kadentsev, V. I., 146, 165(98) Kadowaki, S., 228 Kainuma, K., 382 Kaji, E., 105 Kalin, J. H.,290(24), 292 Kaller, A. L., 388 Kalsbeek, R., 363, 364 Kaluza, G . , 303, 327, 346, 356(111) Kamerling, J. P., 138, 139, 141, 142(62), 143(34), 144(62), 145(82, 86), 146(34, 95), 147(34, 39), 151(82), 152(95), 156(93), 157(93), 158(90), 159, 160(93, 97), 163(34, 82, 94, 95, 97, 105, 148), 164(94), 165(34, 62, 94, 95,

A U T H O R INDEX, VOLUME 40 97, 105, 139, 147), 166(94, 139, 147), 167(139), 169(93), 173(82), 179(105), 180, 183(82, 90), 184(82, 148), 185(34, lOS), 209(34), 216(62) Kanabayashi, J., 240 Kandutsch, A. A., 293, 323(33) Kanfer, J., 203, 247, 272, 281, 282 Kang, M . S., 307, 326, 339, 341(329), 342(134), 343(134, 347), 344(350), 348(350) Kanwar, Y. S., 171, 215 Kaplan, J., 365 Kaplan, R., 175 Karasawa, K., 339 Karlri, K. K., 340 Karkas, J . D., 202 Karlsson, K.-A,, 238 Karnovsky, M. L., 177,215 Karrer, P., 62 Kartenheck, J., 357 Kasper, D., 178 Kathan, H. H., 141, 142(65) Katlic, A. W., 175 Kato, H., 343 Kato, S.,326 Katterman, R., 338 Katunuma, N., 219 Katz, E., 370 Katz, F. N., 319,360 Kaufinan, B., 192, 248, 254, 255, 256, 257, 258(111), 259, 260, 264, 26S( 108, 112), 266 Kanfmann, S. H. E., 142,221(80) Kauss, H., 317 Kawabata, S., 35(323), 114 Kawade, Y., 374 Kawaguchi, M., 228 Kawamllrd, K., 339, 340(324) Kawanami, J., 249 Kawashima, Y., 106 Kay, M. M. B., 226 Kean, E. L., 186, 187(253), 211(253, 262), 311, 342(158), 360 Keberle, J., 62 Keegstra, K., 302(103), 303 Keenan, R. W., 290(15, 20, 21), 291, 315(21), 341, 342(340) Keenan, T. W., 192, 194, 246, 266 Kefurt, K., 19, 120 KefiirtovB, Z., 120 Kehoe, J . M., 175

413

Keilich, G., 133, l67(15), 170, 195(15), 198,209,390 Keiyer, J. H . , 30 Keller, R. K., 293, 296, 297(52),309(52), 323(34), 341,342, 362 Kelley, L. K.. 226 Kelso, C. D., -31 Keinp, R. B . , 215 Kemp, S. F., 245, 255(43), 257, 259, 265 Kennedy, E. l’,, 247 Kennedy, S. I . T,, 353 Kenny, C. P., 139, 169, 170(166, 167) Kent, P. W., 104, 105, 177, 336, 337 Keppler, D. 0. H., 306, 337, 338(299) Kerinen, S., 347 Kern, M., 303 Kern, P., 231 Kerr, A. K. A,, 311 Kessler, J., 209 Kcsssler, N., 20.3 Khomntova, N . I]., 62 Khorlin, A. Y a . , 191, 193(278),202 Kihuris, J., 333 Kidzierska, B . , 134 Kijimoto, S.,249, 250 Kijimoto-Ochiai, S.,250 Kilker, R. D., 302 Kim, U., 193 Kirnelberg, €1. K., 323 King, B. F., 226 Kinoshita, T., 80, 82, 106 Kint, J. A,, 279 Kioipes, T. C., 297, 297(57) Kirchner, G., 202 Kishimoto, Y., 274 Kishore, G . S.,205, 206 Kiss, J., 30, 114(59) Kiss, P., 338 Kitaniikado, M.,240, 242(26) Kitaniura, M . , 230 Klasek, A,, 63 Klatt, D., 225, 226(495) Klein, U., 363 Klemm, W. H . , 181 Klenk, E., 132, 142, 148(3), 154(2),235, 236, 249(4), 253 Klenk, H.-D., 302(105, 107), 303, 321, 327, 329(265), 334(265), 347, 356(229),358(265), 360, 370(105), 372

414

A U T H O R INDEX, V O L U M E 40

Klionsky, B., 249, 279 Klohs, W. D., 194,338 Kloppenburg, M., 144 Knollmann, R., 114 Knowles, R. W., 372 Knox, R. J., 226 Knull, K. R., 351 Kobata, A,, 140, 302 Koch, C. J., 378 Koch, H . U . , 334, 349(290) Kochetkov, N. K., 62, 140, 146, 165(98) Kodama, Y., 339, 340(324) Kottgen, E., 215, 227(419), 231(419) Koga, K., 117, 118, 119(337) Koh, H.-S., 108(312), 109 Kohn, G., 207, 273 Kohno, H . , 105 Kohno, K., 375(542), 376, 377 Kohoutova, J., 42,43(136), 44(136) Koide, N., 352, 353(415), 354 Koistinen, V., 374 Kojima, K., 260 Kolb, H . , 222, 223, 226 Kolb, H . A,, 222, 226 Kolb-Bachofen, V., 222,223, 226 Kolisis, F. N., 212 Kolodny, E. H., 203,266,272,277 Kolpack, F. J., 382, 387 Komatsu, Y., 343 Konowak, A,, 36, 37(88),38, 39(88, 92), 40,41,42,43(136), 44(124, 136, 138), 48, 51(138), 60(138), 113 Korey, S. R., 271 Kornfeld, R., 179, 285, 299, 300(71), 301(71), 302(71) Komfeld, S., 179, 220, 228, 285, 295(117, 119-121), 299, 300(71), 301(71, 73), 302(71),304, 305(117), 307(83, 117), 313(121), 314(119), 333(119, 120, 121), 355, 356(429), 361, 362, 371(427), 376 Kornilov, A. N., 3, 39,41, 44(105), 54 Korte, F., 46, 47, 57(155),59, 60(185) Korytnyk, W., 194, 321, 336(234), 337(234, 304), 338 Koscielak, J., 240 Koskela, S.-L., 138, 147(38) Koviii., J., 107 Kovarik, J., 332 Kowarski, C. R., 79, 84(257) Koyama, G., 34, 127(82)

Kozaki, S., 342 Kozerski, L., 39 Kozikowski, A. P., 79 Koiluk, T., 83 Kratky, Z., 329, 332, 333(286), 337(286), 349(286) Krag, S. S., 307, 313 Kralinina, L. N., 63 Kreger, D. R., 386,390 Kreisel, W., 221 Kribben, B. D., 6 Kriese, A., 226 Krisman, C. R., 245 Kruczek, M . E., 290(20, 21), 291, 315(21) Kmglikova, R. I., 63 Kruisius, T., 166, 189 Ku, C. S. L., 192, 194(285) Kubicka, T., 197 Kubler, D. G., 36 KuCiir, s., 337 Kucera, J., 62 Kudriashov, L. I., 62 Kuehl, W. M . , 319 Kuster, J. M . , 144, 145(86),223 Kuhl, W., 270, 279,280(170) Kuhla, D. E., 65 Kuhlenschmidt, M. S., 282 Kuhn, R., 133, 253 Kulczycki, A,, 361 Kumagai, K., 229, 232(526), 362 Kuniar, V., 209 Kumasaka, M., 210 Kundu, S. N., 181 Kuniak, M . P., 79 Kunieda, T., 84, 85, 87(278, 279, 280), 88, 89(280), 90(276-279, 286), 91(286), 92(279, 282, 287), 93(286), 94, 95(290) Kuo, S. C., 339, 342(325) Kuppel, A., 390 Kuratowska, Z., 197 Kuro, G., 324 Kuroda, M . , 323, 324(240) Kurokawa, M., 216 Kusiak, J. W., 280 Kutchai, H . , 345 Kuwahara, S. S., 157 Kuwert, E . , 210 Kwart, H., 84 Kyner, D., 331

AUTHOR INDEX, VOLWME 40 L Lahat, J., 216 Lacord-Bonneau, M., 194 Laine, R., 249 Lake, W. W., 7 Laliberte, R., 65, 72(211) Lallier, R., 266, 366 Lam, H.-Y., 111 Lambed, J. B., 84 Lampen, J. O., 339, 342(325). 359 Lampfrid, H., 132 Landaw, S. A., 206 Lane, M . D., 362 Langerbeins, H., 154 Lapina, E. B., 191, 193(278) Lasthuis, A.-M., 188 Lauenstein, K., 238, 249(4) Lauer, R. F., 52 Laurell, C.-B., 271 Laver, M. L., 309 Laver, W. G., 356 Lazdins, J., 215 Leavitt, R., 355, 371(427) Lebel, B., 216 LeBlanc, D., 283,284 Ledeen, R. W., 153, 164(118), 253 Ledger, P. W., 347, 365 Lee, E. C . , 5 Lee, J. E., 277 Lee, R. E., 282 Lee, R . T., 208 Lee, S. H., 254, 255(105) Lee, T., 351 Lee, Y. C., 208, 282 Lees, M., 247, 271(45) Lefebvre, Y., 65, 72(211) Legheand, J., 44 Lehle, L., 294, 296(43), 297, 301, 302(81), 310, 311, 312(156), 316, 317, 318,328,329(274), 330(274), 331(274), 341, 349(274) Lei, J., 73 Leibovitz, Z., 272, 273 Lekholm, U., 231 Lelliott, C., 391 Leloir, L. F., 245, 289,294(2), 295(2), 296(2), 297(2), 298(2), 308(2),309(2), 310(2),315, 317 Lemal, D. M . , 14, 114(34) Lemieux, R. U., 31

415

Lemmer, A , , 227 Lengle, E. E., 227 Lenhard, V., 232 Lennarz, W. J., 245, 2W(50), 294, 295(50), 296, 299(50), 300(50), 301, 307, 308(50, 89), 309(50), 310(50, 126), 312(50), 317, 320, 324, 326(148), 341, 347(50), 348(248), 362, 366(339), 367(248, 489) Lenten, L. V., 270 Lepine, M.-C., 122 Leppi, T. J., 171 Lerche, D., 215 Lespieau, R., 3 Letoublon, R., 298 Leube, H., 12 Levandowski, L. J., 371 Levin, E., 293 Levine, A. S., I99 Levitan, D. B., 369 Levkowitz, A., 367 Levy-Benshimol, A,, 375 Lewis, K. G., 66 Lewis, L., 383 Lewis, R. W., 218 Li, E., 295(117),299, 301, 304, 305(117), 307(83, 117), 376 Li, S.-C., 240, 242(26), 249, 252, 2!S4, 271, 272, 274, 278, 279(76), 280, 281(238),282, 283(176, 212, 238), 284,285 Li, S. S.-L., 175 Li, Y.-T., 240, 242(26), 249, 252, 254, 257, 271, 272, 274, 278(176),279(76), 280,281, 282, 283(176, 212), 284, 285(240) Liak, T., 77 Liang, C.-J.,302 Lieberknecht, A., 80, 84(258), 114(258) Lies&, J,, 324 Lim, M., 78 Limanova, 0. V., 12 Lin, W., 139, 144(47), 233(47) Lindberg, E., 132 Lindenmann, A., 62 Lingappa, V., 319 Linnekin, D., 362 Lipke, p. N , , 368 Lis, H., 296, 299(49),309(49), 310(49), 310(49), 318(49), 351 Lis, M., 358

416

AUTHOR INDEX, VOLUME 40

Liska, F., 62 Lissac-Cahu, M., 39 Liu, C.-K., 193 Liu, D. Y., 232 Liu, T., 307, 308 Liu, T.-Y., 185, 194(251) Lo, J. T., 270, 274(169) Lochinger, W., 133, 167(14) Lock, T., 228 Lodish, H. F., 319, 360, 370 Loh, Y. P., 358 Lohmander, L. S., 138 Lohmeyer, J., 347 Lokhande, H. T., 387 Lombardo, A., 205 Lopez, M., 324 Lopez-Solis, R. O., 193 Lord, J. M., 318 Lothrop, D. A,, 323 Loucheux-Lefebvre, M. H., 353, 354(422) Louisot, P., 192, 315, 316 Loures, M. A., 136, 176(26) Lowden, J. A., 207,285 Loyter, A., 367 Luben, G., 225 Luhineau, A,, 37(350), 52(355, 356), 53(356), 124, 125(95), 127 Lucas, H. J., 115 Lucas, J. J., 289, 290(8, I l ) , 293, 317 Ludowieg, J., 158 Ludwig, H., 332,372 Liiftmann, H., 114 Lui, S. W. L., 194 Lukei, R., 110, 119 Lund, P. K., 350 Lundblad, A., 138, 147(38) Lrindin, S. J., 219 Luria, S. E., 351, 373(398) Lu Shun Yan., D., 207 Lutz, H. U., 226 Lutz, P., 133, 167(14) Lynch, R. G., 361 Lysenko, N., 293

M McCarthy, M., 353 McCloskey, M. A,, 193,294, 320(44)

McCluer, R. H., 253 McCormick, J. E., 101, 102(301), 103(302) McCrae, W. M., 274 MacDonald, H. R., 378 McElhinney, R. S., 101, 102(301), 103(302) McFarland, V. W., 266 McCuire, E. J., 176, 188(213), 192(213) McKelvey, H., 275 McKhann, C. F., 227,228(513) McKibbin, J. M., 280 Maclachlan, G., 300, 301(78), 318 Maclaren, N . K., 265, 266(143) McLawhon, R., 347 McPhie, P., 218 McPortland, R. P., 323 Maccioni, H. F., 255 Maccioni, H. J., 257, 258(118), 263(118), 264,265 Macher, B. A , , 240,243(14) Maeda, K., 34, 127(82) Maeda, M., 347 Maeda, S., 121 Maget-Dana, R., 142, 175, 176(204, 206) Magi, S., 33 Magil, A. B., 172 Mahoney, W. C., 341 Maier, H., 211 Makerji, K., 270,274(169) Makin, S. M., 12, 36, 37(89), 39(89),49 Makino, S., 80 Makita, A., 249, 250 Makita, M., 164 Makman, M. H., 377 Makovitzky, J., 144, 172, 174 Maley, F., 351, 354 Malniendier, C. L., 221 Mameli, L., 208 Mandel, P., 208,255, 265 Mandsley, D. V., 230 Mankowski, T., 290(22), 291, 293, 294, 296(43), 316 Manley, R. S. J , , 384 Mann, J., 386 Mansson, J.-E., 257 Manville, J. F., 44 Marchand, N. W., 206 Marchessanlt, R. H., 381, 383(1),384, 389, 390(49),391 Marchmont, R. J.. 219, 345

AUTHOR I N D E X , VOLUME 40 Margalith, E., 370 Margolis, R . K., 219 Margolis, R. U., 219 Marinari, U . M., 345 Marinoni, G., 117, 128 Mariott, M., 311, 316 Markwell, M. A. K., 230 Marley, J . B., 324 Marnell, L. L., 335 Maros, L., 72(240, 241), 73 Maroteaux, P., 138, 207(40) Marshall, M., 363 Marshall, R. D., 312 Martel, A,, 75, 77(249) M5rtensson, E., 248, 249(65) Martin, A,, 139, 169, 170(166, 167) Martin, H. C., 289, 315(5) Martin-Barientos, J., 317 Martinez, G., 212 Masamune, S . , 30 Masojidkova, M., 42,43(136), 44(136) Massamiri, Y., 153, 157, 158, 159(121, 134) Masserini, M., 217 Masters, V. M., 216 Masunari, M., 4,28(10) Masushige, S., 297, 297(57), 298 Matsibora, N. P., 388 Matsnbara, T., 238 Matsnda, Y., 219 Matsukura, S . , 342 Matsumoto, M., 203, 243, 260(27) Matsumoto, T., 27, 32, 35(323), 40(79), 114 Matsuura, T., 88, 92(287) Mattingly, S. J., 195, 206, 211(306) Matiislrinra, M., 354 Max, S. R., 265, 266(143) May, €3. K., 324 Mayarna, M., 343 Mayer, F., 211, 212(408), 213(408) Mazzotta, M. Y., 274, 278, 281(238), 283(212, 238) Meader, D., 389 Meager, A,, 376 Medawar, G., 65, 72(211) Meeks, R.-G., 345 Meera Khan, P., 364 Mehl, E., 281 Meindl, P., 147, 199, 209 Meisler, M., 274, 275

