Carbohydrate Sulphates

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

Carbohydrate Sulfates

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

Carbohydrate Sulfates Richard G . Schweiger, EDITOR

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

Stauffer Chemical Co.

A symposium sponsored by the ACS Division of Carbohydrate Chemistry at the 174th Meeting of the American Chemical Society, Chicago, Illinois, August 30-31, 1977.

ACS

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON,

D. C .

1978

77

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

CIP Data

Library of Congress

Carbohydrate sulfates. (ACS symposium series; 77 ISSN 0097-6156) Includes bibliographies and index. 1. Carbohydrates—Congresses. 2. Sulphates—Congresses. 3. Organosulphur compounds—Congresses. I. Schweiger, Richard G., 1928II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series: American Chemical Society. ACS symposium series; 77. QD320.C37 ISBN 0-8412-0426-8

547'.78 ASCMC8

78-17918 77 1-294 1978

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATESOFAMERICA

ACS Symposium Series

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

Robert F. G o u l d , Editor

Advisory Board Kenneth B. Bischoff

Nina I. McClelland

Donald G . Crosby

John B. Pfeiffer

Jeremiah P. Freeman

Joseph V . Rodricks

E. Desmond Goddard

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Robert A . Hofstader

Raymond B. Seymour

James P. Lodge

Roy L. Whistler

John L. Margrave

Aaron Wold

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.fw001

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.pr001

PREFACE t first glance, the subject "Carbohydrate Sulfates" appears to be so specific and so limited that one may think of it as a small field which can be covered thoroughly in a few articles. The title, however, is misleading, and there is considerably more to it than the title implies. For example, there are polysaccharide sulfates, some of which occur naturally, such as carrageenan, furcellaran, agar, etc., and others, such as the cellulose and starch sulfates, are obtainable only synthetically. Other classes, like the sulfated glycoproteins and sulfated glycolipids, have important biological functions. And there are typical low molecular weight carbohydrate sulfates, many of which occur naturally in plants while others are accessible by synthesis only. Such a variety of compounds obviously requires more than straightforward carbohydrate chemistry. Polysaccharide sulfates are typical polyanions and have colloidal characteristics. Consequently, they involve polyelectrolyte and colloid chemistry. The researcher working with sulfated glycolipids and glycoproteins must be knowledgeable in the general areas of lipids and proteins. Studying biological functions either in the body or in the plant requires familiarity with biological and medical sciences. Many more examples of the complexity of the field of carbohydrate sulfates could be given. This symposium, therefore, cannot possibly present all of today's research in this field, not even a major portion of it. However, it does represent a nice cross-section of present research activities. Together with the references cited, the articles provide a good up-to-date overview of the various areas discussed. Hopefully, the symposium will stimulate the search for additional indepth knowledge, for only basic information of this kind will ultimately create new products for our industry, permit us to cure and prevent diseases, and, some day perhaps, let us understand nature and life more fully. I wish to thank all of the participants, scientists from abroad and from this country, for their efforts and their contributions to bring this symposium to a successful conclusion. Stauffer Chemical Co.

RICHARD G. SCHWEIGER

San Jose, California February, 1978 ix

1 Monosulfate Esters of L-Ascorbic Acid DONALD W. LILLARD, JR. and PAUL A. SEIB

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506

L-Ascorbate 2-Sulfate. In 1972 Halver and his colleagues (1) reported L-ascorbic acid 2-sulfate (AA2S, F i g . 1) was as effective as L-ascorbic acid (AA) in preventing scurvy in rainbow trout and coho salmon. Prior to that time, Ford and Ruoff (2) synthesized AA2S and showed the sulfate ester was more stable than AA to oxidative degradation. Those two reports stimulated the interest of C. W. Deyoe in AA2S because he was engaged in research on formulating catfish feed at Kansas State University. Fish feed is often produced in floating form by extrusion puffing, a process that destroys 70-80% of the L-ascorbic acid in the feed (3). In addition, L-ascorbate 2-sulfate appeared attractive as a potential form of vitamin C since AA2S occurs in Nature; i t is found in the dormant embryo of brine shrimp (4,5), in human urine (6), and in rat l i v e r , spleen, adrenal gland, and urine (7,8). Since there was no commercial supply of AA2S, Prof. Deyoe asked that our group produce a large amount of AA2S. A chronology of events concerning L-ascorbate 2-sulfate is given in Table I. The biological significance of AA2S has not been established. Its antiscorbutic activity appears species-dependent. AA2S is inactive in the guinea pig (14,15), but is active in fish and marginally active in the rhesus monkey (23). It is also p a r t i a l l y active i n insect diets (24). No data are available on man. It has been suggested that AA2S may serve as a storage form of AA, or as a direct sulfate donor in the biosynthesis of s u l fate esters (20,22). Wholebody radioautographs of fish intubated with radioactive L-ascorbate l - 14C or L-ascorbate 2-sulfate-S showed the radioactivity of both compounds was concentrated in the same organs, mainly the skin, fins, lower jaw-plate, kidney, l i v e r , and heart (25). However, in rats and guinea pigs (8), the uptake of AA by organs was different than observed for AA2S. To obtain large amounts of AA2S we f i r s t modified (16) the original procedure of Ford and Ruoff (2) and produced AA2S in 75% yield starting from 5,6-0-isopropylidene-L^-ascorbic acid. But we also found (16) a much better method to prepare AA2S (Figure 2) with the following attractive features; (a) the starting material 35

0-8412-0426-8/78/47-077-001$05.00/0

©

1978 American Chemical Society

Synthesis of 5,6-0-isopropylidene-L-ascorbate-2-sulfate.

Event

3 5

35

35

1

Demonstration of L - a s c o r b i c a c i d s u l f o t r a n s f e r a s e a c t i v i t y i n r a t l i v e r and "colon homogenates which catalyzed format i o n of AA2S- S from L - a s c o r b i c a c i d and 3 -phosphoadenyls u l f a t e - S or sodium " s u l f a t e - S .

1976

Mohamaram et a l . (22)

I n c o r p o r a t i o n of s u l f a t e from AA2S i n t o c h o i n d r o i t i n s u l f a t e . Discovery of widespread d i s t r i b u t i o n of AA2S s u l f hydrolase.

C a r l s o n et a l . (17), Hatanaka et a l . I s o l a t i o n of AA2S s u l f h y d r o l a s e from a marine organism and and mammalian l i v e r . (18) and Roy, et a l . (19)

1974

1975-6 Hatanaka et a l . (20) and F l u h a r t y et a l . (21)

Seib et a l . (16)

1974

Synthesis of AA2S improved.

AA2S gave no a n t i s c o r b u t i c a c t i v i t y i n guinea p i g s .

(14) and Campeau et

1973- 4 Kuenzig et a l . a l . (15)

structure.

Determination of X-ray c r y s t a l l o g r a p h i c

1972- 3 Bond et a l . (5) and McClelland (13)

Discovery of AA2S i n human u r i n e ; d a i l y e x c r e t i o n reported to be 30-60mg of the dipotassium s a l t . Prevention of scurvy i n coho salmon and rainbow t r o u t by equivalent amount of AA2S or L-ascorbate.

Baker et a l . (6,Π.)

1971

I d e n t i f i c a t i o n of AA2S i n Nature from undeveloped cysts of b r i n e shrimp.

1972-3 Halver et a l . (J., 12)

Mead and Finamore (4)

Chu and Slaunwhite (9) and Mumma (10) în VAJUIO o x i d a t i v e s u l f a t i o n of ft-octanol and androsterone during mild o x i d a t i o n of L-ascorbate 2 - s u l f a t e (AA2S) i n non-aqueous media. ~~

Ford and Ruoff (2)

Reference

1969

1968

1965

Date

Table I. Chronology of L-Ascorbate 2-Sulfate

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

1.

LiLLARD AND SEiB

Monosulfate

Esters of

L-Ascorbic

Acid

3

i s L-ascorbic a c i d , (b) the r e a c t i o n solvent i s water, (c) the conversion of AA to AA2S i s 98%, (d) the s u l f a t i o n r e a c t i o n i s r a p i d (< 30 minutes), (e) the s u l f a t i n g agent i s the s u l f u r t r i oxide complex of the l e a s t expensive t e r t i a r y amine ( t r i m e t h y l a mine), (f) the i s o l a t i o n of AA2S i s simple, and (g) a n a l y t i c a l l y pure c r y s t a l s of barium L-ascorbate 2 - s u l f a t e are i s o l a t e d i n 80% yield. Obviously, the chemical synthesis of AA2S presents l i t t l e difficulty (16). L-Ascorbate 6-Sulfate (AA6S). L-Ascorbate 6 - s u l f a t e (AA6S) i s an~intermediate (not i s o l a t e d ) i n t h e production (26) of La s c o r b y l 6-palmitate, a m a t e r i a l that i s used mainly to prevent o x i d a t i v e r a n c i d i t y (27) i n f a t s , o i l s , and dehydrated foods. In a d d i t i o n , L - a s c o r b y l 6-palmitate i s a u s e f u l a d d i t i v e i n breads (28) and cârotenoid c o l o r a n t s (29). L-Ascorbyl 6-palmitate i s synthesized by r e a c t i n g L-ascorbic a c i d TAA) with p a l m i t i c a c i d i n concentrated s u l f u r i c ac"id. The e s t e r i f i c a t i o n of AA i n concentrated s u l f u r i c a c i d i s at f i r s t s u r p r i s i n g , s i n c e L-ascorbic a c i d i s r a p i d l y dehydrated and decarboxylated i n hot a c i d to give almost q u a n t i t a t i v e y i e l d s of f u r f u r a l (30). But the r i n g - s t r u c t u r e of AA i s very s t a b l e i n concentrated s u l f u r i c a c i d at room temperature. We p r e v i o u s l y showed (26) that the absorbance (265nm) of a s o l u t i o n of AA Cx/3xlO" M) i n 95-98% s u l f u r i c a c i d was unchanged over a 46-day p e r i o d . We a l s o found the absorption maximum f o r unionized L-ascorbic a c i d (X 245nm at pH 2) s h i f t e d to longer wavelengtrï ( À 265nm) when AA was d i s s o l v e d i n concentrated s u l f u r i c a c i d , i n d i c a t i n g that a d e l o c a l i z e d c a t i o n had formed i n the s t r o n g l y a c i d i c medium (Figure 3). We f u r t h e r speculated that the primary hyd r o x y l group of AA i s f u l l y s u l f a t e d i n concentrated s u l f u r i c a c i d . Carbon-13 nmr measurements confirmed (26) the formation of the h y d r o x y a l l y l c a t i o n shown i n F i g u r e 3. The ^ C - s p e c t r a of AA i n 99% s u l f u r i c a c i d and of 4-deuterio-L-ascorbic a c i d i n water at pH 2 are shown i n Figure 4. The assignments of the resonance s i g n a l s were made from the r e l a x a t i o n r a t e s of the carbon atoms and from proton-coupled s p e c t r a . The assignments w i l l not be discussed here, s i n c e they are the subject of another paper (31). When AA i s d i s s o l v e d i n 99% s u l f u r i c a c i d , spectrum Β i n Figure 4 shows the r e a c t i o n products a l l have a set of s i x s i g n a l s which c l o s e l y resembles the set of s i g n a l s of AA i n water. Spectrum Β c l e a r l y shows the r e a c t i o n product contains three com­ ponents which are a l l c l o s e l y r e l a t e d to the s t r u c t u r e of AA. Thus, AA does not undergo dehydration and polymerization (32) i n concentrated s u l f u r i c a c i d at room temperature. In t h i s r e p o r t , we describe the i s o l a t i o n and c h a r a c t e r i z a ­ t i o n of the two p r i n c i p a l components formed when AA i s d i s s o l v e d i n concentrated s u l f u r i c a c i d , namely, L-ascorbate 6 - s u l f a t e (AA6S) and L-ascorbate 5 - s u l f a t e (AA5S)T Other references to work on AA6"S" are given i n Table I I ; AA5S has not been reported

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

-

5

m a x

m a x

4

CARBOHYDRATE SULFATES

CH-OH HC0H

OSOl

-0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

Figure 1. -L-Ascorbate 2-sulfate (AA2S)

CH 0H

CH OH ι
9

H-C-0H

(1 ) a l k a l i , p H 10 7 0 ' , 30 m i n . , N

(CH3) N:SO 3

3

n

(2) c a t i o n e x c h a n g e resin (H ) +

(3) b a r i u m

-o

hydroxide

oso: Figure 2.

Synthesis of L-ascorbate 2-sulfate

CH OS0 H

CHoOH

2

Γ

3

HCOH

HCOH 0^

HO

2

.0^

4H S04 2

Γ

ΌΗ

HO

OH

OH + H3O* + 3HS0 ~ 4

Figure 3.

L-Ascorbic acid in concentrated sulfuric acid

Figure 4.

200

Ul

C-3

150

6 , ρ. ρ .τη.

C-2

100

C-4

C-5

C-6

Fourier transform, carbon-13 spectrum of: (A) 4-deuterio-L-ascorbic acid in deuterium oxide at 30°C, ascorbic acid in concentrated sulfuric acid at30°C

C-l

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

50 and (B) L-

DSS

δ:

Ci

ο"

S"

ο

Ci

S!"

Co

Ο Ο

M S

Ο

m a x

T o l b e r t (35)

1975

3

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n N , N dimethylformamide (DMF). AA6S was c h a r a c t e r i z e d by p.m.r. and u.v. spectroscopy. Y i e l d not reported

Hatanaka, Ogawa, and Egami (34)

1974

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n DMF. AA6S was p u r i f i e d by DEAE-cellulose chromatography i n 20% yield. The compound had ε 12 χ 10 , X 265nm (pH 7.0).

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n p y r i ­ dine; AA6S i s o l a t e d by DEAE-cellulose chromatography. Y i e l d not r e p o r t e d . Product chromatographically pure.

Allaudeen and Ramakrishnan (33)

1970

'Event

Reference

6-Sulfate (AA6S)

Date

Table I I . Chronology of L-Ascorbate

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

1.

LiLLARD AND SEIB

Monosulfate

Esters of L-Ascorbic

Acid

7

previously. The i s o l a t i o n of L-ascorbate 6 - s u l f a t e was accomplished as f o l l o w s . A 0.7M s o l u t i o n of L - a s c o r b i c a c i d i n 99% s u l f u r i c a c i d was quenched by dropwise additfion, with s t i r r i n g , of the a c i d i c mixture to e t h y l ether at -65°. A f t e r n e u t r a l i z a t i o n with aqueous barium hydroxide and removal of ether and barium s u l f a t e , i o d i metric t i t r a t i o n showed that 74% of the reducing power i n the s t a r t i n g m a t e r i a l was present i n the aqueous phase. The r e a c t i o n products were separated (35)by ion-exchange column chromatography on DEAE c e l l u l o s e using a gradient of very d i l u t e s u l f u r i c a c i d as e l u t i n g solvent (Figure 5). The percentage of each component i n the r e a c t i o n mixture was determined by i o d i n e t i t r a t i o n and u.v. absorbance at 265nm (Table I I I ) . Component I I I , which accounted f o r 81-85% of the products (u.v. a c t i v i t y ) a p p l i e d to the ion-exchange column, was found to be L-ascorbate 6 - s u l f a t e . The barium s a l t of AA6S was i s o l a t e d as ah amorphous s o l i d i n an o v e r a l l y i e l d of 48% (u.v. on s o l i d s ) . Elemental a n a l y s i s showed the s o l i d contained 81% barium L-ascorbate 6 - s u l f a t e and 19% barium s u l f a t e . No attempt was macTe to f u r t h e r p u r i f y the s o l i d . We found when i s o l a t i n g A A 6 S , the y i e l d of the product can be preserved by using a s l u r r y of barium c a r bonate which had been p r e v i o u s l y de-aerated with n i t r o g e n . Presumably oxygen i s absorbed on the s u r f a c e of the f i n e p a r t i c l e s of barium carbonate, which leads to p a r t i a l o x i d a t i o n of the d e s i r e d product. L-Ascorbate 6 - s u l f a t e was c h a r a c t e r i z e d by u.v. and c.m.r. spectroscopy. The c.m.r. spectrum (Figure 6) shows that, r e l a t i v e to the s h i f t s observed f o r AA, s u l f a t i o n at C-6 s h i f t e d the s i g n a l of C-6 i n water downfield by ^6ppm while the s i g n a l of C-5 moved s l i g h t l y u p f i e l d . Those s h i f t s have been noted by others (36) i n the spectra of sugar s u l f a t e s . The p.m.r. spectrum of AA6S showed that the s i g n a l of the H-6 protons are s h i f t e d downfield by M).5 ppm i n AA6S(ôH-6, H-6 4.14-4.30 at pH 7.0) from those observed f o r L-ascorbate(6 H-6, H-6 3.74 at pH 7). Our method of preparing ana p u r i f y i n g AA6S i s somewhat b e t t e r than those p r e v i o u s l y d e s c r i b e d . When we repeated the method of Allaudeen and Ramakrishnan (33) we found the crude sodium s a l t of L.-ascorbate 6 - s u l f a t e could be i s o l a t e d i n only 26% y i e l d . T o l b e r t (35) obtained AA6S i n 20% y i e l d , which i s probably the same obtained by Hatanaka, et a l . (34). T

1

L-Ascorbate 5 - S u l f a t e . We suspected that Component I I (Figure 5 and Table I I I ) was L-ascorbate 5 - s u l f a t e f o r s e v e r a l reasons: (1) primary hydroxyTs are known to s u l f a t e 3-10 times f a s t e r i n concentrated s u l f u r i c a c i d than are secondary hydroxyls, which explained the small percentage of Component I I ; (2) Component I.I gave one spot on paper chromatography i n the monosulfate r e g i o n , and the spot s t r o n g l y reduced s i l v e r and f e r r i c ions (Table IV); (3) when Component I I was c o l l e c t e d over 6% metaphosphoric a c i d and the a c i d i c s o l u t i o n concentrated at 25° to a s m a l l

ure 5.

r

10 hr. Time

15 hr.

Component III

20 hr.

Separation of reaction products of L-ascorbic acid in 99% sulfuric acid. Products were chromât ο graphed on a column of DEAE-cellulose (35).

5 hr.

Component I I

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

6.0

5.0

1.7

1.7

I

II

III

IV

pH

T o t a l Recovery

1464

1118

570

351

Total e f f l u e n t , ml.

89.5

0.2

85.0

4.0

0.3

uv

Percent of

87.0

< 0.5

81.0

5.0

0.5

. _ a mixture Iodine titration

acid.

Percentage determined by comparison of absorbance or i o d i n e t i t e r of a peak to the t o t a l absorbance or iodine t i t e r of the mixture applied to the column.

Effluent

Component

by d i s s o l v i n g L - a s c o r b i c a c i d i n 99% s u l f u r i c

Table I I I . Ion-exchange column chromatography of r e a c t i o n products formed

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

C-T

\

j|

C-3

J -

Figure 6.

150

13

C-4

Λ

V

/

l!,

[

ί

M

C-5 C-6

50

pH 6 . 5

pH 6 . 7

L-Ascorbic acid 5-sulfate

L-Ascorbic acid 6-sulfate

2.0

7J

pH 10/2

pH

pH

L-Ascorbic acid 2 - s u l f a t e

Comparison of the C chemical shifts of L-ascorbic acid and its derivatives

100 6 , p.p.m.

1

C-2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

ο

Hi

ο a

> w

d

See

Sample

ASC

=

o

b

not

m

i

i

t

y

2

or

The

H-5

is

179.37

116.06

176..14

80.51 69.81

77.64 77.43

under

the

HOD

peak

at

4.5-4.8.

2,2-dimethyl-2-silapentane-5-sulfonate.

266

apparently

(p.m.r.)

signal

acid.

internal

L-ascorbic

D 0.

to

(c.m.r.)

with

relative

external

exchanged

l

from

Experimental.

Downfield

C R

b

244

1 7 5 .,72

71.50

61.41

4.53

4.68

white

113.90

+

177.65

0.5

6-sulfate

267

white

+

0.3

245

4.57

65.12

81.31 72.28

182.,34

113.01

178.08

255

H-4

232

C-6

red

C-5

-

C-4

4.50

C-3

p.m.r .

65.33

C-2

b

80.96 72.26

C-l

pH 7

δ at

i n Water,

c.m.r.,

1 7 7 . ,87

7

2

H 0

Acid

L-Ascorbate

of

Esters

115.70

pH

in

Monosulfate

179.71

λ

the

265

2

,

of

244

pH

u.v.

Properties

white

5-sulfate

Spray

Spray

0.5

Chloride

Nitrate

2-sulfate

Ferric

+

Alcoholic

Silver

a

IV,

Neutral

1.0

ASC°

C h r o m a t o :n ; r a p h y

Unsubstituted

Derivative

R

Paper

Table

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

9

δ at

3.86

3.73

3.74

H-6'

H-6,

D 0,

4.30

14- 4.14- 4.30

4.

_d

4.05

4.02

H-5

in

6.7

6.5

7.0

7.1

pH

pH

7

b

Ci*

1

IT

Co

ο ο

w

I—I

Η

α

>

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

12

CARBOHYDRATE SULFATES

volume, the r e s u l t i n g mixture contained only L - a s c o r b i c a c i d as determined by paper chromatography; and (4) tîïe 5- and 6-hydroxyls of AA would be expected to undergo very l i t t l e d i s u l f a t i o n s i n c e i t i s known (37) that v i c i n a l d i o l s do not form d i n i t r a t e s upon r e a c t i o n with the e l e c t r o p h i l i c nitronium i o n . Presumably, r e a c t i o n of d i o l s with s u l f u r t r i o x i d e i n concentrated s u l f u r i c a c i d would g i v e the same r e s u l t . Attempts to i s o l a t e Component I I from the column e f f l u e n t were u n s u c c e s s f u l , because as we discovered l a t e r , s u l f a t e i s e l i m i n a t e d from AA5S i n the presence of barium i o n . In f a c t , Component I was thought to be the 4,5-unsaturated product formed by the e l i m i n a t i o n r e a c t i o n . The u.v. p r o p e r t i e s of Component I support the extended resonance system of three conjugated double bonds. Compared to L - a s c o r b i c a c i d , the a b s o r p t i o n maximum of Component I was s h i f t e d 15-20nm to longer wavelength ( X 260nm and 285nm at pH 2 and 7, r e s p e c t i v e l y , νΟΛΛίιΛ À 245nm and 265nm f o r AA at the same pH). In a d d i t i o n , Component I was very unstable above pH 7. We t h e r e f o r e decided to prepare AA5S by an unequivocal route, which i s shown i n F i g u r e 7. Reaction of L - a s c o r b i c a c i d i n p y r i dine with two e q u i v a l e n t s of t- b u t y l d i m e t h y l s i l y l c h l o r i d e f o l lowed by s u l f a t i o n with p y r i d i n e - s u l f u r t r i o x i d e gave 2 6-0-bZ&(t-butyldimethylsilyl. )-L-ascorbate 5 - s u l f a t e (AA5S). H y d r o l y t i c removal of the 2,6-blockThg group with 80% a c e t i c a c i d followed by ion-exchange chromatography gave the d e s i r e d AA5S. m a x

m a x

9

We found Component I I was indeed i d e n t i c a l to AA5S. The two m a t e r i a l s co-migrated on paper chromatograms and on column chromatograms packed with 0-(diethylaminoethyl)-cellulose. The s t r u c t u r e of AA5S was r e a d i l y confirmed by u.v., c.m.r., and p.m.r. The carbon-13 spectrum (Figure 6, Table IV) showed that C-5 s h i f t e d downfield by ^6 ppm while C-4 and C-6 s h i f t e d s l i g h t l y upfield. In the p.m.r. spectrum the s i g n a l s of H-4 and H-6 were s i m i l a r to those of L - a s c o r b i c a c i d , whereas the s i g n a l of H-5 was apparently s h i f t e d downfield by 0.5-0.8 p.p.m. under the HOD peak (Table IV). The i s o l a t i o n of barium L-ascorbate 5 - s u l f a t e i n pure s o l i d form i s d i f f i c u l t because of the tendency of the molecule to undergo e l i m i n a t i o n of the s u l f a t e group. In one attempt, a s o l i d was i s o l a t e d that contained mostly AA5S as shown by c.m.r. s p e c t r o scopy. When an aqueous s o l u t i o n of the barium s a l t of AA5S was allowed to stand at room temperature, barium s u l f a t e p r e c i p i t a t e d i n i n c r e a s i n g amounts with time. Experimental General. A l l evaporations were done under reduced pressure below 50°. M e l t i n g p o i n t s were determined with a Thomas-Hoover apparatus. O p t i c a l r o t a t i o n s were obtained using a Swiss-made Kern p o l a r i m e t e r . L-Ascorbic a c i d was obtained from ICN

LiLLARD AND SEiB

Monosulfate

CH OH

CH OH

2

(l)pyridine +

H-Ç-OSO"

RCI

2

80%

acetic

acid, 30° 2hr.

(2)pyridine: SO

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

Acid

CH OR

2

H-C-OH

Esters of L-Ascorbic

CH

3

R= C h U - C - S i 0

r CH,

Figure 7.

Synthesis of L-ascorbate 5-sulfate

H-C-OSO

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

14

CARBOHYDRATE SULFATES

Pharmaceutical, Inc., C l e v e l a n d , Ohio. Concentrated s u l f u r i c a c i d (99%, 37.0±0.15N) was obtained from F i s h e r S c i e n t i f i c , F a i r l a w n , NJ. T h i n - l a y e r chromatography was performed on microscope p l a t e s coated with s i l i c a g e l G (Brinkman Instruments, Inc., Westburg, New York). Compounds were heated by spraying with 50% aqueous s u l f u r i c a c i d followed by c h a r r i n g on a hot p l a t e . Descending paper chromatography was done u s i n g e t h y l acetate, a c e t i c a c i d , and water (6:3:2, V:V). Components were l o c a t e d using one of three d i p reagents. R&agznt A. Strongly reducing components were detected by d i p p i n g a chromatogram i n a s o l u t i o n of 0.7% (by weight) of s i l v e r n i t r a t e i n a mixture of acetone, concen­ t r a t e d ammonium hydroxide, and water (200:1:1). Reagent B. Mod­ erate to weakly reducing components were detected by d i p p i n g i n Reagent A followed by d i p p i n g i n a l c o h o l i c sodium Hydroxide (39). Rtag&nt C. Components with e n o l i c hydroxyls were detected by d i p p i n g i n a 1% f e r r i c c h l o r i d e i n 95% methanol (40). The r e l a ­ t i v e m o b i l i t i e s and c o l o r r e a c t i o n s of L - a s c o r b i c a c i d and i t s monosulfate e s t e r s are given i n Table IV. U.v. s p e c t r a were obtained i n aqueous s o l u t i o n s using a Beckman Model DB-G spectrophotometer. Nuclear magnetic resonance s p e c t r a were recorded with a V a r i a n Model XL-100 spectrometer. For carbon-13 s p e c t r a , the n.m.r. instrument was coupled to a N i c o l e t 1084 pulse F o u r i e r transform system. Chemical s h i f t s are given i n ppm downfield from e x t e r n a l (^C) or i n t e r n a l (^H) sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). L-Ascorbate 6-Sulfate (AA6S). To o b t a i n good y i e l d s of AA6S, molecular oxygen should be e l i m i n a t e d from reagents and s o l v e n t s by b o i l i n g and/or purging with n i t r o g e n . ( P r e - p u r i f i e d grade, Matheson, Ε., Rutherford, New J e r s e y ) . L-Ascorbic a c i d (3g) was d i s s o l v e d with s t i r r i n g at 25° i n 99% s u l f u r i c a c i d (lOmL), and the s o l u t i o n held 4 h at 25°. The v i s c o u s r e a c t i o n mixture was added dropwise with r a p i d s t i r r i n g to e t h y l ether (300mL) a t -65°, which caused the s u l f a t e e s t e r s to p r e c i p i t a t e as a v i s c o u s gum. The ether was maintained a t -65° by o c c a s i o n a l , d i r e c t a d d i t i o n of s o l i d carbon d i o x i d e . While the ether mixture was s t i l l at -65°, aqueous saturated barium hydroxide was added (200mL) and the r e s u l t i n g mixture was allowed to warm to - 5 ° . The ether l a y e r was drawn o f f and washed twice with water (2 χ 150mL). The combined aqueous l a y e r s were kept a t 0-5° and n e u t r a l i z e d by a d d i t i o n of s o l i d barium hydroxide. Barium s u l f a t e was removed by f i l t r a t i o n , and the f i l t r a t e made to volume (lOOOmL). An a l ­ iquot (lO.OmL) of the r e a c t i o n mixture reduced 3.65mL of 0.1N i o d i n e s o l u t i o n , whereas an a l i q u o t (5.0mL) of a known amount (1.015g) of L - a s c o r b i c a c i d i n 6% aqueous metaphosphoric a c i d (100.OmL) recTuced 11.6mL of 0.1N i o d i n e . The t i t e r s showed the aqueous e x t r a c t contained 74% of the e n e - d i o l groups put i n t o the concentrated s u l f u r i c a c i d . The remaining f i l t r a t e (990mL) was evaporated to a small

1.