417

Melanqon, S. B., 208 Melchers, F., 360 Mellor, B., 318 Meloche, H., 212 Melton, L. I]., 170 Mendelsohn, N . , 331 Menon, K. M . J., 230 Mentaberry, A., 290(18), 291 Merat, A., 265 Merrick, J. M., 143, 176(81) Merz, G., 230 Mescher, M. F.,325 Messer, H., 207 Messer, M., 146, 167,208 Mestrallet, M. C., 258 Me\tres-Ventura, P., 181, 186(249), 192(249) Meuzelaar, H. L. C., 167 Meyer, K., 177 Michael, A. F., 216 Michalski, J.-C., 138, 140, 142, 149(55), 150(55),179, 196(55), 197(55), 198(324), 199(55),200(338),201(55), 202, 205(SS), 207(40, 55), 208(55), 209(324) Michl, J., 378 Mieczkowski, J., 42,44(140), 46(147), 51(140), 57(140), 72 Mihich, E., 321, 336(234), 337(234) Milgrom, F., 143, 176(81) Miller, A. L., 276 Miller, C. S., 207 hliller, R. J., 347 hliller-Podraza, H., 240 Milligan, T . W . , 195, 206, 211(306) Mills, J. T., 289, 293(7), 324(7), 34817) Miinaki, K., 4, 28(10) Minke, R., 392, 393(61) Misaki, A., 386 Mitsiii, H., 376, 377, 378 Mitsiio, N., 87, 88, 92(282) Miwa, T., 80, 82, 106 Miyagawa, S., 141, 143(71), 176(71) Miyamoto, C., 344 Miyano, K., 80 Miyatake, T., 249, 271, 279 Miyazaki, S., 228 Mizogrichi, T., 117 MiLrahi, A,, 374 Mizuno, T., 2 h4ochalin, \.. H., 3, 36, 37, 38, 39, 41,

418

AUTHOR INDEX, VOLUME 40

42(90, 94),44(94, 105, 130, 131Muramatsu, T., 352, 353(415), 354(415) 134), 47, 51, 54, 57(166), 58(166) Mnraznmi, N., 339, 341(330) Mock, R., 228 Murphy, L. A,, 308 Moczar, E., 190 Murphy, V. G., 381 Moffatt, J. G., 108, 109 Murray, L. W., 365 Mohos, S., 156 Murray, T. P., 32, 36(77), 40, 41, 44(126), Mohr, E., 198 45, 49(151), 50(151), 51(151), 57(126) Molenaar, E., 30, 280 Murthy, M. S., 228 Molibog, E. V., 202 Mnrty, V. L. N., 140 Moll, M., 110 Mustard, J. F., 222 Molnar, Z., 335 Myhill, M., 195 Molodtzov, N. V., 62 Myers, R. W., 208 Momose, K., 325 Myers-Robfogel, M. W., 188, 194(263) Monaghan, R., 324 Monsigny, M., 175, 176(204, 206) N Montezinos, D., 344 Monti, L., 33 Nadler, H. L., 274 Montrenil, J., 133, 138, 142(37), 147, Nagahashi, J., 318 163(105), 165(105), 167(16), 168, 169, Nagai, Y., 139, 203, 204(360), 230, 257 Nagasawa, J.-I., 2 179(105), 185(105),195(16), 207(40), 299, 301(70), 302(70), 310(70), Nagashima, M., 362 350(70) Nairn, R., 376 Nakagawa, H., 240, 242(26) Mookerjea, S., 194,307,321(124) Nakagawa, M., 4, 7, 19, 26, 28(10), Moorhouse, R., 395,399 114(44), 115, 117(331) Mora, P. T., 266 Nakajima, M., 108(312), 109 Morelis, R., 192, 315, 316 Nakaminami, G., 4, 19, 28(10), 114(44), Morell, A. G., 220, 352 115, 117(331) Morell, P., 248, 265(63) Morgan, B. L. G., 180 Nakamura, K., 302(106), 303, 335, 358 Morgan, E. H., 271 Nakamura, S., 339 Nakamura, T., 278, 285(240) Morin, M. J., 336, 337(304) Morioka, T., 203 Nakamura, Y., 27 Nakanishi, Y., 326 MorrC, D. J., 192, 194, 246, 266, 357 Nakano, M., 248 Morris, E. R., 170 Nakatsukasa, Y., 71 Morrison, T. G., 360, 372 Nakayasu, M., 369 Morton, R. A., 314 Nanni, G., 345 Moschera, J., 174, 218 Nathenson, S. G., 352 Moskal, J. R., 249, 250(83), 253 Natsume, M., 96, 97, 98, 99, 100 Mosman, T. R., 361 Motas, C., 351, 354(406,407) Nebelin, E., 199 Nees, S., 149, 193, 196, 205(286), Mraz, W., 281 211(314), 212(314,408), 213(408) Miihlpfordt, H., 136 Nehrkorn, H., 271,277(178), 278(178) Muellenberg, C. G., 302 Miiller, E., 224 Neiduszynski, I. A., 381 Nemec, J., 119 Miiller, H. E., 196 Muller, M., 216 Ness, G. C., 293,323(34) Neuberger, A,, 148 Muggli, R., 382 Neufeld, E. F., 179, 220, 269, 277, 278, Muh, J. P., 194 363 Muhleisen, M., 217 Nevar, C., 317 Mumford, R. A., 282 Munekata, M., 375(542), 376 Newman, D., 232

AUTHOR INDEX, VOLUME 40 Newman, H., 36, 42(86), 46, 47(86) Newman, M. S., 84 Ng, M. H., 194 Ng, S.-S., 134, 136(19), 137(19),138(19), 139(19), 140(19), 142(19), 143(l Y ) , 203(19), 258 Nguyen, H. T., 366, 367(489) Nicolau, J., 181 Nielsen, J. T., 61, 62 Nieniczura, w., 111 Nigam, V. N., 266 Nii, Y., 94, 95(290) Nilsson, B., 138 Nilsson, G., 197 Nimmerfall, F., 216 Nishi, N., 393 Nishikawa, Y., 378 Nishimura, H., 343 Nishimura, K., 276 Nishimura, M., 375(542), 376 Nishimura, R. N., 258 Nishmo, T., 325 Nista, A,, 227 Nohle, U., 210, 212(401) Noguchi, J., 393 Norden, A. G. W., 274 Nordliny, S., 367 Nordt, F. J., 222, 223(483), 225(483),226 Norris, K., 73 Norrman, B., 167 Northcote, D. H., 318,326 Nose, N., 352, 353(415), 354(415) Novak, P., 63 Novogrodsky, A., 232 Nowoczek, G., 273 Noyori, R., 80 Nydegger. U . E., 224

0 O’Brien, J. R. L., 232 O’Brien, J. S., 207, 274, 275, 276, 277, 282, 283(269), 285 O’Brien, P. T., 179 Ockerman, P. A., 274 Odagiri, T., 210 Odin, L., 153 Ohman, R., 248, 249(65), 265, 272 Ogamo, A., 278, 285(240) Ogasawara, T., 35(323), 114

419

Ogata, T., 71, 109(232), llO(232)

Ogawa, K., 386,389,390(49) Ogawa, M., 100 Ogura, H., 370 Ohanian, S. H., 366 Ohdan, S., 121 Ohkubo, I., 250 Ohlbaum, D. J., 378 Ohno, M., 34, 127(82) Oiwa, R., 342 Oka, S., 386 Oka, T., 342 Okada, S., 275,277 Okamoto, S., 342 Okamoto, T., 121 Okamura, K., 386 O’Keefe, E., 351 Okina, M.,339, 340(324) Okita, T., 80 Olden, K., 366, 375(543), 376 O’Malley, J. A,, 374 Omura, S., 324, 342 Onishi, H. R., 359 Onodera, K., 375(542),376, 378 Orestov, I. L., 2 Brskov, F., 139, 144(47), 233(47) Brskov, I., 139, 144(47),233(47) Orth, R., 278. 281(238), 283(238) Osawa, T., 140, 228 Oster, H., 206 Osuga, D. T., 351 Otsuka, H., 342

P l’acak, J., 42 Pacheco, H., 31, 44 Pacht, P. D., 14, 114(34) Pacini, A , , 221 Packham, M . A,, 222 Pacuszka, T., 258 Paerels, G. B., 156 Paiva, Y. A. S.,181 l’alade, G. E., 172 Palaniarczyk, G., 293, 294, 296(43), 297, 315,316 Palese, P., 208, 209, 210 Pallman, B., 273 Palm, W., 354 Palmer, J. L., 228 Pan, Y. T., 378

420

AUTHOR INDEX, VOLUME 40

Phkova, M., 52 Papahadjopulos, D., 323 Papermaster, D. S., 156 Parker, J. W., 232 Parker, K. D., 390, 392 Parker, T. L., 149,210(112) Parodi, A. J., 245, 289, 294(2), 295(2), 296(2), 297(2), 298(2), 301, 302, 307, 308(2), 309(2),310(2), 315, 317 Parrot, G., 216 Parrot, J. L., 216 Parthasarathy, R., 101 Pastan, I., 346 Patchet, A., 324 Patil, N . B., 387 Paton, J. C., 324 Patt, L. M., 317 Patton, C . L., 319 Pattyn, S. R., 373 Patwardhan, B. H., 209 Paul, B., 336 Paul, R., 30, 31(62) Paulson, J . C., 189, 190(270), 193(267), 229, 230,267 Pauly, J. L., 232 Pavlikovi, J., 63 Pays, M., 153, 157(121), 159(121) Pazur, J. H., 351 Peers, M. C., 138, 207(40) Pendergast, M., 373 Penglis, A. A. E., 336 Pennock, J. F., 314 Perdew, G. H., 295(117a), 305,311(117a) Pereira, M. E. A,, 136, 176(26) Perelmuter, M., 316 Perez, S., 383, 384 Perkins, M. E., 375 Perlin, A. S., 279 Perlitsch, M. J., 209 Peron, F. G., 230 Perona, G., 222 Person, S., 372 Pessina, G. P., 221 Peterkofsky, B., 345, 346(364) Peters, B. P., 175 Peters, S. P., 282 Petersen, J. B., 73 Peterson, E., 316, 321(188) Petschek, K. D., 232 Pfannschmidt. G., 162. 163(142).

223(142), 224(142), 225( 142), 226(142) Pfeil, R., 133, 143, 144, 145(17, 8 2 ) , 146(17, 95), 151(82), 152(95), 158(90), 163(82, 95, 148, 149), 165(95), 166(94), 167(17), 173(82), 183(82, 90), 184(82, 148), 195(17) Pfitzner, K. E., 108, 109 Phelps, C. F., 179 Phillips, M. L., 232 Piancatelli, G., 72 Picard, J., 231 Pierpont, A. A,, 195, 211(306) Pigman, W., 138, 139(41), 140(41), 141, 142(67), 174, 350, 357(391, 392) Pizer, L. I., 370 Planter, J. J., 360 Pless, D. D., 293 Plettenberg, H., 109 Plouhar, P. L., 297 Plummer, T. H., 351 Polyakova, G. V., 388 Poncz, L., 360 Pont Lezica, R., 290(12-14), 291, 292(12, 13),296, 311(46), 317(46), 318, 328, 329(275),330(275), 332, 344, 349(375) Porshnev, J. N., 36, 37,42(90, 94),44(94, 130, 131-134), 47(135) Porter, C., 321, 336(234), 337(234) Porter, C . W., 336 Porter, N. K., 336 Poschman, A,, 206 Potier, M., 207, 208 Potts, J, T., 350 Pouyssegur, J., 346 Powell, J . T., 228 Powell, M. E., 240, 243, 260(28) Power, D., 309 Pratt, E. F., 108 Pratt, R. M., 344, 366, 368(354),375(543), 376 Presper, K. A,, 253 Preston, R. D., 381 Preti, A., 205, 208, 217 Pribilla, W., 218 Price, C . C., 12 Prioer, W. E., 220 Priebe, W., 39, 42(106), 44 Prieels, J.-P., 189

AUTHOR I N D E X . \'OI,UME 40 Privalova, 1. M., 202 Prolxt, W., 217 Proscher, F., 153 Pronzato, M. A,, 345 Priizanski, W., 232 l'iirandare, B. N., 227 Purandare, M. C., 227

Q Quain, C. 0 . M. C., 360 Quesada, L. A,, 292 Quill, H., 298 Quirk, J. hl., 203, 270, 272, 277, 280

H Radin, N. S.,248, 270, 274, 282, 283(269) Radulescu, S., 351, 354(406) Raff, M. C., 360 Ragliavan, S. S., 282 Rahmann, H., 136, 137(28), 138(28), 181, 208, 217(28), 230 Raivio, K. O., 138, 147(38) Rajan, R., 227 Rajfold, Y . E., 12 Rakotoarivony, J., 1'34 Ramey. C . W., 172 Raiiijeesingh, M., 75, 77(249) Rand, M. L., 222 Ranganayakiilu, K., 45, 49(151), SO(151). Sl(151) Rao, S. S., 227 Rao, V. S. R., 169 Rapllael, R. A,, 13, 27, 29 Rapin, I., 284 Rapoport, S. M., 225, 226(495) Rapport, M. M., 249, 270, 272, 274(186) Rappenecker, G., 385 Rasilo, M . L., 367 Ratcliffe, W. A., 148 Ratnam, S., 194 Rauterberg, E., 232 Rauvala, H., 166, 189, 203 Rawls, W. E., 229 Ray, E. K., 369 Ray, M. M., 318 Ray, P. M., 318

42 1

Kay, P. K., 227 Read, G . A , , 345 Kearick, J. I . , 189, 190(270),267 Rt,;tvelley, D. A.. 399 Hres, D. A , , 170, 354 Regan, D. H., 226 Regoeczi, E., 221 Reid, L., 218 Reid, P. E., 135. 170, 171(9l), 172, 173(91) Heijiigoud, D. J . . 335 Keiniers, €1.-J..222 Keinking, A , , 307 Reiter, S.,282 Remold, H . G.. 232 Renkonen, O . , 322, 367 Renliiiid, M . . 138, 147(38) Kennels, M. H . , 265 Hespondek, M., 142, 221(80) Reriter, G., 136, 139, 143, 144(50), 145(82), 146(95),151(82), 152(95), 163(82, 95, 148), 165(85, 95), 166(94), 173(82, 92), 183(82),184(82, 148), 214(85) Krutter, W., 215, 221, 227(419), 231(419), 338, 347(316), 349(316) Reuvers, F., 293, 307, 326 Revol, J. F., 384, 389 Hey. M., 364 Reynolds, L. W., 207, 208 Riceviiti, G., 222 Richard, A,, 158, 159(134) Richard, M., 315 Richardson, C. D., 345 Richardson, C. L., 266 Riesco, B. F., 168 Rietra, P. J. G. hl., 280 Hietz, E., 5, 6(17) Riolie, O., 30, 31 Risse, H. J., 317 Robliins, J. H . , 139, 144(47), 185, 194(251), 233(47) Kobhins, P. W., 295(75), 300, 301(74, 75), 302(108),303(75),307, 308, 313, 320, 328, 329(275),330(275), 349(275), 360 Rol~erts,I,. M., 318 Roliinson, D., 274, 277 Robinson, H. C., 344 Roboz, J. P., 228 Rocha de Morillo, M., 192

422

A U T H O R INDEX, VOLUME 40

Roche, A.-C., 175, 176(204, 206) Roche, E., 389 Rocklin, R. E., 232 Roelcke, D., 232 Rdmer, H., 217 Romer, W., 232 Rosner, H . , 136, 137(28), 138(28), 208, 217(28),230 Roff, J. E., 31(75), 32 Rogers, C. M., 145, 173(92) Rohrschneider, J. M., 358, 372(437) Roldan-Gonzalez, I+ 386 Romanowska, E., 195 Romeo, G., 280 Romero, P. A., 290(13, 141, 291, 292(13), 296, 311(46), 317(46), 318, 344 Ronin, C., 307, 309, 312 Roos, M. H . , 362 Roozendaal, K. J., 227 Rose, U., 178 Roseman, S., 133, 154, 155(127), 177, 178, 186, 187(253), 188, 192, 211(253), 212, 244,248, 250,254, 2SS(l08), 256,257,258(111), 259(108), 260(108), 264(108), 265(108, 112), 266(108), 299, 300(67), 301(67) Rosen, 0. M., 377 Rosenberg, A., 149, 150(110), 195(110), 196, 197(110), 198(110), 199(110), 204(110), 205(110), 207(110), 215, 269, 271(161), 272, 273 Rosenfeld, L., 282 Rosenthaler, J., 216 Rosner, M. R., 302(108), 303 Rosowsky, A., 49 Ross, G. T., 352 Rosso, G. C., 298 Rost, K., 134 Roth, M. G . , 373 Roth, S., 317 Roth, S. H., 335, 345(294) Rothman, J . E., 319, 347, 357(386),360 Rothrock, I., 324 Rott, R., 200, 203, 303, 321, 356( 111, 229), 360, 372 Roux, M., 384 Rowland, F. N., 365 Rowley, E. K., 51 Roxburgh, C. M., 13 Rubin, A . L., 232

Hubin, C. S., 377 Rudigier, J. F. M., 336 Rudney, H., 325 Ruiz-Herrera, J., 331 Russi, M . , 221 Hastum, Y., 336