LiLLARD AND SEiB

Monosulfate

Esters of L-Ascorbic

Acid

15

volume, and made to 25.0mL with water. An a l i q u o t (lO.OmL) was subjected to ion-exchange chromatography on a column (2.6 χ 40cm) of (die thy lamino e t h y l ) - c e l l u l o s e (Whatman DE-52, H. Reeve Angel, Inc., C l i f t o n , New J e r s e y ) , which had been p r e v i o u s l y converted to the hydrogen s u l f a t e form by washing with 0.1M s u l f u r i c a c i d . The column was developed at a flow r a t e of 45ml h~* using a l i n e a r g r a d i e n t (0 -> 0.18M of s u l f u r i c a c i d . The absorbance of the e f f l u e n t was monitored at 254nm, and the components (Figure 5) were c o l l e c t e d i n a v o l u m e t r i c f l a s k over 6% metaphosphoric a c i d . The amount of each component was determined by i t s ab­ sorbance (245nm) and by i o d i m e t r i c t i t r a t i o n (Table I I I ) . Component I I I , which accounted f o r 81% of the r e a c t i o n pro­ ducts, was found to give a s i n g l e spot (paper chromatography, AA 0.59), which co-migrated with a sample of L-ascorbate 6 - s u l ­ f a t e prepared as described by Allaudeen and Ramakrishnan (33). The spot r a p i d l y reduced s i l v e r and f e r r i c i o n s . Both samples had u.v. p r o p e r t i e s i d e n t i c a l to those of L - a s c o r b i c a c i d ( À 245nm at pH 2 and X 265nm at pH 7). ~ In a separate experiment Component I I I was i s o l a t e d as i t s barium s a l t a f t e r s e p a r a t i o n of the products obtained from a r e a c t i o n s t a r t i n g with 2.85g of L - a s c o r b i c a c i d . The column (2.6 χ 40cm) was developed as p r e v i o u s l y d e s c r i b e d , except a t o t a l of 400mL of 0.18M s u l f u r i c a c i d was used i n the l i n e a r , gradient e l u t i o n step, followed by 1800mL of 0.18M s u l f u r i c a c i d . Compo­ nent I I I was c o l l e c t e d i n a slowly s t i r r e d mixture of water (50ml) and barium carbonate that had p r e v i o u s l y been purged with nitrogen. A f t e r removal of barium s u l f a t e , the f i l t r a t e was concentrated to dryness to give barium L.-ascorbate 6 - s u l f a t e (5.0g) as an amor­ phous s o l i d which was 75"% barium AA6S as determined by u.v. analy­ sis . Anal. C a l c u l a t e d f o r 0.73 moles CftHôOgSBa +0.27 moles BaSO^ C, 13.44; H, 1.13; 0, 34.35; Ba, 41.50. Found: C, 13.33; H, 1.13; 0, 35.56; Ba, 47.54. The p.m.r. spectrum of barium AA6S at pH 7 (Table IV) agreed with that reported by Hatanaka, et a l . (34).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

R

m a x

m a x

L-Ascorbate 5 - S u l f a t e . To a p y r i d i n e (lOmL) s o l u t i o n of La s c o r b i c a c i d (1.5g) cooled i n an i c e bath was added 2.1 equival e n t s of ^ - b u t y l d i m e t h y l s i l y l c h l o r i d e (3.0g) ( A l d r i c h Chemical Company, Milwaukee, Wisconsin). A f t e r 3 hr s t i r r i n g at 25°, t . l . c . (benzene, e t h y l acetate 9/1, V/V) showed the r e a c t i o n mixture contained one p r i n c i p a l (R 0.7) and one minor component (Rf 0.8). P y r i d i n e - s u l f u r t r i o x i d e (1.35g) was added, and the s u l f a t i o n r e a c t i o n allowed to proceed overnight at 25°. A f t e r s u l f a t i o n t . l . c . (chloroform, a c e t i c a c i d 3/2, V/V) showed one major spot at Rf 0.7. f

The r e a c t i o n mixture was evaporated to remove p y r i d i n e , and water (20mL) was added twice and the mixture re-evaporated to a t h i c k syrup. The syrup was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (16mL), water (4mL) added, and the r e s u l t i n g mixture was held at 25°. A f t e r 12 hours t . l . c . (chloroform, a c e t i c a c i d 3/2, V/V)

CARBOHYDRATE SULFATES

16

showed the r e a c t i o n mixture contained one p r i n c i p a l product at R.£ 0.2. Evaporation of the h y d r o l y s i s r e a c t i o n mixture to a small volume (lOmL) removed water, a c e t i c a c i d , and ^ - b u t y l d i m e t h y l s i lanol. Aqueous barium hydroxide was added to pH 7.0, and the mix­ ture was concentrated to ^25mL and a p p l i e d to a DEAE-cellulose (H , SO^") column. Developing the column with a gradient of s u l ­ f u r i c a c i d (0 ->• 0.18M) eluted a m a t e r i a l whose m o b i l i t y was the same as that of Component I I (Figure 5). The column e f f l u e n t was c o l l e c t e d over barium carbonate as p r e v i o u s l y described to give 2.03 g of an amorphous s o l i d . The s o l i d barium s a l t of L - a s c o r b i c 5 - s u l f u r i c a c i d had i d e n t i c a l paper and chromatographic p r o p e r t i e s as those of Component I I . The s o l i d product d i d not give an ac­ ceptable elemental a n a l y s i s . The u.v., p.m.r., and c.m.r. s i g n a l s of AA5S are given i n Table IV.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

+

L-Ascorbic A c i d 4-d. The t i t l e compound was prepared by a mod­ i f i c a t i o n of the procedures of .Tolbert (41) and Brenner (42). LAscorbic a c i d (0.05 moles) was d i s s o l v e d and evaporated from de^ aerated deuterium oxide (lOmL) three times. To the dry m a t e r i a l was added 4M sodium methoxide i n methanol-cl (50mL), and the mix­ ture was r e f l u x e d 24 hours. The cooled a l c o h o l i c s o l u t i o n was added to water (lOOmL) c o n t a i n i n g 45g of s t r o n g l y a c i d i c , c a t i o n exchange r e s i n ( H ) . The r e s i n was removed, the f i l t r a t e f r e e z e d r i e d , and the residue d i s s o l v e d i n warm a c e t o n i t r i l e . Cooling gave white c r y s t a l s (2.8g, 32%) which had m.p. and a mixed m.p. of 192-3° with authentic L - a s c o r b i c a c i d . +

Ac knowledgment s The authors thank D. Mueller measurements.

and

J . P a u k s t e l i s f o r c.m.r.

Abstract Methods to prepare L-ascorbate 2-,5-, and 6 - s u l f a t e s are given. The compounds wer e c h a r a c t e r i z e d by 1 H - a n d Cnuclear magnetic resonance spectroscopy and by u l t r a v i o l e t spectroscopy. The b i o l o g i c a l p r o p e r t i e s of L-ascorbate 2 - s u l f a t e are reviewed. 1 3

Literature Cited 1. Halver, J . E . , Johnson, C. L., Smith, R. R., Tolbert, Β. M . , and Baker, F. M. Fed. Proc. Fed. Amer. Soc. Exp. B i o l . , (1972) Abstract No. 2764. 2. Ford, E. A. and Ruoff, P. M. Chem. Commun. (1965) 628. 3. Shenouda, M., M. S. Thesis, Kansas State University (1976); Quadri, S. F . , Liang, Y. T . , Seib, P. Α . , Deyoe, C. W., and Hoseney, R. C. J . Food Sci. (1975) 40 837. 4. Mead, C. G. and Finamore, F. J . Biochemistry (1969) 8 2652.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

1.

LiLLARD AND sEiB

Monosulfate

Esters of L-Ascorbic

Acid

17

5. Bond, A. D., McClelland, B. W., Einstein, J. R., and Finamore, F. J., Arch. Biochem. Biophys. (1972) 153, 207. 6. Baker, Ε. Μ., Hammer, D. C., March, S. C., Tolbert, Β. Μ., and Canham, J. Ε., Science (1971) 173, 826. 7. Mumma, R. O. and Verlangieri, A. J., Biochem. Biophys. Acta (1972) 273 249. 8. Hornig, D., Ann. Ν. Y. Acad. Sci. (1975) 258, 103; Hornig, D. World Rev. Nutr. Dietetics (1975) 23, 225. 9. Chu, T. M. and Slaunwhite, W. R., Jr., Steroids (1968) 12, 309. 10. Mumma, R. D., Biochem. Biophys. Acta (1968) 165, 571. 11. Baker, Ε. Μ., Kennedy, J. Ε., Tolbert, Β. Μ., and Canham, J. E. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1972). Abstr. No. 2760. 12. Halver, J. Ε., Ashley, L. Μ., Smith, R. R., Tolbert, Β. M., and Baker, Ε. M. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1973) Abstr. No. 4010. 13. McClelland, B. W., Acta Crystallogr. (1974) 30, 178. 14. Kuenzig, W., Avenia, R., and Kamm, J. J., J. Nutr. (1974) 104, 952. 15. Campeau, J. D., March, S. C. and Tolbert, Β. Μ., Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1973) Abstr. No. 4008. 16. Seib, P. Α., Liang, Y. T., Lee, C. H., Hoseney, R. C., and Deyoe, C. W. J. Chem. Soc., Perkin I (1974), 1220. 17. Carlson, Β. Μ., Downing, Μ., Tolbert, Β. M. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1974) 33, 1377. 18. Hatanaka, H., Ogawa, Y., Egami, F., J. Biochem. Tokyo. (1974) 75, 861. 19. Roy, A. B., Biochem. Biophys. Acta (1975) 377, 356. 20. Hatanaka, H. and Egami, F. J. J. Biochem. (1976) 80, 1215. 21. Fluharty, A. L . , Stevens, R. L . , Miller, R. T., Shapiro, S. S., and Kihara, H. Biochem. Biophys. Acta (1976) 429, 508. 22. Mohamram, M., Rucker, R. Β., and Hodges, R. E. Biochem. Biophys. Acta (1976) 437, 305. 23. Brin, M., Machlin, Κ. J., Kuenzig, W., and Garcia, F. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1975) 34, 884. 24. Kramer, K. J., Hendricks, L. Η., Liang, Y. T., and Seib, P. A. submitted to J. Ag. Food Chem. 25. Halver, J. Ε., Smith, R. R., Tolbert, Β. Μ., and Baker, Ε. Μ., Ann. New York Acad. Sci. (1975) 258, 81. 26. Cousins, R. C., Seib, P. Α., Hoseney, R. C., Deyoe, C. W., Liang, Y. T., and Lillard, D. W., Jr., J. Am. Oil Chem. Soc. (1977) 54, 308. 27. Cort, W. M. J. Am. Oil Chem. Soc. (1974) 51, 321; Food Tech (1975) 29(11) 46. 28. Hoseney, R. C., Seib, P. Α., and Deyoe, C. W., Cereal Chem. (1977) 54, 1062. 29. Klaüi, H. Wiss. Ver. Deutschen Gesell. fur Ernährung. (1963) 9 390.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch001

18

CARBOHYDRATE SULFATES

30. Feather, M. S. and Harris, J. F. Adv. in Carbohyd. Res. (1973) 28, 161. 31. Tolbert, B. M., Matwiyoff, Ν. Α., Lillard, D. L., Mueller, D. D., Paukstelis J., and Seib, P. A. in preparation. 32. Liler, Μ., "Reaction Mechanisms in Sulfuric Acid, "Aca­ demic Press, New York, N.Y. (1971); Gillespie, R. S., and Robinson, Ε. Α., "Non-Aqueous Solvents," Waddington, T. C., ed. Academic Press, New York (1965), pp. 117-120. 33. Allaudeen, H. S., and Ramakrishnan, R., Arch. Biochem. and Biophys. (1970) 140, 245. 34. Hatanaka, Η., Ogawa, Υ., and Egami, F., J. Biochem. (1974) 75, 861. 35. Tolbert, Β. Μ., Spears, A. H., Isherwood, D. J., Atchley, R. Η., and Baker, Ε. Μ., Abstracts of the Fed. of Exp. Biol. Soc, (1972) 31 (No. 2), Abstr. No. 2761. Tolbert, Β. Μ., personal communication (1974). 36. Honda, S., Yuku, Η., and Takiura, Κ., Carbohyd. Res. (1963) 28, 150. 37. Mahoney, J. F., and Purves, C. Β., J. Am. Chem. Soc. (1942) 64, 9; Yin, T. P., and Brown, R. K. Can. J. Chem. (1959) 37, 444. 38. Lillard, D. L., "L-Ascorbic Acid in Concentrated Sul­ furic Acid; Improved Synthesis of L-Ascorbic Acid 6-Sulfate," M. S. Thesis, Kansas State University (1977). 39. Trevelyan, W. Ε., Parker, C. P., and Harrison, J. S., Nature, (1950) 166, 144. 40. Vestling, C. S., and Rebstock, M. C., J. Biol. Chem. (1948) 161, 285. 41. Tolbert, Β. M. personal communication (1975) 42. Brenner, G. S., Hinkley, D. F., Perkinds, L. Μ., and Wever, W., J. Org. Chem., (1969) 29, 2389. RECEIVED F e b r u a r y 6, 1 9 7 8 .

2 Glucosinolates and Other Naturally Occurring O-Sulfates ANDERS KJAER

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

Department of Organic Chemistry, The Technical University of Denmark, 2800 Lyngby, Denmark

The following discussion will be restricted to sulphates of natural provenance. Carbohydrate sulphates, the legitimate topic of this symposium, quantitatively dominate the class of naturally occurring, organic sulphates but by no means represent it fully. To be sure, the typical C - O - S ester linkage is also encountered in a vast and rapidly increasing number of other known sulphates of animal and plant origin. Moreover, chemical entities exhibiting other structural features, such as P - O - S , N - O - S , and N - S - l i n k a ges, are also known as natural products. The ensuing discussion w i l l c e n t r e on the g l u c o s i n o l a t e s 1, a u n i q u e l y c o n s t i t u t e d and

well-defined class of anions occurring as plant products that are, at the same time, organic sulphates and glucose derivatives, yet assembled in a fashion so unorthodox that the raison d ' ê t r e for their presentation at this symposium may appear tenuous. The chemical character and biological degradation of glucosinolates, however, exhibit features of potential interest to carbohydrate sulphate chemistry; these aspects will be emphasized. The discussion will also include a brief, up-to-date survey of some other organic sulphates, selected to indicate the scope of structural variation encountered within the living world. Glucosinolates The collection of glucosinolates, thus far comprising about seventy-five individual anions, exclusively encountered in higher 0-8412-0426-8/78/47-077-019$05.00/0 © 1978 American Chemical Society

20

CARBOHYDRATE SULFATES

plants, p o s s e s s e s a r e m a r k a b l e s t r u c t u r a l u n i f o r m i t y . T h u s , the β -D-thioglucopyranosidic

l i n k a g e , the h y d r o x y l a m i n e - O - s u l p h a t e

m o i e t y , and the ( Z ) - t h i o h y d r o x i m a t e a r r a n g e m e n t p r e s e n t i n 1 a r e c h e m i c a l f e a t u r e s to w h i c h no known e x c e p t i o n

e x i s t s . Hence, the

s i d e - c h a i n R of 1 c o n s t i t u t e s the s o l e s t r u c t u r a l p a r a m e t e r . T h i s r e m a r k a b l e c h e m i c a l u n i f o r m i t y r e f l e c t s , a l m o s t by n e c e s s i t y , a corresponding s i m i l a r i t y in biosynthetic derivation. Few p a t h w a y s i n h i g h e r p l a n t s have b e e n c l a r i f i e d i n g r e a t e r

anabolic detail

+

than those l e a d i n g f r o m α - a m i n o a c i d s , R* C H ( N H g ) C 0 " ~ , to g l u ­

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

2

c o s i n o l a t e s 1, t h r o u g h a s e q u e n c e of steps o u t l i n e d i n S c h e m e 1.

R 1 Scheme 1 Ox

R C H - (NH ) -C0 3

+

2

> R-CH-(NHOH) -C0 H

Ox

-C02 [R-CH-(NO)-C0 H]

2

R-CH(S-Glu):NOH <

UDPG

R-CH(SH):N0H

PAPS

<

-X +H

- R-CH(SX):NOH <

Ox R-CH(:NOH) 0

RCH -N0

3

Two

XSH

KH

R-CH(S-Glu):N-OS0 -

The

> R-CH(:NOH)

2

2

g l u c o s i n o l a t e s have b e e n a subject of s e v e r a l r e v i e w s .

m a j o r s u m m a r i e s U , _2), c h r o n o l o g i c a l l y f o l l o w e d by a d d i t i o ­

n a l a c c o u n t s (3^, 4) and y e a r l y r e p o r t s (5),

provide

a b r o a d back­

g r o u n d and d e t a i l e d up-to-date status of our knowledge about t h i s c l a s s of n a t u r a l p r o d u c t s . On

this occasion, however, c e r t a i n f a ­

c e t s of the g e n e r a l theme, r e g a r d e d as b e i n g of p a r t i c u l a r i n t e r ­ est, have b e e n s e l e c t e d f o r d i s c u s s i o n .

2

2.

KJAER

Glucosinolates and

Other O-Sulfates

21

Despite reported sporadic o c c u r r e n c e s in taxonomically r e m o t e f a m i l i e s (2, 3), the g l u c o s i n o l a t e s a r e o b v i o u s l y c o n c e n t r a ted i n the l a r g e e c o n o m i c a l l y i m p o r t a n t b o t a n i c a l f a m i l i e s C a p p a r a c e a e and C r u c i f e r a e , but a r e p r e s e n t a l s o i n c e r t a i n s m a l l e r f a milies,

such as R e s e d a c e a e , M o r i n g a c e a e and T o v a r i a c e a e , a l l of

e s t a b l i s h e d p h y l o g e n e t i c a f f i n i t y to the c r u c i f e r s and c a p e r s . V i r t u a l l y e v e r y t a x o n w i t h i n the o r d e r c o m p o s e d of the above f a m i l i e s c o n t a i n s one o r m o r e g l u c o s i n o l a t e s i n t h e i r f r u i t s o r v e g e t a -

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

t i v e p a r t s . In fact, the a b s e n c e o r p r e s e n c e of g l u c o s i n o l a t e s r a n k s a m o n g the m o s t c l e a r - c u t c h e m i c a l c h a r a c t e r s w i t h i n the c l a s s of h i g h e r plants. I n v a r i a b l y , the g l u c o s i n o l a t e s a r e a c c o m p a n i e d i n the n a t u r a l s o u r c e s by s p e c i f i c e n z y m e s , the

myrosina-

ses, c a t a l y z i n g the h y d r o l y s i s of the t h i o g l u c o s i d i c l i n k a g e to p r o duce g l u c o s e and the l a b i l e aglucone, 2a o r 2b. D e g r a d a t i o n of the latter may

p r o c e e d i n m u l t i p l e ways, d e p e n d i n g on

s u c h as the plant s o u r c e and the p H - v a l u e , t o

circumstances

give e i t h e r i s o t h i o -

c y a n a t e s ( m u s t a r d o i l s ) 3, n i t r i l e s 4, or, r a r e l y , t h i o c y a n a t e s 5;in m a n y c a s e s m i x t u r e s of these a r i s e and i n s o m e i n s t a n c e s the i s o t h i o c y a n a t e s u n d e r g o f u r t h e r r e a c t i o n , s u c h as i n t r a m o l e c u l a r c y c l i z a t i o n , to give m o r e stable

end-products. R-CN

US]

3 2a

2b

R-SCN

[•HSO/l

5 A l l o b s e r v e d t r a n s f o r m a t i o n s of the aglucone i n v o l v e f i s s i o n of the sulphate 'ester-bond'. M u c h c o n t r o v e r s y a r o s e s o m e y e a r s

CARBOHYDRATE SULFATES

22

ago c o n c e r n i n g the e n z y m i c , o r n o n - e n z y m i c , c h a r a c t e r of t h i s c l e a v a g e . On the b a s i s of s i m p l e c h e m i c a l r e a s o n i n g , h o w e v e r , the spontaneous c o n v e r s i o n of 2a (or 2b) into 3, a r e a c t i o n r e p r e s e n t i n g the s u l p h u r e q u i v a l e n t of the w e l l - k n o w n

Lossen-rearrange-

ment y i e l d i n g isothiocyanates f r o m h y d r o x a m i c acids, equipped with a s u i t a b l e l e a v i n g group, i s an a l t o g e t h e r a c c e p t a b l e event. In fact, nature's device:to u t i l i z e sulphate as the l e a v i n g group, s e r v e s w e l l i n the o r d i n a r y L o s s e n - r e a r r a n g e m e n t

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

The

{6).

e n z y m i c a l l y i n i t i a t e d p r o d u c t i o n of i s o t h i o c y a n a t e s 3

p r o v i d e s the r a t i o n a l e f o r the c h e r i s h e d p u n g e n c y and f l a v o u r of n u m e r o u s f o o d s t u f f s and c o n d i m e n t s of c r u c i f e r o u s c h a r a c t e r s u c h as m u s t a r d , r a d i s h , w a t e r c r e s s , h o r s e r a d i s h , t u r n i p s and r a p e . E n o r m o u s e c o n o m i c and c o n s i d e r a b l e d i e t a r y p r o b l e m s a r e a s s o c i a t e d with the g l u c o s i n o l a t e contents i n c r u c i f e r o u s c r o p s e m p l o y e d as f o d d e r and f o r h u m a n c o n s u m p t i o n . In J a p a n alone, the amount of f r e s h o r p r o c e s s e d r a d i s h c o n s u m e d by the l o c a l p o p u l a t i o n has b e e n e s t i m a t e d at f i v e m i l l i o n tons p e r y e a r (7). A n o t h e r f a c e t of g l u c o s i n o l a t e c h e m i s t r y i n v o l v i n g the s u l phate g r o u p i n g i s a p p a r e n t i n the f o r m a t i o n of, i n t e r a l i a , m e r o s i n i g r i n upon t r e a t m e n t

of a l l y l - g l u c o s i n o l a t e 6 (the

potassium

s a l t of w h i c h i s the c l a s s i c a l ' s i n i g r i n ' ) with s o d i u m m e t h o x i d e , o b s e r v e d m o r e than 60 y e a r s ago (8).

B a s e d on the now

accepted

g e n e r a l g l u c o s i n o l a t e s t r u c t u r e j., m e r o s i n i g r i n m o s t l i k e l y p o s s e s s e s the s t r u c t u r e 7, a r i s i n g f r o m cular displacement

a base-induced, i n t r a m o l e -

of the sulphate f u n c t i o n by the 2 - O H - g r o u p i n g

of the g l u c o s e m o i e t y (_9).

KJAER

2.

23

Glucosinolates and Other O-Sulfates

S u b j e c t i n g a l l y l - ( 8 ) o r b e n z y l g l u c o s i n o l a t e (9) to s t r o n g aqueous b a s e r e s u l t s i n the f o r m a t i o n

of v i n y l g l y c i n e (10) and

n y l g l y c i n e (1.1), r e s p e c t i v e l y , i n a d d i t i o n to t h i o g l u c o s e and p r o d u c t s . The

phe-

other

f o l l o w i n g pathway, i n v o l v i n g a N e b e r - t y p e r e a c t i o n ,

has b e e n i n v o k e d (9): 'S-Glu

HO" — ι -S-Glu

"CVSO3

4

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

H

K 2°

H0

H

i

Ύ

so 2

R = CH2=CH

9, R = C Hs 6

2 OH"

HN

H,N

2

A

Glu-S"

^/k^S-Glu

CO, >—H 0 2

10,

R =

CH =CH

II

R =

C H

2

6

5

I n t r i g u i n g l y , the a m i n o a c i d s p r o d u c e d c o n t a i n e x c e s s of the L-isomers,

r e f l e c t i n g s o m e s t e r i c c o n t r o l f r o m the g l u c o s e

ring,

the o n l y c h i r a l m o i e t y i n v o l v e d i n the r e a c t i o n . S e v e r a l g l u c o s i n o l a t e s have b e e n s y n t h e s i z e d . A c h a r a c t e r i ­ s t i c f e a t u r e of a l l the a p p r o a c h e s i s the i n t r o d u c t i o n of the s u l ­ p h a t e - g r o u p i n g at the f i n a l stage of the s y n t h e t i c sequence, n o r ­ m a l l y with the S O g - p y r i d i n e c o m p l e x a s a r e a g e n t . In t h i s r e s p e c t the a p p r o a c h s i m u l a t e s the b i o s y n t h e t i c a s s e m b l a g e ; h e r e , i n the v e r y l a s t step ( S c h e m e l ) , t h e sulphate r e s i d u e i s t r a n s f e r r e d f r o m 3'-phosphoadenosine 5'-phosphosulphate to the o x y g e n a t o m of the h y d r o x y l a m i n e g r o u p i n g i n a r e a c t i o n p r o b a b l y

catalyzed

by the s a m e e n z y m e f o r the whole r a n g e of g l u c o s i n o l a t e s (5a). P u r e glucosinolate salts are non-volatile, high-melting

com­

pounds, r e a d i l y s o l u b l e i n w a t e r b y v i r t u e of t h e i r c o m b i n e d g l u c o s i d e a n d salt c h a r a c t e r ; t h e i r r e a d i n e s s to c r y s t a l l i z e i s c a p r i ­ c i o u s and not a l w a y s v e r y p r o n o u n c e d . In no c a s e have

s u g a r s o-

t h e r than g l u c o s e b e e n o b s e r v e d i n the t h i o g l y c o s i d i c l i n k a g e of

CARBOHYDRATE

24

SULFATES

n a t u r a l l y o c c u r r i n g g l u c o s i n o l a t e s . E q u a l l y constant i s the s u l phate r e s i d u e ; the a n a l o g o u s phosphate e s t e r s , a p r i o r i not unr e a s o n a b l e a l t e r n a t i v e s , have n e v e r been e n c o u n t e r e d . l t

i s temp-

t i n g to a s s u m e that the r a i s o n d'être f o r the sulphate g r o u p i n g i n the g l u c o s i n o l a t e i s i t s c h a r a c t e r

of an e x c e l l e n t l e a v i n g group.

P u r s u i n g t h i s l i n e of s p e c u l a t i o n , the t r u e b i o l o g i c a l f u n c t i o n of

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

the g l u c o s i n o l a t e s m a y h e n c e be that of s e r v i n g as d e p o s i t o r i e s of v o l a t i l e r e a c t i o n p r o d u c t s , p o s s e s s i n g

specific biological pro-

p e r t i e s and a r i s i n g f r o m the e n z y m i c a l l y

initiated degration r e -

a c t i o n s d i s c u s s e d above. In fact, m u c h knowledge h a s a c c u m u l a t e d o v e r the y e a r s to i n d i c a t e that i s o t h i o c y a n a t e s , and p e r h a p s other d e g r a d a t i o n

products as well, play a d e c i s i v e r o l e i n seve-

r a l w e l l - e s t a b l i s h e d and s p e c i f i c p a r a s i t e - h o s t r e l a t i o n s w i t h i n i m p o r t a n t p l a n t s of the c r u c i f e r f a m i l y . O b v i o u s l y , s u c h f u n c t i o n s w i l l p e r se a t t r i b u t e to the g l u c o s i n o l a t e s a p h y l o g e n e t i c

signifi-

c a n c e that w o u l d m a k e t h e i r d i s c o n t i n u o u s n a t u r a l d i s t r i b u t i o n w i t h i n the plant k i n g d o m l e s s s u r p r i s i n g . O t h e r O r g a n i c Sulphates of P l a n t O r i g i n A few r e p r e s e n t a t i v e s of other g r o u p s of o r g a n i c

sulphates,

o c c u r r i n g i n h i g h e r p l a n t s s e l e c t e d r a t h e r a r b i t r a r i l y by an o r ganic c h e m i s t p r i m a r i l y c o n c e r n e d with s t r u c t u r a l a s p e c t s , m a y s e r v e as an i l l u s t r a t i o n of the v e r s a t i l i t y and s c o p e of o r g a n i c s u l p h a t e s e n c o u n t e r e d e v e n w i t h i n the l i m i t e d g r o u p of l i v i n g spec i e s m a d e up by h i g h e r p l a n t s . C h o l i n e sulphate ,12 a c c u m u l a t e s , to the extent of m o r e than o n e - t h i r d of the t o t a l amount of sulphate fed, i n the r o o t s of s u l p h u r - d e f i c i e n t p l a n t s of c o r n , b a r l e y , and s u n f l o w e r , e v e n u n d e r s t e r i l e c o n d i t i o n s . In n o n - d e f i c i e n t r o o t s , and i n a l l l e a v e s , it a c counts f o r 5-15 % of the t o t a l , s o l u b l e s u l p h u r compounds. A n i m p o r t a n t r o l e of 1.2 a s an e f f e c t i v e t r a n s p o r t agent f o r p a s s a g e t h r o u g h the c e l l m e m b r a n e h a s b e e n s u g g e s t e d (11). A d r a m a t i c d e v e l o p m e n t i n o u r knowledge of f l a v o n o i d s u l phates i n h i g h e r plants s h o u l d not p a s s u n n o t i c e d h e r e . M o r e than

2.

KJAER

Glucosinolates and

25

Other O-Sulfates

40 y e a r s ago, p e r s i c a r i n ( i s o r h a m n e t i n

3-sulphate) 13 was

repor-

t e d as a constituent of w a t e r - p e p p e r ( P o l y g o n u m h y d r o p i p e r ) (12); the i s o l a t i o n of only few

a d d i t i o n a l and

scattered flavonoid

sulpha-

t e s m a d e t h e s e s t r u c t u r e s i n t r i g u i n g enough to p r o m p t the s t a t e ment, i n 1971, that 'flavonoid b i s u l p h a t e s a r e a m o n g s t the r a r e s t of n a t u r a l l y o c c u r r i n g f l a v o n o i d s ' (_13). In the s a m e y e a r , h o w e v e r , a v e r i t a b l e l a n d s l i d e of d i s c o v e r y

set in, t r i g g e r e d by D r . J . B.

H a r b o r n e and h i s a s s o c i a t e s and r e s u l t i n g i n a p r o f o u n d r e v i s i o n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

of our v i e w of these c o m p o u n d s . F l a v o n o i d

s u l p h a t e s now

r e g a r d e d , not as p h y t o c h e m i c a l o d d i t i e s , but r a t h e r as an

must

be

impor-

tant c l a s s of n a t u r a l c o m p o u n d s . T h u s , i n a r e c e n t r e v i e w (14) i n dividual flavonoid sulphates

'detected v a r i o u s l y i n o v e r 200

spe-

c i e s f r o m 20 plant f a m i l i e s ' a r e l i s t e d , and undoubtedly m a n y r e a r e l i k e l y to a p p e a r . T h i s e x a m p l e i l l u s t r a t e s how l a r g e g r o u p of n a t u r a l p r o d u c t s m a y

mo-

readily a

be o v e r l o o k e d b e c a u s e of

n e g l e c t on the p a r t of the c h e m i s t , i n the p r e s e n t c a s e undoubtedl y c a u s e d by the s a l t c h a r a c t e r

and o v e r - a l l i n c o n s p i c u o u s n e s s

of the compounds. F l a v o n o l s and f l a v o n e s , as s u c h o r i n g l y c o s i d i c f o r m , with one the 3-OH

and 7-OH,

o r m o r e sulphate

r e s i d u e s attached, m o s t l y at

but o c c a s i o n a l l y a l s o l i n k e d to the

sugar

m o i e t y , a r e p r e v a l e n t a m o n g the f l a v o n o i d s u l p h a t e s known thus far. The

p o s s i b i l i t y of f l a v o n o i d s u l p h a t e s h a v i n g an i m p o r t a n t

f u n c t i o n i n salt uptake and m e t a b o l i s m , i n a d d i t i o n to that of c o n f e r r i n g w a t e r s o l u b i l i t y to o t h e r w i s e i n s o l u b l e f l a v o n o i d s , h a s b e e n s u g g e s t e d i n v i e w of t h e i r p r e f e r r e d o c c u r r e n c e w i t h i n p l a n t s with aquatic, often s a l i n e h a b i t a t s .