S

Sable, H . Z., 45 Sack, J., 290(24),292 Sadler, J. E., 189, 190(270),267 Saito, M., 140, 203, 204(360) Saito, T., 199 Saito, Y., 323 Sakai, M., 96 Sakata, K., 46 Sakata, T., 108(312), 109 Sakazaki, R., 342 Sakurai, A,, 46 Saltz, B., 232 Sami, S., 87 Samokhvalov, G. I., 36, 37, 42(90, 94), 44(94, 130, 131-134), 47(135) Samuelsson, B. E., 238 Sander, M., 142, 144(77),145(77), 155(141),161, 162(77), 163(141), 164(141), 166(141), 172(141),175, 176(204), 197, 200, 202(343), 203(77), 205(325), 208(141), 209(325) Sandermann, H., 292 Sanders, H., 31, 35 Sandhoff, K., 271,273, 277(178), 278, 284 Sanford, B. A., 378 Sam, T., 377 Saraste, J., 847 Sarel, S., 79, 84(257) Sargeant, T. E., 323 Sarko, A., 382, 383(13, 14, 15, 16), 384, 388, 390 Saroli, A,, 31 Sarris, A. H., 172 Sartnrelli, A. C., 181 Sasak, W., 293, 297,298(56) Sasaki, F., 34, 127(82) Sasaki, S., 19 Sassen, M . M. A,, 318 Sato, K., 340 Sato, T., 80

AUTHOI-I INDEX. VOLUME 40 Sattler, M., 275 Sauerheber, R. D., 345 Saxen, L., 367 Scandehari, C., 222 Scanlon, E. F., 228 Scettri, A., 72 Schaap, T., 207, 273 Schachter, H., 176, 188(214), 192(214), 299, 300(67),301(67) Schauer, R., 133, 134, 136, 137(29). 138, 139(18), 140, 141(29), 142(29, 62, 69), 143(34, 74), 144(50, 62, 77), 145(17, 74, 77, 86, 89), 146(17, 33, 34, 74, 95), 147(34, 39), 148(69, 106, 107), 149(55), I50(55, 107), 151(74, 82, 107), 152(95), 153(107), 154(107), 155(89, 107, 109, 141), 156(74, 89, 93, 107), 157(93, 107), 158(74, 90, 107), 159(135), 160(89, 93, 97, 107), 161, 162(74,77, 107), 163(34, 74, 82, 94, 95, 97), 105, 107, 141, 163(142, 148, 149), 164(94,107, 141, 142), 165(34, 62,85, 94, 95, 97, 105, 107, 139, 146, 147), 166(94, 107, 139, 141, 142, 147), 167(16, 17, 139), 169(17, 93), 170(18), 171, 172(89, 141, 182), 173(92, 182), 174(142, 182), 175, 176(204),177(216), 178, 179(74, 105, 107, 226, 228, 2:30), 180(233), 181(216, 231, 232), 182(216, 226, 229-231), 183(33,82,90,216,230). 184(33, 82, 148, 216, 228, 230, 24X), 18*5(34,105, 238), 186(33, 249, 252). 187(18, 72, 252), 190, 191(258, 259), 192(249, 257), 193, 195(16, 17), 196(55),197(55,312), 198(324), 199(55),200(89, 338),201(55, 1It5), 202(89, 115, 260, 343), 203(77), 204(359), 205(55, 286, 312, 325), 206, 207(55), 208(55, 141, 368), 209(34, 115, 312, 324, 325, 371), 210(112, 113), 211(18, 107, 113, 229, 314), 212(314, 401, 408), 213(233,408), 214(455), 215(85, 106, 229), 216(62), 217(28, 415), 218(229, 415, 418), 219(415), 221(80), 221, 223(142, 483, 4841, 224(142), 225(142, 483), 226(142), 227(229,415),229(229, 4 15) Schechter, N., 325 Scheele, C. M., 370

423

Scheffler, I. E., 314 Scheid, A., 229 Sclreinthal, B. M., 151 Schengrund, C.-L., 149, 150(110), 195(110), 196, 197(110), 198(110), 199(110), 204(110), 205(110), 207(110), 215, 269, 271(161), 273 Scher, M. G., 290(17, 25), 291, 292, 301, 307 Schick, H. J., 225 Schiller, H., 208 Schindelhauer, M., 206 Schlepper-Schlfer, J., 223 Schlesinger, M. J , , 347, 358 Schlesinger, P. H., 335 Schlesinger, S., 220, 295, 301(73), 31.3, 355, 356(429),371(427) Schmale, J. D., 228 Schmid, H., 7 Schmid, K., 142, 168, 169, 268 Schmidt, G., 225, 226(495) Schmidt, J. W., 378 Schmidt, M. F. G , , 302(105), 303, 322, 326(237), 327, 328(272), 328(274), 330(237, 274), 331(274), 332, 333(286),337(286), 349(274, 2861, 358, 370( 105), 372(437) Schmidt, R. R . , 49, 80, 84(258), 114(258) Schmidtberger, R., 225 Schmiechen, R., 115 Sclimied, R., 193 Schmitt, J. W., 304 Scliniitz-Moorniann, P., 172 Schneersori, R., 139, 144(47),233(47) Schneider, E. G., 366, 367(489) Schneider, G., 26, 29(53) Sclineider, 1. H., 352 Schnitzer, T. J., 370 Sclrbnharting, M., 2.30 Scholtissek, C., 321, 326(233), 327( 328(272),333, 335(233),336(233), 338(233),349(290), 360 Sclionne, E., 374 Schoop, H . J., 160, 178, 179(226), 182(226) Scliram, A. W . , 280 Schramm, G . , 198 Scliraven, J., 273 Sclireiber, C., 331 Sclireiber, G., 362 Sclireiber, J . R . , 298

424

AUTHOR INDEX, VOLUME 40

Schudt, C., 222 Schulman, J., 209 Schultz, A. M., 248 Schulze, I. T., 356 Schur, B. D., 317 Schut, J., 156 Schutzbach, J. S., 309, 310(146), 311 Schwaiger, H., 362 Schwartz, E. L., 181 Schwarz, R. T., 295(122), 302(105, 107), 303, 304, 306, 307(116), 310(116), 321, 322(116), 326(228, 237), 327, 328(228, 266, 272), 329(265, 273, 274, 275), 330(228,237, 274-276), 331(274), 332(116, 266), 333(122, 286), 334(265, 266), 335(266), 337(116, 286), 339(228), 346(122), 349(116,266,274-276, 286, 290), 356(11l), 358(265), 360, 370(105), 372(437) Schwarzmann, G., 203, 204(359), 208 Schulman, J. L., 210 Scott, M. E., 209 Scriba, M., 374 Seaman, G . V. F., 226 Sedlacek, H. H., 225, 227 Sefton, B. M., 301, 302(103), 302(104, 303), 303(82) Seger, R., 206 Segler, K., 136, 137(28), 138(28), 208 Seidah, N. G., 358 Seiler, F. R., 225, 227 Sekine, Y.,99 Selitrennikoff, C . P., 341 Sene, C., 175, 176(206) Sentandreu, R., 331 Serafini-Cessi, F., 193 Seth, P., 229 Seyama, Y., 278 Seyfried, T. N., 200 Sezzi, M. L., 227 Shapiro, D., 281 Shapiro, L. J., 269 Sharma, C . B., 310 Sharma, M., 336 Sharon, N., 240, 277, 296, 299(49), 309(49), 310(49), 311(49), 318(49), 350, 351(393),357(393) Sharpless, K. B., 52, 54 Shattil, S. J., 323 Shavrygina, 0. A., 36, 37(89), 39(89),49

Shechtnian, N. M., 37 Sheehan, J. C., 62 Sheehan, J. K., 393 Sherman, M. I., 367 Shibaev, V. N., 321, 337(235), 338 Shibuya, M., 19, 114(44) Shichi, H., 219, 302 Shier, W. T., 194 Shimaoka, N., 343 Shimizu, F., 186 Shimizu, K., 339 Shimizu, S., 174 Shininger, T. L., 318 Shinohara, S., 342 Shioi, S., 19, 26, 114(44) Shirai, S., 240 Shiu, R. P. C., 346 Shoji, S., 342 Shore, G., 318 Shreffler, D. C., 362 Shukla, A. K., 158, 159(135), 202 Shukla, S. H., 387 Shiilinan, M.L., 191 Siddiqui, B., 138, 139(43),237, 240(9), 243(9), 249, 253, 279(76) Sidell, N., 228 Siewart, G., 325 Sigler, G. F., 342 Sijoholm, I., 351 Silbert, D. F., 323 Silverman-Jones, C . S., 298 Silverstein, C., 378 Simon, P., 336 Simons, K., 320 Singer, H. H., 361 Singer, P. A,, 361 Singh, U. P., 41, 44(126), 45, 49(151), 50(151),51(151),55, 57(126, 160, 161),58(160, 161) Sirokman, F., 30, 114(59) Simmons, R. L., 227,228(513) Simone, I. V., 222 Sinionneaii, M., 217 Simpson, D., 360 Simpson, D. L., 351,372 Sinay, P., 146 Sinha, A. K., 323 Sinha, S. K., 338 Sinigaglia, F., 225 Sirbasku, D. A,, 212 Siuta, P. B., 362

AUTHOR INI)EX, VOLUME 40 Skehan, P., 335, 346 Skoza, L., 156 Skutelsky, E., 171 Sloan, H. R., 275 Sloane-Stanley, G. H., 247, 271(45) Sloniiany, A,, 140 Sloniiany, B. L., 140 Sly, W. S., 363 Sniellie, J. G., 350, 357(389) Smets, L. A,, 227 Smilowitz, H . , 347 Smirnova, G. P., 140, 146, 165(98) Smith, C. H., 226 Smith, D. F., 176, 337 Smith, I. C. P., 139, 169, 170(166, 167) Smith, M . J., 297, 298(57) Smith, P., 139 Smith, R. E., 371 Smith, T. L., 178 Smolina, Z. I., 51, 57(166), SS(l66) Snetting, M. C., 157 Snider, M. D., 320 Sciderlund, H., 356 Sohiir, P., 26, 29(53) Solland, J., 44 Sominers, L. W., 187 Sonnino, S . , 139, 144(50), 217, 240 Sonogashira, K., 7 Sorrell, M. F., 345 s o s s o , M. c.,222 Sotiroudis, T. G., 212 Soupart, P., 215, 219(425) Soyagimi, H., 99 Spataro, A. C., 188, 194(263) Spaulding, D. R., 323 Speake, B. K., 300, 301(76, 77), 30:3(76), 304(76) Spencer, J. P., 310, 311(152), 326, 342, 343(347), 344(350), 348(350) Spicer, S. S., 171 Spiegel, S., 171 Spiegel, Y., 171 Spik, G., 138, 142(37),207(40) Spiro, M. J., 289, 293(6), 299(6), 301, 303(6), 307, 308(86, 135), 348(6) Spiro, R. G., 188, 244, 289, 293(6), 299(6), 301, 303(6), 307, 308(86, 135), 348(6) Spoormaker, T., 133, 139(18), 170(18), 187(18), 211(18) Spratrgcr, J . W., 207

425

Springer, G . F., 228, 352 Springer, J., 324 Silringfield, J. D., 309, 310(146), 311 Sreenivasan, S., 387 Sriv;tstava, R. M., 33, 36, 40, 41, 42(85), 44(129) Srivastava, S. K., 270, 274(169), 277(167), 278 Srogl, J., 62, 63, 64(205) Stdil, P. D., 335 Staiiek, J., Jr., 3 , 42 Stnneloni, R. J., 308 Stanley, P., 176, 313, :322(170) Stannard, B. S.,363 Stapley, E., 324 Stark, N. J., 346 S t d i n g , J. J., 337 Steeti, G. O . , 238 Steer, C. J., 220 Steers, E., 269 Steigerwald, J. C., 2.56, 265(112) Stein, A,, 271 Stein, O., 378 Stein, Y., 378 Steiner, S. M,, 370 Stellner, K., 252 Stenflo, J., 344 Stenzel, K. H., 232 Stephen, A. M., 391 Sternian, R., 338 Stcmi, W., 372 Stetson, B., 308 Steward, W. E., 374 Steyn-Parve, E. P., 316, 326(191) Stillor, I., 62. 63. 64(205) Stilxinovic, A. J., 388 Stirling, J. L., 277 Stii-m, S., 396 Stockcrt, R. J., 220 Stotfyn, A., 245, 246, 247(44), 249, 254, 255(106), 259, 260(41, 441, 268(41) Stoffyn, P. (J.), 245, 246, 247(41), 248(51), 249, 254, 255(106), 258(51), 259, 26W-11, 44), 268(44) Stohrri-, R., 371 Stoll, A., 62 Stone, K. J., 292, 325 Sttroltiiiller. A . C., 246, 255(13), 257, 259, 265 Stratitiegird, O., 230 Strating, J., 30

426

AUTHOR INDEX, VOLUME 40

Straw, D. C., 195, 206, 211(306) Strecker, G., 138, 142(37), 147, 163(105), 165(105), 169, 179(105),185(105), 207(40), 363 Streicher, H.-J., 222 Strickler, J. E., 319 Strijland, A., 363, 364 Strmen, J., 337 Strominger, J. L., 290(16), 291, 292(16), 325(16), 347(16) Struck, D. K., 245, 290(50), 295(50), 296, 299(50), 300(50), 308(50), 309(50), 310(50), 312(50), 317, 347(50), 362 Sirdo, T., 176 Sufrin, J. R., 336, 337(304) Sugano, K., 203, 204(360) Sugimori, T., 156, 195, 196(129), 198(129), 203(315) Sugimiira, T., 369 Sugita, M., 140, 146(57), 165(57), 240, 243 Sugiyama, Y., 19, 114(44) Suhara, Y., 34, 127(82) Sulkowski, E., 374 Siilzman, L. A,, 320 Summers, D. F., 301 Sundaramjan, P. H., 381, 383(1), 389, 390(49) Sung, J . S.-S., 280 Supp, M., 133, 167(15), 195(15) Siirani, M. A. H., 367 Srrthanthiran, M., 232 Sutherland, E. W., 303 Sutherland, I. W., 396 Siittajit, M., 172, 202, 208(186) Suttie, J. W., 344 Sutton, A,, 139, 144(47), 233(47) Suzuhi, S., 309, 326 Suzuki, C., 249 Suzuki, K., 203, 205(354), 265, 270, 271, 274(169), 275(205, 206), 278, 283, 284,340 Suzuki, S., 235, 236, 249(5), 277(5) Suzuki, Y., 203, 260, 274, 275(206) Svennerholm, E., 162 Svennerliolm, L., 137, 139, 151, 153(30), 154(30), 155(30,120), 162, 230, 231, 243, 248, 249(65), 257, 265, 272 Svoboda, M., 52 Swank, G. D., 362 Swanson, A. L., 178

Sweeley, C. C., 138, 139(43), 164, 237, 240(9), 243(9, 14), 249, 279, 280 Sweet, F., 31, 32, 35, 36(65), 40(84), 42(65, 76, 84), 44(65, 76) Szakiics-Pinter, M., 72(240, 241), 73 Szarek, W. A., 3, 119 Szechner, B., 65, 67, 69(217), 70, 72

T Tabas, I., 299, 300, 301(73), 307(83), 363 Tiibora, T., 322 rrafei,J., 2 Tager, J . M., 280,335,363, 364 Taigel, G., 133, 167(14) Takahata, H., 88, 90(286), 91(286), 93(286) Takahashi, K., 27 Takai, M., 388 Takatsuki, A., 321, 339, 340(322, 324), 366, 374, 375(542), 376, 377 Takeda, H., 240 Taketomi, S., 231 Taki, T., 243, 260(27) Takizawa, T., 84, 85, 87(278, 279, 28O), 88, 89(280), 90(276-279, 286), 91(286), 92(279, 282, 287), 93(286), 94, 95(290) Tallman, J. F., 197, 257, 265, 266(143), 269, 270, 272(160), 277, 281 Tamari, K., 344 Tamura, G., 321, 339, 340(324), 366, 369, 374, 375(542), 376,377, 378 Tamura, S., 46 Tamura, T., 84, 85, 87(279), 88, 90(276, 279), 92(279) Tanabe, T., 190, 220 Tanaka, H . , 274, 275(205), 342 Tanaka, J., 265 Tanaka, K., 394 Tanaka, Y., 343 Tanenbaiini, S. W., 209, 210 Taniguchi, M., 118, 119(337) Tanner, W., 294, 296(43), 297, 309(51), 310, 311, 316, 317, 318, 341, 362, 372(468, 469) Tannert, C., 225, 226(495) Tanzawa, K., 323, 324(240) Tanzer, M. L., 347, 365

AUTHOR INDEX, \’OI,LJME 10 Tappel, A . L., 269 Tarentino, A. L., 351 Tartakoff, A. M., 301, 329(95),347, 349(95), 357(383) Tanc, L., 217 Taylor, A,, 368 Taylor, N . F., 104 Taylor, P. V., 227 Tegelaers, F. P. W., 363 Telser, A,, 344 Tenrin, S. C., 40 Tennant, L., 274 Tenner, A. J., 314 Terada, M., 369 Tettamanti, G., 139, 144(50),197, 205, 217, 240, 272 Thieffry, A , , 125, 127 Tlroni, D., 170 Thoinas, G. H., 207, 208 l h o m a s , M .A. W., 218 Thompson, J. K., 121 Thorne, K. J. I., 289, 315(5) Thornton, E. R., 169, 202, 204 TIiraenliart, O., 210 Thrum, H., 339 Thudichum, J. L. W., 235 Tick, N. T., 228 Tiesjema, K. H., 167 Tiinpl, K., 367 Tipson, R . S., 124 Tkacz, J. S., 302, 339, 340, 359 Toknra, S., 393 Tolksdorf; M., 207 Toinida, M . , 377 Tomita, K., 9, lO(24) Tompkins, W. A . I?., 228, 229 Tonegnzzo, F., 320 Tonew, E., 339 Toncxw, M., 339 Tonn, S. J., ,321 Torssell, K., 21, 22(47), 23, 38(48), 44(48j, 46(48) Tragerman, L. J., 238 Trantz, V., 84 Trepanier, D., 289, 290(10) Trirnble, R. B., 351, 354 Trotter, J. T., 194 Trowbridge, I. S., 295(120),305,306(118), 313. 333(118, 120) Troy, F. A,, 193, 294, 320(44) Tsai, C.-M., 176

427

‘Tschesche, H . , 356 ‘l‘siitla, M., 231 T\iiji, A , , 219 Tsnji, H., 228 Tsiiji, M., 326 ‘I‘siikatla,Y., 156, 195, 196(129), 198(129),20.3(315) Tiicker, L. C . N., 35

D. K. P., 205 liima, D. J., ,345 Tnppp, H., 147, 199, 208, 209 Tnrco, S. J., 300. 301(72j, 308, 346 Tiisiynski, G. l’., 352 Tyagi, M. l’,, 21, 22(47), 38(48),44(48). Tillsiani,

16(48)

Tytgat, F., -31.5

U L’chida, N., :347 Clclrida, Y., 1.38, 195, 196(129),198(129), 203(315),208 “(la, Y., 280 I’cno, K., 178, lHl(227) Llgalde, R., 308 Llhlenl)ruck, C., 132, 142 Ulrlendorf, R. W., 275 L[llrich, K., 364 Llnletsll, N., 344 Uinezawa, I I . , 34, 127(82) Irnger, F. M., 152, 178 lliikovski, B. W., 51, 57(166),58(166) I~rl)an,P.-F., 208, 255, 265

v \’uheri, A., 367 Vaitukaitis, J . L., 3.52 Valenta, M., 63 v a n Beek, W. P., 227 \’;ince, D. E., 345 Van deCastlr, J. F., 108 Van den Bergli, F. A. J. T. M., 280 van Den Berghe, D. A., 373 vim den Eijnden, 1).H., 168, 188, 211, 268 VIIII Diik, W., 188, 211

428

AUTHOR INDEX, VOLUME 40

van Doorninck, W., 226 van Halbeek, H., 133, 145(17), 146(17), 157(17), 159, 165(139), 166(139), 167(17, 139), 168, 169(17), 195(17), 204, 205(364), 268 van Hall, E., 352 van Heyningen, W. E., 230, 351 van Hoof, F., 138, 147(39),286 Vanier, M. T., 265 Vann, W. F., 185, 194(251) Van Rietschoten, J., 312 Varpakhouskaya, I. S., 38, 39, 41, 44(105) Vass, G., 122 Vassalli, P., 301, 329(95), 347(95), 347, 349(95) Vatele, J.-M., 52(355, 356), 53(356), 127 Veerkamp, J. H., 318 Veh, R. W., 142, 144(77), 145(77), 149, 155(141), 161, 162(77), 163(141), 164(141), 166(141), 171, 172(182), 173(182), 174(182, 193), 175, 176(204), 184, 192(250), 195, 197(312), 199, 200(338), 202(343), 203(77), 205(312, 325), 207(55), 208(55, 141, 368), 209(312, 325), 210(112) Veluraja, K., 169 Venerando, B., 205 Verbert, A,, 292, 317(27), 325(27) Verheijen, F. W., 207 Verizzo, D., 290(24), 292 Versluis, C., 138, 143(34), 145, 146(34), 147(34), 159, 163(34, 94), 164(94), 165(34, 94, 139, 147), 166(98, 139, 147), 167(139), 185(34),209(34) Vertiev, Yu. V., 149, 210(114), 230 Vessey, D. A., 293 Veyrieres, A,, 117 Vijay, I. K., 295(117a), 305, 311(117a) Vikek, J., 374 Villalba, M., 277 Villalta, F., 136, 176(26) Villemez, C. L., 296, 297(47), 309(47), 343, 349(351) Vischer, P., 302 Vliegenthart, J. F. G., 133, 138, 139(18), 141, 142(62), 143(34), 144(62), 145(17, 82, 86), 146(34, 95), 147(34, 39), 151(82), 152(95), 156(93), 157(17, 93), 158(90), 159, 160(93, 97), 163(34, 82, 94, 95, 97, 105, 148),

164(94), 165(34, 62, 85, 94, 95, 97, 105, 139, 147), 166(94, 139, 147), 167(16, 17, 139), 168, 169(17, 93), 170(18), 173(82), 179(105), 180, 183(82, 90), 184(82, 148), 185(34, 105, 238), 187(18), 195(16,17), 202, 204, 205(364), 209(34), 211(18), 215(85), 216(62), 268 Vogt, D., 223 Vogt, P. K., 373 Volk, B. A,, 221 Volk, B. W., 277 von Bonsdorff, C.-H., 347 von During, M., 222, 223(484) von Figura, K., 363, 364 von Nicolai, H., 195, 196(311) Vosburgh, W. G., 84 Voss, B., 364 Vulfson, A. N., 36, 39,41, 42(90), 44(105) Vuong, R., 387, 391 Vyns, D. M., 38, 42, 119

w Wada, M., 96, 97, 98, 100 Wade, R. H., 388 Waechter, C. J., 245, 290(17, 25), 291, 292, 293, 296, 301, 307, 309, 3 10(147) Wagle, S. R., 338 Wagner, D. O., 375 Waheed, A., 363 Walkinshaw, M.D., 399 Walling, C., 87 Walton, D. J., 121 Wan, C. C., 254, 274, 278, 281(238), 283(212, 238) Wang, A., 339 Wang, F. F.C., 351, 354(405) Wang, M.-C., 111,323 Warburg, O., 346 Ward, J. B., 339, 341(326), 348(321) Ward, W. E., 238 Wardell, S., 203 Warner, G. A , , 252 Warren, C. D., 309 Warren, L., 134, 136, 137, 138, 140, 142(20), 143(20), 146, 153, 154, 155(22, 99), 156(22), 157(22), 176,

AUTHOH I N D E X . \.'OLUME 40 177(215), 178(215), 179(22), 186(2lS), 187(99, 215), 188(215), 211(215), 352, 375 Watanabe, K., 139, 238, 240, 243, 260(28),376 Watanabe, M . , 80 Watkins, W. M., 195 Watson, D., 178 Watson, H. A., Jr., 65 Watson, J. A,, 323 Wax, S. D., 206 Wasman, S., 193, 331 Wehb, W. R., 206 Weber, E. J., 236 Welier, P., 171 Welister, J., 216 Wedgwood, J. F., 290(16), 291, 292(16), 32S(16), 348(16) Weeks, D. I., 141, 142(65) Weeks, P. D., 65 Weidemann, G., 327 Weidinger, A,, 388 Weigel, P. H., 220 Weih, M., 387 Weil, H., 374 Weill, J., 194 Weinfeld, H., 323 Weinreb, N . J., 269 Weintravb, B. D., 363 Weintraul), S., 372 Weise, M.,227 Weiss, A. H ~2, Weias, L., 215 Weitzman, S., 303 Wellard, H. J., 386 Wellner, R. B., 289, 290(8, 11) Wells, M . A., 247 Wells, W. W., 164 Welten-Versteegen, G. W., 316, 326(101) Weltner, W., Jr., 157, 167(132), 168 Wcinber, M., 136, 137(29), 138, 141(29), 142(29), 143(34), 144, 145, 146(33, 3 3 ) , 147(34, 106), 156(93),157(93), 158, 160(93, 97), 163(34, 97), 165(31, 85, 97), 169(93), 179, 181(232), 183(33), 184(33, 248), 185(34), 186(33), 187, 190, 191(258, 2591, 192(257), 199, 200(338),202(343), 203, 205, 206, 208(368),209(34, 371 ) 212. 21x85. 106) Wenger, 11 A,, 203, 274, 275, 282

429

Werner, I., 137, 147(32), 153 Werner, J., 210 Wertz, G. W., 335 Wrneinann, W., 216 Wessler, E., 137, 147(32) Westerveld, A,, :364 Westwood, J . H.. 333, 337 Weygand, F., 12, 115 Whistler, R. L., 101 White, D. A,, 300, 303(76),304(76) Wliiteliouse, M. W., 132 W i h , M., 293 Wickner, W., 319 Wiedemann, H. R., 207 Wiegandt, H.. 142, 144(77), 145(77), 162(77), 199, 203(77),204(337, 359), 205(364),217, 230, 237, 240(10), 242(10), 244, 253, 273 Wieniann, R., 17 Wilchek, M., 171 Williams, C. S.,:32, 36(77) Williams, K., 375, 376(541) Williams, R. E., 170 Williains, T., 247, 250!46), 257(46) Willimison, A. H., 361 Willoughby, E., 292 Wilson, 0. S., 316, 321(188) Wingard, L. B., Jr., 210, 211(404) Winick, M . , 180 Winkler, J.. 295(122), 306, 333(122), 346( 122) Winter, W. T., 381, 393, 394, :395, Winterbourne, D. J., 336, 337 Winterburn, P. J., 179 Winzler, K.J., 172, 202, 208(186) \~'iranowska-Steward, M., 374 Wirtli, D. F . , 300, 301(72), 360 Wirtz-Peitz, F.,141, 142(69), 148(69). 154, 155(109), 158 Wistar, R., Jr., 176 Wittei-, D. E., 344 Wlodecki, B., 65 Woda, B. H . , 346 Wnllert, W., 360 Wolt; G., 297, 298(57) Wolfe, L. S., 279 Wolf-UlliSll, c.,398 Wolkoff, A. W., 220 W o m a c k , J . E., 207 W o n g . C.-M.. 111 Won#, R., 219

AUTHOR INDEX, VOLUME 40

430

Wong, Y. P., 361 Wood, W. A,, 212 Woodruff, J. J., 221 Woods, G. F., 31, 35, 40 Woodward, R. B., 14, 114(34) Wos, B., 142, 221(80) Wright, J . A , , 313 Wu, A. M., 218 WU, H.-C. H., 382, 383(13, 14, 15, 16) Wu, K. K., 192, 194(285) Wyke, A. W., 339, 348(321) Wyke, J . A,, 324 Wykes, A , , 362

Yonehara, II., 339 Yoshida, A., 270, 277(167), 278(167) Yoshida, H., 71 Yoshikane, M., 71, 109(232), 110(232) Yoshizaki, H., 240 Yosizawa, Z., 249 Yu, R. K., 153, 164(118), 176, 178, 181(227), 200, 240, 253, 254, 255( IOS), 258 Yurev, Y. K., 79, 84(256) Yuspa, S. H . , 298 Yusufi, A. N. K., 255 Z

X Xodo, P., 222

Y Yamada, K., 174 Yainada, K. M., 346, 366, 375(543), 376 Yamada, S., 117, 118, 119(337) Yamada, T., 240, 242(26) Yamakawa, T., 139, 141, 143(71), 176(71), 235, 236, 244, 249(5), 277(5), 278, 279 Yamaniori, S., 339, 341(330) Yamamoto, Y., 377, 378 Yamanaka, T., 284 Yamane, N., 210 Yamashita, K., 302 Yamashita, M., 71, 109(232), llO(232) Yaniashita, Y . , 384 Yamazaki, S., 374 Yang, H.-J., 253 Yarnell, M. M., 228 Yasuda, S., 32, 35(323), 40(79), 114 Yasue, S., 141, 143(71), 176(71) Yeh, A. K., 208 Yeh, hl., 187 Yeung, K.-K., 249, 250(83) Yguerabide, J., 346 Yip, M. C . M., 257, 258, 259 Yoh, M., 117 Yohe, H. C., 178, 181(227), 258 Yokosawa, N., 250

Zabos, P., 331 Zadarlik, K., 143, 176(81) Zakim, D., 293 Zakstelskaya, L. Ya., 202 Zamhotti, V., 197, 272 Zimochy, J., 337 Zarnojski, A , , 18, 36, 37(88), 38, 39(88, 92), 40,41,42(106, 123), 43(136), 44(123, 124, 136, 138-140), 46(123, 147), 47, 48(146), 49(146), 50(146), Sl(138-140, 146), 54, 56, 57(140, 179), 58(182), 60(138),65, 66, 70, 72, 83, 113, 128(215) Zivada, J., 52 Zdebska, E., 240 Zefirov, N. S., 37, 79, 84(256) Zeigler, M., 207 Zeile, K., 61 Zemek, J., 337 Zen, S., 105 Zhukova, I. G., 146, 165(98) Ziegler, D., 133, 167(15), 195(15), 198, 209, 210 Ziegler, M . , 273 Ziegler, W., 273 Zilliken, F., 132, 195, 196(311),216 Zimmermann, D. H., 303 Zobicovi, A,, 110 Zopf, D. A,, 176 Zugenmaier, P., 385, 386 Zurabyan, S. E., 191 Zwierzchowska, Z., 65 Zwisler, O., 230

SUBJECT INDEX FOR VOLUME 40

A

Alkanes, sugar substrates, 109 Alkenic precursors, for sugar syntheses,

Acetic acid, ethoxalylfluoro-, ethyl ester, carbohydrate substrate, 104, 105 -, (ethylenedinitrilo)tetra-, inhibitor o f protein glycosylation, 297 -, vinyl-, see 3-Butenoic acid Acetylenic precursors, for sugar synthcses, 3 , 4 Acosamine, N-acetyl-DL-, synthesis, 24,

4 -30 Allitol, 2,3:4,5-dianhydro-D~-,synthesis,

26 Allofuranoside, methyl 5,6-di4-acetyl2,3-O-isopropylidene-D~-, synthesis,

108 Allofuranosiduronic acid, methyl 5 0 acetyl-2,3~)-isopropylidene-P-DL-, methyl ester, synthesis, 108 -, methyl 2,3C)-isopropylidene-, methyl ester, synthesis, 75, 76 -, methyl 2,3,5-tri~-acetyl-P-DL-, methyl ester, synthesis, 108 Allonic acid, S-amino-5,6-dideoxy-~~-, synthesis, 112 Allopyranoside, methyl 5-amino-S-Nbenzoyl-5-deoxy-DL-, synthesis, 100,

25 -, N-acetyl-L-, synthesis, 115 Acrolein, DL-threonic acid synthesis, 5 Acrylaldehyde, reactions with nitro alcohols, 105, 106 N-Acylneuraminate-9(7)-O-acetyl transferase, 184 Acylneuraminate pyruvate-lyase, 158, 177, 211-214,219 Alanine, L-, carbohydrate precursor, 117 Alcohols nitro, reactions with acrylaldehyde, 105, 106 reactions with sodium glyoxylate, 106, 107 Ald-3-enopyranoside, alkyl 3,4-dideoxy-

101 -, methyl 6-deoxy-P-DL-, synthesis, 69 -,

methyl 2,3,4,6-tetraO-acetyl-~-DL-, synthesis, 71 Allopyranuronic acid, 1,2,3,4-tetraa-acetyl-P-DL-, methyl ester, synthesis,

108 Allose, 2,5-anhydro-3,40-isopropylidene-DL-, synthesis, 75 Alloside, 20-acetyl-1,6-anhydro-P-D~-, synthesis, 55 Ally1 alcohol, DL-threonic acid synthesis,

DL-

epoxidation, and oxirane-ring opening, 56-59 cis-hydroxylation, 54, 55 synthesis, 48-59 Alcl-2-enopyranosidif-ulose,methyl, re6 duction, selectivity, 71 Altropyranoside, methyl ~ - D L -synthesis, , AId-2-enosif-ulose, synthesis, 65 70 Aldopyranose, 2,6-diamin0-2,3,4,6-tetr:i-, methyl 5-amino-5-N-l~enzoyl-5-deoxydeoxy-, synthesis, 47, 48 DL-, synthesis, 101 Aldopyranoside, alkyl 3 , 4 - a n h y d r o - ~ ~ -, methyl 4 , 6 - d i - O - a c e t y l - a - ~ ~synthe-, oxirane-ring opening, 57-59 sis, 70, 71 synthesis, 56 Altrose, DL-, synthesis, 93 -, alkyl 2,3-anhydroil-deoxyAmicetose configuration, 44 DL-, synthesis, 24 ring opening reactions, 44-47 L-, synthesis, 129 -, inethyl 3,4-dideoxy-3-(dimethyl~1mino~-4rnicetoside, methyl a - ~ - synthesis, , DL-, N-oxide, Cope degradation, Sl 117 4inino acids, optically active, as precurAldopyranosuloses, unsaturated, synthesors for sugars, 117-119 sis, 64-72 4 31