•

OMe

26

CARBOHYDRATE SULFATES

O t h e r p l a n t s p h e n o l i c s , r e c o g n i z e d as sulphate

conjugates,

i n c l u d e the i s o m e r i c c h l o r o g e n i c a c i d O - s u l p h a t e s 14 (14); O - s u l p h a t e s d e r i v i n g f r o m 1 - c a f f e y l g l u c o s e ,15 and p - c o u m a r y l glucose

16 (14), as w e l l a s the 3/-Q,?) (.15) and 6'-Ο-sulphate 18

(16) of betanin, a p i g m e n t c h a r a c t e r i s t i c of m e m b e r s of the taxo-

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

nomically restricted order

Centrospermae.

1~L R = OH 16

R =Η

A p p l i c a t i o n of p a p e r e l e c t r o p h o r e s i s technique

to e x t r a c t s of

s e v e r a l s p e c i e s of the f a m i l y P o l y g o n a c e a e r e c e n t l y l e d to the r e c o g n i t i o n of c o v a l e n t l y bound sulphate i n h a l f of 27 s p e c i e s stu­ died, b e l o n g i n g to the genus R u m e x . F r o m one of these a n O - s u l ­ phate of e m o d i n 1 (or 8 ) - g l u c o s i d e ,19 and a d i g l u c o s i d e s u l p h a t e of the c o r r e s p o n d i n g

e m o d i n dianthrone

w e r e c h a r a c t e r i z e d , both

c o n t a i n i n g the sulphate r e s i d u e i n the g l u c o s e m o i e t i e s

Η

V7_ R = S 0 " j R = Η 1

3

Jj^

R = H , R

1

= SO3"

(17).

2.

KJAER

Glucosinolates and

27

Other O-Sulfates

Conclusion Our

knowledge about c o v a l e n t l y bound s u l p h a t e s i s o b v i o u s -

l y i n i t s i n f a n c y . We

know that s u l f o h y d r o l a s e s , e n z y m e s c a t a l y -

z i n g the h y d r o l y s i s of o r g a n i c sulphates, a r e w i d e - s p r e a d throughout the w o r l d s of m i c r o o r g a n i s m s , p l a n t s and a n i m a l s . our u n d e r s t a n d i n g of these and r e l a t e d e n z y m e s h a s r e m a r k a b l y r e c e n t l y we

Although

increased

a r e s t i l l left with the r a t h e r p a r a d o x i c a l

situation: many e n z y m e s lack w e l l - r e c o g n i z e d n a t u r a l substrates

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

and, at the s a m e t i m e , n a t u r a l s o u r c e s p r o v i d e o r g a n i c

sulphates

f o r w h i c h no e n z y m i c a p p a r a t u s s e e m s to e x i s t . O b v i o u s l y , c o n t i n u e d e f f o r t s to c l a r i f y the d i s t r i b u t i o n , c h e m i s t r y

and

enzymic

t r a n s f o r m a t i o n of n a t u r a l l y o c c u r r i n g s u l p h a t e s cannot f a i l to be r e w a r d i n g to those p o s s e s s i n g the i m a g i n a t i o n , s k i l l and

perseve-

r a n c e n e e d e d to h e l p us u n d e r s t a n d the i m p o r t a n c e and

biological

f u n c t i o n of these

compounds.

Abstract The

chemistry

of the g l u c o s i n o l a t e s , a c l a s s of n a t u r a l l y oc-

c u r r i n g t h i o g l u c o s i d es c o n t a i n i n g an

O - s u l p h a t e grouping, i s

b r i e f l y d i s c u s s e d . T h e i r e n z y m i c d e g r a d a t i o n to i s o t h i o c y a n a t e s , and n i t r i l e s ,

as w e l l as other r e a c t i o n s i n w h i c h the

sulphate

g r o u p i n g p a r t i c i p a t e s , a r e r e v i e w e d . O t h e r c l a s s e s of O - s u l p h a tes, o c c u r r i n g i n h i g h e r p l a n t s , a r e l i s t e d , i n c l u d i n g f l a v o n o i d sulphates. cussed

The

p o s s i b l e r ô l e of o r g a n i c s u l p h a t e s i s b r i e f l y

and future r e s e a r c h a s p e c t s

dis-

adumbrated.

Literature Cited 1. 2.

Kjær, A., Fortschr. Chem. Org. Naturst., (1960) 18, 122. Ettlinger, M. G. and Kjær, A. in "Recent Advances in Phytochemistry", Vol. I, p. 89, Mabry, T. J., Alston, R. E. and Runeckles, V. C., Ed., Appleton-Century-Crofts, New York, Ν. Y. (1968).

28

3.

4.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch002

5.

6. 7. 8. 9.

10. 11. 12. 13. 14.

15. 16. 17.

CARBOHYDRATE SULFATES

Kjær, A. in "Chemistry in Botanical Classification", Nobel Symposium 25, p. 229, Bendz, G. and Santesson, J., Ed., Academic Press, New York and London (1974). Kjær, A. in "The Biology and Chemistry of the Cruciferae", p. 207, Vaughan, J. G., MacLeod, A. J. and Jones, B. M. G., Ed., Academic Press, London, New York, San Francisco (1976). Kjær, A. and Olesen Larsen, P. in "Biosynthesis", Specialist Periodical Report, The Chemical Society, London, (a) (1973) 2, 95; (b) (1976) 4, 200 (c) (1977) 5, in press. Daniher, F. A. J. Org. Chem., (1969) 34, 2908. Maeda, Y. personal communication. Schneider, W. and Wrede, F. Ber. Deut. Chem. Ges., (1914) 47, 2225. Lundeen, A. J. "The Structure of the Mustard Oil Glucosides and Synthesis of the Glucotropaeolate Ion" Ph. D. Thesis, The Rice Institute, Houston, Texas (1957). Friis, P., Olesen Larsen, P. and Olsen, C. E. J. Chem. Soc., Perk. I, (1977) 661. Nissen, P. and Benson, A. A. Science, (1961) 1759. Kawaguchi, R. and Kim, K. W. J. Pharm. Soc. Japan, (1937) 57, 108. Saleh, N. A. M . , Bohm, B. A. and Ornduff, R. Phytochemistry (1971) 10, 611. Harborne, J. B. in "Progress in Phytochemistry", Vol. IV, p. 189, Reinhold, L., Harborne, J. B. and Swain, T., Ed., Pergamon Press, Oxford, New York, Toronto, Sydney, Paris and Frankfurt (1977). Imperato, F. Phytochemistry, (1975) 14, 2526. Wyler, Η., Rosler, H . , Mercier, M. and Dreiding, A. S. Helv. Chim. Acta (1967) 50, 545. Harborne, J. B. and Mokhtari, N. Phytochemistry (1977) 16, 1314.

RECEIVED February 6, 1978.

3 Sulfate Ester Groups as Potential Informational Regulators in Glycoproteins P. W. K E N T , C. J. C O L E S , J. R. C O O P E R , and N. R. M I A N

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

Glycoprotein Research Unit, Durham University, Durham D H 1 3 L H , England

The presence of ester sulphates i n carbohydrate macromolecules i s a feature widespread both i n plants and animals. Considerable speculation exists about the biological functions played by the acidic ester groups, whether these groups solely serve to augment the anionic character of the macromolecule concerned or whether they exert more specific and perhaps more subtle biological effects. The purpose of this paper i s to consider these questions, in particular i n terms of sulphated macromolecules present in animal tissues. The p a r a l l e l aspects i n plants are no less important, bearing i n mind the location of sulphated polysaccharidic material i n c e l l wall structures, their potential role i n relation to water-retaining mechanisms and their capacity to interact with other macromolecules. Sulphated Glycosaminoglycans In animals, relevant macromolecules examined i n greatest detail have been the sulphated glycosaminoglycans (mucopolysaccharides) associated with connective tissues eg. chondroitin sulphates, keratosulphate, heparan sulphate, derman sulphate. In p r i n c i p l e , the sulphate ester groups are located i n defined positions on oligosaccharide chains of regular repeating sugar sequences and these i n turn are attached by a l k a l i -label linkages to a protein (Table I ) . Thus i n chondroitin 4-sulphate, sulphate ester groups are envisaged i n the ideal situation as being attached to C-4 of every N-acetylgalactosaminyl residue and to C-6 of the same aminosugars i n chondroitin 6-sulphate. Nevertheless considerable variations i n practice have been noted i n the actual extent of sulphate between chondroitin sulphate types of different biological o r i g i n . Chondroitin sulphate from shark cartilage, for example, exhibits the abnormally high sulphate content of 33.7% while that from bovine c o r t i c a l bone has 10.3% sulphate.

0-8412-0426-8/78/47-077-029$05.00/0 © 1978 American Chemical Society

Composition of glycosaminoglycans

D-glucosamine

D-gluco samine

D-galactose

D-glucuronic a c i d or L-iduronic acid

Keratan sulphate

Heparin

sulphate

D-glucuronic a c i d o r L-iduronic acid

D-galacto samine

L-iduronic acid or D-glucuronic a c i d

Dermatan sulphate

Heparan

D-galacto samine

D-glucuronic a c i d

Chondroitin 6-sulphate

D-gluco samine

D-galacto samine

D-glucuronic a c i d

Chondroitin 4-sulphate

D-gluco samine

D-glucuronic a c i d

Hyaluronic acid

D i s a c c h a r i d e repeating u n i t Hexosamine Hexuronic a c i d

Table I .

O-sulphate and N-sulphate

O-sulphate and N-sulphate

0-sulphate

0-sulphate

0-sulphate

0-sulphate

Sulphate

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

D-xylose, D-galactose

D-xylose, D-galactose

D-mannose, D-fucose, s i a l i c acid, D-galacto samine

D-xylose, D-galactose

D-xylose, D-galactose

D-xylose, D-galactose

Other sugar r e s i d u e s , i n c l u d i n g those i n the l i n k a g e r e g i o n

H M

>

d

>

κ! ϋ

w ο

> w

η

GO Ο

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

3.

K E N T ET AL.

Sulfate Ester Groups

31

I n c o n t r o v e r t i b l e such e s t e r sulphate can c o n t r i b u t e to the e l e c t r o p h o r e t i c a n i o n i c c h a r a c t e r , and under experimental c o n d i t i o n s a t l e a s t , endow the macromolecule with dye-binding p r o p e r t i e s . Studies on the induced c o t t o n e f f e c t s o f a n i o n i c dye-mucopolysaccharide complexes i n the a d s o r p t i o n band o f the dye eg. methylene blue (Stone, 1964* 1965) have been i n t e r p r e t e d on the b a s i s o f polymer conformations, i n d i c a t i v e of both random and h e l i c a l forms (Hirano and Onodera, 1 9 6 7 ) · Whereas c h o n d r o i t i n ( s u l p h a t e - f r e e ) e x h i b i t e d random conformation, c h o n d r o i t i n 4-sulphate ( l i k e derman sulphate and hyaluronate) had h i g h degrees o f h e l i c a l s t r u c t u r e s o f the L e f t Screw sense. By c o n t r a s t , c h o n d r o i t i n 6-sulphate, h e p a r i n and h e p a r i t i n sulphate were h e l i c a l conformations o f the R i g h t Screw sense. I n t e r e s t i n g l y , c h o n d r o i t i n polysulphate ( c h o n d r o i t i n 4 * 6 sulphate) had the dye-binding c h a r a c t e r i s t i c s o f c h o n d r o i t i n 4-sulphate. A d d i t i o n a l s u l p h a t i o n o f c h o n d r o i t i n 6-sulphate (to OH-4 o f the aminosugar r e s i d u e s ) on the other hand l e a d s to conformational i n v e r s i o n . I n the h e p a r i n s e r i e s , de Ν-sulphation o f t h a t m a t e r i a l o r o f h e p a r i t i n sulphate d i d not b r i n g about change o f conformation, though complete desulphation caused the h e l i c a l s t r u c t u r e to disappear. The primary s t r u c t u r e p l a y s a d e c i s i v e p a r t i n conformational determination, the l i n k e d p o s i t i o n o f each e s t e r sulphate being important i n r e l a t i o n to hydrogen-bonding c a p a b i l i t y . Elegant X-ray a n a l y t i c a l s t u d i e s by Rees (1969) and by A t k i n s and Laurent (1973) have e s t a b l i s h e d f i n e d e t a i l s o f ordered conformation o f these mucopolysaccharides. Detailed reviews o f these aspects have been presented by Kirkwood (1974) and Rees (1975). The presence o f e s t e r sulphate groups i n these d e f i n e d and r e i t e r a t e d p o s i t i o n s i n sequence nevertheless does not n e c e s s a r i l y impair the a c t i o n o f dégradative enzymes. H y d r o l y t i c endohexosaminidases eg. hyaluronidase a c t r e a d i l y on c h o n d r o i t i n 4 - and 6-sulphates, as on hyaluronate, g i v i n g corresponding d i s a c c h a r i d e and t e t r a s a c c h a r i d e products. In g e n e r a l , i t would be concluded t h a t i n these i n s t a n c e s the i n - b u i l d i n g o f e s t e r sulphates i n a r e g u l a r p e r i o d i c i t y does not seemingly i n f l u e n c e the metabolic s t a b i l i t y nor t h e i r immunological p r o p e r t i e s , but r a t h e r the macromolecular shape and, by i n f e r e n c e , t h e i r i n t e r - m o l e c u l a r a s s o c i a t i o n s . Sulphated G l y c o p r o t e i n s E s t e r sulphates are however widely found i n other s i t u a t i o n s , e s p e c i a l l y i n a wide v a r i e t y o f sulphated g l y c o p r o t e i n s (Table I I ) . These were f i r s t shown to e x i s t i n g a s t r o - i n t e s t i n a l e p i t h e l i a l mucins and c o r n e a l g l y c o p r o t e i n s . The degree o f sulphate here, too, i s v a r i a b l e , though a value

G-i tract

Human G a s t r i c carcinoma

Human G a s t r i c tissue

Human G a s t r i c juice

A.

Source

Sialic acid

5.8 3-2 3.1 3.8

2.5

2.9 1.3 2.3

8.5

Sulphate

2.1 3.8 6.5 7.1

5.1

4-3 4-4 4.9

1.8 7-4

21.1 1.4

28.9 30.2 28.5

13.6 10.2 10.2 32.2

13.6 10.2 10.2

34.1

-

22.6

16.2

17.3

Galactose

26.3 25.4 25.0 22.0

GlcN GalN

2.0 1.9 1.9 2.3

Hexosamine

36.2 35.8 32.8 33.2

18.2 17.3 15.4

Fucose Ref.

Kimoto e t a l (1968 )

) ) M a r t i n e t a l (1968)

M a r t i n e t a l (1967)

) ) Hakkinen e t a l (1965 ) )

(g/lOOg d r y wt)

Table l i a Composition o f E p i t h e l i a l Sulphated G l y c o p r o t e i n s

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

w

H

>

c!

X

« o

> o

submaxillary gland

References

Pig Colonic muco sa

1.3 3.0 6.5 7.4 7.4 8.8 7.0 5.5

+0 0.06 1.84

3.2 3.8 3.8 3.4

11.8 24-5

Sialic

52.3 51.2 48.1 21.3 22.4 21.4 29.3

5.6 3.2 5.2 11.2 8.8 21.4 5.5

( 2 ) Slomiary and Meyer (1972)

( 4 ) Inoue and Yosizawa (1966)

( 3 ) Coles and Kent (1977)

32.1

18.0 24.5

6.2 5.0 12.5

Hexosamine

Fucose

(1966)

acid

3 2 2 3

0.9 0.97 0.75

-

_

GlcN GalN

21.7 25.6 23.2 20.4

13.9 14.6 13.0

45

9.4 19.6

Galactose

L

)

(2)

(

8.0 ) 8.7 )

8.0 )

)

u

'

) (3)

)

(

Ref

55.4 ) , ) 40.0 )

Peptide

(g/lOOg d r y wt)

of E p i t h e l i a l Sulphated G l y c o p r o t e i n s (Pooled p r e p a r a t i o n s )

3.5

Sulphate

Composition

: (1) Katzman and E y l o r

Pig Duodenal tissue

Pig g a s t r i c mucosa

G-i t r a c t

Pig

Source

Table l i b .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

CO 00

-*

o Si

es

8"

C/3

M H W H

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

34

CARBOHYDRATE SULFATES

of about 2$ i s common i . e . about 20 sulphate residues per 100,000 d a l t o n s . Unlike the mucopolysaccharides, g l y c o p r o t e i n s show l i t t l e evidence of ordered sugar-repeating sequences and the task o f a l l o c a t i n g exact p o s i t i o n s to such sulphate residues i s c o n s i d e r a b l e . I n a study o f g l y c o p r o t e i n s (sulphate content between 2.8 and 5 · 9$) o f p i g g a s t r i c mucosa, Slomiany and Meyer (1972) showed that a t l e a s t one N - a c e t y l glucosamine residue was sulphate, a t p o s i t i o n 6 , and l o c a t e d i n c l o s e proximity to the peptide attachment ( F i g . l a ) . T h i s m a t e r i a l i s f r e e from u r o n i c and s i a l i c a c i d s . A s i m i l a r arrangement has been i n d i c a t e d i n the sheep e p i t h e l i a l g l y c o p r o t e i n i n which s i a l i c a c i d residues are a l s o present (Kent, 1 9 7 0 ) . ( F i g . l b ) . The p i g g a s t r i c g l y c o p r o t e i n e x h i b i t s strong (A 4- H) blood group a c t i v i t y and i t i s apparent that a s i n g l e sulphate e s t e r group remote from the immuno-determinant groups has no demonstrable e f f e c t on the blood group a c t i v i t y . The l a r g e m a j o r i t y o f sulphated g l y c o p r o t e i n s have not y e t been investigated i n s u f f i c i e n t d e t a i l to a s s i g n the s t r u c t u r a l p o s i t i o n of the sulphate r e s i d u e s . I n t h i s r e s p e c t , the chemical study o f 3 5 s - l a b e l l e d g l y c o p r o t e i n s obtained by biochemical i n c o r p o r a t i o n techniques, and w e l l c h a r a c t e r i s e d by customary p h y s i c a l means, o f f e r s s u b s t a n t i a l advantages. I t remains a matter f o r s p e c u l a t i o n whether i n c e r t a i n o f these cases the presence of the small number o f e s t e r sulphate groups nevertheless a c t as ' i n f o r m a t i o n a l b l o c k s ' e i t h e r of immunologically important s i t e s o r o f substrate s p e c i f i c i t y i n enzymic degradation. Semi-synthetic

Studies

Means o f gathering i n f o r m a t i o n about these important p o s s i b i l i t i e s can be envisaged i n other ways. I n p a r t i c u l a r , the e f f e c t s are open to i n v e s t i g a t i o n s o f i n t r o d u c i n g sulphate groups, ^ S - l a b e l l e d otherwise, i n t o a w e l l - c h a r a c t e r i z e d g l y c o p r o t e i n by chemical means. While i t must be i n e v i t a b l y expected t h a t m u l t i p l e s i t e s w i l l be e s t e r i f i e d even with low degrees o f s u l p h a t i o n , nevertheless s u f f i c i e n t l y s e l e c t i v e chemical and enzymic techniques are now a v a i l a b l e t o make e x p l o r a t i o n worthwhile. A p a r t i c u l a r study has been made o f the g l y c o p r o t e i n s obtained from pooled p i g duodenal t i s s u e by papain d i g e s t i o n and f r a c t i o n a t i o n i n i t i a l l y by i o n exchange ( B e r t i l l i n i e t a l . 1 9 7 1 ) · The i n i t i a l mixed glycopeptides had a sulphate content between 1.04$ and 1.11$ f o r a range o f successive p r e p a r a t i o n s , while the s i a l i c a c i d content v a r i e d between 1.9$ and 2 . 6 $ . F r a c t i o n a t i o n by a n i o n i c exchange chromatography (Watman DE52) was e l u t e d with sodium acetate b u f f e r followed by a sodium chloride gradient ( F i g . 2 ) . T h i s enabled s i x components to be o

r

Gal

3 )

GalNAc

I ?

Fuc

Fuc

(4)

?

Gal

(6) .Gal.

(6) 4 Gal.

X-CHAIN

Gal(44)

Fuc (16)

GalNAc

GalNAc

Gal Gal-

I T

GlcNAc 16 Gal Y-CHAIN

-(GlcNAc)

Sulfate (47)

a

sulfated glycoproteins

SER

OR

THREO

PEPTIDE

H)

Gal—i- G l c N A c — ^ G l c N A c — G a l — ^ - G l c N A c

6-SULFATE (3)

Suggested structure of the carbohydrate chains of hog stomach blood group (A +

H

Fuc I 2(

Fuc

(3)

Abbreviations: Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, ^-acetylgalactosamine.

Figure lb. Proposed oligosaccharide structure of desialated colonic goblet cell glycoprotein H (sheep). Numbers in parentheses are number of residues per average molecule. For each polypeptide, 30 main X-chain oligosaccharides are present, each bearing two or three side Y-chains (taken from Kent (1970)).

Figure la.

(6)

G a l N A c — G a l — G l c N A c — Gal [2 ft

(4)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

36

CARBOHYDRATE

SULFATES

separated (Table I I I ) . The p r i n c i p a l f r a c t i o n (A) was a nearn e u t r a l PAS-t- m a t e r i a l accounting f o r 45$ of the o r i g i n a l m a t e r i a l , f o l l o w e d by two l e s s e r , but more a c i d i c g l y c o p r o t e i n s (C and D). I n a d d i t i o n , a c h o n d r o i t i n sulphate f r a c t i o n (F) and h y a l u r o n i c a c i d (E) were p r e s e n t . Of the g l y c o p r o t e i n components, a l l three showed some (A and H) blood group a c t i v i t y and the t h i r d component (D) had i n a d d i t i o n an e s t e r sulphate content of 1.84$ (Table I V ) . I n a s t r u c t u r a l examination of the p r i n c i p a l glycopeptide A, a l k a l i n e borohydride cleaved the o l i g o s a c c h a r i d e r e s i d u e s , with concomitant d e s t r u c t i o n of s e r y l and t h r e o n y l residues of the peptide c h a i n and q u a n t i t a t i v e formation of N-acetylgalactosaminoL The o l i g o s a c c h a r i d e side chains were of a t l e a s t three types, t e r m i n a t i n g i n s i a l i c a c i d , fucose and g a l a c t o s e , attached to the peptide c h a i n by O - g l y c o s y l r e s i d u e s . The second g l y c o p e p t i d e (C ) d i f f e r e d from A notably by i t s higher content of s i a l i c a c i d s , w h i l s t glycopeptide (D) had both s i a l i c a c i d and sulphate groups w i t h i n i t . Though the s i t e s of s u l p h a t i o n (227 groups per molecule, m.wt. 3.5 x 10^) have not y e t been i d e n t i f i e d p r e c i s e l y , i t i s known from the work of Smith t h a t the groups are not i n the t e r m i n a l regions of the o l i g o s a c c h a r i d e side branches but i n those regions proximal to the carbohydrate-peptide j u n c t i o n s , as i n the other e p i t h e l i a l g l y c o p r o t e i n s mentioned e a r l i e r . Complete s u l p h a t i o n ( i . e . > 96$) o f mixed duodenal g l y c o p e p t i d e p r e p a r a t i o n s (or of the c o n t r i b u t i n g p u r i f i e d GLP-A o r GLP-C) has been achieved ( P r i n o , L i e t t i and P a g l i a l u n g a , 1971). Though the product i s reported to possess some a n t i coagulant a c t i v i t y i n v i t r o , and i t s b i o l o g i c a l p o t e n t i a l as an a n t i - u l c e r agent i s c o n s i d e r a b l e , the l a t t e r property i s common to a number of c h e m i c a l l y sulphated macromolecules eg. dextran sulphate ( R i c k e t t s 1952, R i c k e t t s and Walton 1 9 5 2 ) , amylopectin sulphate ( f o r example, San and Ryan 1 9 7 0 ) , h y a l u r o n i c a c i d sulphate ( P a n t l i t s c h k o et a l . 1951 )> but with the added advantage t h a t the parent p i g g l y c o p e p t i d e d e r i v e s from an e p i t h e l i a l system r e l a t e d to the ABO blood group system. ~Microtechniques have been devised f o r the S-sulphation of the mixed duodenal g l y c o p e p t i d e s , as w e l l as f o r the component f r a c t i o n s , u s i n g 3 5 SO^Cl i n p y r i d i n e . Near q u a n t i t a t i v e s u l p h a t i o n (> 96$ or a v a i l a b l e h y d r o x y l s i t e s ) produced a h i g h l y a n i o n i c d e r i v a t i v e (GLPS) i n which the sugar composition was i n other r e s p e c t s undisturbed. This material (and a l s o sulphated GLP-A) was a modest i n h i b i t o r of p u r i f i e d human and p i g pepsins i n v i t r o (Table V ) . I t a l s o was a powerful i n h i b i t o r of c e l l growth of Swiss 3T3 f i b r o b l a s t s and of c e l l adhesion. I t i s d o u b t f u l however whether t h i s p r o p e r t y i s s p e c i f i c to the d e r i v a t i v e and other r e l a t e d H

A l c i a n Blue

PAS

identity

neutral glycopeptide

16$

45$

neutral glycopeptide

C

A

sulphated glycopeptide

31$

D

3$

E

l 2

chondroitin sulphate e t c .

f o r about

hyaluronic a c i d (E-^)

E

(Component Β i s of low molecular weight and accounts 1$ of the i n i t i a l m a t e r i a l )

$ of i n i t i a l m a t e r i a l

Component

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

CARBOHYDRATE SULFATES

38

T a b l e IV* C o m p o s i t i o n o f P i g D u o d e n a l

Glycopeptides

Çtimoles/g. d r y Component

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

NANA NGNA Fucose Gal GalNAc GluNAc

) )

106

D

C

A ) )

342 772 1260 1140

wt)

230 199 812 1170 1140

) )

263 316 721 1240 926

( X y l , Mann, G l u c u r o n i c a c i d not detected)

Asp Thre Ser Glu Pro Gly Ala Val I-Leu Leu His Lys Arg Tyr Phe

55 567 310 85 369 63 151 93 17 66 64

16 39

ND ND

55 533 304 87 331 66 138 88 14 56 59 15 33

ND ND

49 711 290 66 447 67 72 53 34 41 35 12 17

ND ND

( C y s and M e t n o t Sulphate

l e s s than 0.3

7.4

227

detected)

3.

Sulfate Ester Groups

K E N T ET AL.

39

Ε

S o'

5

50m M Sodium Acetate 1-2M NaCI pH 4 0 —— •

A

CO

\

(/) g

ΟΙΟ-

Q _l < 005Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

^

lOmM Sodium Acetate pH 4 0

Λ ν/Λ α

o^S^

C

F

D

y^

100

500

400 ELUTION VOLUME (ML)

Figure 2. Fractionation of duodenal glycopeptides by ion exchange. in 5mL starting buffer was applied to a column of Whatman DE 52 (120 X 1.25) equilibrated with lOmM sodium acetate, pH 4.0 (starting ionic gradient terminating with 50mM sodium acetate, 1.2M sodium was applied. Flow rate, 10mL/hr.

Table V.

200 mg of GLP anion exchanger buffer). A linear chloride, pH 4.0

E f f e c t of sulphated duodenal glycopeptides on p u r i f i e d

pepsins

( i n c o l l a b o r a t i o n with Dr. W. H. T a y l o r ) H i g h l y p u r i f i e d p i g o r human pepsins were incubated a t 3 7 ° with glycopeptide

(2.5mg/ml).

30 min, pH 4 · 0 ( a c e t a t e ) ; haemoglobin s u b s t r a t e . Values expressed

at % of glycopeptide-free c o n t r o l .

glycopeptide

human * 118.6

native glycopeptide sulphate ( f u l l ) glycopeptide sulphate (33%)

Pig 112

substrate

91

72

inhibition

-

90

inhibition

(average o f 3 experiments)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

40

CARBOHYDRATE SULFATES

p o l y s u l p h a t e s eg. dextran sulphate behaved s i m i l a r l y , both q u a l i t a t i v e l y and q u a n t i t a t i v e l y ( F i g . 3 ) towards f i b r o b l a s t s i n culture. M i l d a c i d i c h y d r o l y s i s r e s u l t s i n cleavage o f two s i a l i c a c i d d e r i v a t i v e s , separated by t . l . c . on c e l l u l o s e and i d e n t i f i e d as the tetra-O-sulphates o f N-acetylneuraminic a c i d and as the penta-O-sulphate o f N - g l y c o l l y l n e u r a m i n i c a c i d . Sulphated forms of fucose have not y e t been i d e n t i f i e d though presumably a r e cleaved simultaneously. N e i t h e r o f these products a r e o x i d i s e d by sodium metaperiodate though they may be detected by the Svennerholm procedure. Not s u r p r i s i n g , the f u l l y sulphated glycopeptide (GLPS 9 6 $ ) i s not a substrate f o r cholerae neuraminidase and there i s some evidence t h a t i t may be a noncompetitive i n h i b i t o r o f t h i s enzyme towards other s i a l o p r o t e i n s . The i n h i b i t i o n i s not considered t o be s p e c i f i c but r a t h e r a consequence o f the p o l y a n i o n i c c h a r a c t e r o f the substance. I t i s o f i n t e r e s t t o examine the s i t u a t i o n a r i s i n g from much lower degrees o f s u l p h a t i o n o f the g l y c o p e p t i d e and e m p i r i c a l l y two d e r i v a t i v e s r e p r e s e n t i n g 3 4 $ and 1 1 $ s u l p h a t i o n of a v a i l a b l e hydroxyl s i t e s have been prepared. The low s u l p h a t i o n d e r i v a t i v e (GLP-S 1 1 $ ) i s h y d r o l y s a b l e by d i l u t e s u l p h u r i c a c i d , with r e l e a s e o f s i a l i c a c i d s (NANA and NGNA) i n which predominantly one hydroxyl group i s r e p l a c e d by sulphate. (The d e r i v a t i v e s a r e o x i d i s e d by I 0 7 , suggesting t h a t the sulphate e s t e r i n NANA i s a t Cy o r C ^ j . T h i n - l a y e r chromatography o f the h y d r o l y s a t e showed the presence o f a t l e a s t three sulphates and i t appears l i k e l y t h a t a sulphated fucose i s a l s o r e l e a s e d . I t i s estimated t h a t 16$ o f the t o t a l sulphate content i s present i n s i a l i c a c i d and fucose t e r m i n a l r e s i d u e s . U n l i k e the f u l l y sulphated d e r i v a t i v e (GLP-S 9 6 $ ) , d e r i v a t i v e s with lower s u l p h a t i o n had l i t t l e e f f e c t on f i b r o b l a s t c e l l growth o r on p e p s i n a c t i v i t y . The sulphated d e r i v a t i v e (GLP-S 1 1 $ ) a l s o i s not a s u b s t r a t e f o r V. cholerae neuraminidase and indeed shows some i n h i b i t i o n o f the enzymic h y d r o l y s i s w i t h a known s u s c e p t i b l e substrate (Table V I ) . I t i s b e l i e v e d t h a t t h i s i s a competitive i n h i b i t i o n and a t t r i b u t a b l e t o the presence o f O-sulphate r e s i d u e ( s ) i n t e r m i n a l s i a l i c a c i d s , s i n c e the sulphated g l y c o peptide residue a f t e r m i l d a c i d i c h y d r o l y s i s was not such an inhibitor. I t i s o f i n t e r e s t t o c o n s i d e r whether the i n h i b i t i o n can be r e l i e v e d by metabolic i n t e r v e n t i o n o f a p p r o p r i a t e enzymes, and t h i s would suggest a p a r t i c u l a r r o l e f o r the sulphatases, lysosomal systems known t o be capable o f a c t i o n both on g l y c o p r o t e i n and g l y c o l i p i d carbohydrate sulphate e s t e r s . I n a p p r o p r i a t e c o n d i t i o n t h i s step c o u l d represent a f u r t h e r c o n t r o l mechanism r e g u l a t i n g the metabolic turnover o f important t e r m i n a l sugar r e s i d u e s . A p a r a l l e l s i t u a t i o n e x i s t s with 0 - a c e t y l d e r i v a t i v e s o f NANA and NGNA r e s i d u e s i n bovine, p o r c i n e and equine submaxillary gland g l y c o p r o t e i n s (reviewed

3.

Sulfate Ester Groups

KENT ET AL.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

2

-2

-I

Ο

+1

+2

Log (^g/ml) Glycopeptide Concentration

Figure 3.

Table VI

Effect of sulfated glycopeptides on 3T3 fibroblast growth

Sulphated D e r i v a t i v e s of P i g Duodenal Glycopeptide

(sodium s a l t s )

(g/lOOg dry wt)

glycopeptide

%

sulphate

-

degree of sulphation

fully sulphated

49 > 96%

5.0

2.3

Gal

24.8

13.4

Fuc

11.0

5.1

Hexosamines

37.5

20.1

NANA ) NGNA )

partly sulphated SI 14.2

partly sulphated S2 4.7 ca.ll$

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

42

CARBOHYDRATE

SULFATES

by Schauer, Buscher and C a s a l s - S t e n z e l , 1 9 7 4 ) · In. the n a t i v e m a t e r i a l those t e r m i n a l s i a l i c a c i d s c a r r y i n g a 4-O-acetyl s u b s t i t u e n t a r e reported to be i n s e n s i t i v e to V. cholerae neuraminidase and here a l s o an i n t e r e s t i n g r e g u l a t o r y r o l e may be e x e r c i s e d by d e - a c e t y l a s e s . The i n t a c t nature o f t e r m i n a l s i a l i c a c i d s i s now e s t a b l i s h e d as an important i n f o r m a t i o n a l s i g n a l r e g u l a t i n g c e r t a i n forms o f b i o l o g i c a l r e c o g n i t i o n of g l y c o p r o t e i n s . I n p a r t i c u l a r , t h i s new concept o f the r o l e o f carbohydrates i s i n v o l v e d i n r e g u l a t i n g the serum s u r v i v a l time o f plasma g l y c o p r o t e i n s (reviewed by Ashwell and M o r e l l 1 9 7 4 ) · Hydrolysis of t e r m i n a l s i a l i c a c i d s by the a c t i o n o f neuraminidase, even i n p a r t , exposes subterminal sugar galactose^and the consequent a l t e r a t i o n i n molecular i d e n t i t y leads to the removal of the g l y c o p r o t e i n by s p e c i f i c h e p a t i c b i n d i n g s i t e s (Lunney and Ashwell, 1 9 7 6 ; Kawasaki and Ashwell 1 9 7 6 ) . Thus any f u r t h e r m o d i f i c a t i o n of t e r m i n a l sugars by a d d i t i o n a l subs t i t u t i o n c l e a r l y has important i m p l i c a t i o n s f o r t h i s recogn i t i o n process. I t i s a p p r e c i a t e d t h a t as y e t , amongst the g l y c o p r o t e i n s , a d d i t i o n a l s u b s t i t u t i o n by s u l p h a t i o n r a r e l y occurs on e x t e r i o r sugar s i t e s . Presence of these e s t e r groups i n the i n t e r i o r p a r t s o f the carbohydrate side-branches nevertheless can be envisaged as having i n f o r m a t i o n a l s i g n i f i c a n c e of two k i n d s . The f i r s t aspect of t h i s hypothesis a k i n to the above s i t u a t i o n can be considered as i n f l u e n c i n g the s u s c e p t i b i l i t y of the monosaccharide concerned towards exoglycosidases i n the course of metabolic turnover. The second aspect i n v o l v e s the changed acceptor status o f a sulphated monosaccharide residue towards sugar n u l e o t i d e t r a n s f e r a s e s . The p o s s i b i l i t y e x i s t s t h a t these s p e c i f i c enzymes may w e l l d i s c r i m i n a t e between the non-sulphated and sulphated residues o f a g i v e n sugar w i t h i n a s i n g l e g l y c o p r o t e i n molecule with s i g n i f i c a n t a l t e r a t i o n i n the p a t t e r n s of b i o s y n t h e s i s o r r e s y n t h e s i s of o l i g o s a c c h a r i d e c h a i n s . Accumulated evidence suggests t h a t i n v i v o sugar residues i n the more e x t e r i o r p a r t s o f the g l y c o p r o t e i n s t r u c t u r e are more r e a d i l y exchangeable than the deeper i n t e r n a l regions o f the molecule. I n t h i s , O-sulphation may be one c o n t r i b u t i n g f eature. The hypothesis t h a t O-sulphation may have i n f o r m a t i o n consequences f o r synthesis and b i o s y n t h e s i s of g l y c o p r o t e i n s i s not s o l e l y r e s t r i c t e d to those processes. The extensive change i n a n i o n i c charge and the conformational e f f e c t o f i n t r o d u c i n g a s i n g l e sulphate e s t e r i n t o a carbohydrate residue i n v o l v e d i n an immunological determinant s i t e may be expected a l s o t o l e a d to a l t e r a t i o n o f antigen-antibody i n t e r a c t i o n s . The o v e r a l l evidence suggests f u r t h e r r o l e s f o r the p a r t

3.

K E N T ET AL.

Sulfate Ester Groups

43

played by low l e v e l s of s u l p h a t i o n i n a v a r i e t y o f processes. I t p o i n t s to the importance o f i d e n t i f y i n g p r e c i s e l y the p o s i t i o n s occupied by O-sulphates i n g l y c o p r o t e i n s and o f f i n d i n g new and s p e c i f i c means o f i n t r o d u c i n g such e s t e r s a t d e f i n e d p o s i t i o n s of o l i g o s a c c h a r i d e s . The sulphatases would appear to be enzyme systems of considerable p o t e n t i a l importance i n respect to c o n t r o l o f i n f o r m a t i o n a l content o f g l y c o p r o t e i n s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

References Ashwell, G. and Morell, A.G. (1974) Advanc. Enzymol. 41 99-128 Atkins, E.D.T. and Laurent, T.C. (1973) Biochem. J. 133,605-606 Bertillini, G., Butti, Α., Piantanida, B., Prino, G., Riva, Α., Rossi, A. and Rossi, S. (1971) Drug Res. 21, 244-248 Hirano, S. and Onodera, K. (1967) Life Sciences 6, 2177-2183 Kawasaki, T. and Ashwell, G. (1976) J. biol. Chem. 251, 1296-1302 Kent, P.W. (1970) Exposés Ann. Biochem. Méd. Sér. 30, 98-120 Kent, P.W. (1974) in Topics in Gastroenterology II (ed.) S.C. Truelove and J. Trowell, Blackwell, Oxford, U.K. pp. 77-99 Kirkwood, S., (1974) Annu. Rev. Biochem. 43, 401-417 Lunney, J. and Ashwell, G. (1976) Proc. Nat. Acad. Sci. USA 73, 341-343 Pantlitscko, M., Schmid, J., Seelich, F. and Kaiser, E. (1951) Monatsh. Chem. 82, 380-386 Prino, G., Lietti, A. and Paglialunga, S. (1971) Drug Res., 21, 918-921 Rees, D.A., (1969) Advanc. Carbohydrate Chem. 24, 267 Ricketts, C.R. (1952) Biochem. J. 51, 129-135 Ricketts, C.R., and Walton, K., (1952) Chem. Ind. 869 Schauer, R., Buscher, H-P., and Casals-Stenzel, J. (1974) in Metabolism and Function of Glycoproteins ed. R.M.S. Smellie and J.G. Beeley Biochem. Soc. Symposium No.20 pp. 87-116 Slomiany, B.L. and Meyer, K., (1972) J. biol. Chem. 247, 5062-5070 Stone, A.L., (1964) Biopolymers 2, 315 Stone, A.L., (1965) Biopolymers 3, 617 Sun, D.C.H., and Ryan, M.L. (1970) Gastroeneterology 58, 756-761 Winterbourne, D.J. and Kent, P.W. (1977) Trans. Biochem. Soc. 5, 439-440 Yosizawa, Z. (1972) in Glycoproteins ed. A. Gottschalk pp. 1000-1018 Elsevier, Amsterdam RECEIVED February 6,

1978.

4 Studies on the Synthesis of Sulfur-Containing Glycolipids ("Sulfogylcolipids") ROY GIGG

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

Laboratory of Lipid and General Chemistry, National Institute for Medical Research, Mill Hill, London, NW7 1AA

A sulphur-containing l i p i d was f i r s t detected in brain by Thudichum (1) about one hundred years ago and in 1910 Koch (2) postulated that this l i p i d was a complex between a phospholipid and a sulphated cerebroside. Later Levene (3-5) obtained a sulphur-containing brain l i p i d free from phosphate but it was not until 1933 that Blix (6) obtained a pure sample for which the analytical data indicated that it was a sulphate ester of a cerebroside (1). The presence of the sulphate group on the 3-position of the galactose residue was established in 1962 by Yamakawa (7-8) and Stoffyn (9) by methylation studies and by showing that the compound was stable to periodate oxidation. The β - c o n f i g u r a t i o n of the galactosidic-linkage was established by degradative studies (10) and thus the complete structure (11) of the sulphate ester of cerebroside ("sulphatide") was estab­ lished. (For reviews see references 11-14). Interest in the sulphatides has increased considerably since the demonstration (15-16) that these compounds accumulate in tissues in the inherited disease known as metachromatic leukodystrophy. This accumulation is due to the deficiency of a sulphatase which normally degrades the sulphatide (11) to a cerebroside (1). In 1963, Martensson (17) isolated a new sulphated l i p i d , from human kidney, which contained both glucose and galactose and this was later (18,19) characterised as the sulphate ester (IV) of the lactosyl ceramide (III). Compound (IV) has also been detected in testicular tissue (20). Another ceramide-contaiηing sulphoglycolipid was detected in hog gastric mucosa in 1974 (2J_) , and this was identified as a triglycosyl ceramide sulphate with the probable structure (VI). The trigly cosyl ceramide (V) is a normal tissue constituent and accumulates in tissues in Fabry's disease due to the deficiency of an α - g a l a c t o s i d a s e . In 1972 a new sulphoglycolipid was reported to be a major component of the glycolipids of mammalian testes and spermatozoa (22-24). This l i p i d ("seminolipid ) differed from the known M

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

mammalian s u l p h o g l y c o l î p i d s i n t h a t i t c o n t a i n e d 1-0-alky1-2-0a c y l -L,-g 1 y c e r o l i n s t e a d o f c e r a m i d e i n t h e l i p i d m o i e t y . The c o m p l e t e s t r u c t u r e (VI1) was e s t a b l i s h e d ( 2 £ , 2 £ ) f o r s e m i n o l i p i d and t h e compound was l a t e r d e t e c t e d i n r a t b r a i n ( 2 7 , 2 8 ) but was f o u n d t o be a b s e n t f r o m b i r d and f i s h t e s t e s (20) . In b r a i n an a n a l o g o u s d i a c y l d e r i v a t i v e a l s o o c c u r s (28-30). Kates (2Jj r e p o r t e d the p r e s e n c e of a s u l p h a t e d g l y c o l i p i d i n t h e membrane o f t h e e x t r e m e l y h a l o p h i l i c b a c t e r i u m H a l o b a c t e r î u m c u t i rubrum. L i k e the p r e v i o u s l y d e s c r i b e d l i p i d s from t h i s m i c r o o r g a n i s m , t h e su 1 p h o g l y c o l i ρ i d c o n t a i n e d 2 , 3 - d i - j 0 p h y t a n y l - L - g l y c e r o l i n t h e l i p i d m o i e t y as o p p o s e d t o 1 , 2 - d i 0_-acyl - L - g l y c e r o l o r 1 - 0 - a l k y l - 2 - 0 - a c y l - L - g 1 y c e r o l w h i c h i s p r e s e n t in most o f t h e known p h o s p h o l i p i d s and g l y c o l i p i d s based on g l y c e r o l . M a n n o s e , g l u c o s e and g a l a c t o s e 3 - s u l p h a t e were a l s o i d e n t i f i e d and t h e c o m p l e t e s t r u c t u r e (V111) was l a t e r e s t a b l i s h e d (12) by m e t h y l a t i o n s t u d i e s and by e n z y m i c h y d r o l y ­ sis. In 1959 ( 1 1 ) , a s u l p h o l i p i d was d e t e c t e d i n a v i r u l e n t s p e c i e s o f M y c o b a c t e r i urn t u b e r c u 1 o s î s . A family of acylated d e r i v a t i v e s o f t r e h a l o s e 2 - s u l p h a t e was l a t e r i d e n t i f i e d by G o r e n ( 2 4 , 1 £ ) and t h e s t r u c t u r e s o f some o f t h e s e were e s t a b lished. The s t r u c t u r e ( I X ) , which r e p r e s e n t s a 2 , 3 , 6 , 6 " - t e t r a 0,-acyl - α : α ' - D - t r e h a l o s e 2 ' - s u l p h a t e was p r o p o s e d (36) f o r t h e principal glycolipid. T h e s e compounds may p r e v e n t membrane f u s i o n o f l y s o s o m e s w i t h phagosomes and t h u s h i n d e r t h e h o s t ' s normal method o f d e s t r u c t i o n o f i n v a d i n g m i c r o o r g a n i s m s (37). S l o m i a n y (38) has r e c e n t l y r e p o r t e d t h e p r e s e n c e o f s u l ­ phated g l y c e r o g l y c o l i p i d s i n human g a s t r i c s e c r e t i o n . The m a j o r compound was c h a r a c t e r i s e d as a g l y c o s i d e (X) o f a t r i g l u c o s y l s u l p h a t e w i t h an a l k y l - a c y l - g l y c e r o l . The p r e s e n c e o f s u l p h a t e e s t e r s o f g l u c o s y l diglycerides i n seaweeds has a l s o been r e p o r t e d ( 3 9 ) . In 1959, Benson (40,4]_) r e p o r t e d t h e o c c u r r e n c e o f a s u l ­ phur c o n t a i n i n g l i p i d in p l a n t s . The s u l p h u r was n o t r e l e a s e d as s u l p h a t e on a c i d i c h y d r o l y s i s and t h e s t r u c t u r e was s u b s e ­ q u e n t l y e s t a b l i s h e d (k2) as a g l y c o s i d e o f 6-deoxy-6-su1pho glucose with a 1 , 2 - d i - 0 - a c y l - L - g l y c e r o l (XI). This structure was c o n f i r m e d ( 4 l ) by X - r a y c r y s t a l 1 o g r a p h i c s t u d i e s on t h e rubidium s a l t of the d e a c y l a t e d s u l p h o n o g l y c o l i p i d . This l i p i d i s a m a j o r component o f p l a n t g l y c o l i p i d s and o c c u r s i n t h e c h l o r o p l a s t membrane ( f o r a r e v i e w s e e r e f e r e n c e 1 3 ) . O t h e r s u l p h o - and su 1 p h o n o g 1 y c o l i ρ i d s h a v e been i s o l a t e d f r o m seaweeds (22) . Previous

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F l o w e r s (kk) r e p o r t e d t h e s y n t h e s i s o f a g l y c o s i d e (XVI) of β - g a l a c t o p y r a n o s y 1 3 - s u l p h a t e w i t h racemic d î h y d r o c e r a m i d e in 1966. He p r e p a r e d t h e a c e t y l a t e d g a l a c t o s y l b r o m i d e (XI11) c o n t a i n i n g a f r e e 3 - h y d r o x y - g r o u p , from 4,6-0_-ethyl idene-1 , 2 - 0 -

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CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

î s o p r o p y l î d e n e - a - D - g a l a c t o p y r a n o s e ( X 1 1 ) by t h e r o u t e shown. Compound ( X l l l ) was c o n d e n s e d w i t h r a c e m i c 0 - b e n z o y l - N - o c t a d e canoyl d î h y d r o s p h i n g o s î n e to g i v e the β - g a l a c t o p y r a n o s y 1 d e r i ­ v a t i v e (XIV). S u l p h a t i o n o f compound (XIV) w i t h t h e p y r i d i n e s u l p h u r t r i o x i d e c o m p l e x and s u b s e q u e n t b a s i c h y d r o l y s i s g a v e the d i h y d r o s u l p h a t i d e (XVI). In v i e w o f t h e known e a s e o f migration of acetyl groups, i t is s u r p r i s i n g that t h i s p r e p a r a ­ t i o n was a c h i e v e d w i t h o u t p r o b l e m s due t o t h i s t y p e o f r e ­ arrangement. S h a p i r o (4£) has r e p o r t e d t h a t a t t e m p t s t o a p p l y t h i s synthesis to the preparation of sphingosine containing s u l p h a t i d e s were u n s u c c e s s f u l . J a t z k e w i t z and Nowoczek (46) and Yamakawa and h i s c o w o r k e r s (8) h a v e d e s c r i b e d t h e p r e p a r a t i o n o f c e r e b r o s i d e s s u l p h a t e d on t h e 6 - p o s i t i o n o f g a l a c t o s e by t h e a c t i o n o f c h l o r o s u l p h o n i c a c i d on c e r e b r o s i d e ( l ) . M i y a n o and Benson (kj) synthesised the deacylated d e r i v a ­ t i v e (XXI) o f t h e p l a n t su 1 p h o n o l î ρ î d but a t t e m p t e d a c y l a t i o n (42) o f t h i s d i d n o t g i v e t h e s u l p h o n o l i p i d . The 6 - d e o x y - 6 s u l p h o - J ) - g l u c o s e d e r i v a t i v e (XVI11) was p r e p a r e d by r e p l a c e m e n t o f t h e t o s y l g r o u p o f compound ( X V I l ) . Compound (XVI11) was c o n v e r t e d i n t o t h e α - a l l y l g l y c o s i d e (XIX) and t h e a 1 1 y1 g r o u p was h y d r o x y l a t e d w i t h p e r m a n g a n a t e t o g i v e t h e c r u d e d i o l ( X X ) . C r y s t a l l i s a t i o n o f t h e c r u d e compound (XX) g a v e t h e p u r e i s o m e r (XXI) i d e n t i c a l w i t h t h e m a t e r i a l p r e p a r e d by d e a c y l a t i o n o f the n a t u r a l l i p i d . Other attempts to a c y l a t e d e r i v a t i v e s of t h e a 1 1 y1 g l y c o s i d e (XIX) (kj) were a l s o u n s u c c e s s f u l . A p p l i c a t i o n o f a G e n e r a l Method o f O l i g o s a c c h a r i d e to the Synthesis of S u l p h o g l y c o l i p i d s

Synthesis

We h a v e r e c e n t l y d e v e l o p e d and r e v i e w e d (48) a g e n e r a l method o f o l i g o s a c c h a r i d e s y n t h e s i s i n w h i c h a l l y l e t h e r s a r e u s e d f o r ' t e m p o r a r y p r o t e c t i o n and b e n z y l e t h e r s a r e u s e d f o r ' p e r s i s t e n t ' p r o t e c t i o n o f h y d r o x y - g r o u p s and h a v e f o u n d t h i s method o f p a r t i c u l a r v a l u e f o r t h e s y n t h e s i s o f g l y c o l i p i d s e . g . t h e d i g a l a c t o s y l d i g l y c e r i d e ( X X I l ) (42), t h e t r i g a l a c t o s y 1 d i g l y c e r i d e ( X X l l l ) (£0) and t h e g l u c o s y l d i g l y c e r i d e (XXIV) 1

(51). In t h i s method a p a r t i c u l a r h y d r o x y - g r o u p , t e m p o r a r i l y p r o t e c t e d as an a l l y l e t h e r , can be l i b e r a t e d by removal o f t h e a l l y l group w h i l s t the remaining hydroxy-groups o f the o l i g o ­ s a c c h a r i d e r e m a i n p r o t e c t e d as b e n z y l e t h e r s t h u s a l l o w i n g a f u r t h e r g 1 y c o s i d a t i o n t o be c a r r i e d o u t s p e c i f i c a l l y a t t h e f r e e h y d r o x y - g r o u p . It was a p p a r e n t t h a t o t h e r s u b s t i t u t i o n s c o u l d a l s o be c a r r i e d o u t s p e c i f i c a l l y a t t h e l i b e r a t e d h y d r o x y g r o u p and s u l p h a t i o n a p p e a r e d t o be an a p p r o p r i a t e r e a c t i o n t o i n v e s t i g a t e so t h a t s p e c i f i c a l l y s u l p h a t e d o l i g o s a c c h a r i d e s o r g l y c o l i p i d s c o u l d be p r e p a r e d .

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

S i n c e t h e s u c c e s s o f t h i s method depends l a r g e l y on t h e e a s e o f p r e p a r a t i o n o f s p e c i f i c a l l y b e n z y l a t e d and a l l y l a t e d derivatives o f c a r b o h y d r a t e s , new methods f o r a c h i e v i n g this are important. We h a v e r e v i e w e d (48) o u r d e v e l o p m e n t o f t h e a l l y l e t h e r s ( p r o p - 2 - e n y l , p r o p - l - e n y l , b u t - 2 - e n y l and 2 m e t h y l p r o p - 2 - e n y l e t h e r s ) as p r o t e c t i n g g r o u p s and shown t h e i r v a l u e in the p r e p a r a t i o n o f b e n z y l a t e d c a r b o h y d r a t e d e r i v a t i v e s and r e c e n t l y some o t h e r d e v e l o p m e n t s h a v e o c c u r r e d w h i c h a r e u s e f u l in t h i s r e s p e c t . A u g e , D a v i d and V e y r i e r e s ( £ 2 ) h a v e shown t h a t t h e a x i a l - e q u a t o r i a l 0,0_-d i b u t y l s t a n n y l î dene d e r i v a t i v e ( X X V ) , ( w h i c h i s r e a d i l y p r e p a r e d by a z e o t r o p i c d i s t i l l a t i o n o f w a t e r f r o m a m i x t u r e o f t h e d i o l and d i b u t y l t i n o x i d e i n b e n z e n e ) was b e n z y l a t e d on t h e e q u a t o r i a l 3-hydroxy-group, t o g i v e t h e d e r i v a t i v e (XXVI) w i t h c o m p l e t e r e g i o s p e c i f i c i t y , when i t was t r e a t e d w i t h b e n z y l b r o m i d e i n d i m e t h y l f o r m a m i d e a t 100 . This confirmed

high r e g î o s p e c i f i c i t y , by N a s h e d and A n d e r s o n

i n t h e g a l a c t o s e s e r i e s , was (£2) when t h e y showed t h a t a

s i m i l a r reaction occurred with a l l y l i o d i d e . Thus t h e 0 , 0 d i b u t y l s t a n n y l î d e n e d e r i v a t i v e (XXV11) g a v e t h e 3 - 0 - a l l y l d e r i v a t i v e ( X X V I 1 1 ) w i t h o n l y t r a c e amounts o f t h e i s o m e r i c 4 - 0 - a l l y l d e r i v a t i v e being formed. A s i m i l a r a l k y l a t i o n of the a x i a l - e q u a t o r i a l s y s t e m o f t h e m y o - i n o s i t o i d e r i v a t i v e (XXIX) a g a i n g a v e t h e e q u a t o r i a l a l l y l e t h e r (XXX) w i t h h i g h r e g i o s p e c i f i c i t y (£2.see a l s o 54,55.) . We h a v e a l s o a p p l i e d ( £ 6 ) t h i s r e a c t i o n t o t h e 0 , 0 - d i b u t y l s t a n n y l i d e n e d e r i v a t i v e (XXXI) o f 1, 6 - a n h y d r o - 2 - 0 _ - b e n z y l - p - D _ - g a l a c t o p y r a n o s e and h a v e p r e p a r e d t h e 4 - 0 - b e n z y l ( X X X l l ) and 4 - 0 - a l l y l (XXX111) d e r i v a t i v e s w i t h high r e g i o s p e c î f i c i t y . We h a v e p r e v i o u s l y (52) p r e p a r e d t h e d e r i v a t i v e s ( X X X l l and X X X I 1 1 ) by more i n v o l v e d p r o c e d u r e s . R e c e n t l y a l s o a v a l u a b l e new t e c h n i q u e has been d e v e l o p e d f o r t h e p r e p a r a t i o n o f 4 - 0 - b e n z y l e t h e r s o f c a r b o h y d r a t e s by N a n a s i and L i p t a k (58-62). They h a v e shown t h a t 4 , 6 - 0 - b e n z y 1 i dene d e r i v a t i v e s o f c a r b o h y d r a t e s , w h i c h c o n t a i n a b u l k y p r o t e c t i n g g r o u p on t h e 3 h y d r o x y - g r o u p ( e . g . b e n z y l ) , a r e h y d r o g e n o l y s e d by l i t h i u m a l u m i n i u m h y d r i d e - a l u m i n i u m c h l o r i d e t o give 4-0-benzyl d e r i v a t i v e s with high r e g i o s p e c i f i c i t y . Thus t h e 4 , 6 - 0 - b e n z y l î d e n e d e r i v a t i v e (XXXIV) g a v e t h e 4 - 0 - b e n z y l d e r i v a t i v e (XXXV) i n 9 2 % y i e l d u n d e r t h e s e c o n d i t i o n s . It has been shown a l s o t h a t t h i s r e a c t i o n can be c a r r i e d o u t s a t i s f a c t o r i l y i n t h e p r e s e n c e o f a l l y l e t h e r s (63,64). We have e n v i s a g e d t h a t a s i m i l a r r e a c t i o n s h o u l d o c c u r on the h y d r o g e n o l y s î s of a c r o l e i n a c e t a l s t o g i v e a l l y l (or p r o p - l enyl) ethers. T h e a c r o l e i n a c e t a l ( X X X V l l ) was r e a d i l y p r e p a r e d f r o m t h e d i o l (XXXVI) by t h e a c t i o n o f a c r o l e i n d i e t h y l a c e t a l and an a c i d c a t a l y s t i n b e n z e n e a t room t e m p e r a t u r e . Hydrogenol y s i s o f compound ( X X X V l l ) w i t h l i t h i u m a l u m i n i u m h y d r i d e a l u m i n i u m c h l o r i d e g a v e ( £ 6 ) i n 2 0 % y i e l d , a m i x t u r e o f t h e 40 - a l l y l d e r i v a t i v e ( X X X V l l 1 ) and t h e 4 - 0 - ( p r o p - 1 - e n y 1 ) derivative (XXXIX) ( i n a r a t i o o f c a 3 : 2 ) w h i c h w e r e c h a r a c t e r i s e d by c o m -

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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( X X X I X ) , m.p. 7 6 - 7 7 ° , [a] j ^ - 2 2 . 8 ° (C 1 i n CHC1 )] . Only t r a c e s o f t h e c o r r e s p o n d i n g 6-jO-subst i tu t e d d e r i v a t i v e s w e r e o b s e r v e d s h o w i n g t h a t t h e r e a c t i o n o c c u r s w i t h t h e same regiospecificity as w i t h t h e b e n z y l i d e n e d e r i v a t i v e . Different conditions for t h e h y d r o g e n o l y s i s a r e b e i n g i n v e s t i g a t e d in a t t e m p t s t o i m p r o v e t h e y i e l d s o f compounds (XXXVI11 and X X X I X ) . S i n c e o u r p r e v i o u s r e v i e w (48) on t h e u s e o f a l l y l e t h e r s as p r o t e c t i n g g r o u p s , Boss and S c h e f f o l d (65) have shown t h a t a l l y l e t h e r s can be i s o m e r i s e d t o p r o p - l - e n y l e t h e r s by t h e a c t i o n o f p a l l a d i u m on c h a r c o a l and i t has a l s o been shown ( 6 6 , 67) t h a t some h y d r o g é n a t i o n o f t h e a l l y l g r o u p t o a p r o p y l g r o u p may o c c u r when t r i s t r i p h e n y l - p h o s p h i n e r h o d i u m (1) c h l o r i d e i s u s e d f o r t h e i some.r î s a t i o n . The t r a n s f e r o f h y d r o g e n f r o m c e r t a i n s o l v e n t s t o u n s a t u r a t e d compounds i s a w e l l known p r o p e r t y of the rhodium c a t a l y s t ( 6 8 , 6 9 ) . Several other reports on t h e u s e o f a l l y l e t h e r s as p r o t e c t i n g g r o u p s i n c a r b o h y d r a t e and o t h e r b r a n c h e s o f c h e m i s t r y h a v e a l s o a p p e a r e d ( 5 2 - 5 5 , 6 3 , 6 4 , 6 6 , 6 7 , 7 0 - 8 1 ) s i n c e o u r p r e v i o u s r e v i e w . See a l s o (107,108).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

6

In c o n s i d e r i n g t h e a p p l i c a t i o n o f t h i s g e n e r a l method t o t h e s y n t h e s i s o f c a r b o h y d r a t e s u l p h a t e s i t was n e c e s s a r y t o show t h a t b e n z y l e t h e r s c o u l d be removed s a t i s f a c t o r i l y f r o m t h e corresponding sulphated benzyl d e r i v a t i v e s . It has been r e p o r t e d (82) t h a t t h e b e n z y l and b e n z y l i d e n e g r o u p s o f compounds (XL) and ( X L l ) c o u l d n o t be removed by c a t a l y t i c h y d r o g e n o l y s i s a l t h o u g h no d e t a i l s o f t h e e x p e r i m e n t a l c o n d i t i o n s u s e d w e r e reported. T u r v e y ( 8 1 , 8 4 ) has a l s o r e p o r t e d p r o b l e m s i n t h e h y d r o g e n o l y s i s of benzyl e t h e r s in the p r e s e n c e of s u l p h a t e g r o u p s and has u s e d p r o l o n g e d h y d r o g é n a t i o n t i m e s i n aqueous ethanol at elevated p r e s s u r e s . We d e c i d e d t h e r e f o r e t o s t u d y t h e h y d r o g e n o l y s i s o f t h e b e n z y l g r o u p s f r o m some model compounds b e f o r e t h i s g e n e r a l a p p r o a c h t o s u l p h o g l y c o l i p i d s y n t h e s i s was further considered. The c r y s t a l l i n e b e n z y l a t e d d e r i v a t i v e s (XL 11) and (XLV) o f b e n z y l a-D.-ga1 a c t o p y r a n o s i de w e r e a v a i l a b l e s i n c e t h e y had been p r e p a r e d p r e v i o u s l y (85) f o r i n v e s t i g a t i o n s o f t h e g e n e r a l oligosaccharide synthesis. T h e s e compounds w e r e r e a d i l y c o n v e r t e d i n t o t h e c o r r e s p o n d i n g s u l p h a t e s ( X L l 1 1 ) and ( X L V l ) by t h e p y r i d i n e - s u l p h u r t r i o x i d e c o m p l e x and h y d r o g e n o l y s i s o f t h e barium s a l t s over p a l l a d i u m - c h a r c o a l at atmospheric p r e s s u r e i n g l a c i a l a c e t i c a c i d d u r i n g t h r e e d a y s g a v e p r o d u c t s (XL1V) and ( X L V l 1 ) w h i c h c o - c h r o m a t o g r a p h e d w i t h a u t h e n t i c s t a n d a r d s (56). Thus t h i s g e n e r a l a p p r o a c h a p p e a r e d t o be f e a s i b l e and a synthetic route to a t y p i c a l s u l p h o g l y c o l i p i d ('seminolipid') c o n t a i n i n g a g a l a c t o s e 3 - s u l p h a t e r e s i d u e was i n i t i a t e d .