432

SUBJECT INDEX, VOLUME 40

Amphomycin, inhibition of protein glycosylation, 342, 343 Amylose A-, crystal structure bibliography, 383 B-, crystal structure bibliography, 383, 384 -, tri-0-ethylchloroform complex, crystal structure bibliography, 385 crystal structure bibliography, 386 dichloromethane complex, crystal structure bibliography, 385 nitromethane complex, crystal structure bibliography, 385 Amylose- 1-butanol complex, crystal structure bibliography, 384 Anabolic sphingolipidosis-type CM3, ganglioside deficiency disease, 266 Anesthetics effect on ganglioside degradation, 273 on protein glycosylation, 345 Antibiotic 24010, effect on protein glycosylation, 339, 341 Antibiotics aminopolydeoxy sugars, synthesis, 106 component syntheses, 46 inhibition of protein glycosylation, 321,339-344 Antibodies, sialic acid analysis, 175, 176 Antigenicity, glycoprotein, effect of sugar side-chains, 352, 355, 356 Anti-recognition effect, sialic acids, 220229 Apiose DL-, synthesis, 2, 13, 14, 81, 84 L-, synthesis, 115 Arabinitol, DL-, synthesis, 27 Arabinono-l,4-lactone, 5-deoxy-DL-, synthesis, 19 , Arabinopyranoside, methyl ~ - D L - synthesis, 69 Arabinose, DL-, synthesis, 8-10, 73, 91 Asparticin, inhibitor of protein glycosylation, 342 6-Azapseudouridine, synthesis, 80 B Bacillus Zicheniforniis peptidoglycan, crystal structure bibliography, 399

Bacitracin, inhibition of protein glycosylation, 325, 326 Bacteria, sialic acid occurrence, 134 Bibliography, of crystal structures of polysaccharides, 381-399 Bicyclic precursors, sugar synthesis, 7480 Biological functions, sialic acids, 214232 Biosynthesis glycosphingolipids, 244-268 enzyme preparation and enzyme assay, 245-247 lipid-linked oligosaccharides, 288-321 sialic acids, 170-194 1,3-Butadiene, 1-alkoxy-, carbohydrate substrates, 36-38 -, tmns,truns-l,4-diacetoxy-, sugar substrate, 49 -, 1,4-dimethoxy-, sugar substrate, 49 1,3-Butadienyl ethers, sugar, cycloaddition, 123- 128 1,3-Butanediol, 2,4-difluoro-, synthesis, 104 2-Butena1, see Crotonaldehyde 1-Butene, 4-acetoxy-3-(acetoxymethyl)-1ethoxy-, DL-apiose synthesis, 13 cis-2-Butenoic acid, see Isocrotonic acid truns-2-Butenoic acid, see Crotonic acid 3-Butenoic acid, precursor for sugar synthesis, 6 l-Buten-3-yne, 1-methoxy-, precursor for sugar synthesis, 11, 12 2-Butyne-1,4-diol, substrate for carbohydrate synthesis, 27-29 2-Butyn-4-01, 1,l-diethoxy-, precursor for sugar synthesis, 7 Butyraldehyde, 2,4-dihydroxy-3-(hydroxymethy1)-, synthesis, 13, 14

C Carbohydrates protein-bound, biological significance, 287-379 synthesis of optically active, 112-129 from chiral precursors, 115-1 19 from natural products, 119-123 by resolution of racemates, 113-115

SUBJECT INDEX. VOLUME 40 stereo-differentiating, 123- 129 Carbon-13 nuclear magnetic resonaiwc spectroscopy, of sialic acids, 169 Carcinoscorpin, sialic acid analysis, 175 Catabolism glycosphingolipid, 268-286 enzyme preparation and enzyme assay, 269-271 protein activators, 281-286 Cell death, programmed, 368 Cell differentiation, effect of glycosylation inhibition, 366-369 Cell growth, ganglioside effect, 231 Cell mutants effect on biosynthesis of lipid-linked oligosaccharides, 312-314 effect o n protein glycosylation, 347 Cells transformed, fibronectin content, 375, 376 virus-transformed, glycospliingolipid biosynthesis, 266 Cell-surface glycoproteins, glycosylatioii inhibition effect, 374, 375 Cellrilose alkali, ciystal structure bibliogriipliy.

388 biosynthesis, 311 chain packing, 382 native, crystal striicture bibliography,

386 Celliilose I, tri-0-acetyl-, crystal strricture bibliography, 388 Cellulose 11, crystal structure bibliography, 387 -, tri-0-acetyl-, crystal structure 1)iI)liog-

raphy, 389 Cellulose I I , , crystal structure I>ibliogr;iphy, 387 Cellulose III,, , crystal structure bibliography, 387 Cellulose lV, , crystal structure bibliography, 387 Cellulose-N, N’-diinethyl-1,3-propanedianiine complex, crystal structure bibliography, 388 Cellulose trinitrate, crystal structure I ) i b liography, 389 Cephalochordates, sialic acids ncciirrence, 137

433

Ceramide, nionosaccliaride attachments, 238 Cerebroside, discovery, 235 Crrulenin, inhibition of protein glycosylation, 324 Clialcose, DL-, synthesis, 22 Clialcoside, methyl DL-, synthesis, 46 Chiral precursors, for sugar syntheses, 115-123 Chitin, regenerated, crystal striictiirr bib 1i o graph y , 393 cu-Chitin, crystal structure l>iI)liography, 392,393 p-Chitin, crystal structure l>il>liography, 393 Cliloroform, complex with tri-0-ethylamylose, ciystal structure bibliography, 385 Cholera toxin, biological response, 230, 231 Cholesterol, 25-hydroxy-, inhibition of protein glycosylation, 323,324 Chondroitin 4-sulfate crystal structure I>ibliography,394 sodium salt, crystal structure hlhliography, 395 Chromatography gas-liquid, of sialic acids, l M , 165 ion-exchange, of sialic acids, 150 partition on cellulose, of sialic acids,

151 thin-layer, of r i d i c acids, 162-164 Cinerulose A, synthesis, 65 Circular dichroism, of sialic acids, 170 Cloning, effect of tunicamycin, 376 Clostridial infections, sialidase role, 214, 2 19 Clostridial sialidascx, 272 CMP-N-acetylrieuraminic acid, biosynthesis, 186- 188 Cr\.IP-N-acetylneuraminic acid hydrolase, 211 CMP-sialates, enzymic synthesis, 186-

188 Coglucosidasc, activator, 283 Collagen, biosynthesis, glycosylation cffects, 364-366 Coloriy-stimulating factor, 377 Colorimetric analysis, of sialic acida, 153-160

434

SUBJECT INDEX, VOLUME 40

Compactin, inhibition of protein glycosylation, 323, 324 Conformation macromolecules, effect of sialyl residues, 218,219 protein, effect of' sugar side-chains, 353 Cope degradation, 50, 51, 53 Coumarin, inhibition of protein glycosylation, 344 Crotonaldehyde, precursor for sugar syntheses, 7 Crotonic acid, precursor for sugar synthesis, 4-7 Crustaceans, sialic acids occurrence, 136 Crystal structure, of polysaccharides, bibliography, 381-399 Critscum, activator for enzymic hydrolysis of glycosphingolipids, 281-285 Cymarose, DL-, synthesis (attempted), 22,23 Cymaroside, methyl WDL-, synthesis, 40 Cytidylyltransferases, 186- 188

D Daunosamine, synthesis, 16 -, 4 - d e o x y - ~ ~synthesis, -, 111

Daunosaminide, isopropyl N-acetyl-aDL-, synthesis, 17 -, methyl DL-, synthesis, 27 -, methyl 3-N-acetykhdeoxy-a-DL-, synthesis, 18 Desosamine DL-, synthesis, 22,46, 106 L-, synthesis, 120 Desosaminide, ethyl D-, synthesis, 47 -, methyl DL-, synthesis, 46, 107 Deuterostomes, sialic acids occurrence, 137 Dibucaine, effect on protein glycosylation, 345 Diels- Alder condensation of alkoxy-1,3-butadienes, in sugar syntheses, 36-38,49 in sugar syntheses with fiiran, 74, 75 Digalactosylceramide, biosynthesis, 248 2,7-Dioxabicyclo[3.2.0]hept-3-ene, 6methyl-, substrate for 3-deoxystreptose, 83

6,8-Dioxabicyclo[3.2.l]oct-3-ene,synthesis, 35, 36 1,3,2-Dioxaphospholene, carbohydrate substrate, 122, 123 1,3-Dioxolane, 2-(2-furyl)4,4,5,5-tetramethyl-, substrate for carbohydrate synthesis, 73 1,3-Dioxol-2-one, carbohydrate substrate, 84 -96 -, 4-bromo-5-(trichloromethyl)-, synthesis, 85, 86 -, 4-chloro-5-(trichloromethyI)substrate for monosaccharide synthesis, 84-96 teloniers, 85, 86 Disaccharide precursors, synthesis, 128 Disaccharides, synthesis, 123-129 Disialosyllactosylceramide, biosynthesis, 254,255,262 Diiimycin, inhibition of' protein glycosylation, 343, 344 Dolichol biosynthesis, 288-299, 323 composition, 288 cytological and topological aspects, 314-321 siibcellr~l;~r localization, 315-318 -, 2,3-dehydro-, phosphates, 289 Dolichol phosphate biosynthesis, 289-299 inlrihitors of formation, 322-326 Dopamine P-hydrolase, sialic acid effect, 2 19

E Enkephalins, effect on glycosylation, 347,350 Enzymes, sialylated, 219 Enzymic hydrolysis, protein activators, 281-285 Epoxidation, in sugar synthesis, 42-44, 56-59 Epoxides, ring opening reactions, in sugar synthesis, 44-47, 56-59 Erythritol, synthesis, 29 -, 2-amino-%deOXy-D-, synthesis, 114 -, %amino-2-deoxy-DL-, synthesis, 29, 30 -, 2,3-anhydro-, synthesis, 29

SUBJECT INDEX, VOLUME 40 -, 2-deoxy-2-fluoro-DL-, synthesis, 104 Erythrocytes, desialylated, 222-226 Erytlirofiiranaside, methyl 3-C-(hytlro\ipinethyl)-2,30-isopropyiidene-P-DL-, synthesis, 81, 82 Erythronic acid, 4 - l ~ r o t n o 4 - d e o x y - ~ ~ - , synthesis, 5 -, 4-chloro4-deoxy-DL-, synthesis, 5 -, 4-deoxy-DL-, synthesis, 5 -, 2-deoxy-2-fluoro-DL-, methyl ester, synthesis, 105 Etythrono-1,4-lactone, DL-, synthesis, 6 Erytliropoietin, desialylated, 221 Erythrose D-, synthesis, 121, 122 DL-, synthesis, 6, 7, 90 Esclrericliia coli polysaccharide mutarit M4 1, crystal structure bil)Iiograpli>~,

395 Ethane, l-(2-f~iryl)-1,2-dihydl.oxy-,sul)strate for carbohydrate synthesis, 70 Etlranol, effect on glycosylation, 3 4 s 1,2-ECtlienediyl carbonate, ,see 1,3-Dioxol2-one Ethers, sugar substrates, 109 Everniicose D-, synthesis, 114 DL-, synthesis, 20 Exoglycosidases glycospliingolipid catabolism, 268 nonienclatrire, 285 Ezoaminouroic acid methyl glycosidr. \yntliesis, 4 6

F Fal~iy'sdisease, 279, 280 Fil)ronectin, in trarisformed cells, 375. 376 Forosainine, D-, synthesis, 114 Forosaniinide, metliyl, synthesis, 106 Fructose D-, synthesis, 2 L-, synthesis, 2 Fructoside, methyl 0-DL-, synthesis, 71 Fucosylceramide, isolation, 238 ~ - ~ - F i i c o s y l t r a n s f e r a s252 e, 2-Firraldehyde, substrate for carboliydrate synthesis, 7 2 , 73 -, 5-methyl-, substrate for carhohytlr;ttt. synthesis, 74

43s

Fiiran, 2,5-di;ilkoxy-2,S-diliy~lr~~-, siijia -,

substrates, 61 2,3-dihydi-o-, carbohydrate sril)strate,

80-84 -, 2,5-dihydro-, transformations, s r i g u syntheses, 61-74 -, 2,5-dihytlro-2,5-dimetlioxycarbohydrate substrate, 64 -72 ci.y- and trcitw, Iiydroxylatioii, 61, 62 -, 3-(1,1-dinietlioxyetliyl)-2,S-dihydro2,5-dimethoxy-, synthesis, 63 -, 2-ethylll,5-diliydro-S-oxo-, Iiexitol precursor, 111 -, tetraliydro-',l-dihyciroxy-3-( 1-11ydroxyetl~yl)-2,S-dirnethoxy-,synthesis, 6 3 F'rirfuryl alcolrol, substrate f o r car1)oIiydrate syntlicsis, 72 3-Friroic acid, 2,3-dihydro-, car1,oIiytlrate siil)str;ite, 80, 81

G G;ilactitol, 2.3 :4,.5-diantiydro-l,6-di-()inesyl-, synthesis, 26 C~;tlactomaririan,crystal structure 1)iI)liography, 391, 392 C;iilactopyl.anosidc, methyl 6-deoxy-uDL-, synthesis, 70 Galactose, DL-, synthesis, 93 -, ~~~)-(2-acct~tlliido-2-deoxy-cu-D-~~i~~1ctopyranoavl)-2-O-cu-~-fiicopyranosvl-~,-, synthesis, 127 p-D-Calactosidasc. acidic and neritrul, 274-276 cr-D-Galactositl;iae A, 279, 280 cu-u-Galactosicl;c\e B, 279, 280 <;dactosidr, 241-acetyl- 1,6-anliydro-pDL-, sytitheis, SS Galactosylct.riiiiide I)iosynthesis, 247, 248 catabolisiii, 274 glycosphingolipitls, 238 isolation, 238 G~ilactosylcercuiiidt.sulfate sulf:it:tsc, itctivator tor enzymic Iiydrolysis, 281, 283 a-Galactosvl transfefiise, 249, 252 p-D-Galactosyltraiisferase, 248 Gang1iosi d e h asialo CM1, I)iosyntheais, 259, 263

436

SUBJECT INDEX. VOLUME 40

catabolism, 274-276 asialo GM2, biosynthesis, 259, 263 catabolism, 276-279 biosynthesis, 253-265 deficiency disease, 265, 266 transient intermediate, 264 definition, 237 degradation by sialidase, 271-274 discovery, 235 GD1, biosynthesis, 260, 263 GDla, biosynthesis, 258, 261 C D l h , biosynthesis, 257,258, 262 GD2, biosynthesis, 256, 257, 262 GD3, biosynthesis, 254, 255, 262 GM1, biosynthesis, 257, 258, 261, 263 catabolism, 274-276 sialidase action, 272 GMlb, biosynthesis, 263 GM2, biosynthesis, 255-257, 261 catabolism, 276-279 sialidase action, 272 GM3, biosynthesis, 254, 261 sialidase action, 273 GTla, biosynthesis, 258, 259, 261 GTlb, biosynthesis, 258, 262 sialidase action, 271, 273, 274 isolation, 235, 242, 243 sialic acids occurrence, 139, 140 sialylation, 217 structure, nomenclature, and abhreviations, 242, 243 toxin binding, 230, 231 Gangliosidosis, GM1, 274-276 Ganglio-type gangliosides, biosynthesis, 255-265 Garosamine, DL-, synthesis (attempted), 23 Gaucher’s disease, 282 Globoid leukodystrophy, see Kral)lie’s disease Globopentaosylceramide, biosynthesis, 250 Globoside, discovery, 236 Globotetraosylceramide biosynthesis, 249 catabolism, 276-279, 286 Globotriaosylceramide, biosynthesis, 249 D-Glucan, from Acetobacter xyhnuni, crystal structure bibliography, 388 (1 3)-a-D-Glucan crystal structure bibliography, 386

-

polymorphs, crystal structure bihliography, 386 (1 + 3)-p-D-Glncan, crystal structure bibliography, 389 Glucofuranose, 3G-(benzyl 4-deoxy-p-~gulopyranosid4-ylj-1,2:5,6-di-O-isopropylidene-a-D-, synthesis, 126 Glucopyranoside, methyl CI-D-, synthesis, 113 -, methyl ~ - D L - ,synthesis, 70 Glucopyranosylamine, 2-acetaniido-l-N(L-aspart4-oyl)-2-deoxy-/3-D-, biosynthesis, 287, 288 Glucose, DL-, synthesis, 93 -, 2-arnino-2-deoxy-~-,inhibition of lipid-linked oligosaccharide formation, 326, 334-336 -, 2-deoxy-~-,see Hexose, 2-deoxy-~(1 ra bino-, 2-deoxy-2-fluoro-~-,inhibition of lipid-linked oligosaccharides, 326, 332,333 Glucosylceramide biosynthesis, 247, 248 glycosphingolipids, 239, 240 isolation, 238 Glutamic acid, L-, carbohydrate substrate, 118 Glyceraldehyde, 2-deoxy-2-fluoro-DL-, synthesis, 105 -, 2,3-O-isopropylidene-~-, carbohydrate substrate, 120-122 Glycoconjngates, sialic acids occurrence, 138, 140 Glycolic acid, 2-(2-f~1rylj-,resolution of racemates, 113 Glycophorin A, inhibition of protein glycosylation, 339 Glycoproteins antifreeze, 351 antigenicity, effect of sugar sidechains, 352, 355, 356 crystallographic structures, effect of sugar side-chains, 354 desialylated, elimination, 220-223 fbnction of sngar side-chains, 350-353 physicochemical properties, effect of‘ sugar side-chains, 355 proteolysis, glycosylation effect, 356359 routing, secretion, recognition, and up-