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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Seminolipid

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

With a l l n a t u r a l l y o c c u r r i n g l i p i d s a spectrum o f l o n g c h a i n a c y l or a l k y l groups ( o f t e n u n s a t u r a t e d ) e x i s t s in a p a r t i c u l a r s p e c i e s b u t i t has been shown ( 2 £ , 2 6 ) t h a t i n s e m i n o l i p i d , from a d u l t t e s t e s , t h e a c y l group i s p r e d o m i n a n t l y h e x a d e c a n o y l and t h e a l k y l g r o u p i s p r e d o m i n a n t l y h e x a d e c y l as shown i n f o r m u l a (LX) . Thus t h e a l m o s t c o m p l e t e a b s e n c e o f u n s a t u r a t e d a l k y l c h a i n s from t h i s l i p i d i n d i c a t e d t h a t a r o u t e u s i n g b e n z y l e t h e r s (which a r e removed by h y d r o g e n o l y s i s ) would be u s e f u l f o r p r e p a r i n g m a t e r i a l i d e n t i c a l i n a l l r e s p e c t s w i t h the natural m a t e r i a l . The r o u t e u s e d by F l o w e r s (kk) f o r the synthesis of the d î h y d r o s u l p h a t i d e (XVI) w h i c h i n v o l v e d , i n t h e f i n a l s t a g e , an a l k a l i n e h y d r o l y s i s t o remove t h e a c e t y l p r o t e c t i n g g r o u p s i s a l s o l e s s u s e f u l " i n t h e c a s e o f an e s t e r i f i e d g l y c e r o l d e r i v a t i v e s i n c e t h e l o n g - c h a i n a c y l g r o u p would a l s o be removed a t this stage. T h e r e a r e , however, examples in t h e l i t e r a t u r e ( 8 6 - 8 9 ) where a c e t y l g r o u p s have been c l e a v e d p r e f e r e n t i a l l y f r o m a c e t y l a t e d g l y c o s y l d i g l y c e r i d e s by h y d r a z i n o l y s i s . Howe v e r , t h e p o s s i b i l i t y o f a c e t y l m i g r a t i o n s on t h e g a l a c t o s e r e s i d u e d u r i n g m a n i p u l a t i o n s o f t h e i n t e r m e d i a t e s and t h e p o s s i b i l i t y o f h y d r a z i n o l y s i s (^Q.) o f t h e s u l p h a t e g r o u p s u g g e s t e d t h a t an a p p r o a c h u s i n g t h e s t a b l e a l l y l and b e n z y l e t h e r s w o u l d be more d e f i n i t i v e i n t h i s c a s e . The .ideal i n t e r m e d i a t e f o r t h i s a p p r o a c h a p p e a r e d t o be the b e n z y l a t e d d e s u 1 p h o - s e m i n o l î ρ i d d e r i v a t i v e ( L V l l l ) which on s u l p h a t i o n and h y d r o g e n o l y s i s w o u l d g i v e s e m i n o l i p i d (l_X) . The r o u t e shown, s t a r t i n g f r o m t h e g l y c e r o l d e r i v a t i v e (XLVl11), was d e v e l o p e d (56) f o r t h e s y n t h e s i s o f ( L V l l l ) u s i n g t h e 'temporary p r o t e c t i n g p r o p e r t i e s o f a l l y l , p r o p - l - e n y l and but-2-enyl groups. T h e s t a r t i n g a l c o h o l ( L X V 1 1 1 S X L V l 1 1 ) ) c o u l d be p r e p a r e d r e a d i l y f r o m c o m m e r c i a l l y a v a i l a b l e c h i m y l a l c o h o l (LX1X) by t r i t y l a t i o n , c r o t y l a t i o n and s u b s e q u e n t a c i d i c h y d r o l y s i s . The commercial chimyl a l c o h o l i s f a i r l y crude ( c o n t a i n i n g o n l y ca 80% o f h e x a d e c y l c h a i n s ) and t h e r e f o r e a p u r e l y s y n t h e t i c r o u t e was d e v e l o p e d ( £ 6 ) t o compound ( L X V l l l ) , u s i n g a g a i n t h e ' t e m ­ p o r a r y ' p r o t e c t i n g p r o p e r t i e s o f t h e a l l y l e t h e r s , so that a p u r e s t a r t i n g m a t e r i a l was a v a i l a b l e . 1

1 , 2 - £ - 1 s o p r o p y l i d e n e - L - g l y c e r o l ( L X l ) , which is the s t a n ­ dard s t a r t i n g material f o r s y n t h e t i c g l y c e r o l i p i d chemistry, and i s r e a d i l y p r e p a r e d f r o m 1,2:5,6-di-0-isopropylidene-J)m a n n i t o l (21) , was c o n v e r t e d i n t o t h e a l l y l e t h e r and t h i s was h y d r o l y s e d with a c i d t o g i v e 3-0-al 1 yl-L_-gl y c e r o l ( L X l l ) (92) . Compound ( L X l l ) was t r i t y l a t e d and t h e p r o d u c t was c o n v e r t e d i n t o t h e b u t - 2 - e n y l e t h e r ( L X l 1 1 ) w h i c h was h y d r o l y s e d w i t h a c i d t o g i v e 3~0-al1 y 1 - 2 - 0 - ( b u t - 2 - e n y l ) - L - g 1 y c e r o l (LX1V). Compound (LX1V) was a l i q u i d w i t h p r o p e r t i e s s i m i l a r ( b u t w i t h o p p o s i t e o p t i c a l r o t a t i o n ) t o t h o s e o f t h e 1 , 2 - d i -0_- ( b u t - 2 -

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e n y l )-L_-gl y c e r o l w h i c h we h a v e d e s c r i b e d p r e v i o u s l y (22.) . A l k y l a t i o n o f compound (LX1V) w i t h p u r e h e x a d e c y l b r o m i d e g a v e t h e h e x a d e c y l e t h e r (LXV) and t h i s was t r e a t e d w i t h p o t a s s i u m t b u t o x i d e i n d i m e t h y l s u l p h o x i d e w h i c h c l e a v e d (24) but-2e n y l g r o u p and i s o m e r i s e d (25) the a l l y l group, to a p r o p - l enyl group, to g i v e the a l c o h o l ( L X V l ) . Compound ( L X V l ) was c o n v e r t e d i n t o t h e b u t - 2 - e n y l e t h e r ( L X V l 1 ) w h i c h was t r e a t e d w i t h d i l u t e a c i d t o remove t h e p r o p - l - e n y l g r o u p g i v i n g t h e required alcohol (LXVl11). T h e a l c o h o l ( L X V l 1 1 ) was c h a r a c t e r i s e d by removal o f t h e b u t - 2 - e n y l g r o u p by p o t a s s i u m t b u t o x i d e i n d i m e t h y l s u l p h o x i d e t o g i v e p u r e 1 - O - h e x a d e c y l -L_g l y c e r o l (LX1X) whose b i s - p - p i t r o b e n z o a t e had t h e e x p e c t e d p r o p e r t i e s [ m . p . 6 0 - 6 2 ° , [a] £ - 3 3 . 3 ° (C 1 i n C H C l J ] .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

t

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The a l c o h o l ( X L V l 1 1 ) was c o n d e n s e d w i t h acetooromogalactose u s i n g t h e H e l f e r i c h m o d i f i c a t i o n (26) of the Koenîgs-Knorr react i o n , t o g i v e t h e β - g a l a c t o s i d e (XL1X) . T h i s r e a c t i o n uses m e r c u r y (11) c y a n i d e as a c a t a l y s t and we h a v e shown (2Z) that m e r c u r y (11) c h l o r i d e r e a c t s s l o w l y w i t h t h e d o u b l e bond o f allyl ethers. We were t h e r e f o r e c o n c e r n e d p r e v i o u s l y (22) that t h i s a d d i t i o n might a l s o o c c u r w i t h the mercury s a l t s in t h e Helferich condensation. H o w e v e r , G a r e g g and N o r b e r g (Jk) had u s e d t h e H e l f e r i c h method i n a g l y c o s i d a t i o n r e a c t i o n w i t h a l l y l 2 , 4 - d i - 0 - b e n z y l - α - L - f u c o p y r a n o s i d e a t 40 d u r i n g twenty h o u r s and had i s o l a t e d a d i s a c c h a r i d e i n 7 0 % y i e l d and S i n a y and J a c q u i n e t (28) had a l s o c a r r i e d o u t a H e l f e r i c h c o n d e n s a t i o n i n t h e p r e s e n c e o f a l l y l e t h e r s , w i t h o u t p r o b l e m s and we w e r e t h e r e f o r e encouraged to t r y the r e a c t i o n in the presence of a but-2-enyl group. We h a v e shown (5£) t h a t the b u t - 2 - e n y l group r e a c t s a t a p p r o x i m a t e l y t h e same r a t e as t h e a l l y l g r o u p w i t h m e r c u r y (11) c h l o r i d e i n a q u e o u s - a c e t o n e whereas t h e 2 - m e t h y l a l l y l g r o u p r e a c t s a t a v e r y much h i g h e r r a t e . The c r u d e a c e t y l a t e d β - g a l a c t o s î d e (XL1X) was t r e a t e d w i t h b a s e w h i c h removed t h e a c e t y l g r o u p s t o g i v e t h e crude a l c o h o l (L). T h i s was t r e a t e d d i r e c t l y w i t h a c i d i c a c e t o n e t o g i v e t h e crude î s o p r o p y l i d e p e d e r i v a t i v e ( L l ) . The p u r e p r o d u c t (Ll) [ m . p . 6 3 - 6 5 ° , [alt + 1 2 . 2 ° (C 1.9 i n CHC1 )] crystallised r e a d i l y f r o m l i g h t p e t r o l e u m t h u s making t n i s a c o n v e n i e n t s t a g e f o r p u r i f i c a t i o n f r o m any b y - p r o d u c t s f o r m e d d u r i n g t h e g l y c o s i d a t i o n r e a c t i o n (26) and f r o m any 4,6-0-isopropylidene d e r i v a t i v e w h i c h can be f o r m e d i n t h e a c e t a l i s a t i o n r e a c t i o n (5Z>22)· Compound ( L l ) was r e a d i l y c o n v e r t e d i n t o t h e d i o l ( L l l ) by b e n z y l a t i o n and s u b s e q u e n t a c i d i c h y d r o l y s i s . The d i o l ( L l l ) was c l e a v e d by p e r i o d a t e t h u s i n d i c a t i n g t h a t c o m pound ( L l ) was i n d e e d a 3 , 4 - 0 - î s o p r o p y l î d e n e d e r i v a t i v e . The d i o l ( L l l ) was c o n v e r t e d i n t o t h e 0.,0-di b u t y l s t a n n y l i dene d e r i v a t i v e ( L l l l ) w h i c h was t r e a t e d w i t h a l l y l b r o m i d e in d i m e t h y l f o r m a m i d e a t 100 d u r i n g 5 hours to g i v e the 3 - Q - a l l y l derivative (LlV). T h i n l a y e r c h r o m a t o g r a p h y showed t h e p r e s e n c e o f a n o t h e r p r o d u c t ( c a 5% o f t h e m a j o r p r o d u c t ) , w h i c h i s p r o b a b l y t h e 4 - 0 - a l l y l d e r i v a t i v e , b u t t h i s was s e p a r a t e d f r o m

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

t h e m a j o r p r o d u c t by c h r o m a t o g r a p h y on a l u m i n a . Nashed and A n d e r s o n (53) r e p o r t e d t h a t a l l y l b r o m i d e r e a c t e d s l u g g i s h l y w i t h an 0.,,0-di b u t y l - s t a n n y l î d e n e d e r i v a t i v e and t h e r e f o r e u s e d a l l y l iodide: we p r e f e r t h e g r e a t e r s t a b i l i t y o f t h e a l l y l b r o m i d e a l t h o u g h t h e r e a c t i o n may t a k e l o n g e r . T h e p u r i f i e d a l l y l e t h e r (L1V) was b e n z y l a t e d t o g i v e t h e f u l l y p r o t e c t e d d e r i v a t i v e (LV) w h i c h was t r e a t e d w i t h p o t a s s ium t . - b u t o x i d e i n d i m e t h y l s u l p h o x i d e t o c l e a v e t h e b u t - 2 - e n y l g r o u p (94) and i s o m e r i s e t h e a l l y l g r o u p (95) g i v i n g t h e p r o p l-enyl ether (LVl). H y d r o l y s i s of a p o r t i o n of the product (LVl) w i t h d i l u t e a c i d gave c r y s t a l l i n e 2 , 4 , 6 - t r i - 0 - b e n z y l J)-gal a c t o p y r a n o s e , i d e n t i c a l w i t h t h e m a t e r i a l p r e p a r e d p r e v i o u s l y ( 9 4 ) « thus p r o v i n g the s u b s t i t u t i o n p a t t e r n of the galactose moiety. A c y l a t i o n of the a l c o h o l (LVl) with hexadecanoyl c h l o r i d e i n p y r i d i n e g a v e t h e p a l m i t o y l e s t e r ( L V l 1 ) w h i c h on t r e a t m e n t w i t h m e r c u r y (11) c h l o r i d e (97) g a v e t h e a l c o h o l (LVl11). S u l p h a t i o n o f compound ( L V l 1 1 ) w i t h t h e p y r î d i n e - s u l p h u r t r i o x i d e c o m p l e x (90 J 00) g a v e t h e t r î - 0 - b e n z y l s e m i n o l i p i d d e r i v a t i v e (L1X) . T h i s was h y d r o g e n o l y s e d i n t h e p r e s e n c e o f p a l l a dium on c h a r c o a l i n a c e t i c a c i d t o g i v e s e m i n o l i p i d (LX) w h i c h c o c h r o m a t o g r a p h e d w i t h an a u t h e n t i c s a m p l e k i n d l y p r o v i d e d by P r o f e s s o r Yamakawa. It has been shown ( l 0 1 ) t h a t , i n t h e d i r e c t s u l p h a t i o n o f J ) - g a l a c t o s e , p r e f e r e n t i a l s u l p h a t i o n o f t h e 6- and 3- p o s i t i o n s occurs. T h i s w o u l d i n d i c a t e a more r a p i d r o u t e t o s e m i n o l i p i d f r o m t h e i n t e r m e d i a t e (LXX) w h i c h i s r e a d i l y a v a i l a b l e f r o m t h e key i n t e r m e d i a t e ( L l ) . C l e a v a g e o f the b u t - 2 - e n y l group from (LXX) s h o u l d g i v e t h e a l c o h o l ( L X X l ) w h i c h on a c y l a t i o n w o u l d g i v e t h e e s t e r (LXX11). A c i d i c h y d r o l y s i s of the i s o p r o p y l i dene g r o u p f r o m (LXX11) s h o u l d g i v e t h e d i o l (LXXl11) and t h i s s h o u l d be s u l p h a t e d p r e f e r e n t i a l l y on t h e 3 h y d r o x y - g r o u p t o g i v e the s u l p h a t e (LXX1V). Alternatively sulphation [like a c y l a t i o n (£2)] o f t h e 0 , 0 - d i b u t y l s t a n n y l i d e n e d e r i v a t i v e o f t h e d i o l ( L X X l 1 1 ) s h o u l d a g a i n g i v e (LXX1V) p r e f e r e n t i a l l y . H y d r o g e n o l y s i s o f (LXX1V) w o u l d t h e n g i v e s e m i n o l i p i d (LXXV) . Any o f t h e i s o m e r o f s e m i n o l i p i d , w i t h t h e s u l p h a t e g r o u p on t h e 4 - p o s i t i o n , t h a t was f o r m e d i n t h e s e r e a c t i o n s c o u l d be p r e f e r e n t i a l l y d e s t r o y e d by t h e a c t i o n o f p e r i o d a t e t o g i v e a pure sample of s e m i n o l i p i d . These a l t e r n a t i v e routes are b e i n g i n v e s t i g a t e d s i n c e t h e y w o u l d a l s o be o f v a l u e i n t h e s y n t h e s i s of o t h e r n a t u r a l l y o c c u r r i n g s u l p h o g l y c o l î ρ î d s which contain galactose 3-sulphate residues. The 1 - 0 - h e x a d e c y 1 - 2 - 0 ( b u t - 2 - e n y l ) - L - g l y c e r o l ( L X V l 1 1 ) s h o u l d a l s o s e r v e as an i n t e r m e d i a t e f o r the s y n t h e s i s of the g l u c o s e c o n t a i n i n g s u l p h o g l y c o l i p i d o f human g a s t r i c s e c r e t i o n ( 3 8 ) .

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

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the Synthesis

Su]phonoliρid

In s t u d i e s aimed (48) a t t h e s y n t h e s i s o f t h e i m m u n o l o g i ­ c a l l y i n t e r e s t i n g g l y c o l i p i d s o f S t r e p t o c o c c i (102) and t h e glucuronosyl diglyceride o f Pseudomonas d i m j n u t a ( 103) we p r e p a r e d (21) t h e c r y s t a l l i n e 3 - 0 - ( 3 , 4 - d i -0_-benzyl - α - D - g l u c o p y ranosyl)-l,2-0-isopropylidene-L^-g1ycerol (LXX1X) . T h i s was o b ­ t a i n e d by c o n v e r t i n g t h e b i s - p - n i t r o b e n z o a t e ( L X X V l ) (104) i n t o t h e c h l o r i d e (LXXVl1) w h i c h was c o n d e n s e d w i t h 1 , 2 - d i - 0 - ( b u t - 2 e n y l ) - L - g l y c e r o l , u n d e r c o n d i t i o n s shown p r e v i o u s l y (105) t o give predominantly 1 , 2 - c i s - g l y c o s i d i c l i n k a g e s , t o form t h e crude glycoside (LXXVl11). Compound (LXXVl11) was c o n v e r t e d i n t o t h e c r y s t a l l i n e g l y c o s i d e (LXX1X) and t h i s was s u b s e q u e n t l y (51) c o n v e r t e d i n t o 3 - 0 - ( α - D - g l u c o p y r a n o s y l ) - 1 , 2 - d i - 0 - o c t a d e c a n o y l - L - g l y c e r o l , a 'glucosyl d i g l y c e r i d e . A r o u t e from compound (LXX1X) t o t h e p r o t e c t e d g l v c o s y l d i g l y c e r i d e (LXXX) was a l s o p l a n n e d s i n c e t h e l a t t e r compound s h o u l d s e r v e as an i n t e r m e d ­ iate f o r the synthesis of 3 - 0 - ( α - D - g 1 u c u r o n o p y r a n o s y l ) - 1 , 2 - d i - 0 a c y l - L - g l y c e r o l (a g l u c u r o n o s y l d i g l y c e r i d e ' ) and o f a g l u c o s y l d i g l y c e r i d e p h o s p h o r y 1 a t e d on t h e 6 - p o s i t i o n o f g l u c o s e such as o c c u r s i n S t r e p t o c o c c i (102) . M o r e o v e r compound (LXXX) s h o u l d a l s o s e r v e as an i n t e r m e d i a t e f o r t h e s y n t h e s i s o f t h e p l a n t s u l p h o n o l i p i d s i n c e c o n v e r s i o n i n t o t h e s u l p h o n a t e e s t e r (LXXX1X) and r e p l a c e m e n t w i t h s u l p h i d e s h o u l d g i v e t h e c o r r e s p o n d i n g 6t h i o d e r i v a t i v e (XC) . O x i d a t i o n o f t h e t h i o l and h y d r o g e n o l y s i s o f t h e b e n z y l g r o u p s s h o u l d t h e n g i v e t h e p l a n t su 1 p h o n o l î ρ i d (XC1). 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

of the Plant

1

1

1

We s u b s e q u e n t l y f o u n d (5Z) t h a t t r e a t m e n t o f d e r i v a t i v e s o f 1 , 6 - a n h y d r o - s u g a r s w i t h a c e t y l c h l o r i d e - h y d r o g e n c h l o r i d e gave t h e c o r r e s p o n d i n g 6-0_-acetyl g l y c o s y l c h l o r i d e s (a r e a c t i o n w h i c h we have termed c h 1 o r a c e t o l y s i s ) and t h i s i n d i c a t e d a more c o n v e n i e n t r o u t e t o t h e d e r i v a t i v e (LXXX) (51). 1,6-Anhydro-2, 3 , 4 - t r i - 0 - b e n z y l - β - D - g l u c o p y r a n o s i d e ( L X X X V l l l ) was t r e a t e d w i t h acetyl chloride-hydrogen c h l o r i d e to give the crude 6 - 0 - a c e t y l 2,3,4-tri-0-benzyl-J)-glucopyranosyl c h l o r i d e ( L X X X V l l ) and t h i s was c o n d e n s e d w i t h 1 , 2 - d i - 0 - ( b u t - 2 - e n y l ) - L - g 1 y c e r o l to g i v e the crude α - g l y c o s i d e (LXXXVl). Compound ( L X X X V l ) was t r e a t e d w i t h p o t a s s i u m t - b u t o x i d e i n d i m e t h y l s u l p h o x i d e w h i c h removed t h e a c e t y l and b u t - 2 - e n y l g r o u p s t o g i v e t h e c r y s t a l l i n e t r i o l (LXXXV) . T h e s t r u c t u r e o f compound (LXXXV) was c o n f i r m e d by a f u r t h e r s y n t h e s i s f r o m compound (LXX1X) whose s t r u c t u r e had been e s t a b l i s h e d p r e v i o u s l y ( 9 3 ) . 1

1

The c r y s t a l l i n e t r i o l ( L X X X V ) was c o n v e r t e d v i a t h e i s o p r o p y 1 i d e n e d e r i v a t i v e (LXXX1V) i n t o t h e a l l y l e t h e r (LXXXlll). A c i d i c h y d r o l y s i s o f t h e i s o p r o p y l i d e n e g r o u p and s u b s e q u e n t i s o m e r i s a t ion o f t h e a l l y l group gave t h e p r o p - l - e n y l e t h e r (LXXX11) w h i c h was a c y l a t e d t o g i v e t h e b i s - o c t a d e c a n o y 1 ester (LXXXl). H y d r o l y s i s o f t h e p r o p - l - e n y l group w i t h mercury ( l l ) c h l o r i d e gave t h e p r o t e c t e d g l u c o s y l d i g l y c e r i d e (LXXX). Experi­ ments on t h e c o n v e r s i o n o f compound ( L X X X ) . i n t o t h e p l a n t s u l -

4.

GiGG

Sulfur-Containing Glycolipids

61

l+O (ucxx)

ft-©μ

R=eSo,,Me

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

CUXXMX")

exci)

I

(XClll) R ' ; C ^ P k

;,C.W

( X c i v ) R Ï C U ^
«/εν·

.V* ,.1

ο

©βΛ

CrtjSo

I I

OH

v

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch004

62

CARBOHYDRATE SULFATES p h o n o l î p i d ( X C l ) , by t h e method d e s c r i b e d a b o v e , a r e i n p r o g r e s s . The c h l o r i d e (LXXXVl1) t o g e t h e r w i t h t h e a l k y l g l y c e r o l d e r i v a t i v e ( L X V l 1 1 ) s h o u l d s e r v e as i n t e r m e d i a t e s f o r t h e s y n t h e s i s o f t h e s u l p h o g l y c o l i p i d o f human g a s t r i c s e c r e t i o n ( 3 8 ) . The a v a i l a b i l i t y o f t h i s r o u t e a l s o s u g g e s t e d a more d i r e c t approach t o the plant s u l p h o n o l i p i d v i a the intermediate (LXXX1V). C o n v e r s i o n o f t h e a l c o h o l (LXXX1V) i n t o a s u l p h o n a t e e s t e r and r e p l a c e m e n t o f t h i s w i t h p o t a s s i u m t h i o l a c e t a t e i n dimethylformamide should give a t h i o a c e t a t e d e r i v a t i v e . Alkal i n e h y d r o l y s i s o f t h e t h i o a c e t a t e and o x i d a t i o n o f t h e t h i o l produced w i t h i o d i n e should g i v e t h e o x i d i s e d t h i o l (XCl1) [ c f . r e f e r e n c e (106)1. A c i d i c h y d r o l y s i s o f compound ( X C l 1 ) t o t h e a l c o h o l ( X C l 1 1 ) and a c y l a t i o n s h o u l d g i v e t h e e s t e r (XC1V) . O x i d a t i o n o f t h e e s t e r (XC1V) t o t h e s u l p h o n i c a c i d (XCV) and subsequent h y d r o g e n o l y s i s o f t h e benzyl groups should g i v e t h e plant sulphonolipid (XCVl). The n a t u r a l l y o c c u r r i n g p l a n t su 1 p h o n o l i ρ i d c o n t a i n s u n s a t ­ u r a t e d a c y l g r o u p s (42) b u t t h e r o u t e s p r o p o s e d a b o v e w i l l g i v e o n l y m a t e r i a l c o n t a i n i n g s a t u r a t e d a c y l groups s i n c e t h e benzyl g r o u p s a r e removed by h y d r o g e n o l y s i s . However, s i n c e s u l p h o n i c a c i d s f o r m m e t h y l e s t e r s w h i c h c a n be c l e a v e d by sodium i o d i d e i t s h o u l d be p o s s i b l e t o c o n v e r t a s y n t h e t i c d i a c e t y l d e r i v a ­ t i v e (XCVl1) v i a t h e m e t h y l e s t e r p r o t e c t e d w i t h t e t r a h y d r o p y r a n y l g r o u p s (XCVl11) i n t o a d e r i v a t i v e (XC1X) c o n t a i n i n g unsaturated acyl groups. M i l d a c i d h y d r o l y s i s s h o u l d then g i v e t h e s u l p h o n a t e e s t e r (C) w h i c h w i t h s o d i u m i o d i d e s h o u l d give the unsaturated s u l p h o l i p i d ( C l ) .

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RECEIVED

February 6, 1978.

5 Spectrofluorimetric Methods for Estimating and Studying the Interactions of Polysaccharides in Biological Systems R. B. CUNDALL and D. MURRAY Department of Chemistry and Applied Chemistry, University of Salford, England

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

G. O. PHILLIPS School of Natural Sciences, North Wales Institute, Clwyd, Wales, CH5 4BR, U.K.

The analysis of polyanions using metachromasia which accom­ panies dye binding has been successfully applied to plant sulphated polysaccharides using absorption and fluorescence emission spectroscopy. The l a t t e r i s more useful because of its greater s e n s i t i v i t y at low polyanion concentrations, and because i n fluorescence the spectral properties of cationic dyes such as acridine orange simplify into two well separated peaks corres­ ponding to free dye (λmax 525 nm) and metachromatically bound dye (λmax 640 nm) (Fig. 1). By measuring the monomeric dye fluores­ cence at 540 nm (when excited at 400 nm) at successively increas­ ing concentrations of a polyanion (carrageenan), a t i t r a t i o n profile (Fig. 2) i s obtained. The intercept with the polymer concentration axis gives the overall stoichiometry of the inter­ action with one dye molecule binding to each anionic s i t e . Successive experiments with known polyanion concentrations can relate the intercept with the reciprocal of the anionic sites concentration (Fig. 3) and so allowing the rapid determination of the anionic s i t e concentrations of unknown solutions. Additives such as simple electrolytes, proteins and l i p i d s etc. interfere with the dye binding properties of polyanions. Added salt 3, 4, 5 can release dye from the complex. We have found that polymeric cations remove dye quantitativelyÊ- and more e f f i c i e n t l y than s a l t s , forming polyanion-polycation complexes which are stoichiometric i n basic to acidic s i t e s , and which are not influenced by added s a l t s . This observation i s the basis of the procedures we describe here to study interactions of polyanions with a range of synthetic and biological polymers. From our experience of such interactions, procedures have been devised for the quantitative estimation of polyanions, particularly sulphated polysaccharides in biological systems and so eliminating the interfering effects of associated materials such as s a l t s , proteins and l i p i d s . 1

0-8412-0426-8/78/47-077-067$07.00/0 © 1978 American Chemical Society

2

68

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CARBOHYDRATE SULFATES

Wavelength nm. Figure 1. (Top) Absorption spectral shifts of acridine orange with aggregation of the dye on a polyanion, κ-carrageenan. (Bottom) Fluorescence shifts of acridine orange with aggrega­ tion (X = 400 nm). ex

CUNDALL ET AL.

Spectrofluorimetric

Methods

%

ree

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Dye

r a t i o of polyanion s i t e s added (Ρ) t o c a t i o n i c dye m o l e c u l a r (D).

Figure 2.

Titration of carrageenan vs. 10~ M acridine orange in salt-free solution 5

ο

Figure 3.

Calibration curve relating microlitre endpoints and poly­ anion concentrations

CARBOHYDRATE SULFATES

70

Analysis

of polysulphates

i n the presence of

polycarboxylates.

A s t a b i l i s e r m i x t u r e e x t e n s i v e l y used i n d a i r y p r o d u c t s consists o f one o r more o f v a r i o u s c a r r a g e e n a n s , c a r b o x y m e t h y l c e l l u l o s e s , p e c t i n s , l o c u s t b e a n , g u a r and o t h e r gums, n e u t r a l p o l y s a c c h a r i d e s and e m u l s i f i e r s . The p a r t i c u l a r m a t e r i a l s and r e l a t i v e p r o p o r t i o n s a r e chosen a c c o r d i n g t o t h e s p e c i f i c p r o p e r t i e s d e s i r e d f o r t h e p a r t i c u l a r product. A n a l y s i s o f s u c h complex m i x t u r e s i s d i f f i c u l t and t i m e c o n s u m i n g , b u t can be a c h i e v e d i f s u i t a b l e f r a c t i o n a t i o n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

°

A A _ A Ο

procedures are u t i l i s e d — — . Here we d e s c r i b e a more r a p i d procedure f o r such m i x t u r e s . The e f f e c t o f pH on t h e s t o i c h i o m e t r y o f p o l y a n i o n - a c r i d i n e o r a n g e complexes i s shown i n ( F i g . 5) a f t e r c o r r e c t i o n f o r v a r i a t i o n s o f d y e - f l u o r e s c e n c e w i t h pH. For polycarboxylates dye r e l e a s e i s e v i d e n t n e a r t h e c a r b o x y l pKa (~ pH 4 ) . When o n l y c a r b o x y 1 g r o u p s a r e p r e s e n t (CNC and HA] t h e dye r e l e a s e i s c o m p l e t e b e l o w pH 2 . 0 , b u t t h e p o l y s u l p h a t e s (CAR and PSS) show c o n s i d e r a b l y l e s s dye r e l e a s e a t t h i s pH. Hence we can q u a n t i t a ­ t i v e l y d i s t i n g u i s h between them. By t i t r a t i n g s t a b i l i s e r m i x t u r e s a t pH v a l u e s a c i d and a l k a l i n e t o t h e c a r b o x y l pK^ i t i s p o s s i b l e t o d e t e r m i n e s e p a r a t e ­ l y t h e s u l p h a t e g r o u p s and t o t a l i o n i c s i t e c o n c e n t r a t i o n , hence g i v i n g a l s o a measurement o f c a r b o x y l s i t e s c o n c e n t r a t i o n s . For a p r e p a r e d 1 : 1 m i x t u r e o f CAR and CNC, b a s e d on a n i o n i c s i t e s , e s t i m a t e d s i t e c o n c e n t r a t i o n s were w i t h i n 2% o f t h e p r e d i c t e d values. Complex i c e - c r e a m s t a b i l i s e r m i x t u r e s c o u l d be s i m i l a r l y analysed s a t i s f a c t o r i l y (Table 2). The p r e s e n c e o f e m u l s i f i e r s and n e u t r a l p o l y s a c c h a r i d e s had no e f f e c t on t h e a n a l y s i s o f commercial carrageenan ( S t a b i l i s e r A ) . F i g . 6 shows t h a t t h e dye b i n d i n g o f S t a b i l i s e r Β and i t s component c a r r a g e e n a n v a r i e s w i t h pH, i n d i c a t i n g t h e h i g h p e r c e n t a g e o f CMC p r e s e n t . Carrageenans used c o m m e r c i a l l y p r e s e n t a p a r t i c u l a r d i f f i c u l t y i n a n a l y s i s b e c a u s e t h e y may c o n t a i n d i f f e r i n g amounts o f a p a r t i c u l a r i s o m e r . We p r e v i o u s l y showed t h a t t h e s e i s o m e r s d i f f e r s i g n i f i c a n t l y i n t h e amount o f dye bound p e r u n i t w e i g h t o f p o l y m e r . Therefore, u n l e s s s t a n d a r d s a r e a v a i l a b l e , o r t h e r a t i o s o f i s o m e r s known, a f r a c t i o n a t i o n . s t e p must be u n d e r t a k e n f o r c o m p l e t e a c c u r a c y . However, when κ - c a r r a g e e n a n o n l y i s p r e s e n t as i n S t a b i l i s e r B, i t s p r o p o r t i o n can be d e t e r m i n e d f r o m t h e r a t i o o f t h e t i t r a t i o n end p o i n t s i n a c i d . The r e s u l t s shown i n T a b l e 2 c o r r e s p o n d t o 13.3% κ - c a r r a g e e n a n i n S t a b i l i s e r B. The p e r c e n t a g e o f CNC i n t h e s a m p l e can be d e t e r m i n e d f r o m t h e d i f f e r e n c e between t h e a n i o n i c s i t e c o n c e n t r a t i o n s o f n e u t r a l and a c i d pH. Although some v a r i a t i o n i n dye b i n d i n g p r o p e r t i e s o c c u r s w i t h c h a n g i n g d e g r e e o f s u b s t i t u t i o n (DS) t h e e f f e c t i s r e l a t i v e l y s m a l l . Of c o u r s e , t h e DS o f t h e CNC must be known t o c o n v e r t t h e d e t e r ­ mined s i t e c o n c e n t r a t i o n i n t o w e i g h t o f p o l y m e r . Conversely the DS o f any CNC, o r o t h e r p o l y m e r o f v a r i a b l e DS, can be d e t e r m i n e d by t i t r a t i o n u s i n g a known w e i g h t o f s a m p l e .

Figure 4a.

Effect of simple electrolytes on polyanion-AO

binding; 1 NaCl

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

h-

1

Co

a.

ο

Η*

g-

Ι"

•§> ο -*

Ο

<^

"β

>

Μ Η

Ρ

>

α

η

pi

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

72 CARBOHYDRATE SULFATES

%

DYE BINDING

Spectrofluorimetric

Methods

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL ET AL.

Figure 5. Variation of polyanion—AO binding with pH. luronic acid; (Φ) chondroitin sulfate; (U) CMC; (V) nan; (O) heparin; (Y) PSS.

Hya­ κ-carragee-

74

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

100

ol

.

1

,

2

,

.

.

3

U

5

L.J

6

PH Figure 6.

pH Dependence of dyehinding to two commercial icecream stabilizer mixes

5.

CUNDALL ET AL.

Materials

Spectrofluorimetric

Methods

75

and Methods

A c r i d i n e Orange was d y e - l a s e r g r a d e p u r c h a s e d f r o m K o d a k E a s t m a n , and was f u r t h e r r e p e a t e d l y r e c r y s t a l l i s e d f r o m e t h a n o l . A s t o c k dye s o l u t i o n o f 10 M was s t a b l e f o r up t o 1 y e a r i n a b l a c k e n e d f l a s k , and dye was w i t h d r a w n when r e q u i r e d u s i n g a microlitre s y r i n g e and s u b s e q u e n t l y d i l u t e d t o t h e r e q u i r e d concentration. U n l e s s s t a t e d , a l l dye s o l u t i o n s used were 10" M. H y a l u r o n i c a c i d (HA) and c h o n d r o i t i n s u l p h a t e (CS) were p u r c h a s e d f r o m Sigma B i o c h e m i c a l s ( L o n d o n ) . Kappa-carrageenan (CAR) was p r o v i d e d by t h e Copenhagen P e c t i n Co. and s o d i u m h e p a r i n (HEP) by A b b o t t L a b s , N o r t h C h i c a g o , w i t h a n t i c o a g u l a n t a c t i v i t y o f 150 u n i t s p e r ' mg. C a r b o x y m e t h y 1 c e l l u l o s e 7 MP (CMC) was a medium v i s c o s i t y g r a d e o f d e g r e e o f s u b s t i t u t i o n (DS) 9 . 7 t o 0 . 8 f r o m H e r c u l e s I n c . and s o d i u m p o l y ( s t y r e n e s u l p h o n a t e ) (PSS) was t h e i s o t a c t i c c o n f o r m e r f r o m Dow C h e m i c a l Co. I n c . Poly(p-xylyl v i o l o g e n ) — was p r e p a r e d by D r . G. A s h w e l l a t N o t t i n g h a m U n i v e r s i t y , E n g l a n d , and was p o l y c a t i o n i c o v e r t h e e n t i r e pH r a n g e . Poly-Ll y s i n e h y d r o b r o m i d e was o f m o l e c u l a r w e i g h t 1 5 , 0 0 0 - 3 0 , 0 0 0 , and was p u r c h a s e d f r o m Sigma ( L o n d o n ) . Samples o f c h a r a c t e r i s e d Kappa c a s e i n s were p r e p a r e d by D r . R. G. M o r l e y — . Calf-skin, a c i d s o l u b l e c o l l a g e n was o b t a i n e d f r o m Sigma ( L o n d o n ) . The i c e - c r e a m s t a b i l i s e r m i x t u r e ( S t a b i l i s e r B) and c o m m e r c i a l c a r r a g e e n a n ( S t a b i l i s e r A) were p r o v i d e d by D a r i Tech Corporation, Atlanta. F l u o r e s c e n c e i n t e n s i t i e s w e r e r e c o r d e d on a f u l l y compensated s p e c t r o f l u o r i m e t e r - . I n t h e dye b i n d i n g p r o f i l e s o f s t a n d a r d s o l u t i o n s , t h e χ - a x i s i s r e p r e s e n t e d by P/D, t h e r a t i o o f p o l y m e r s i t e s t o t h e t o t a l c o n c e n t r a t i o n o f dye molecules.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

5

Results

and D i s c u s s i o n

B i n d i n g Strengths- o f P o l y a n i o n s . F o r a P/D = 1.0 complex o f p o l y a n i o n and 10~^M dye ( F i g . 4) shows t h e r e l a t i o n s h i p o f f r e e dye w i t h l o g . o f s a l t c o n c e n t r a t i o n f o r a s e r i e s o f p o l y a n i o n s and two e l e c t r o l y t e s ( N a C l and C a C l ) . I n t e n s i t i e s were c o r r e c t e d f o r s a l t - i n d u c e d a g g r e g a t i o n o f t h e f r e e - d y e , and f o r q u e n c h i n g o f t h e dye f l u o r e s c e n c e by t h e c h l o r i d e i o n . The r e s u l t s a r e i n agreement w i t h t h o g e o b t a i n e d by p u l s e r a d i o l y s i s f o r h e p a r i n and m e t h y l e n e b l u e — . Most o f t h e dye r e l e a s e o c c u r s w i t h i n a decade o f l o g . s a l t c o n c e n t r a t i o n . The c r i t i c a l e l e c t r o l y t e c o n c e n t r a t i o n s — ' — can be f o u n d by e x t r a ­ p o l a t i o n t o c o m p l e t e dye r e l e a s e , and v a l u e s a r e g i v e n i n T a b l e 1. The o r d e r o f i n c r e a s i n g dye b i n d i n g s t r e n g t h i s : 2

HA
76

CARBOHYDRATE SULFATES

Table 1. Critical Electrolyte Concentrations for Polyanion—Acridine Orange Complexes from Fluorescent Intensity Data

Critical

Electrolyte

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Concentration

( M ).

POLYANION NaCl H yalu roni c Acid

CMC

C hond roit in

3-9*

Ca C l

10

3 7 * 10~

7 *1u~

2

- 3

2

2

8·5χ10~

i*io~

5

3

6*10~

3

Sulphate

Heparin

Κ -

Carrageenan

NaPSS

0-26

-2 2-2* 10

1-2

0-53

1-6

10

CUNDALL ET AL.

Table 2.

Spectrofluorimetric

Methods

Titrations at Varying pHs of Solutions Containing Polymer-Bound COO and S0 Groups 4

A

Ice Cream

PH

End Point

A

3.07

69

A

2. 57

69

)

A

6.0

41.2

>

Stabiliser

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Stabilisers

Β

6.0

160

Β

2.57

520

Β

3.07

520

% Carrageenan

98%

) ) )

13.3%

Heparin

PH

Theoretical

Experimental

End Point

End Point (Average of 3)

7.0

40

39

3.0

56

57

CARBOHYDRATE SULFATES

78

E s t i m a t i o n o f C a r b o x y l t o S u l p h a t e g r o u p s p r e s e n t i n t h e same polyanion. C e r t a i n g l y c o s a m i n o g l y c a n s c o n t a i n b o t h c a r b o x y l and s u l p h a t e e s t e r groups i n a r e g u l a r r e p e a t i n g s t r u c t u r e along the p o l y s a c c h a r i d e backbone. The v a r i a t i o n w i t h pH o f t h e s t o i c h i o metry o f p o l y a n i o n - a c r i d i n e o r a n g e b i n d i n g i s shown i n F i g . 5 f o r two s u c h p o l y s a c c h a r i d e s , h e p a r i n and c h o n d r o i t i n s u l p h a t e . The b e h a v i o u r i s i n t e r m e d i a t e between t h a t o f t h e p o l y s u l p h a t e s and p o l y c a r b o x y l a t e s w i t h i n c o m p l e t e dye r e l e a s e near t h e c a r b o x y l pK^. R e c e n t w o r k e r s ^ ' IS have n o t e d che v a r i a b i l i t y o f h e p a r i n structure* To i n t e r p r e t o u r r e s u l t s we have a d o p t e d t h e s t r u c t ­ u r a l u n i t s p r o p o s e d by Kennedy — . The c a r b o x y l t o s u l p h a t e r a t i o i s a p p r o x i m a t e l y 2 t o 5. Heparin (10~ N i n t o t a l a n i o n i c s i t e s ] , was t i t r a t e d a g a i n s t 1 0 ~ Ι Ί dye a t pH 7.0 and pH 3.0. The r e s u l t s shown i n T a b l e 2B c o n f i r m t h e c a r b o x y l t o s u l p h a t e r a t i o 2 : 5 w i t h t o t a l s i t e s g i v i n g an o v e r a l l a c c u r a c y o f 2% i n i n d i v i d u a l measurements. 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

5

E s t i m a t i o n of carrageenan i n milk products. Bovine m i l k , c a r r a g e e n a n - f r e e baby f o o d and c h o c o l a t e m i l k were doped w i t h 1 mg p e r ml o f κ - c a r r a g e e n a n . We have a d o p t e d v a r i o u s p r o c e d u r e s t o e s t a b l i s h t h e s t a n d a r d t i t r a t i o n c u r v e f o r a 1 mg p e r ml c a r r a g ­ eenan s o l u t i o n f r o m each s a m p l e . F i g . 7 shows t h e d i r e c t t i t r a t i o n o f t h e s t a n d a r d c a r r a g e e n a n , doped baby f o o d and undoped baby f o o d , and i n d i c a t e s t h a t t h e c a r r a g e e n a n c a n n o t be a s s a y e d d i r e c t l y . U s i n g model s y s t e m s , t h e e f f e c t o f l i p i d s ( l a r d ) and p r o t e i n (BSA) on t h e t i t r a t i o n were i n v e s t i g a t e d , a n d , as shown i n F i g . 8, t h e p r e s e n c e o f m o d e r a t e c o n c e n t r a t i o n s o f each v i r t u a l l y d e s t r o y e d t h e q u a n t i t a t i v e n a t u r e o f t h e dye b i n d i n g . Using mixtures of BSA and M - c a r r a g e e n a n i t was f o u n d t h a t i n t e r f e r e n c e f r o m t h e p r o t e i n c o u l d be removed by d i g e s t i o n w i t h a m i x t u r e o f t r y p s i n , a- c h y m o t r y p s i n and C a r l s b e r g s u b t i l i s i n a t a l k a l i n e pH. Prior t o d i g e s t i o n i t was n e c e s s a r y t o remove a l l l i p i d m a t e r i a l f r o m the d a i r y p r o d u c t . T h i s c o u l d b e s t be a c h i e v e d by v i g o r o u s s h a k i n g w i t h c h l o r o f o r m and m e t h a n o l . The m i x t u r e a f t e r c e n t r i f u g a t i o n had t h r e e p h a s e s , a q u e o u s , o r g a n i c , and between them t h e denatured p r o t e i n l a y e r . A n a l y s i s o f the v a r i o u s l a y e r s confirmed t h a t a l l t h e p o l y a n i o n was s e d i m e n t e d a l o n g w i t h t h e d e n a t u r e d protein. A f t e r f r e e z e d r y i n g t h i s l a y e r and d i g e s t i o n w i t h t h e 3-enzyme s y s t e m , p r o d u c t s were w a t e r - s o l u b l e . F i g . 9 shows t h e optimal conditions f o r recovery of polyanion. D i a l y s i s of the d i g e s t i o n p r o d u c t s gave 9 8% r e c o v e r y o f c a r r a g e e n a n f r o m b o t h b o v i n e m i l k and baby f o o d . C h o c o l a t e p i g m e n t s have p r e v i o u s l y been f o u n d t o i n t e r f e r e w i t h a n a l y t i c a l procedures developed f o r non-pigmented m i l k s l l r l . , and t h e scheme we d e v e l o p e d C F i g . 9] was no e x c e p t i o n s i n c e t h e c h o c o l a t e p i g m e n t s i n a c t i v a t e d t h e p r o t e o l y t i c enzymes. This d i f f i c u l t y was overcome u s i n g t h e p r o c e d u r e s shown i n F i g . 10. The c a r r a g e e n a n was e x t r a c t e d f r o m t h e d e n a t u r e d p r o t e i n l a y e r , a f t e r d e - f a t t i n g , using s a l i n e a l k a l i n e b u f f e r , since the c h o c o l a t e p i g m e n t e d p r o t e i n s were i n s o l u b l e compared t o t h o s e 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL ET AL.

0

Spectrofluorimetric

1

Methods

2

3

P/D Figure 7. Doping of babyfood with carrageenan. Dyebinding titrations of doped and undoped sample and standard carragee­ nan. (O) Undoped SMA babyfood; (χ) standard 10~ M carra­ geenan; (Φ) doped sample. 3

Ratio

P/D

Figure 8. Effect of protein and lipid on the titration of acridine orange with 10~ M carrageenan. (χ) Standard carrageenan; (O) carrageenan with emul­ sified lard, 1 mg/mL; (Φ) carrageenan with BSA, 0.5 mg/mL. 3

CARBOHYDRATE SULFATES

To 20 ml S a m p l e : -

Add

30 ml C h l o r o f o r m 40 ml M e t h a n o l

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Saturated

NaCl

SPIN 4000 rpm f o r 10

Lyophilise Solid Add

mins

Layer

20 ml TRIS/HCL B u f f e r

pH

5 mg T r y p s i n 5 mg

γ-Chymotrypsin

5 mg S u b t i l i s i n DIGEST § 40°C

for

12

hrs

FILTER t h r o u g h a c t i v a t e d diatomaceous e a r t h

1.

Dialyse

2.

Titrate

Figure 9.

vs

10~ N 5

AO

Procedure for the analysis of polyanion-containing solu­ tions

CUNDALL ET AL.

Spectrofluorimetric

Methods

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

To 1 0 m l . p r o d u c t , a d d : CHL0R0F0RM

(15ml.)

METHANOL

(20ml.)

NaCl

MOOmg.)

C e n t r i f u g e a t 4000 rpm. for

10 m i n u t e s .

Lyophilise s o l i d layer, add:TRIS / HC1 BUFFER,

pH

9.0

4M. SODIUM CHLORIDE

(5ml.) (5ml.)

Shake f o r v a r y i n g t i m e

and

t e m p e r a t u r e d e p e n d i n g on sample.

1.

Filter

2.

Dialyse versus

d i s t i l l e d w a t e r , 3 changes.

3.

T i t r a t e versus

3 χ 10~ M.

Figure 10.

t h r o u g h Watmans

No.42.

5

a c r i d i n e orange.

Procedure for the analysis of pigmented dairy products

polyanion-stabilized

82

CARBOHYDRATE SULFATES

p r e s e n t i n baby f o o d . The e x t r a c t a f t e r f i l t r a t i o n r e m a i n e d s l i g h t l y p i g m e n t e d , and i n t e r f e r e d s l i g h t l y w i t h t h e t i t r a t i o n , g i v i n g r e d u c e d s h a r p n e s s o f t h e end p o i n t w i t h 1 χ 1 0 ~ Μ d y e . However, u s i n g a dye c o n c e n t r a t i o n o f between 3 t o 5 χ 10 M i n c r e a s e d t h e a c c u r a c y s i n c e dye b i n d i n g t o c a r r a g e e n a n i s stronger at t h i s higher c o n c e n t r a t i o n - ^ . Polyanion recovery v a r i e d w i t h t h e t e m p e r a t u r e o f t h e e x t r a c t i o n s t e p , t o a maximum of -96%. The optimum t e m p e r a t u r e v a r i e s w i t h t h e s a m p l e .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

5

Carrageenan Standards. T a b l e 5 shows t h e n u m e r i c a l v a l u e s o f the e q u i v a l e n t w e i g h t s o f s e v e r a l samples o f carrageenan i s o m e r s , d e t e r m i n e d by C u n d a l l e t a l — . I n t h e s i t u a t i o n when we a r e a s s a y i n g f o r c a r r a g e e n a n on a w e i g h t p e r volume b a s i s , t h e s t a n d a r d c a r r a g e e n a n s a m p l e must be o f t h e same r a t i o o f i s o m e r s as t h e unknown s a m p l e . F a i l u r e to account f o r t h i s could l e a d t o e r r o r s up t o 30% i n t h e e x t r e m e case o f p u r e lambda and kappa i s o m e r s on a w e i g h t b a s i s , a l t h o u g h t h e c o n c e n t r a t i o n e x p r e s s e d i n e q u i v a l e n t s o f a n i o n i c s i t e s p e r l i t r e w o u l d be u n a f f e c t e d . A s i m i l a r p r o b l e m o c c u r s w i t h s t a n d a r d s f o r CNC, as a l r e a d y noted. The d i f f i c u l t y h e r e a r i s e s f r o m t h e v a r i a b l e d e g r e e o f s u b s t i t u t i o n (DS) o f c o m m e r c i a l CHC s a m p l e s . Here a l s o c o n v e r s i o n o f c o n c e n t r a t i o n i n t e r m s o f i o n i c s i t e s , as d e t e r m i n e d by dye binding t i t r a t i o n , i n t o a weight of s t a b i l i s e r , necessitates a knowledge o f t h e DS o r c a l i b r a t i o n w i t h s t a n d a r d CNC s a m p l e s . I n t e r a c t i o n o f p r o t e i n and p o l y a n i o n s . The o b s e r v a t i o n t h a t c a t i o n i c p o l y p e p t i d e s q u a n t i t a t i v e l y d i s p l a c e a c r i d i n e o r a n g e f r o m NaPSS, i n t h e s e l a b o r a t o r i e s ^ i s h e r e u t i l i s e d t o s t u d y i n t e r a c t i o n s w h i c h may o c c u r i n c o n n e c t i v e t i s s u e s and t o i n v e s t i g a t e t h e mechanism o f t h e s t a b i l i s a t i o n o f d a i r y p r o d u c t s by a n i o n i c gums. The n o r m a l t i t r a t i o n o f dye by p o l y a n i o n i s shown i n F i g . 1 1 ( s o l i d l i n e ) . The s y s t e m can be q u a n t i t a t i v e l y r e v e r s e d by t i t r a t i o n w i t h p o l y - L - l y s i n e . The c o n c e n t r a t i o n χ - a x i s i s g i v e n as t h e r a t i o PC/PA, ( p o l y c a t i o n s i t e s added t o t o t a l p o l y a n i o n s i t e s ) , w i t h PA c o n s t a n t a t 2 χ 10 i . e . P/D = 2 . The dye r e l e a s e c u r v e c l o s e l y p a r a l l e l s t h e dye b i n d i n g c u r v e w i t h a l m o s t q u a n t i t a t i v e dye r e l e a s e o c c u r r i n g between PC/PA 0 t o 1. I n i t i a l stoichiometric inter­ a c t i o n o c c u r s , t h e r e f o r e , between p o l y c a t i o n and p o l y a n i o n n o t a s s o c i a t e d w i t h d y e , f o l l o w e d by i n t e r a c t i o n o f t h e p o l y c a t i o n w i t h dye p o l y a n i o n c o m p l e x , t o g i v e a complex w i t h e q u i v a l e n c e o f a c i d and b a s e s i t e s and d i s p l a c e m e n t o f t h e d y e . W i t h p r o t e i n s t h e i n t e r a c t i o n i s more complex s i n c e a c i d i c and b a s i c amino a c i d r e s i d u e s can be p r e s e n t . The a c i d i c r e s i d u e s of the p r o t e i n w i l l modify the e x t e n t o f i n t e r a c t i o n w i t h p o l y a n i o n , and t h e s e c o n d a r y and h i g h e r s t r u c t u r e o f t h e p r o t e i n w i l l a f f e c t the a v a i l a b i l i t y of the b i n d i n g s i t e s . The n e t c h a r g e o f t h e p r o t e i n can be v a r i e d by c h a n g i n g t h e pH, and a t pH 3 most p r o t e i n s have a n e t p o s i t i v e c h a r g e . The i n t e r ­ a c t i o n between t h r e e p r o t e i n s and one model p o l y m e r , p o l y v i n y l

Spectwfluorimetric

Methods

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL ET AL.

Figure 11. General effect of the time dependence of polyanion-polyanion interaction on binding profiles. (Φ) Points recorded instantane­ ously; (O) points recorded after 1.5 hr.

84

CARBOHYDRATE SULFATES

p y r r o l i d o n e , w i t h a P/D = 2.0 complex o f PSS and a c r i d i n e o r a n g e a t pH 3.0 i s shown i n F i g . 1 2 . The b e h a v i o u r i s n o t u n i f o r m . PVP does n o t d i s p l a c e dye a t any pH i n d i c a t i n g t h a t t h e a m i d e - l i n k a g e g r o u p does n o t p a r t i c i p a t e i n t h e b i n d i n g . W i t h κ - c a s e i n an i n i t i a l dye r e l e a s e was o b s e r v e d , b u t t h e dye was r e - b o u n d w i t h increasing protein concentration. A l l p r o t e i n s used were 1 0 ~ M i n b a s i c s i t e s , t h e b a s i c e q u i v a l e n t w e i g h t b e i n g d e f i n e d as t h e w e i g h t o f p r o t e i n c o n t a i n i n g one b a s i c amino a c i d r e s i d u e , i . e . either l y s i n e , arginine, h i s t i d i n e or hydroxylysine. B o v i n e Serum A l b u m i n (BSA) d i s p l a c e d dye l i n e a r l y , i n d i c a t ­ i n g a f i n a l s t o i c h i o m e t r y o f a b o u t 6 t o 1, BSA t o PSS s i t e s . By u s i n g s t r u c t u r e b r e a k i n g s o l v e n t s s u c h as u r e a 15- i t i s p o s s i b l e t o i n c r e a s e t h e e x t e n t o f BSA i n t e r a c t i o n . A t pH 3 and an u r e a c o n c e n t r a t i o n o f 4M, w h i c h i s t h e maximum u r e a l e v e l a t w h i c h r e p r o d u c i b l e dye b i n d i n g o c c u r r e d , t h e s t o i c h i o m e t r y i n c r e a s e s t o 2 : 1 , BSA t o PSS. These f i n d i n g s d e m o n s t r a t e t h e i m p o r t a n c e o f secondary s t r u c t u r e o f such g l o b u l a r p r o t e i n s i n r e g u l a t i n g interactions. The i n t e r a c t i o n o f PSS and c o l l a g e n i s a g a i n d i f f e r e n t . A l m o s t q u a n t i t a t i v e dye r e l e a s e i s a p p a r e n t a t pH 3 and i n t h e absence o f a s t r u c t u r e b r e a k i n g s o l v e n t . T h i s c o u l d be due t o the p r o p o s e d ! ^ heterogeneous charge d i s t r i b u t i o n o f c o l l a g e n i n w h i c h g r o u p s o f amino a c i d r e s i d u e s o f l a r g e l y h y d r o p h i l i c c h a r a c t e r a r e s e p a r a t e d by r e g i o n s o f p r e d o m i n a n t l y h y d r o p h o b i c character. T h i s e f f e c t w o u l d be a l l i e d t o t h e more ' o p e n ' n a t u r e o f t h e c o l l a g e n t r i p l e h e l i x , where c h a r g e d g r o u p s a r e l i k e l y t o be e x t e r n a l f o r s t e r i c r e a s o n s , when compared w i t h t h e g l o b u l a r p r o t e i n s , where r e s i d u e s a r e b u r i e d deep w i t h i n t h e f o l d e d s t r u c t u r e as shown by BSA. The method c o u l d , t h e r e f o r e , o f f e r a means o f i n v e s t i g a t i n g d i s t r i b u t i o n o f c h a r g e w i t h i n t h e p r o t e i n tertiary sturcture. Changes i n s t o i c h i o m e t r y o f c o l l a g e n - g l y c o s a m i n o g l y c a n (GAG) c o m p l e x e s w i t h pH were s t u d i e d by i n i t i a l l y f o r m i n g 1 : 1 c o m p l e x e s a t a c i d pH, and o b s e r v i n g t h e u p t a k e o f dye by t h e p o l y a n i o n as t h e complex i s d i s s o c i a t e d w i t h t h e a d d i t i o n o f b a s e . The i n t e r a c t i o n s o f h y a l u r o n i c a c i d and c h o n d r o i t i n s u l p h a t e a r e shown i n F i g . 12 i n c o m p a r i s o n w i t h t h o s e f o r model p o l y a n i o n s , CMC and PSS. A l l i n t e n s i t i e s were c o r r e c t e d f o r t h e a c i d d i s s o c i a t i o n o f t h e d y e - p o l y a n i o n complex u s i n g d a t a f r o m F i g . 5 , and f o r t h e v a r i a t i o n o f dye f l u o r e s c e n c e i n t e n s i t y w i t h pH. At a c i d pH t h e d i s s o c i a t i o n c l o s e l y f o l l o w s t h e h y d r o g e n - i o n t i t r a t i o n curve f o r c o l l a g e n — . The i n t e r a c t i o n i s v i r t u a l l y a b s e n t a t n e u t r a l pH f o r t h e s u l p h a t e d p o l y m e r s . Interactions w i t h t h e p o l y c a r b o x y l a t e s a r e s l i g h t l y more r e s i s t a n t t o a l k a l i n e dissociation. -5 The r e s u l t s g i v e n i n F i g . 13 were o b t a i n e d u s i n g 10 M polymer c o n c e n t r a t i o n s . I n c r e a s i n g the c o n c e n t r a t i o n s o f each p o l y m e r i n c r e a s e d t h e s t a b i l i t y o f t h e complex t o b a s e d i s s o c i a t ­ i o n , as a l s o d i d t h e a d d i t i o n o f 10 M calcium chloride f o r chondroitin sulphate.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL ET AL.