S U B J E C T INDEX, VOLUME 40 take, glycosylation effects, 359364 siigar side-chains, effect on confortnntion, 353, 354 Glycosaminoglycans, crystal structure hibliography, 392-395 Glycosidases glycosphingolipid catabolism, 268 isolation and pcirification, 269, 270 specificities, 285 Glycosides nitro, synthesis, 106 thio, synthesis, 101-104 Glycosphingolipids, 235-286 abbreviations, 241-244 hiosynthesis, 244-268 enzyme preparation and enzyme assay, 245-247 of globo and isoglobo series, 24% 251 of Iacto and neolacto series, 250, 252, 253 in pathological conditions, 265, 266 in virus-transformed cells, 266 i n citro, 266-268 catabolism, 268-286 enzyme preparation and enxymc~ assay, 269-271 with a-linked galactose, 279-280 with P-linked galactose, 274-276 protein activators of enzymic Iiytltolysis, 281-285 classification, 238-244 enzymic hydrolysis, protein activators, 281-286 identification, 267, 268 nomenclature, 237, 238, 241-241 strrictriral features, 236-244 Glycosylation protein, biological effects, 350-378 inhibition b y sugar starvation, 336 inhibition effect on cell differentiation, 366-369 inhibition effect on viruses, 369-37:3 inhibitors, 321 -350 lipid pathway, 287-321 sidedness, signal theory, and tlicor) of menibrarie-triggered folding, 319-321 Glycosyltransferases, 251 acceptor specificities, 246, 267, 268

437

blocking, 266 glycospliingolipid biosyn thesis, 24524 7 Glyoxylic acid, sodium salt, reaction with nitro alcohols, 106, 107 Gulonic acid, 5-amino-5,6-dideoxy-D~-, synthesis, 112 (;iihpyranoside, methyl 6 - t i e o x y - a - ~ ~ - , synthesis, 69

H IHematositle, discovery, 235 Hemichordates, \ialic acids occurrence, 137 Heptitols, synthesis, 04 Heptose, 7-deoxy-glllcc.ro-grrlo-, synthesis, 95 Heptuloside, methyl u - D L - ~ ~ w o - hyn, thesis, 71 IHerpes infections, glycosyliction inhibition effect, 369, 370 2;1-IIexadiene-l,6-diol, substrate h r sugar synthesis, 26 2,4-€lexadienoic acid, .see Sorbic acid l,.l-Hexadieti-~3-one,1-chloro-, sulistrate tbr carholiydrate synthesis, 27 2-Hexenoic acid, cis3,5-epoxy-, I-esoliition, 114 4-Hexenoic acid, frutis-3-liydroxy-:methyl-, methyl ester. precut-sor tor sugar synthesis, 21 -, 3 ( R ) - h y d r o s y 3 ( H ) - n r e t l i y l - ,preparation, 114 Hex-2-enotio-1,5-lact01ie, 2,3,4,6-tetradeoxy-DL-glycero-, see Parasorbic acid Hex-3-enopyranose, 1,6-~1nlrydro-:3,-l-dideoxy-fl-DL%rrJt/iro-, \ yn the si s, 49 Mex-2-enol)yranoside, methyl 2,3,6-trideoxp-ru-DL4r!/t/iro-, synthesis, 32, 67, 69 -, methyl 2 , 3 , 6 - t r i d e o x y - p - ~ ~ ~ r I / t / i r o - , synthesis, 67, 69 -, methyl 2,3,6-trideoxy-~u-DL-tl,rc.o-, synthesis, 67, 69 -, methyl 2,3,6-trideoxy-p-DL-tl,rc.o-, synthesis, 67 Hex-3-enopyranoside, alkyl 3,4-dideoxy-, synthesis, 52

438

SUBJECT INDEX, VOLUME 40

-, benzyl 2,3,4,6-tetradeoxy-u-DL-glycero-, synthesis, 53,54 -, methyl 3,4,6-trideoxy-a-~~+ryfhro-, synthesis, 74 -, methyl 3,4,6-trideoxy-a-~~-threo-, synthesis, 74 Hex-2-enopyranosid-44-rllose, methyl 2,3,6-trideoxy-u-~~-, reduction, 67 -, methyl 2,3,6-trideoxy-p-DL-, reduction, 67 -, methyl 2,3,6-trideoxy-~-glycero-,synthesis, 117 Hex-2-enopyranuronic acid, 1,4-di-O-acetyl-2,3-dideoxy-u-DL-erythn,-, butyl ester, synthesis, 4 9 -, 1,4-di-O-acetyl-2,3-dideoxy-u-DLtlzreo-, biityl ester, synthesis, 49 Hexitol, 2-deoxy-DL-riho-, synthesis, 109 -, 5 , 6 - d i d e o x y - ~ ~ - r i b osynthesis, -, 110 Hexofuranoside, methyl 5-deoxy-2,3-0isopropylidene-P-DL-ribo-, synthesis, 109 -, methyl 5,6-dideoxy-2,3-isopropylidene-p-DL-ribo-, synthesis, 109 Hexofiiranosid-2-ulose, methyl l-deoxy3-C-methyl-p-D-ribo-, synthesis, 123 Hexonamide, 3-arnino-3,4,6-trideoxy-~xylo-, synthesis, 120 Hexonolactone, 4,6-dideoxy-~-riho-,synthesis, 119 Hexopyranose, 3-acetamido-2,3,6-trideoxy-DL-aruhino-, see Acosamine, N-acetyl-, 3-amino-2,3,6-trideoxy-3-C -methylD L - ~ ~ x o see - , Vancosamine

~ r l l ~ ~ O ) - u - D L ~ ~ usynthesis, h ~ n o - , 46, 47 -, methyl 5-aniino-5-N-benzoyl-4,5-dideoxy-ribo-, synthesis, 101 -, methyl 4-aniino-2,3,4,6-tetradeoxy-uDL+ryfkro-, synthesis, 106 -, methyl 4-amino-2,3,4,6-tetradeoxy-pDL-e ry t h ro -, synthesis, 106 -, methyl 2,6-his(acetamido)-2,3,4,6-tetradeoxy-u-DL-, synthesis, 35 -, methyl 3,6-bis(acetamido)-2,3,4,6-tetradeoxy-u-DL-threo-, synthesis, 18 -, methyl 2,4-diamino-2,3,4,6-tetradeoxy-Lu-DL-aruhino-, synthesis, 34 -, methyl 2,3,6-tri-0-acetyl4-deoxy-a-r>x y l o - , synthesis, 113 IIexopyranosid-2-ulose, methyl l-deoxy3-C-methyl-p-~-riho-,synthesis, 123 Hexofiiranosidiiroriic acid, methyl 5deoxy-2,30-isopropylidene-P-DLriho-, methyl ester, synthesis, 109 fi-Hexosaminidases, isolation and characterization, 277-279 Hexose, 3-ainino-2,3,6-trideoxy-DL-lyxo-, .see Daunosaniine -, 3-benzamido-2,3,6-trideoxy-D-xylo-, synthesis, 118 -, 3-benzainido-2,3,6-trideoxy-~-xylo-, synthesis, 117 -, 2-deoxy-D-arnbinoeffect on immunoglobulin secretion, 361 inhibition of lipid-linked oligosaccharide formation, 327-331, 378 -, 3-deoxy-D~-,synthesis, 17, 18 -, 1,6:2,3-dianhydro-4-deoxy-p-~~-lyxo-, -, 2,6-dideoxy-D~-,synthesis, 17 ring opening, 45 -, 3 , 6 - d i d e o x y - ~ ~synthesis, -, 17, 18 -, 3,6-dideoxy-~~-clruhitio-, synthesis, -, 2,3,6-trideoxy-DL-ert~~ro-, ,see Amice33 tose -, 3 , 6 - d i d e o x y - ~ ~ - r i b osynthesis, -, 33 Hexoses -, 2,6-dideoxy-3C-methyl-~~-ribo-, see deoxy, synthesis, 17, 18 Mycarose 2,4-dideoxy, synthesis, 4 1 -, 4,6-dideoxy-3-0-methyl-~~-xylo-, see 4-Hexyn-1-al-3-01, 3-methyl-, dimethyl Chalcose acetal, precursor for DL-mycarose, -, 3,4,6-trideoxy-3-(dimethylamino)-DL14, 15 xylo-, see Desosamine Hexopyranoside, henzyl 2,6-dideoxy-uHormones, ganglioside effect, 231 Hyaluronic acid D L - ~ Y X O -synthesis, , 54 calcium and strontium salts, crystal -, henzyl 2,6-dideoxy-u-~~-riho-, synthesis, 54 structure bibliography, 393, 394 crystal structure bibliography, 393 -, ethyl 3,4,6-trideoxy-3-(dimethyl-

S U B J E C I I N D E X , V O L U M E 40 I Idopyranoside, methyl 5-benzamido-5deoxy-, synthesis, 97, 98 -, methyl 6-deoxy-a-DL-, synthesia, 70 Idose, DL-, synthesis, 93 Immunogenicity, sialic acids, 176 Imrnunoglobulins secretion, effect of glycosylation, 361 structural effects, 354 Inhiliition, glycosylation, biological rifeects, 350-379 Inhibitors, of protein glycosylation, 321 -

439

Klebsiellu K25 polysaccharide, cry\tal structure Iiihliograpliy, 397

Klebsiella K30 polysaccharide, crystal structure Iiihliograpliy, 396 Klebsiellu KM polysaccharide, c v s t a l stnicture bibliography, 398 Klebsielln K57 polysaccharide, crystal structure I,ibliography, 396 Klebsiella K63 polysaccharide, crystal structure bil)liography, 398 Krabbe’s disease, 274 -276

350 Inositol, D ~ - l , 2 : 3 , 4 - d i ~ - i s o p r o p y l i c l e i r ~ L 5O-niethyl-ccpi-, carbohydrate sill)Lactosylceramide strate, 108 biosynthesis, 248 Inositols, carbohydrate substrates, 108, catabolism, 274 -276 109 Lactotetraos ylceramide Ins II li n biosynthesis, 251, 252 glycosylation inhibition effect, 377 catabolism, 274 -276 receptor, 231 I,aspartoniycin, inhihitor o f protein glyInterferon cosylation, 342 glycosylation inhibition effect, 373, Lectin 374 anti-recogiiition glycoprotein, 220 inactivation, 221 sialic acid analysis, 175 Iodine tris(trifluoroacetat~),reaction\ Lentinan, crystal structure Iiibliography. with alkanes and ethers, 109 390 Isocrotonic acid, precursor for sugar syriLidocaine, effect on glycosylation, 345 thesis, 4-7 Limulin, sialic acid analysis, 175 Isoglohotetraosylceramide, biosyntlitxsis, Lincosaminide, methyl 7-deoxv-a-DL-, 24 9 synthesis, 6 0 Isoglobotriaosylcerainide, biosynthesis, Lipase 24 9 in cultured cells, 377, 378 effect of sugar side-chain, 351 Lipoprotein, desialylation, 221 K Lymphocytes, desialylated, 221 Kanosaminide, methyl N-acetyl-2,4,6-triLysosomes, glycosphingolipid catalioO-acetyl-a-~-,synthesis, 72 lism, 269 Kasugaininide, methyl D-, synthesis, 114 Lyxotiiranoside, methyl 5-deoxy-3-C:-(hy-, methyl DL-, synthesis, 34 droxymetliyl)-2,3-O-isopropyl ideneKasuganobiosamine, synthesis, 35 P-DL-, synthesis, 82 (-)-Kasuganobiosainine, synthesis, 127 Lyxopyranoside, methyl a-DL-, synthesis, Ketones, conjugated, substrates for car67 bohydrate synthesis, 26, 27 -, methyl P-DL-, synthesis, 68 Klebsiellu K5 polysaccharide, clystal -, methyl 2 , 3 - a n h y d r o - a - ~ ~synthesis, -, structure bibliography, 398 68, 6 9 Klehsiellu K 9 polysaccharide, crystal -, methyl 2,3-atihydro-P-u~-,synthesis, structure bibliography, 397 69 Klehsiellu K16 polysaccharide, crystal -, methyl 2,3,4-tri-<)-acetyl-5-amino-5structure bibliography, 397 deoxy-5-N-jmetlroxy)-, synthesis, 100

SUBJECT INDEX, VOLUME 4 0

440

Lyxose, DL-, synthesis, 8, 9

Mycarose, DL-, synthesis, 14, 15, 20, 21,

-, 5-bromo-5-deoxy-DL-, synthesis, 90 -, 5,5,5-trichloro-5-deoxy-~~-, synthesis,

59 -, L-, synthesis, 114, 129 -, 3-ep,i-DL-, synthesis, 20, 21, 59, 60 -, 5-C-methyl-~L-,synthesis, 6 0 -, 5-C-methyl-3-epi-DL-, synthesis, 60

92

M

Macromolecules, sialic acid effect on Mycaroside, methyl-DL-, synthesis, 14, structure, 2 18-220 15, 114 D-Mannan, crystal structure bibliograMycospocidin, inhibition of protein glyphy, 389 cosylation, 339,340 Mannitol, DL-, synthesis, 2 Mannopyranoside, methyl a-D-, syntheN sis, 113 -, methyl 6-acetamido-2,3,4-tri-O-acetyl- Negamycin, synthesis, 18 6 - d e o x y - a - ~ ~synthesis, -, 72 Neolactotetraosylceramide -, methyl 6-deoxy-a-DL-, synthesis, 69 biosynthesis, 251, 252 -, methyl 6 - d e o x y - 6 C - n i t r o - u - ~ ~syn-, catabolism, 274-276 thesis, 72 Neosaminide C, methyl 4-deoxy-D~-, -, methyl 4,6-di-O-acetyl-a-DL, synthesynthesis, 47 sis, 70 Neuraminic acid Mannose, D-, synthesis, 2 anti-recognition effect, 220 -, L-, synthesis, 2 history, 132 methyl P-glycoside, isolation, 132, 148 -, 2-deoxy-2-fluoro-D-, inhibition of occurrence, 141 lipid-linked oligosaccharides, 326, 333,334 -, N-acetylhiosynthesis, 176-181 Mass spectrometry, of sialic acids, 165enzymic modification, 181-185 167 isolation, 132 Megosamine, DL-, synthesis, 23,25, 26 Megosaminic acid lactone, synthesis, 2 3 occurrence, 14 1 reversible cleavage, 158,211-214 Methane, dichloro-, complex with tri-0structure, 132 ethylaniylose, crystal structure bib-, N-acetyl-l-0-acetyl-, occurrence, 145 liography, 385 -, N-acetyl-7-O-acetyl-, occurrence, 144 -, nitro-, complex with tri-0-ethylamy-, N-acetyl-8-O-acetyl-, occurrence, 145 lose, crystal structure bibliography, 385 -, N-acetyl-9-O-acetyl-, occurrence, 143, Methylenitan, synthesis, 2 144, 145 -, N-acetyl-4-O-acetyl-9-0-la~tyl-,occurMevalonic acid, inhibition of protein glycosylation, 324 rence, 145, 160 -, N-acetyl-9-azido-9-deoxy-, synthesis, Mevinolinic acid, inhibition of protein 178 glycosylation, 324 Molluscs, sialic acids occurrence, 136, -, N-acetyl-l,Y-di-0-acetyI-, occnrrence, 137 145 Monensin, effect on glycosylation, 347 -, N-acetyl-7,9-di-O-acetyl-, occtirrence, 143, 144, 145 Monosaccharides -, N-acetyl-8,9-di-O-acetyl-, occurrence, dolichol-linked, biosynthesis, 288-299 144, 145 syntheses from 2,5-dihydrofitrans, 61-, 4-O-acetyl-N-glycoly1-, occurrence, 74 145 from vinylene carbonate, 84 -96 Mucous secretions, viscosity, sialic acid -, 7-O-acetyl-N-glycolyl-, occurrence, effect, 218 145 Mycaminoside, methyl DL-, synthesis, 3 2 -, 9-0-acetyl-N-glycolyl-, occurrence, -, methyl WL-, synthesis, 117 143, 144

SUBJEC3' INDEX, VOLUME 40

-, N-acetyl-4-O-methyl-, synthesis, 146 -, N-acetyl-80-methyl-, occnrrence, 146 -, N-acetyl-7,8,9-tri-O-acetyl-, occurrence, 143, 145

-, 4,9-di-O-acetyl-N-glycolyl-, occ'iirrence, 145 -, 7,9-di-O-acetyl-N-glycolyl-, occiirrence, 143 -, 8,9-di-O-acetyl-N-glycolyl-, occurrence, 145 -, N-glycolylbiosynthesis, 181, 183 isolation, 132 occurrence, 142, 143 -, N-glycolyl-8-O-methyI-, occurrencc, 146 -, N-glycolyl-8-O-sulfo-, occurrence, 146 -,

7,8,y-tri-O-acetyl-N-glycolyl-, occiir-

rence, 143, 145 Neuraminidases, see Sialidases Nrwcastle disease, effect of glycosyLtion inhibition, 372 Nigeran, crystal structure bibliograplry. 384 Nojirimycin, synthesis, 96 Nomenclature exo-glycosidases, 285 glycosphingol ipid, 237 -244 Non-carbohydrate substrate, see Sul) strate Non-2-enopyranulosonic acid, 5-aceta-

mido-3,5-dideoxp-D-alyc.ero-D-~~1locto-, occnrt-ence, 147, 185

44 I

D L - ~ ~ ! I C ~ , . ~ - D L - ~ ( ~ synthesis, ~ U C ~ O - , 60 Octose, DL-threo-DL-ido-, synthesis. 95 Oleandroside, methyl DL-, synth -, methyl WDL-, synthesis, 40 -, methyl a-L-,synthesis, 117 Oligosaccharide preciirsors, lipid-linked, biosynthesis pathways, 295 Oligosaccharides lipid-linked, assernl)ly and transfer to protein in citro, 307-312 assembly and transfer to protein in oioo, 299-306 biosynthesis, 288-321 cell mutants in biosynthesis, 3123 14 inhibitors of formation, 326-342 sialic acids occui-rence, 138- 140 Olivomycose DL-, synthesis, 20 L-, synthesis, 129 Oncology sialic acid tunction, 232 sialidase treatment, 228

7-Oxabicyclo[2.2.l]hept-2-ene-exo-5,6diol, 5,6~)-isopropylidene-,carbohydrate substrate, 75, 76 1,3,4-0xadiazole, 3 - a m i n o - 5 - p - ~ ~ - r i h o furanosyl-. synthesis, 77 1,2-Oxazine, 3,6-dihydro-cis-6-nietlioxy3-methyl-, substrate for hexonic acids. 112

P

l'achyman, 0-acetyl-, crystal structure 2-Non cilopyranos- 1-onic acid, 5-anlii~O:3,5-dideoxy-D-gl!/cer-o-D-~~1/~1~~~~)-, bibliography, 390 Parasorbic acid Neiiraminic acid DL-, reduction, 38 Norleucine, 6-diazo-5-oxo-~-,inlril)ition synthesis, 21, 22 of protein glycosylation, 344, 368 (S)-Parasorbic acid, carbohydrate subNoviose, synthesis, 70 strate, 119 Nuclear magnetic resonance spectros3-Pentadienone, siibstrate for carbohycopy, of sialic acids, 167 drate synthesis, 27 Nucleoside analogs, synthesis, 77-80 2,4-Pentanedione, 3-henzoyloxy-, precursor for 4-deoxy-DL-darinosaniine, 111 0 Pentaric acid, 2,3-di-O-acetyl4-deoxy-~3,5-0ctadiyne-2,7-diol, 2,7-dimettiyl-, threo-. dirnethyl ester, synthesis, 116 reaction with sngars, 124 4-Pentenal. precursor for sugar synthesis, 12 Octopyranoside, inethyl 6-acetamidoycero-D~iillo-, 6,7,8-trideoxy-u-~~-gl 2-Penten-1,5-dial4-one, synthesis, 73 synthesis, 60 3-Pentenoic acid, truns-2-trydroxy-, pre-, methyl 6-acetainido-6,7,8-trideoxy-ncursor tor sugar synthesis, 19 sc'(2

442

SUBJECT INDEX, VOLUME 40

4-Pentenoic acid, 3-hydroxy-, precursor glycero-, synthesis, 33 Pentopyranosid4-ulose, methyl 2,3-difor sugar synthesis, 19 -, 3(R)-hydroxy-, preparation, 114 deoxy-(lS)-D~-,reaction with p-tolPent-2-enopyranosid4-ulose, 2,3-diuenesulfonylhydrazide, 109, 110 deoxy-DL-, synthesis, 66, 72 erythro-Pentopyranosil-dose hydrate, 1-Penten-3-yn-5-01, 1-ethoxy-, precursor synthesis, 62 for sugar synthesis, 9 Pentose, 2-deoxy-~-erythro-,synthesis, 120 Pentitol, 2-deoxy-~~-erythro-, synthesis, 109 -, 2-deoxy-DL+rythro-, 2-deoxy-2-fluoro-, synthesis, 104, 105 diethyl acetal, synthesis, 12 Pentofuranose, 3-deoxy-t/ireo-, tris(trisynthesis, 4, 11, 12, 18, 19, 20, 27, 28 fluoroacetate), synthesis, 83 -, 2-deoxy-~-erythro-,synthesis, 114 -, 2,3-dideoxy-a,P-DL-glycero-, synthe-, e - d e o x y - ~ ~ - t h r e o - , sis, 12, 13 diethyl acetal, synthesis, 12 -, 2,3,4-trideoxyllC-(phenylphossynthesis, 12 phinyl)-DL-glycero-, synthesis, and -, 4-deoxy-2,3-0-isopropylidene-~~1,5-diacetate, 110 erythro-, synthesis, 109 Pentofuranoside, ethyl 3-amino-3-deoxy-, 2,5-dideoxy-~~-threo-, synthesis, 20 p-DL-arubino-, synthesis, 10, 11 -, 3,5-dideoxy-~-erytlzro-, synthesis, 119 -, methyl 2,3-anhydro-5-0-benzyl-a-~- Pentoses Iyxo-, synthesis, 118 2-deoxy, syntheses, 11-13 -, methyl 2,3-anhydro-5-0-benzyl-fi-~- 2,4-dideoxy, synthesis, 41 lyno-, synthesis, 119 syntheses, 8-17 -, methyl 2,3-anhydro-5-0-benzyl-a-~- 4-Pentulose, 5-deoxy-3C-(dimethoxyribo-, synthesis, 119 methyl)-DL-erythro-, dimethyl ace-, methyl 2,3-anhydro-5-0-benzyl-P-Dtal, synthesis, 64 3-Pentulosonic acid, 2-deoxy-2-fluororibo-, synthesis, 118 Pentonic acid, 2-amino-2-deoxy-~-,syn4,5-0-isopropylidene-DL-, ethyl thesis, 121 ester, synthesis, 104 4-Pentulosonic acid, 2,3-di@-acetyl-5-, 2-amino-2-deoxy-~-,synthesis, 121 Pentono-1,4-lactone, 2-deoxy-DLdeoxy-L-threo-, methyl ester, syntheerythro-, synthesis, 19 sis, 116 -, 2,5-dideoxy-~~-threo-, synthesis, 20 2-Pentyn-l-al, 5-hydroxy-, substrate for Pentopyranose, 1-0-acetyl-3-deoxy-2-Ccarbohydrate synthesis, 36 methyl-5-tliio-~~-erythro-,phenyl2-Pentyn-1-01, 5,5-dimethoxy-, precursor for sugar synthesis, 12 boronate, synthesis, 103, 104 -, l-O-acetyl-3-deoxy4C-methyl-5-thio- 3-Pentyn-2-01, l,I-ethoxy-5-(tetrahydropyran-2-yloxy)-, precursor for sugar DL-Lrythro-, phenylboronate, synthesis, 103, 104 synthesis, 8, 9 -, 3,4-dideoxy-1,2-di-O-(trifluoroacetyl)- Peptidoglycan DL-, synthesis, 109 Bocillus licheniforrnis, crystal structure -, 1,2,4-tri-0-acetyl-3-deoxy-2-C-methylbibliography, 399 biosynthesis, 339 5-thio-DL-threo-, synthesis, 103, 104 -, 1,2,4-tri-0-acetyl-3-deoxy4-C-methyl- Phagocytosis, desialylated erythrocytes, 223 5 - t h i o - ~ ~ - t h r e osynthesis, -, 103, 104 Phenobarbital, effect on glycosylation, Pentopyranoside, methyl 2-l)romo-2,3-dideoxy-l-0-methyl-a-DL-erythro-, 345 Pliosphatidylserine, activator for enzysynthesis, 33 mic hydrolysis, 283 -, methyl 2-deoxy-DL-eryt/aro-, synthesis, 54 Plants, sialic acid occurrence, 134 -, methyl 2,3-dideoxyll-O-methyl-a-~~- Plasmapexin, sialic acid binding, 216

SUBJECT I N D E X , VOLUME 40 Poly saccharides bacterial, crystal structure bibliography, 395-399 crystal structure l)il)liography, 38 I -

399 sialic acids occurrence, 138, 139 Polysialylgangliosides, fomiation, 217 Procollagen, conversion into collagen, 364 -366 1,3-Propanediam ine, N , N ‘-dimethy I-, complex with cellulose, crystal structure bibliography, 388 Propenal, see Acrolein Protein activators, enzymic hydrolysis of glycosphingolipids, 281 -286 Protein glycosylation, see Glycosylatioil Proteoglycans, biosynthesis, 364 -366 Proteolysis, glycoprotein, glycosylatioli effect, 356-359 Protozoa, sialic acid occurrence, 136 Psicoside, methyl a-DL-, synthesis, 71 Psychosine, biosynthesis, 247 Pummerer rearrangement, 101-104 Purpurosamine B, synthesis, 47, 4 8 Prirpnrosaminide C, methyl diacetylDL-, synthesis, 35 -, methyl diacetyl-DL-e’pi-, synthesis, 35 Pristulan, crystal structure bibliogr;q)liy,

443

I-Pyrroline-2-carl,oxylic acid, 4-h ydroxy5-(D-clrci/,i?io-tetritol-l-yl)-, 141

R

Receptor D-galactose-specitic, 220 sialic acids a s components, 229-232 Resolution, of racemic carbohydrates and substrates, 113-117 Rihitol, 2-dcoxy-2-flrlol.c,-DL-, synthesis, 105 I~ilmfiiraii~isc., S - d e o x y - 3 - ( : - ( l i ~ ( l r o-\ ~ n1ethyi)-1. ~ ~ ~ - i s o ~ , i - o p y 1 i ( ~ e i i ~ ~ - ~ ~ - D I ~ - , synthesis, 82 -, 2,3-O-isoprol,ylide1ie-DL-, synthesis, 69 I~ibono-l,4-Lactone,5-deoxy-DL-, synthesis, 19 Hibopyranoside, methyl p-DL-,synthesis, 68 -, methyl 2,3,4-tri0-acetyl-5-amino-5deoxy-5-~-(methoxycar~~onyl)-, synthesis, 100 Ribose, DL-, synthesis, 8-10, 73 -, 5-deoxy-3-C-(diuiiethoxymethyl~-~~-, dimethyl acetal, synthesis, 64

391 2H-Pyran, 2-alkoxy-5,6-diliydrochemical and physical properties, 38-41 epoxidation, 42-44 cis-hydroxylation, 41, 42 synthesis, 35-38 -, 3,4-dihydro-, in sugar synthew,. 30-

35 -, 5,6-dihydro-, in sugar syntheses, 3 5 -

60 -, truns-5,6-dihydro-6-(hydroxyni~tlryl)2-niethoxy-, resolution of race In;itrs,

113 -, 2-ethoxy-5,6-dihydro-6-methyl-, p r e p

aration, 36

-, tetrahydro-, reaction with iodine tris(triflrioroacetate),109 Pyrazofurin A, DL-2-epi-, synthesis, 78 Pyrazole, 3-(carboxaniido)-4-~-D~-r.il,ofuranosyl-, synthesis, 77 Pyridine derivatives, carbohydrate s u b strates, 96-101

S Serotonin, sialic acid binding, 216, 217 Showdom yci n inhibition of protein glycosylation, 343,344 synthesis, 80 -, DL-8-epi-, synthesis, 78 -, DL-2-deoxy-, synthesis, 78 Sialic acids, 131-234 abbreviations, 134, 135 analysis, 152-176 circular dichroism, 170 colorimetric methods, 153- 160 gas-liquid clironiatogr;ll,liy, 1 6 4 , 165 histochemical, 171-176 lectins and antibodies, 175, 176 mass spectrometry, 165-167 nuclear magnetic resonance spectroscopy, 167-170 periodate oxidation, 157, 160- 162 spin-litbelling, 170, 171

SUBJECT INDEX, VOLUME 4 0

444

thin-layer chromatography, 162- 164 X-ray crystallography, 170 biological significance, 2 14-234 bios ynthe s i s, 176- 188 cation binding, 216 enzymic release froin glycosidic linkages, 195-211 ftinction, anti-recognition effect, 220229 due to negative charge, 215-218 effect on macromolecular structure, 2 18-220 history, 132 immunogenicity, 176 isolation, acid hydrolysis of glycosidic bonds, 147, 148 enzymic hydrolysis of gl ycosidic bonds, 149, 150 metabolism, 183, 207 nucleotide esters, enzymic synthesis, 186-188 occurrence, 134-147 purification, chromatography on cellulose, 151, 152 ion-exchange chromatography, 150,

151 receptor components, 229-232 reversible clea\cage, 158, 211-214 tumor antigen masking, 227-229 Sialidases amino acid sequence, 198 deficiency, 207 degradation of gangliosides, 271 -274 immobilized, 149, 210 inhihitors, 209 molecular weights, 198 occurrence, 195 pathophysiological significance, 206211 purification, 149, 150, 196, 198 substrate specificities, 200-210 viral, 210 Sialidoses, 207 Sialoglycoconjujiates, calcium binding of bone tissue, 217 Sialosylgalactosylcerainide,biosyntliesis, 253 Sialosyllactos ylcerarnide biosynthesis, 254 sialidase action, 273 Sialosylneolactasylceramide, biosynthesis, 253

Sialosyltransferase, 253-255 activity, 192-194 ganglioside, 192 occurrence, 188 purification, 189 specificity, 190, 191 Sodium taurodeoxycholate, activator for enzymic hydrolysis of glycosphingolipids, 281-285 Sorbic acid, precursor for sugar synthesis, 24-26 Sorboside, methyl WDL-, synthesis, 71 Sphingolipidosis, 265,266, 268 Sphingosine, structure, 236, 237 Streptose, DL-, tetramethyl acetal, synthesis, 64 -, 3-deoxy-, synthesis, 83 -, dihydro-, synthesis, 82 -, a,p-DL-dihydro-, synthesis, 8 2 Streptovirudin, effect on protein glycosylation, 339, 341 Substrate, non-carbohydrate, sugar synthesis, 1-129 Sugar analogs inhibition, of lipid-linked oligosaccharide formation, 326-338 of protein glycosylation, 336-338 Sugars acetylenic precursors, 3, 4 alkenic precursors, 4-30 5-amin0, synthesis, 99-101 branched-chain, synthesis, 13- 17, 8084 1,3-butadienyl ethers, cycloaddition, 123- 128 4-deoxy, synthesis, 41-48 synthesis from non-carbohydrate substrates, 1-129 thio, synthesis, 101-104 Sugar starvation, effect on glycosylation, 346 Sulfatides, see Sulfoglycosphingolipids Sulfoglycospliingolipids, 237

T Tagatoside, methyl a - D L - , synthesis, 71 Talofuranosiduronic acid, methyl 2,3-0isoi.,ropylidene-p-DL-, methyl ester, synthesis, 75, 76 Talopyranoside, methyl 6-deoxy-a-D~-, synthesis, 69

SUBJECT INDEX, VOLUME 40 Tay-Sachs disease, 276-279 Tetritols, 2-deoxy-2-fluoro. synthesis, 104, 105 Tetrose, 3-acetamido-2,3-dideoxy-D-g/!/cero-, synthesis, 117 -, 3-acetainido-2,3-dideoxy-~~-gl!yr.er.o-. synthesis, 117 %Tetrulose, DL-glycero-, synthesis, 26, 29 3,4-Thiolanediol 1-oxide, esters, tliio carbohydrate substrates, 101-10.4 2H-Thiopyran, 5,6-dihydro-, c a r h l i ydrate substrate, 38 Threaric acid, L-, precursor for sugar syntheses, 115-117 Threitol DL-, synthesis, 29 L-, synthesis, 115 -, 2,3-anhydro-, synthesis, 29 -, 2-deoxy-2-fluoro-DL-, synthesis, 1" -, 4-deoxy-2,3-0-isopropyIidene-u-, S ~ I I . thesis, 117 Threonic acid DL-, synthesis, 5 , 6 L-, synthesis, 115 -, 4-bromo-k~eoxy-DL-, synthesis, 5 -, 4-chloro4-deoxy-DL-, synthesis, 5 -, 4-deoxy-DL-, synthesis, 5 Threonine, L-, carbohydrate sul)stratr, 117 Threono-l,4-lactone, DL-, synthesis, 6 Threose D-, synthesis, 121, 122 DL-, synthesis, 5 , 6, 7, 8, 90 L-, synthesis, 117 Thrombocytes, desialylated, 222 Tolyposamine, synthesis, 106 Toxins, binding by sialic acids, 230, 231 1,2,4-Triazole, 3-amino-5-rihofurariosyl-. synthesis, 77 Trisaccharides, synthesis, 126 Trisialosylganglioside, see Cangliositlea Tsushiniycin, inhibitor of' protein glycosylation, 342 Tubocurarine, sialic acid binding, 217 Tumor cells, sialic acid masking, 227, 228 Tunicamine, inhibition of protein glycosylation, 340 Tunicarnycin effect on cell differentiation, 366-369 on collagen biosynthesis, 365, 366