Spectrofluorimetric

Ratio

Methods

P o l y c a t i o n i c s i t e s added Polyanion s i t e s i n solution

Figure 12. Dye release profiles for the interaction of three proteins and polyvinyl pyrrolidone) with a P/D 2.0 complex of NaPSS-acridine orange at pH = 3.0 in salt-free solution. (X) Polyvinyl pyrrolidone); (\7) kappa casein; (O) bovine serum albumin; (%) collagen.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CARBOHYDRATE SULFATES

Figure 13. Variation of polyanion-collagen binding with pH. (V) Choindroitin sulfate; (O) poly(styrene sulfonate); (X) CMC; (Φ) hyaluronic acid.

5.

CUNDALL E T A L .

Spectrofluorimetric

Methods

87

The r e l a t i v e s t r e n g t h s o f t h e c o l l a g e n - p o l y a n i o n c o m p l e x e s were a s s e s s e d by t h e ' c r i t i c a l e l e c t r o l y t e c o n c e n t r a t i o n ' method o f S c o t t — ' —. T a b l e 3 shows t h e s a l t c o n c e n t r a t i o n s w h i c h were n e c e s s a r y t o d i s s o c i a t e s t o i c h i o m e t r i c 10 Π complexes o f c o l l a ­ gen p o l y a n i o n . V a l u e s f o r PSS and h e p a r i n c o u l d n o t be o b t a i n e d since the high s a l t concentrations f o r d i s s o c i a t i o n p r e c i p i t a t e d the c o l l a g e n . Those f o r h y a l u r o n i c a c i d and c h o n d r o i t i n s u l p h a t e i n d i c a t e t h a t a l l t h e complexes e x i s t i n p h y s i o l o g i c a l c o n d i t i o n s o f i o n i c s t r e n g t h , and a b o v e .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Investigation

o f t h e s t r u c t u r e o f model p o l y e l e c t r o l y t e complexes.

U s i n g t h e t e c h n i q u e we have d e s c r i b e d o f dye d i s p l a c e m e n t f r o m a dye p o l y a n i o n complex by t h e a d d i t i o n o f p o l y c a t i o n s — , we have s t u d i e d t h e s t o i c h i o m e t r y o f t h e complexes f o r m e d between t h r e e p o l y a n i o n s ( P S S , CNC and CAR) w i t h two p o l y c a t i o n s (PPXV and P L L ) . A w i d e r a n g e o f c o n d i t i o n s o f pH and s a l t c o n c e n t r a t ­ i o n s were e m p l o y e d . Complexes were f o u n d t o e x h i b i t a 1 t o 1 p a i r i n g o f p o s i t i v e and n e g a t i v e i o n i c s i t e s between pH 4 and 1 0 , and i n s o l u t i o n s o f h i g h s a l t c o n c e n t r a t i o n , up t o 2Π s o d i u m c h l o r i d e f o r c o m p l e x e s i n v o l v i n g p o l y s t y r e n e s u l p h o n a t e . The s a l t - i n d u c e d dye r e l e a s e l i m i t e d t h e s a l t c o n c e n t r a t i o n w h i c h c o u l d be e m p l o y e d . A s t r o n g t i m e - d e p e n d e n c e o f t h e b i n d i n g was o b s e r v e d a t p o l y m e r c o n c e n t r a t i o n s a p p r o a c h i n g t h e s t o i c h i o m e t r i c r a t i o , as shown i n F i g . 1 1 , w i t h binding u s u a l l y b e i n g c o m p l e t e a f t e r about 1 hour. A t t h i s p o i n t c o m p l e t e e l e c t r o n e u t r a l i t y o f t h e complex is achieved. I t i s p r o b a b l e t h a t t h e i n i t i a l p o l y c a t i o n added i s bound i n s t a n t a n e o u s l y , s i n c e l a r g e numbers o f p o l y a n i o n s i t e s a r e a v a i l a b l e . I t has been o b s e r v e d — t h a t when t h e p o l y e l e c t r o ­ l y t e c o u n t e r i o n s a r e H and OH t h e b i n d i n g i s i r r e v e r s i b l e , and the contact c o n f i g u r a t i o n i s r e t a i n e d , s i n c e the counterions r e a c t t o g i v e H2O. However, t h e presence o f o t h e r c o u n t e r i o n s s u c h as N a and C1~ s c r e e n s t h e p o l y e l e c t r o l y t e s i t e s t o an e x t e n t w h i c h e n a b l e s t h e r e - o r i e n t a t i o n o f t h e complex t o g i v e a l o w e r f r e e - e n e r g y s t a t e , as t h e c o u n t e r i o n s d i f f u s e o u t . T h i s i s c o n s i s t e n t w i t h t h e slow d y e - r e l e a s e at around t h e s t o i c h i o m e t r i c r a t i o observed here. The p r e s e n c e o f some c r o s s - l i n k i n g must be p r e d i c t e d , i n v i e w o f t h e m o l e c u l a r w e i g h t d i f f e r e n c e s and p o l y d i s p e r s i t y of the polymers. V i s c o s i t y measurements ( F i g . 143 on t h e CAR - P L L c o m p l e x , and measurement o f t h e S t e r n - V o l m e r q u e n c h i n g c o n s t a n t [ T a b l e 5, F i g s . 15 and 16) o f dye r e l e a s e d by complex f o r m a t i o n a r e c o n s i s t ­ ent w i t h the complete d e s t r u c t i o n o f t h e e l e c t r i c f i e l d o f t h e polyelectrolytes. In the former i n s t a n c e , the s o l u t i o n v i s c o s i t y o f t h e p o l y a n i o n i s d r a m a t i c a l l y r e d u c e d by t h e a d d i t i o n o f polycation. Polyanion v i s c o s i t y i s i n t i m a t e l y r e l a t e d to the degree o f e x t e n s i o n o f t h e polymer c h a i n , which i s i n t u r n a f u n c t i o n o f t h e degree o f i o n i s a t i o n . The r e d u c t i o n o f v i s c o s i t y w o u l d , t h e r e f o r e , a p p e a r t o be a s s o c i a t e d w i t h t h e n e u t r a l i s a t i o n +

+

88

CARBOHYDRATE

Table 3.

SULFATES

Critical Electrolyte Concentrations of Some Poly anion—Collagen Complexes Obtained by the Method of Scott (7)

Polyanion

C r i t i c a l electrolyte concentration (M) CaCl NaCl

pH

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

Hyaluronic Acid Chrondroitin Sulphate

Table 4.

3.0

0.15

0.06

3.0

(-0.5)

0.125

Stern-Volmer Quenching Constants for 10" M Acridine Orange Containing Stoichiometric Polyelectrolyte Complexes 5

_

sv TO 6k

_

k-Carrageenan C h o n d r o i t i n sulphate NaPSS CMC Hyaluronic A c i d

Table 5.

Κ

Polycation

Polyanion

Solutions

PLL PLL PLL PPXV PPXV

61

69

Equivalent Weights of Repeating Units of Several Carrageenan Samples

Carrageenan sample.

Lambda ( i ) . Lambda ( i l ) . Kappa Iota

(2)

E q u i v a l e n t weight per sulphate group. From sulphate By a c r i d i n e content. orange t i t r a t i o n . 286 258 370 296

298 266 390 290

Spectrofluorimetric

Methods

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL E T A L .

Figure 14. Ostwald viscometer flow times for a viscous polyanion, κ-carrageenan, at varying concentrations of simple (O) and polycationic (Φ) electro­ lytes

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CARBOHYDRATE SULFATES

[ I O D I D E ],M

Figure 15. Stern-Volmer plots for the iodide quenching of dye released by polyanion—PPXV interaction. (Φ) Carboxymethyl cellulose; (O) hyaluronic acid.

Spectrofluorimetric

Methods

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CUNDALL E T A L .

Ο

-025

05

-075

[ I O D I D E ], M

Figure 16. Stern-Volmer plots of dye released by polyanion/poly--L-ly sine interaction. (V) Acridine orange; (Φ) κ-carrageenan; (O) chondroitin sulfate; (X) polystyrene sulfonate.

92

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

CARBOHYDRATE SULFATES

Ο Polyelectrolyte

Figure 17.

·

c o m p l e x and f r e e

counterions

Interaction of poly anions and poly cations—a simplified picture of polymer conformations and counter-ion distribution

5. cundall et a l .

Spectrofluorimetric Methods

93

of charged groups. Similarly measuring the Stern-Volmer quench­ ing constants of the released dye indicates that the dye molecules are truly 'free* and under no constraint from the polyelectrolyte complex, since binding of monomeric dye to polyanions greatly reduces the value of the quenching constant-. A simplified picture consistent with such observations i s indicated in F i g . 17. More direct evidence for this more compact structure of the complex comes from ORD/CD spectroscopy—. The presence of acid glycosaminoglycans had an a - h e l i c a l or random c o i l - d i r e c t i n g effect on cationic polypeptides which are normally in the charged-coil form at neutral pH.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

ABSTRACT The fluorescence characteristics of Acridine Orange bound to naturally occurring and synthetic anionic polysaccharides can be distinguished from free Acridine Orange, and i s the basis for the quantitative estimation of these materials. By suppress­ ing the ionization of individual anionic groups, sulphated poly­ saccharides can be estimated in the presence of carboxylated polysaccharides, and the proportions of the O- and N- sulphate groups relative to carboxylic groups within the same polyanion (e.g. i n heparin) determined. Proteins, l i p i d s , fats, and s a l t s , which can occur in biological systems and food products, i n t e r ­ fere with the basic procedure, but modifications have been introduced to allow the analysis to be undertaken i n the presence of these additivies. The method has permitted the quantitative study of interactions between proteins and glycosaminoglycans. The extent to which such interactions occur under physiological conditions i s assessed. 'Literature Cited' 1.

Stone, A . L . , Childers L . G . , & Bradley, D.F. Biolpolymers, (1963) 1, 111. 2. Cundall, R . B . , Rowlands, D.Ρ., & Phillips, G.O. Analyst, (1973), 98. 857. 3. Scott, J.E., Biolchem.Soc.Transactions, (1973) 1, 787. 4. Mowry, R.W., & Scott, J . E . Histochemie, (1967), 4, 73. 5. Davies, J . V . , Dodgson, K . S . , Moore, J.S. & Phillips, G.O. Biochem. J., (1969) 113, 465. 6. Rowlands, D.P. PhD Thesis (1974) University of Salford. 7. Factor, Α., & Heinsohn, F . , Polymer Letters, (1971) 9, 298. 8. Morley, R.G. PhD Thesis, (1972) University of Salford. 9. Cundall, R . B . , & Evans, G.B. J . S c i e n t i f i c Instruments. (1968), 1, 305. 10. Jooyandeh, F . , Moore, J . Α . , & Phillips, G.O. J . C . S . Perkin II (1974), 1468. 11. Norley, R . G . , P h i l l i p s , G.O., Power, D.M. & Morgan, R . E . , The Analyst, (1973), 98, 813.

94

CARBOHYDRATE SULFATES

12. 13. 14. 15. 16. 17.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch005

18. 19. 20. 21. 22.

Morley, R.G., Phillips, G.O., Power, D.M. & Morgan, R.E., The Analyst, (1972), 97, 315. Graham, H.D. J.Agr. & Dairy Science, (1968) 33, 390. Jaques, L.B. 'Polyelectrolytes and their applications.' (Ed. Rembaum Α., & Seligny, E) (1975) Reidal, Dordrecht Holland, Boston, U.S.A. 145. Johnson, E.A. Nature, (1977) 266, 305. Kennedy, J . F . , Chem. Soc. Reviews, (1973) 2, 355. Cundall, R.B., Murray, D., & Phillips, G.O. (unpublished results) Tanford, C., J.A.C.S., (1974) 86. 2050. Ε. A. Balazs(Ed.) 'Chemistry and Molecular Biology of the Intercellular Matrix.' 2, 1201. Academic Press, London & New York, (1970). Veis, Α., 'Treatise on Collagen' (1967) 1, Ed.Ramachandran Academic Press, London & New York. Michaels, A.S., Min. L., Schneider N.S., J . Phys.Chem. (1975), 69, 1447. Gelman, R.A., Blackwell, J., Biopolymers (1974), 13, 139.

RECEIVED

February 6, 1978.

6 Sulfated Glycosaminoglycans Obtained by Chemical Modification of Polysaccharides D E R E K H O R T O N and T A I C H I U S U I

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Department of Chemistry, Ohio State University, Columbus, OH 43210

As part of the general theme of the symposium devoted to sulfated carbohydrates, this report presents some of the work from our laboratory concerned with the natural, sulfated glycosaminoglycan, heparin, both from the standpoint of structural elucidation and more especially from that of synthetic production of similarly constituted, sulfated polysaccharides that have biological properties resembling those of heparin. There is not time to present a detailed historical development of the entire story of the biological role and efforts to establish the chemical structure of heparin, but i t has been known for many years that this biopolymer contains residues of a uronic acid and 2-amino-2-deoxy-D-glucose in approximately equal proportion, and the material is highly sulfated, both at the amino positions and also at certain of the hydroxyl positions (1). The compound is widely distributed in many connective tissues, where i t appears to be produced as an intracellular component in mast cells. It is widely used in therapy as an anticoagulant in cardiovascular disorders resulting from thrombosis, and i t also displays antilipemic (clearing factor) properties. Much current evidence indicates that heparin as used therapeutically is the carbohydrate portion alone of a proteoglycan that is the native tissue component; a long, single, protein chain may be envisaged as being substituted by a large number of lateral chains of the sulfated polysaccharide, attached by a bridge region containing two D-galactose and a D-xylose residue, attached via L-serine to the peptide chain (2). Not only has the total constitution of this native proteoglycan not been elucidated, but controversies still remain concerning the exact constitution of the glycan chains that are detached during the processes used for extraction of heparin on the commercial scale for therapeutic use as an anticoagulant. Details of the exact procedure used in the commercial isolation of heparin from such rich sources as hog gastric mucuosa and beef lung are not readily available, but in general terras, the process involves autolysis of the tissue with concomitant 0-8412-0426-8/78/47-077-095$05.00/0 © 1978 American Chemical Society

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

96

CARBOHYDRATE

SULFATES

degradation of the non-heparin components, f o l l o w e d by a l k a l i n e treatment t o remove as much as p o s s i b l e of the p r o t e i n component, and subsequent p u r i f i c a t i o n by use of c a t i o n i c detergents. The g l y c a n m a t e r i a l thus i s o l a t e d i s of much lower molecular weight than the parent proteoglycan, and the product most widely used i n therapy has a molecular weight i n the region of 12,000, with a d i s t r i b u t i o n of molecular weights above and below t h i s f i g u r e . The molecular weight i s d i f f i c u l t t o estimate a c c u r a t e l y unless c a r e f u l a t t e n t i o n i s p a i d t o the e f f e c t s of the p o l y a n i o n i c c h a r a c t e r of the compound, but the accompanying graph ( F i g u r e l ) from a recent a r t i c l e (3) shows the r e s u l t s of molecular weight determinations on v a r i o u s h e p a r i n p r e p a r a t i o n s by u l t r a c e n t r i f u g a t i o n methods i n comparison with v i s c o s i t y measurements; i t may be seen t h a t the more r e a d i l y a p p l i e d v i s c o s i t y technique may be used at l e a s t as a rough approximation i n estimating the molecular weight of a h e p a r i n sample. The a n t i c o a g u l a n t a c t i v i t y of such heparin preparations i s c r i t i c a l l y dependent on the l e v e l of s u l f a t i o n ; commercial products assayed by a c o n v e n t i o n a l technique u s i n g sheep plasma t y p i c a l l y show a c t i v i t i e s i n the range of 110 t o l 8 0 I n t e r n a t i o n a l U n i t s per m i l l i g r a m f o r m a t e r i a l c o n t a i n i n g approximately 5 s u l f a t e residues per nominal t e t r a s a c c h a r i d e u n i t of the polymer. P a r t i a l d e s u l f a t i o n leads t o lowering of the b i o l o g i c a l a c t i v i t y , without n e c e s s a r i l y causing changes of more-profound s i g n i f i c a n c e i n the polymer; milder i s o l a t i o n techniques l e a d i n g t o products having s t i l l h i g h e r degrees of s u l f a t i o n have d i s p l a y e d a c t i v i t i e s at higher l e v e l s than those encountered normally i n commercial material. E l u c i d a t i o n of the chemical c o n s t i t u t i o n of the heparin chain has proved remarkably d i f f i c u l t ; the high degree of s u l f a t i o n renders the m a t e r i a l i n t r a c t a b l e i n many of the c o n v e n t i o n a l methods f o r p o l y s a c c h a r i d e structure-determination. I t has been e s t a b l i s h e d with l i t t l e doubt that the u r o n i c a c i d component involvesD-glucuronic a c i d and L - i d u r o n i c a c i d , but the exact p r o p o r t i o n s of these, and t h e i r r a t i o i n the n a t i v e proteoglycan, s t i l l remain c o n t r o v e r s i a l . I t i s by no means c e r t a i n t h a t the r a t i o of these u r o n i c a c i d s present i n commercial heparin r e f l e c t s t h a t present i n the o r i g i n a l proteoglycan, as these two u r o n i c a c i d s c o u l d i n t e r c o n v e r t by C-5 e p i m e r i z a t i o n under the a l k a l i n e c o n d i t i o n s used i n the i s o l a t i o n procedure. In the b i o s y n t h e s i s of the polymer chain, i t i s very probable that t h i s C-5 i n v e r s i o n occurs at the polymer l e v e l (U), and i n the n a t i v e s t a t e there may e x i s t an e n t i r e spectrum of glycan chain-compositions ranging from f r a c t i o n s r i c h i n D-glucuronic a c i d on the one hand t o f r a c t i o n s r i c h i n L - i d u r o n i c a c i d on the other. Working on the premise of a l i n e a r , a l t e r n a t i n g chain of amino sugar and u r o n i c a c i d components, work from t h i s l a b o r a t o r y (5.) i n v o l v i n g W and 0 - d e s u l f a t i o n of commercial heparin, f o l l o w e d by c a r b o x y l r e d u c t i o n w i t h diborane and then s e l e c t i v e fragmentation of the r e s u l t a n t , reduced, d e s u l f a t e d heparin l e d ,

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

HORTON AND usui

Sulfated

Gly cosaminogly cans

Figure 1. Relation between intrinsic viscosity and molecular weight (sedimen­ tation) from [ ] = J.35 Χ 10~ Μ · in aqueous sodium chloride medium. Data from Ref. 2. v

5

0 9

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

98

CARBOHYDRATE SULFATES

according t o the h y d r o l y t i c procedure used, t o two disaccharides, as i l l u s t r a t e d i n F i g u r e 2. One of these d i s a c c h a r i d e s , c h a r a c t e r i z e d on a c r y s t a l l i n e b a s i s , had a s t r u c t u r e t h a t i n d i c a t e d an α-ρ-(ΐ-*»*0 linkage between 2-amino-2-deoxy-D-glucose and D-glucuronic a c i d i n the o r i g i n a l polymer. The second d i s a c c h a r i d e "maltosamine", f o r which a reference sample was synthesized from maltose, i n d i c a t e s from i t s s t r u c t u r e the occurence of an a - D - ( l - * 4 ) linkage between g l u c u r o n i c a c i d and 2-amino-2-deoxy-D-glucose i n the o r i g i n a l polymer. This information l e d t o the formulation of a p a r t i a l backbone s t r u c t u r e as shown i n F i g u r e 2, and subsequent unequivocal i d e n t i f i c a t i o n of L - i d u r o n i c a c i d residues i n some preparations of heparin (6) l e d t o the type of g e n e r a l formulation shown i n F i g u r e 3, which d e p i c t s a h y p o t h e t i c a l t e t r a s a c c h a r i d e r e p e a t i n g - u n i t f o r heparin based on the occurence of L - i d u r o n i c a c i d residues and D-glucuronic a c i d residues, complete s u l f a t i o n of the amino groups, complete s u l f a t i o n of the primary hydroxyl groups, and a d d i t i o n a l s u l f a t e residues at one of the secondary p o s i t i o n s , probably p o s i t i o n 2, i n the L - i d u r o n i c a c i d component; the p r e f e r e n t i a l s u s c e p t i b i l i t y of the D-glucuronic a c i d residues toward degradation by periodate provided evidence that the Dg l u c u r o n i c a c i d residues were l a r g e l y non-sulfated at the 273 positions. Since the-time of these formulations, other workers have proposed on the b a s i s of enzymic s t u d i e s with degradation fragments from heparin that some or a l l of the linkages of the g l y c u r o n i c a c i d components have the β-D or α-L c o n f i g u r a t i o n (7), and support f o r these proposals ( F i g u r e h) has been provided from i n s p e c t i o n of n. m.r. spectra of the polymer (8) and a l s o from X-ray d i f f r a c t i o n studies (9) on the o r i e n t e d polymer. The X-ray d i f f r a c t i o n studies do accord with the general idea of f a i r l y short, l i n e a r chains f o r the macromolecule. However, unequivocal s t r u c t u r a l proof by degradation and synthesis of fragments at a l e v e l beyond t h a t of d i s a c c h a r i d e components i s s t i l l l a c k i n g , as i s such evidence obtained from n a t i v e proteoglycan m a t e r i a l that has not been subjected t o an u n s p e c i f i e d l e v e l of p o s s i b l e a l k a l i n e treatment during a commercial i s o l a t i o n process. Our continuing studies on heparin have been d i r e c t e d along two f r o n t s , f i r s t of a l l the systematic s t r u c t u r a l e l u c i d a t i o n of components of the polymer chain at a l e v e l beyond t h a t of the d i s a c c h a r i d e , together w i t h procedures f o r determining uronic a c i d r a t i o s and p o s i t i o n s of s u l f a t i o n i n the chain. In p a r a l l e l i n v e s t i g a t i o n s , we have examined the chemical synthesis of modified polymers whose s t r u c t u r e simulates that of heparin, to a f f o r d p o t e n t i a l replacements f o r the expensive n a t u r a l m a t e r i a l as a t h e r a p e u t i c anticoagulant, and t o provide a product f o r attachment t o the surface of s y n t h e t i c objects f o r implantation i n the c i r c u l a t o r y s y s t e m , with t h e o b j e c t i v e of c o n f e r r i n g a b i o l o g i c a l l y acceptable, nonthrombogenic s u r f a c e - c h a r a c t e r i s t i c f o r such devices. I t i s the l a t t e r aspect of chemical synthesis

HORTON AND usui

Sulfated

The

Linkage

C0 H

!

2

OH

:

Glycosaminoglycans Sequence

CH 0H

in

;

2

NHO

Heparin

C0 H

;

2

OH

:

,0 — Desulf ated

CH 0H 2

NH

:

Heparin

carboxyl reduction (diborane)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Carboxyl — reduced

Heparin

hydrolysis CH OH

CHgOH

2

CH OH 2

OH OH

HO^ta^l

NK, NH^CI

NH3CI

maltosamin e Figure 2.

Disaccharides from carboxyl-reduced heparin

CH OS0 Na 2

3

HNS0 Na 3

CO^Na

CH 0S0 Na 2

OH

3

HNS0 Na

0

3

H, S 0 N a 3

Component R e s i d u e s Figure 3.

Present

i n Heparin

Component residues present in heparin

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

100

CARBOHYDRATE SULFATES

of h e p a r i n - l i k e p o l y s a c c h a r i d e s that c o n s t i t u t e s the p r i n c i p a l theme of t h i s p r e s e n t a t i o n , but the f o l l o w i n g d i s c u s s i o n presents a b r i e f survey of some of the more recent e f f o r t s t o degrade commercial heparin i n t o o l i g o s a c c h a r i d e s above the d i s a c c h a r i d e l e v e l that might be u s e f u l i n s t r u c t u r e - d e t e r m i n a t i o n studies. The next scheme ( F i g u r e 5) i l l u s t r a t e s a degradative method based on the recognized s u s c e p t i b i l i t y of D-glucuronic a c i d residues i n heparin t o a t t a c k by periodate? Exposure of heparin t o 1 molar equivalent of p e r i o d a t e per t e t r a s a c c h a r i d e u n i t gives a product i n which the D-glucuronic a c i d components are s e l e c t i v e l y degraded; subsequent borohydride r e d u c t i o n of t h i s m a t e r i a l , f o l l o w e d by m i l d h y d r o l y s i s w i t h a p o l y s t y r e n e s u l f o n i c a c i d of high molecular weight c o n f i n e d w i t h i n a d i a l y s i s membrane, shows t h a t degradation t o a low molecular weight, t r i s a c c h a r i d e t e t r o n i c a c i d component does take p l a c e by t h i s procedure. The recovery of the degraded, d i a l y z a b l e components i s r e l a t i v e l y low i n p r o p o r t i o n t o the t o t a l product t h a t s t i l l r e t a i n s polymeric c h a r a c t e r (10). T h i s r e s u l t tends t o argue against the idea t h a t the p e r i o d a t e - l a b i l e u r o n i c a c i d residues are u n i f o r m l y d i s t r i b u t e d along the chain. The d i a l y z a t e , a f t e r p u r i f i c a t i o n by p r e p a r a t i v e paper-chromatography, y i e l d s a m a t e r i a l that appears t o be a homogeneous t e t r a s a c c h a r i d e whose c o n s t i t u t i o n may be t e n t a t i v e l y d e p i c t e d as i n F i g u r e 6. I f the p e r i o d a t e - o x i d i z e d , borohydride-reduced heparin i s f u r t h e r subjected t o carboxyl-group r e d u c t i o n , and the product i s then subjected t o a c i d h y d r o l y s i s under c o n d i t i o n s where the h i g h l y a c i d - r e s i s t a n t 2-amino-2-deoxy-D-glucosyl residues would be expected t o remain attached t o t h e i r aglycons, a mixture of products i s obtained from which the aglycons may be detached by deamination w i t h n i t r o u s a c i d . This procedure gives e r y t h r i t o l a r i s i n g from the o r i g i n a l D-glucuronic a c i d r e s i d u e s that had been cleaved by periodate,"and L - t h r e i t o l a r i s i n g from any Li d u r o n i c a c i d r e s i d u e s t h a t had~undergone C - 2 — C - 3 cleavage~by p e r i o d a t e ( F i g u r e 7). Although the observed r a t i o (10) of these two products (2:1) i s s t r o n g l y i n f a v o r of e r y t h r i t o l , the f a c t t h a t some t h r e i t o l i s a l s o formed i n d i c a t e s that some of the Li d u r o n i c a c i d residues i n the o r i g i n a l polymer were not s u l f a t e d at p o s i t i o n s 2 and 3. The q u a n t i t a t i v e r e s u l t s obtained by t h i s sequence of experiments depend t o a l a r g e extent on the method of the treatment of heparin w i t h periodate; there i s no c l e a r - c u t , l i m i t i n g consumption of p e r i o d a t e at the l e v e l of 1 mole per t e t r a s a c c h a r i d e , and the use of an excess of p e r i o d a t e leads to a p r o g r e s s i v e i n c r e a s e i n the t o t a l periodate uptake. The p o s s i b i l i t y that other s u l f a t e d amino sugar polysaccha r i d e s might have anticoagulant p r o p e r t i e s has a t t r a c t e d a good d e a l of a t t e n t i o n . In an e a r l y a p p l i c a t i o n , Wolfrom and Shen Han (11) u t i l i z e d N-deacetylated c h i t i n (chitosan) and s u l f a t e d i t w i t h p y r i d i n e - c h l o r o s u l f o n i c a c i d t o produce an N-sulfated, p a r t i a l l y O - s u l f a t e d product ( F i g u r e 8). This compound had r e l a t i v e l y h i g h a n t i c o a g u l a n t a c t i v i t y , although t h i s was not so

6.

HORTON A N D usui

Sulfated

Gly cosaminogly cans

101

η

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Figure 4.

Heparin: a suggested tetrasaccharide repeating-unit

HEPARIN

1) i o " 4

2) BH ~ 4

POLYALCOHOL pH 2 . 1 dialysis

^58° MIXTURE Sephadex G-15

void-volume fraction

(70%)

with polystyrenesulfonic Figure 5.

tetrasaccharide fraction (18*)

lower fragments (-10*)

acid

Degradative scheme for heparin based on selective periodate cleavage of Ό-glucuronic acid residues

102

CARBOHYDRATE SULFATES

1) i o 2) BH4 4

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

3)

H ,S0

Figure 6.