445

on inimiinoglobuliii secretion, 36 1

on virus infections, 370, 371 inhibition of protein glycosyl a t'Ion, 339-342 isolation and structure, 340

V L'ancosaniine, DL-, synthesis, 15 L'ertehrates, sialic acids occurrence, 137 L'esicular stornatitis virus, glycoproteins, effect of sugar side-chains, 355, 356 Vinylene carlmnate, see 1,3-Dioxol-2-one Viral heniagplutinin, synthesis, effect of tunicaniycin, 378 Viruses erythrocyte agglutination, effect of sialyl linkages, 229, 230 glycosylation inhibition effect, 369-

373 sialic acids occurrence, 134, 136 \'iscosity, sialic acid residue effect, 218 Vitamin '4 derivatives, role in protein glycosylation, 297-299 \'itamin B,,, sialic acid effect, 219

w Warfarin, inhibition of protein glycosylation, 344, 345 Wotsonin pyrunridatci, xylan, crystal structure bibliography, 391 Wheat-germ agglutinin effect on cellular transport, 376 sialic acid analysis, 175

X Xnnthornoncrs polysaccharide, crystal structure I)il,liography, 399 Xylan, Wcitsonici pyruniitlutci, crystal structure bibliography, 391 (1 + 3)-P-D-XyIan, crystal struetiire bibliography, 390 Xylopyranose, 5-aniino-5-deoxy-, derivative, syntliesis, 97 Xylose, DL-, synthesis, 8, 9, 73, 91 -, 5-bromo-5-deoxy-DL-, synthesis, 90 -, 5,5,5-trichloro-5-deoxy-, synthesis, 92 Xylosylceramide, isolation, 238

CUMULATIVE AUTHOR INDEX FOR VOLS. 36-40" A ANGYAL,STEPHENJ., [Obituaty of] John Archer Mills, 36, 1-8

F FINNE,JUKKA.See Rauvala, Heikki. FLOWERS, HAROLDM., Chemistry and ~, Biochemistry of D- and L - F L W O S39, 279-345

B BALLOU,CLINTONE., [Obituary of] Karl Paul Gerhardt Link, 39, 1-12 BANASZEK, ANNA.See Zamojski, Aleksander. BARNETT,JAMESA,, The Utilization of Disaccharides and Some Other Sugars by Yeasts, 39,347-404 BINKLEY,ROGERW., Photochemical Reactions of Carbohydrates, 38, 105-193 BINKLEY,WENDELLW., [Ol)ituary of] Joseph Vincent Karabinos, 36, 9-13

G GELAS,JACQUES,T h e Reactivity of Cyclic Acetals of Aldoses and Aldosides, 39, 71-156 GORIN,PHILIP A. J., Carbon-13 Nriclear Magnetic Resonance Spectroscopy of Polysaccharides, 38, 13-104 GRYNKIEWICZ, GRZEGORZ.See Z a mojski, Aleksander.

H C CRAWFORD,SALLYANN.See Crawford, Thomas C. CRAWFORD,THOMASC., Tlie Gulono1,4-lactones: A Review of Their Synthesis, Reactions, and Related Derivatives, 38, 287-321 CRAWFORD,THOMAS C., and C U W FORD, SALLYA", Synthesis of L-Ascorbic Acid, 37, 79-155

IIAINES,ALAN H., Tlie Selective Removal of Protecting Groups in Carbohydrate Chemistry, 39, 13-70 HEW, ANTHONY,[Obituary of] William Ward Pigman, 37, 1-5

I IKEHARA,MORIO,OHTSUKA,EIKO,and F., Tlie MAFWHAM,ALEXANDER Synthesis of Polynucleotides, 36, 135-2 13

D DATEMA,HOELF. See Schwarz, Ralph T. DEY, PRAKASH M., Biochemistry of a-DGalactoaidic Linkages in the Plant Kingdom, 37,283-372 E

EL KHADEM,HASSANS., [Obituary of] Emil Hardegger, 38, 1 - 11

J JARNEFELT,JOHAN.See Rauvala, Heikki. JEFFREY,GEORGEA,, and SUNDARALINGAM, MUTTAIYA,Bibliography of Crystal Structures of Carbohydrates, Niicleosides, and Nucleotides (1976), 37,373-436; (1977 and 1978), 38,417-529

* Starting with Volume 30, a Cumulative Author Index covering the previous 5 volumes will be published in every 5th volume. That listing the authors of chapters in Volumes 1-29 inay be found in Volume 29. 446

CUMULATIVE AUTHOR INIIEX FOR VOLS. 36-40

447

K

R

KARKKAINEN, JORMA.See Rauvala, Heikki. KEGLEVIC,DINA, Glycosidrironic Acids and Related Compounds, 36, 57- IC34 KHAN,RIAZ,The Chemistry of Maltose, 39,213-278 KRUSIUS, TOM.See Rauvala, Heikki.

MUVALA,HEIKKI,FINNE,JUKKA, KRUSIUS, TOM, K.4R&INEN, JORMA,and J ~ R N E F E L JOHAN, T, Methylation Technique\ in the Striictural An& ysis of Glycoproteins and Glycolipids, 38, 389-416

L

S

LEE, YUAN CHUAN.See Stowell, Christopher P. LI, SU-CHEN.See Li, Yu-Teh. LI, Yu-TEH, and LI, SU-CHEN,Biosyiithesis and Catabolism of Glycosptiingolipids, 40, 235-286

M MACALLISTER,ROBERTV., NiitritiL-c, Sweeteners Made from Starch, 36,

15-56 MARCHESSAULT, ROBERTI f . , S w Siirrdararajan, Pudiipadi R. MARKHAM, ALEXANDER F. See Ikelrara, M o ri o . MONTREUIL, JEAN,Primary Structrirc, 01' Glycoprotein Glycans: Basis foi- t l r r Molecular Biology of Glycoproteilrs. 37, 157-223

0 OHTSUKA, EIKO.Sec Ikehara, Morio.

SANDFORD, PAULA , , Exocellular, Microbial Polysaccharides, 36, 265-313 SCHAUER,HOLAND, Clrcniistry, Metabolism, antl Biological Functions of' Sialic Acids, 40, 131-234 SCHUERCH,CONRAII,Synthesis m t l Polymeri.c.ation of Antrydro Sirgars, 39, 157-212 SCIIWARZ,HALPH T., and DATEMA, ROELF, The Lipid Pathway of Protein Glycosylation, and Its 1irIiil)itors, antl 'The Biological Significance of Protein-l)oiind Car1,olrytlratc.s. 40, 287-379 SONNTAG, CLEMENSVON, Free-Radical Reactions of Carbohydrates a s Studied b y Radiation Techniques, 37, 7-

77 STOWELL,CHRISTOPHERP., ant1 LEE, YUAN CHUAN,Neoglycoproteins: antl Applicatioi~of The Prep~~ration Synthetic Glycoproteinh. 37, 22528 1 SUNDARALINGAM, MUTTAIYA. S w Jeffrey, George A. SUNDARARAJAN, PUDUPADIK . , a n d MARCHESSAIJL?', ROBE~<TI]., Bihliography of Crystal Structures of' Polysaccli;+rides (1976), 36, 315-332; (1977- 1979), 40,381-399

P PALUR, JOHNI I . Affinity Chrotnatogr:c-

p h y of Macromolecular Sul)stancc~ on Adsorbents Bearing Carbohydr;ctc~ Ligands, 39,405-447 PENGLIS,ANNA A. E., Fluorinatcd <:aIbohydrates, 38, 195-285

U

UNGER,FRANK M., Thc~Clienristi?; and Biological Significance of 3-Deouy-

448

CUMULATIVE AUTHOR INDEX FOR VOLS. 36-40 ~-manno-2-octulosonic Acid (KDO), 38,323-388

w WILKIE,KENNETH C. B., The Hemicelluloses of Grasses and Cereals, 36, 215-264

Z

ZAMOJSKI, ALEKSANDER, BANASZEK, ANNA,and GRYNKIEWCZ, GRZEGORZ, The Synthesis of Sugars from Non-carbohydrate Substrates, 40, 1129

CUMULATIVE SUBJECT INDEX FOR VOLS. 36-40" A Acetals, cyclic, of aldoses and aldosides: reactivity of, 39, 71-156 Adsorbents, bearing carbohydrate ligands, affinity chromatography of macromoleciilar substances on, 39,405-147 Affinity chromatography, of macromolecular substances on itdsorbents hearing carbohydrate ligands, 39,405-447 Aldoses, and aldosides, reactivity of cyclic acetals of, 39, 71-156 Analysis, structural, of glycoproteins and glycolipids, methylation techniques for,

38,389-416 Anhydrides, of sugars, synthesis and polymrrization of, 39, 157-212 L-Ascorbic acid, synthesis of, 37, 79-155

Biological significance, and chemistry, of3-deoxy-D-)facinfro-2octulosoriic acid (KDO), 38,323-

388 of protein-hound carbohydrates, 40,

287-379 Biology, molecular, of glycoproteins, 37, 157-

223 Biosynthesis, and catabolism, of glycosphingolipids,

40,235-286

C Carbohydrates. See cilsn, Aldoses, Disaccharides, Glycans, Hemicelluloses, Polysaccharides, Starch, Sugars. bibliography of crystal structures of,

(1976),37,373-436 (1977 and 1978), 38,417-529 chemistry of', selective removal of protecting groups in the, 39, 13-70 fluorinated, 38, 195-285 free-radical reactions of, radiation techniques for the study of, 37, 7-

B Bibliography, of crystal structures of carbohydrates, nucleosides, and nucleotides

(1976),37,373-436 (1977 and 1978), 38,417-529 of crystal structures of polysaccharides

(1976),36,315-332 (1977-1979), 40,381-399 Biochemistry, and chemistry, of D- and ~ - f i i c o s e39, ,

279-345 of a-D-galactosidic linkages in the plant kingdom, 37, 283-372 Biological functions, and chemistry and metabolism, of sialic acids, 40, 131-234

77 as ligands, on adsorbents, for affinity chromatography of macromolecular substances, 39,405-447 photochemical reactions of, 38, 105193 protein-bound, biological significance of, 40,287-379 Carbon-13, nuclear magnetic resonance spectroscopy, of polysaccharides, 38, 13104 Catabolism, and biosynthesis, of glycosphingolipids, 40, 235-286

* Starting with Volume 30, a Cumulative Subject Index covering the previous 5 volumes will be published in e \ ' e r y 5th volume. That listing the chapters in \.olrimes 1-29 may be found in Volume 29. 449

450

CUMULATIVE SUBJECT INDEX FOR VOLS. 36-40

Cereals, hemicelluloses of, 36, 215-264 Chemistry, and biochemistry of D- and L-fucose, 39,279-345 and biological significance, of3-deoxyD-munno-2-octulosonic acid (KDO), 38,323-388 of carbohydrates, selective removal of protecting groups in, 39, 13-70 of maltose, 39,213-278 ofsialic acids, 40, 131-234 Chromatography, affinity of macromolecular substances on adsorbents bearing carbohydrate ligands, 39,405-447 Compounds, related to glycosiduronic acids, 36, 57134

synthetic, preparation and application of, 37,225-281 Glycosiduronic acids, and related compounds, 36,57-134 Glycosphingolipids, biosynthesis and catabolism of, 40, 235-286 Grasses, hemicelliiloses of, 36, 215-264 Gulono-l,4-lactones, a review of their synthesis, reactions, and related derivatives, 38,28732 1

H Hardegger, Emil, obituary of, 38, 1-11 Hemicelluloses, of grasses and cereals, 36,215-264

D Disaccharides, utilization of, by yeasts, 39, 347-404

F Fluorinated carbohydrates, 38, 195-285 Fucose, D- and L-, chemistry and biochemistry of, 39,279-345

G a-D-Galactosidic linkages, biochemistry of, in the plant kingdom, 37,283-372 Gl ycans, of glycoproteins, primary structure of, 37, 157-223 Glycolipids, methylation techniques in the striict u r d analysis of, 38, 389-416 Glycoproteins, glycans of, primary structure of, 37, 157-223 methylation techniques in the structural analysis of, 38, 389-416 molecular biology of, 37, 157-223 neo-, preparation and application of, 37,225-281

I Inhibitors, o f t h e lipid pathway of protein glycosylation, 40, 287-379

K Karabinos, Joseph Vincent, obituary of, 36, 9-13

L Link, Karl Paul Gerhardt, obituary of, 39, 1 - 12 Linkages, a-D-galactosidic, biochemistry of, in the plant kingdom, 37,283-372 Lipid, pathway, of protein glycosylation, and its inhibitors, 40, 287-379 Lipids, glycosphingo-, biosynthesis and catabolism of, 40, 235-286

M Macromolecular substances, affinity chromatography of, on adsorb-

CUMULATIVE Sl1BIEC‘T INDEX FOH \’C)LS. 36-40 ents bearing carbohydrate ligands, 39, 405-447 Maltose, chemistry of, 39, 213-278 Metabolism, and chemistry and biological functions, of sialic acids, 40, 131 -234 Methylation, techniques for, in the structural analysis of glycoproteins and glycolipids, 38, 389-416 Microbes, exocellular polysaccharides of, 36, 265-313 Mills, John Archer, obituary of, 36, 1-8

N Neogl ycoproteins, the preparation and application of synthetic glycoproteins, 37, 225-281 Nuclear magnetic resonance spectrosCOPY, carbon-13, of polysaccharides, 38, 13104 Nucleosides and nucleotides, bibliography of crystal structures of, (1976), 37,373-436 (1977 and 1978), 38,417-529 Nucleotides, p l y - , synthesis of, 36, 135-213

45 1

significance of protein-t)ountI carbohydrates, 40, 287-379 I’hotochemical reactions, of carbohydrates, 38, 105-193 Pigman, William Ward, ol)ituary of, 37, 1-5 Pl;tnts, the kingdom of, biochemistry of WDgalactosidic linkages in, 37, 28.3372 Polymerization. of anhydro sugars, 39, 157-212 I’ol ynucleotides. synthesis of, 36, 135-213 l’olysaccharides. See cilso, Carbohydrates, Glycans, Hemicellriloses, Starch. bibliography of crystal strnctures of, (1976), 36,315-332 (1977-1979), 40,381-399 carbon-13 nuclear magnetic resonance spectroscopy of; 38, 13-10.1 exocellular, microbial, 36, 265-3 13 Protecting groups, selective rerrioval of, in carbohydrate chemistry, 39, 13-70 Proteins, carhohydrate-l,ound, biological significance of, 40,287-379 glycosylation of, b y the lipid patlrway, and its inhil)itors, 40, 287-379

0 Obituary, of Emil Hardegger, 38, 1- 11 of Joseph Vincent Karabinos, 36, Y - 1 3 of Karl Paul Gerhardt Link, 39, 1- 12 of John Archer Mills, 36, 1-8 of William Ward Pigman, 37, 1-5 2-Octulosonic acid, 3-deoxy-~-mrrrriio-,

(KDO), chemistry and biological sigt1ific;inc.c of, 38,323-388

P Pathway, the lipid, of protein glycosylatiori, and its inhibitors, and the biologicd

H Radiation, techniques of, for study of free-radical reactions of carl)oliyclrates, 37, 777 Heactions, free-radical, of carl)ohydriites, a s stndied b y radiation techniques, 37, 777 of the giilono-1,4-lactones, 38, 28732 1 photocheniical, of carliohydrates, 38, 105-19.3 Hcactivity, of cyclic acetals of aldoses and aldosides, 39, 71-156

452

CUMULATIVE SUBJECT INDEX FOR VOLS. 36-40

Removal, selective, of protecting groups in carbohydrate chemistry, 39, 13-70 Review, of the synthesis, reactions, and related derivatives of the gnlono-1,4-lactones, 38,287-321 S

Sialic acids, chemistry, metabolism, and biological functions of, 40, 131-234 Sphingolipids, glyco-, biosynthesis and catabolism of, 40,235-286 Starch, nutritive sweeteners made from, 36, 15-56 Structure, primary, of glycoprotein glycans: basis for the molecular biology of glycoproteins, 37, 157-223 Substrates, non-carbohydrate, synthesis of sugars from, 40, 1-129 Sugars. See ulso, Disaccharides, Carldiydrates. anhydro, synthesis and polymerization of, 39, 157-212 synthesis of, from non-carbohydrate substrates, 40, 1-129

utilization of certain, by yeasts, 39, 347-404 Sweeteners, nutritive, made from starch, 36, 15-56 Synthesis , and polymerization, of anliydro sugars, 39, 157-212 of L-ascorbic acid, 37, 79-155 of' the gulono-1,4-lactones, 38,28732 1 of polynucleotides, 36, 135-213 of sugars from non-carbohydrate s u b strates, 40, 1-129

T Techniques, of methylation, in the structural analysis of glycoproteins and glycolipids, 38, 389-416

U Utilization, of disaccharides and some other srigars, by yeasts, 39, 347-404

Y Yeasts, utilization of disaccharides and some other sugars by, 39,347-404