H +

3

Tetrasaccharide fragment from periodate-oxidized, borohydride-reduced heparin

1)

0.25 mol

I0

4

2) B H ~ OXIDIZED

,

REDUCED

HEPARIN

I ) CMC 2) B H "

3)

CH OH Q

CH 0H

J~°\

+

CH 0H

o

CH 0H

o

/ ~ \

,θΝ_^5\ίΙ/

H

' ° H2

HoMYLi H C

N H

2

H

I CH^OH

o

/ Α

'

Η2θΗ

Ho\_/UcH 1. HNOg

/ ~ \

Α ί « ν

Ηθ\_/1—OS!-/

(20°)

2. B H 6LUCIT0L +

2 , 5 - ANHYDROMANNITOL + T H R E I T O L

+

ERYTHRITOL

+

IDITOL

Figure 7. Alditol components from heparin after sequential periodate oxidation, borohydride reduction, carboxyl reduction, acid hydrolysis, nitrous acid deamination, and borohydride reduction

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

6.

HORTON AND usui

Sulfated

Glycosaminoglycans

103

high as that of heparin i t s e l f . Part of the reason f o r t h i s d i f f e r e n c e may probably be t r a c e d t o the s t r u c t u r a l d i s s i m i l a r i t y between the c h i t o s a n backbone and the a l t e r n a t i n g backbone of heparin i t s e l f , but another important f a c t o r i s undoubtedly the f a c t t h a t the s u l f a t e d c h i t o s a n had a molecular weight some 20 t o hO times higher than the range considered optimal f o r high a c t i v i t y i n heparin. The m a t e r i a l showed acute t o x i c i t y i n the mouse very s i m i l a r t o t h a t of heparin, although there i s evidence f o r a disadvantageous, delayed t o x i c i t y with t h i s m a t e r i a l . In subsequent s t u d i e s by W h i s t l e r and Kosick (12), a s i m i l a r approach was used, but i n a d d i t i o n , the procedure i n c l u d e d an o x i d a t i o n step by use of d i n i t r o g e n t e t r a o x i d e or oxygen—platinum t o introduce some c a r b o x y l groups i n t o the polymer ( F i g u r e 9). This m a t e r i a l l i k e w i s e showed a c t i v i t y of the same order of magnitude as that obtained by Wolfrom and Shen Han ( l l ) , and i t was shovm that s u l f a t e d and o x i d i z e d products were somewhat more a c t i v e than m a t e r i a l s that were s u l f a t e d only; i n a l l instances the a c t i v i t y i n c r e a s e d with i n c r e a s i n g l e v e l s of s u l f a t i o n , and products of the highest a c t i v i t y were obtained when the sequence was allowed t o give s u f f i c i e n t degradation t o shorten the polymer chain t o molecular-weight l e v e l s (osmometry) of the same order of magnitude as t h a t of heparin. In view of the somewhat n o n s p e c i f i c and degradative c h a r a c t e r i s t i c of d i n i t r o g e n t e t r a o x i d e as an oxidant, an e f f o r t was made i n our l a b o r a t o r y ( F i g u r e 10) t o achieve o x i d a t i o n of c h i t o s a n at C-β with a high degree of s p e c i f i c i t y (15). To accomplish t h i s o b j e c t i v e , c h i t o s a n was converted i n t o i t s p e r c h l o r a t e s a l t , f o l l o w i n g the hypothesis that the s t r o n g l y c a t i o n i c group at C-2 should p r o t e c t the hydroxyl group at C-3 against o x i d a t i o n and allow s p e c i f i c and complete o x i d a t i o n at C-6. The l a t t e r step was indeed achieved by use of chromium t r i o x i d e , and a product was obtained t h a t was completely carboxylated; i t presumably e x i s t s as an inner s a l t . S u l f a t i o n of the l a t t e r product gave the f u l l y N-sulfated, C-6 carboxylated c h i t o s a n analog. The p r o p e r t i e s of t h i s product are d i s p l a y e d i n F i g u r e 11, where i t may be observed that the product does have anticoagulant p r o p e r t i e s , although the procedure used l e d i n l a r g e measure t o r e t e n t i o n of the high-polymeric nature of the o r i g i n a l polysaccharide, and so the molecular-weight range of the product thus obtained l a y above t h a t considered optimal f o r h e p a r i n - l i k e activity. C o n t r o l l e d degradation p r i o r t o s u l f a t i o n of t h i s product would o f f e r promise as a p o t e n t i a l method f o r obtaining a s y n t h e t i c h e p a r i n o i d of higher a c t i v i t y . The former approaches, based as they are upon the c h i t o s a n s t r u c t u r e , do s u f f e r from the fundamental drawback t h a t the polymer chain m a n i f e s t l y d i f f e r s from t h a t i n heparin i t s e l f . In view of t h i s disadvantage, we i n i t i a t e d a new s e r i e s of i n v e s t i g a t i o n s based on an abundant ( l - * * h ) - l i n k e d a-D-glucan, namely amylose. In the i n i t i a l s e r i e s of s t u d i e s (15), amylose was converted by o x i d a t i o n of i t s 6 - t r i t y l ether with methyl

CARBOHYDRATE SULFATES

CHpOH

ÇH OS0 -Na +

, CHN (A)

CIS0 H

5

3

J 2

5

3

( heterogeneous) -

\P

or HCONMe ' S 0 (B) 2

3

( homogeneous) Method A'. D. P. 1280 Anticoagulant activity 56 Iu/mg LD Wolfrom

5 0

( mouse, IV) 380mg/kg

and Shen Han (1959) Method Β '.

D. P. 530

Anticoagulant activity 50 Iu/mg

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

LD50 Figure 8.

CH (OH)OS0 H

2

2

f~

HCONMegS0 / HCONMe

N0 2

NH2

4

7

t

or Pt/0

vST /

2

(CO^MCHgOSOjH)

3

°\T^

3

ONlL/

775mg/kg

Sulfation of chitosan

CH OH

X

(mouse, IV)

\ £

2

3

NHSO^

Whistler

and Kosi k , 1971

Figure 9.

Sulfation and oxidation of chitosan

CHOH 2

-o

HCI0

4

NH CI0

NHAc

+

3

4

Chitosan

Chit i η C0 H 2

Cr0

I) CHN

3

5

5

3" NaOH v

NH+CI0

w

5.8 x l O

Figure 10.

3

NHS0 Na

+

3

Disodium ( l - 4 ) - 2 - d e o x y - 2 sulf oamino-/3-D-glucopyranuronan

d.s. 1.0 by carboxyl M

4

5

M , u

4.3 χ I0

5

Specific C-6 oxidation and ^-sulfation of chitosan

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

6.

HORTON AND usui

Sulfated

Gly cosaminogly cans

105

s u l f o x i d e — a c e t i c anhydride t o give a product l a r g e l y o x i d i z e d t o the 2-keto d e r i v a t i v e at c e r t a i n of the residues ( F i g u r e 12). Conditions a f f o r d i n g a degree of s u b s t i t u t i o n of approximately 0. 5 were selected. This product was s u c c e s s i v e l y oximated and then reduced with l i t h i u m aluminum hydride t o give, a f t e r t r i t y l a t i o n , an aminated amylose i n which the amino groups had been l a r g e l y i n c o r p o r a t e d at the 2 - p o s i t i o n i n the D-gluco c o n f i g u r a t i o n , as demonstrated by a c i d h y d r o l y s i s . "The o x i d a t i o n procedure used a l s o introduced (methylthiο)methyl ether groups at 0-3, at l e a s t i n c e r t a i n of the residues. T r i f l u a r o a c e t y l a t i o n of the f r e e amino groups and the r e s i d u a l hydroxyl groups i n the r e d u c t i o n product, followed by d e t r i t y l a t i o n , exposed the h y d r o x y l groups at C-6, and these were subjected t o o x i d a t i o n by the a c t i o n of oxygen—platinum. In view o f the f a c t that t h i s o x i d a t i o n proceeds much more r a p i d l y i n D-glucose residues than i n those of 2-amino-2-deoxy-D-glucose, i t was hoped that the procedure would l e a d t o a polymer i n which residues of 2-amino-2-deoxy-D-glucose would be l a r g e l y unoxidized at the C-6 p o s i t i o n , whereas those of the non-aminated, D-glucose residues would be o x i d i z e d t o the corresponding D-glucuronic a c i d residues. Removal of r e s i d u a l p r o t e c t i n g groups, followed by s u l f a t i o n of t h i s polymer, gave a compound c o n t a i n i n g the r e q u i s i t e f u n c t i o n a l groups f o r a h e p a r i n l i k e s t r u c t u r e , although, as shown i n F i g u r e 12, the anticoagulant a c t i v i t y of t h i s m a t e r i a l was low. This f a c t o r may a r i s e from the f a c t t h a t the product was of lower molecular weight than the values considered optimal f o r h e p a r i n - l i k e a c t i v i t y . This degradation occurred p r i n c i p a l l y at the stage where r a t h e r severe c o n d i t i o n s were r e q u i r e d t o remove the 0-(methylthiο)methyl groups t h a t had become introduced during the o x i d a t i o n step with dimethyl s u l f o x i d e — a c e t i c anhydride. In view of the drawbacks of the use of t h i s p a r t i c u l a r oxidant, Dr. Usui performed a new s e r i e s of experiments i n which 6 - 0 - t r i t y l a m y l o s e was o x i d i z e d by mixtures of N,N-dicyclohexylcarbodiamide (DCC) and dimethyl s u l f o x i d e , i n v a r i o u s proportions. A range of o x i d i z e d products was obtained, as shown i n F i g u r e 13, and these were f r e e of s u l f u r . Reduction of these polymers by borohydride, followed by a n a l y s i s of the sugar composition of the products by means of the a l d i t o l acetates, revealed t h a t o x i d a t i o n had taken place s e l e c t i v e l y at the 2 - p o s i t i o n at low l e v e l s of oxidation. At higher l e v e l s of oxidation, the 3 p o s i t i o n s were a l s o a f f e c t e d , as shown by the nature of the a l d i t o l s produced i n the a n a l y t i c a l sequence of r e a c t i o n s . Oximation of the 6 - 0 - t r i t y l a r a y l o s e s that had been o x i d i z e d t o a f a i r l y low degree of s u b s t i t u t i o n gave the corresponding oximes, whose n i t r o g e n content corresponded t o the l e v e l of o x i d a t i o n determined p r e v i o u s l y by r e d u c t i o n of the o x i d i z e d products w i t h sodium borodeuteride, f o l l o w e d by a n a l y s i s f o r the deuteriumc o n t a i n i n g sugar component ( F i g u r e 1*0. The oximated product was i n turn a c e t y l a t e d t o a c y l a t e the f r e e hydroxyl groups i n the polymer chain and a l s o the h y d r o x y l group of the oxime f u n c t i o n .

CARBOHYDRATE SULFATES

106

LD

(intraperitoneal , mouse), 237 mg/kg

5 0

Anticoagulant

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Leukemia

activity ,

L - 1210 i£ vitro 50%

Figure

26 I.U./mg

at

inhibition,

0.2 μ,Μ ( 100 >xg/ml)

11. Biological properties of C-6 oxi­ dized, N-sulfation chitosan

CHgOTr 2

2

2. H 0 N H , C H N 2

— 0

5

5

OH

5 5 H

2

(NH ) (OH) 2

· 3»°5 5 5.CIS0 H,

4 N

δ 0

Η

3

( NHCOCFa) (NH ) 2

HORTON Figure 12.

—0

(NOH) ( HOH)

2

(CF C0) 0 C

tetrahydrofuran

/(OH) ^OCHgSMefl

1. 0 , Pt 2. H + 3. OH"

CHoOTr 3

-0

Li AIHL

1. M e S 0 , A c 0

and

J U S T , 1973

(NHCOCF) (OH)

C5H5N

Ν

— 0 (NHSO^Na+î (OH) Anticoagulant a c t i v i t y , l7IU/mg

Conversion of amylose into a 6-carboxyl, 2-sulfoamino analog

HORTON AND usui

Sulfated

CH OH

ChUOTr

9

A

Gly cosaminogly cans

.

DCC , C H N , C F C O ^ 5

5

3

Me S0 , 1 . 4 - dioxane 2 o

—Ο

OH Amy I ose (Super I ose)

D.S.

of

products

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

controlled

by

proportions

of

DCC

3

and

—0

CF C0 H

0'

2

x

max

OH 5.8/wn

Suif u r Figure 13.

free

Alternative oxidation procedure for 6-O-trityhmylose

ANALYTICAL

A.

METHOD

Oximation ( Ν - content)

8. NaBH^

reduction

Det r i tylat ion 1

Η

and

l 3

C n.m.r

C. Reduction Hyd roly si s G. I.c. — m. s of a l d i t o l acetates

Figure 14.

Procedures for analysis of oxidized tritylamyloses

6-0-

CARBOHYDRATE SULFATES

CH 0Tr

}H OT -0

CHoOTr

2

-Q

1

NH«OH k

2

-ο

AcoO

r

OH

— ο By DCC method

NOH

NOAc

D.S. 0.35 by ox i me Sulfur - f ree

\ηοχ · . · >"" 5

6

5

7

1. H"

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

1

THF

2. NaOMe

—ο NH

NH

C

D.S. 0.35 by N H Figure 15.

Preparation of 2-amino-2-deoxyamylose of d.s. 0.35

Properties Ninhydrin:

NH

( aid i toi

acetates

from

Soluble

H 0 ,Me SO

Glc

74%

acid

GlcN

23%

2

0.35)

Analysis positive

Jodine: blue complex in

dilute

Aminated amylose (D.S.

2

Aminated amylose

2

I.r,

:

2

2

Ή n.m.r

base

no NHAc,

δ 5.34 ( H - l of GlcN),

by DCC route

5.25 ( H - l

of GlcN)

hydrolyzate)

ManN

3%

AJIN-3 0 % GlcN-3

0%

Mol. wt. : 4 , 0 0 0 - 20,000 ISephadex ID Figure 16.

G - 2 5 , G-75)

7xlO" M (L-I2I0 cells) 6

5 0

Characterization and properties of aminated amylose

Sulfated

HORTON AND USUI

Gly cosaminogly cans

CH OSO~Na

CHUOH

+

2

CHgOSO^Na +

so? Me SO 2

dialysis (Na D.S.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

[ex]

salt)

0.35 + 168 ( H 0) 2

NHSO~Na D.S. 0.35 by

sulfoamino

D.S.

sulfate

1.8

by

Toluidine Blue test: positive Ninhydrin test: negative All primary OH sulfated [ C nmr] Mol. wt. 3,000 - 20,000 Anticoagulant activity* 45 IU/mg ,3

Figure 17.

OSiMe Figure 18.

Sulfation of aminated amylose

3

OSiMe

3

OH

Preparation of 6-O-acetylamylose

+

λ

and Ε η. m. r. a n a l y s i s

1

- Superlose 39 +55^.

- Superlose 1^997^.

trace

U$

k

o f reduced, d e t r i t y l a t e d product.

trace

1$

3$

- By gel-permeation chromatography o f reduced, d e t r i t y l a t e d product.

C

99$

20$

1:0.25

1 3

95$

35$

2:0. 5

- Supported by

93$

80$

h:l

2^

21$

h%

75$

R e s u l t s of g. 1. c. a n a l y s i s — Man All Glc

D.S. by oximation

70$

2

2

5:1

3

DCC/CF C0 H per mol.

3

Analysis of D C C — C F C 0 H - O x i d i z e d 6-O-Tritylamylose

1*

Expt. no.

Table I.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

+ +

~ 20,000 ~ 20,000 35,000

Iodine

8,000

Mol. vrt. —

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

6.

HORTON AND usui

Sulfated

Gly cosaminogly cans

111

Reduction of the l a t t e r product by diborane proved t o be a much more s a t i s f a c t o r y method f o r i n t r o d u c i n g the amino group than the sequence p r e v i o u s l y used t h a t i n v o l v e d d i r e c t r e d u c t i o n of the oxime with l i t h i u m aluminum hydride. The sequence (Figure 15) gave, a f t e r d e t r i t y l a t i o n , an aminated amylose whose degree of s u b s t i t u t i o n corresponded t o the o r i g i n a l oxime content of the precursor, and the p r o p e r t i e s of the keto products at d i f f e r e n t degrees of s u b s t i t u t i o n and as obtained by d i f f e r e n t means of p r e p a r a t i o n are shown i n Table I. A c i d h y d r o l y s i s of the aminated amylose showed that the net amination had been achieved with high r e g i o - and stereoselectivity. At a d. s. l e v e l of 0.35, e s s e n t i a l l y a l l of the amino groups had become i n c o r p o r a t e d at p o s i t i o n 2 i n the D-gluco c o n f i g u r a t i o n ; no s i g n i f i c a n t amino sugar components a r i s i n g from o x i d a t i o n at C-3 were detected i n the product at t h i s degree of s u b s t i t u t i o n . D e t r i t y l a t i o n of the product was r e a d i l y achieved by use of methanolic hydrogen c h l o r i d e followed by treatment with sodium methoxide i n methanol to remove the r e s i d u a l 0 - a c e t y l groups. The product (Figure 16) was r e a d i l y s o l u b l e i n water, i n d i l u t e a c i d , and a l s o i n d i l u t e a l k a l i , s t i l l shewed the i o d i n e - s t a i n i n g a b i l i t y c h a r a c t e r i s t i c of amylose, but a l s o showed the n i n h y d r i n r e a c t i o n c h a r a c t e r i s t i c of the presence of amino groups; the i n f r a r e d spectrum, showing absence of carbonyl absorption, e s t a b l i s h e d t h a t no migration of a c e t y l groups from oxygen t o n i t r o g e n had occurred during the s a p o n i f i c a t i o n step. F i g u r e 17 shows the r e s u l t s of s u l f a t i o n of t h i s aminated amylose by use of dimethyl s u l f o x i d e — s u l f u r t r i o x i d e . This product, obtained i n 7^$ y i e l d a f t e r d i a l y s i s , was s o l u b l e i n water, gave a negative n i n h y d r i n t e s t f o r f r e e amino groups, and a p o s i t i v e T o l u i d i n e Blue t e s t f o r sulfoamino groups. Its anticoagulant a c t i v i t y , as assayed with sheep plasma, was I n t e r n a t i o n a l U n i t s p e r m i l l i g r a m , namely about hO^o of t h a t of commercial heparin. The C n.m. r. spectrum of the product showed that the primary a l c o h o l groups were e s s e n t i a l l y completely s u l f a t e d , because the CH resonance normally occurring near 6 l . 5 p. p.m. was s h i f t e d downfield by about 6 p. p. m. by the s u b s t i t u e n t e f f e c t of the s u l f a t e groups. The product was q u i t e p o l y d i s p e r s e , and i t s molecular-weight range (3,000-20,000) i n d i c a t e d that some measure of degradation had occurred during the t o t a l sequence u t i l i z e d . Approximately one h a l f of the m a t e r i a l i n t h i s product had a molecular weight below the range considered optimum f o r heparin a c t i v i t y . Continuing work i n our l a b o r a t o r y i s concerned with the f r a c t i o n a t i o n of the m a t e r i a l j u s t described and assay of the anticoagulant a c t i v i t y of products containing t h e i r major component i n the 10 t o 12,000 molecular weight range, with v a r i o u s l e v e l s of amination at the C-2 p o s i t i o n . In a f u r t h e r development of p o t e n t i a l u t i l i t y ( F i g u r e 18) a procedure has been e s t a b l i s h e d (15) f o r s p e c i f i c 6 - s u b s t i t u t i o n of amylose by an a c e t y l group; l 3

2

CARBOHYDRATE

112

SULFATES

t h i s 6-0-acetylamylose and analogous primary monoesters of p o l y s a c c h a r i d e s may prove more u s e f u l than the t r i t y l ethers as s t a r t i n g p o i n t s f o r s y n t h e s i s of heparin analogs. Acknowledgments This work was supported by Grant No. HL-11U89 from the N a t i o n a l Heart, Lung, and Blood I n s t i t u t e , N a t i o n a l I n s t i t u t e s of Health, Department of Health, Education, and Welfare, Bethesda, Md. 2001U.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Literature Cited 1 Jeanloz, R. W., in W. Pigman and D. Horton, Eds., "The Carbohydrates", 2nd ed., Vol. IIB, pp. 609—615, Academic Press, New York, 1970; Lindahl, U., MTP Int. Rev. Sci. Ser. Two, Vol. 7, Carbohydr. (1976) 283—312. 2 Jaques, L. Β., Methods Biochem. Anal. (1977) 24, 203—312; Muir, Η., and Hardingham, Τ. Ε., MTP Int. Rev. Sci. Ser. One, Vol. 5 Biochem. Carbohydr. (1975) 153—222. 3 Johnson, E. A. and Mulloy, Β., Carbohydr. Res. (1976) 51, 119— 127. 4 Hőők, Μ., Lindahl, U., Bäckstrőm, G., Malmstrőm, A., and Fransson, L. -Å., J. Biol. Chem. (1974) 249, 3908—3915. 5 Wolfrom, M. L., Vercellotti, J. R., and Horton, D., J. Org. Chem., (1964) 29, 540—550; Wolfrom, M. L., El Khadem, H. S., and Vercellotti, J. R., ibid. (1964) 29, 3284—3286; Wolfrom, M. L., Tomomatsu, Η., and Szarek, W. Α., ibid. (1966) 31, 1173—1178. 6 Wolfrom, M. L., Honda, S., and Wang, P. Υ., Carbohydr. Res. (1969) 10, 259-265. 7 Hovingh, P., and Linker, Α., Biochem. J. (1977) 165, 287—293; but see also Silva, Μ. Ε., Dietrich, C. P., and Nader, Η. Β., Biochim. Biophys. Acta (1976) 437, 129—141. 8 Perlin, A. S., Mackie, D. Μ., and Dietrich, C. P., Carbohydr. Res. (1971) 18, 185—194. 9 Nieduszynski, I. Α., and Atkins, E. D. T., Biochem J. (1973) 135, 729—731. 10 Horton, D., Liav, A., and Toman, R., unpublished data. 11 Wolfrom, M. L., and Shen Han, Τ. -Μ., J. Am. Chem. Soc. (1959) 81, 1764—1766. 12 Whistler, R. L., and Kosik, Μ., Arch. Biochem. Biophys. (1971) 142, 106—110. 13 Horton, D., and Just, Ε. Κ., Carbohydr. Res. (1973) 29, 173— 179. 14 Horton, D., and Just, Ε. Κ., Carbohydr. Res. (1973) 30, 349— 357. 15 Horton, D., and Lehmann, J., Carbohydr. Res. (1978) 61, 553-556. RECEIVED

M a y 8,

1978.

7 Heparin Derivatives of High Molecular Weight L . M E S T E R , A . A M I T A M A Y A , and M . M E S T E R

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

Institut de Chimie des Substances Naturelles, C.N.R.S., 91190 Gif-Sur-Yvette, France

Many attempts have been made in the last two deca­ des (1-6) to modify the s t r u c t u r a l features of the hepa­ r i n molecule (7,8) in order to produce changes in i t s b i o l o g i c a l a c t i v i t i e s (9,12), but only very few, to mo­ dify the size of the molecule (10,11,13). High molecu­ lar weight heparin preparations are now obtained through methacrylation of heparin and polymerization of the he­ parin methacrylate monomer. Methacrylation and polymerization Heparin (0.1 mMol) is dissolved in 0.5 Ν sodium hydroxide solution and methacryl chloride (4 m M o l s ) is added with s t i r r i n g at room temperature and then heated to 80°C for 2 hours. The solution is neutralized with acetic acid and evaporated under reduced pressure. The heparin methacrylate ester (Figure 1) is poly­ merized by heating with a z o d i i s o b u t y r o n i t r i l e as cata­ lyst in 1,4-dioxane solution at pH = 5. The polymeriza­ tion is stopped by adding hydroquinone and the solution is evaporated under reduced pressure. Operating in this way, the main product is a water soluble heparin methacrylate polymer having a molecular weight of about 40.000, as shown by u l t r a c e n t r i f u g a t i o n . The molecular weight of the initial heparin being 18.000, the polymer is a dimer of heparin. The polymerized hepa­ r i n is isolated from unchanged heparin by passage through a Sephadex G-200 column, equilibrated with an aqueous ammonia solution to pH = 8. The heparin-rich fractions are detected by their methacromatic effect with toluene blue. The fractions containing the water soluble heparin polymer are l y o p h i l i z e d and investigated by u l t r a c e n t r i fugation.

0-8412-0426-8/78/47-077-113$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

Figure 1.

Methacrylated heparin segment

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

(73

Η Μ

>

d

Η Μ

ω

ο

>

ο

I—«

7.

Heparin

MESTER E T A L .

Derivatives

115

Water i n s o l u b l e m e t h a c r y l a t e d h e p a r i n p o l y m e r s are o b t a i n e d when more m e t h a c r y l c h l o r i d e i s u s e d f o r e s t e r i f i c a t i o n and when t h e p o l y m e r i z a t i o n i s c a r r i e d o u t a t pH = 3. The i n s o l u b l e h e p a r i n p r e p a r a t i o n s a r e w a s ­ h e d t h r e e t i m e s w i t h i c e c o l d w a t e r , c e n t r i f u g a t e d and l y o p h i l i z e d . The m o l e c u l a r w e i g h t o f t h e i n s o l u b l e h e p a ­ r i n p o l y m e r s i s h i g h e r t h a n 2 0 0 . 0 0 0 . The w a t e r i n s o l u ­ b l e p o l y m e r s may be u s e d f o r c o a t i n g m e t a l s u r f a c e s w i t h l a y e r s showing a n t i t h r o m b i c activity.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

Ultracentrifugation U l t r a c e n t r i f u g a t i o n o f h e p a r i n ( I ) and o f m e t h a c r y l a t e d h e p a r i n p o l y m e r ( I I ) was c a r r i e d out i n a s u c r o s e g r a d i e n t o f 5 % t o 20 %, i n SPINC0 C e n t r i f u g (41.000RPM, 30 h o u r s , 2 ° C ) , u s i n g s e r u m a l b u m i n (SA) and c y t o c h r o m (CY) as i n t e r n a l r e f e r e n c e s . On ' F i g u r e 2, f r a c t i o n I i s h e p a r i n w i t h a c o n s t a n t o f s e d i m e n t a t i o n S = 2 c o r r e s p o n d i n g to a m o l e c u l a r w e i g h t o f 18.000, f r a c t i o n I I i s a p o l y m e r o f m e t h a c r y l a t e d h e p a r i n , i s o l a t e d on S e p h a d e x G-200 c o l u m n and s h o w i n g a s e d i m e n t a t i o n c o n s t a n t S = 4, w h i c h c o r r e s ­ ponds to a m o l e c u l a r w e i g h t of about 40.000. C o p o l y m e r i ζat i o n C o p o l y m e r i z a t i o n of m e t h a c r y l a t e d h e p a r i n with v i ­ n y l o r m e t h a c r y l monomers i s an u n i q u e m e t h o d to c h a n g e t h e g e o m e t r y and p h y s i c o - c h e m i c a l p r o p e r t i e s o f t h e h e ­ p a r i n m o l e c u l e . F i g u r e 3 shows : A. a f r a g m e n t o f t h e m e t h a c r y 1 a t e d h e p a r i n p o l y m e r and _B. a f r a g m e n t o f a c o ­ polymer of m e t h a c r y l a t e d h e p a r i n w i t h b u t y l m e t h a c r y l a t e . C o p o l y m e r i z a t i o n of m e t h a c r y l a t e d h e p a r i n w i t h v i n y l laur a t e gave l i p o s o l u b l e p o l y m e r s . Copolymerization with r e t i c u l a n t s , like divinylbenzene or Ν , Ν - m e t h y 1 e n e b i s - a c r y l a m i d e i s e s p e c i a l l y e f f e c t i v e to change the geometry of the p o l y m e r i z e d he­ parin molecule. T

Biological

activities

of

the

polymers

One o f t h e most i m p o r t a n t c u r r e n t a i m s f o r c h e m i c a l m o d i f i c a t i o n of the s t r u c t u r e of h e p a r i n i s the d i s s o c i a ­ t i o n o f i t s a n t i t h r o m b i c and a n t i l i p e m i c a c t i v i t i e s . A d e c r e a s e i n t h e f i r s t a n d / o r an i n c r e a s e i n t h e s e c o n d has b e e n o b s e r v e d e a r l i e r by p a r t i a l h y d r o l y s i s (4^) , p e ­ r i o d a t e o x i d a t i o n (6^), i r r a d i a t i o n (.5) o r m o d i f i c a t i o n o f t h e N - s u l f ο g r o u p s (2^^3) . A s i m i l a r d i s s o c i a t i o n o f t h e two a c t i v i t i e s i s r e p o r t e d t h r o u g h p o l y m e r i z a t i o n and c o p o l y m e r i z a t i o n methacry1ated heparin.

now of

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

116

Figure 2. IJltracentrifligation in sucrose gradient 5-20% (Spinco, 41.000 RPM, 30 hr, 2°C); heparin (I) and methacrylated heparin polymer (II).

CHI CH - C I CO - HEPARIN

CHI CH - C I CO - HEPARIN

CHI CH - C (!o - HEPARIN

CHI CH - C (!o-0-CH CH CH CH

3

0

η Q

Figure 3.

2

3

?

2

2

2

2

(A) Polymer of heparin methacrylate; and (B) co-polymer of heparin methac­ rylate with butyl methacrylate

3

7. mester et al.

Heparin Derivatives

117

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

Table I shows thrombin-time and anti1ipemic a c t i v i t y of the methacrylated heparin polymer (II) and of the copolymer of methacrylated heparin with butyl methacrylate ( I I I ) , compared with the corresponding values obtained with heparin (I) in rats by intravenous i n j e c tion of 1 mg/kg doses of the compounds. A l l samples were dissolved in physiological sodium chloride s o l u t i o n . Polymer (II) shows an increased antithrombic a c t i v i t y of short duration and a decreased antilipemic a c t i v i t y . The butyl methacrylate copolymer (III), distinguished i t s e l f through a considerably increased antilipemic a c t i v i t y with s l i g h t l y decreased antithrombic a c t i v i t y . Similar results were obtained through intravenous administration of 2 mg/kg doses of the compounds in rabb i t s , as shown in Table I I . In subcutaneous administration (5 mg/kg) most of the polymers and copolymers show a h e p a r i n - l i k e a c t i v i ty, however, their antilipemic a c t i v i t y was decreased or considerably delayed. C i r c u l a r dichroism measurements 14 C i r c u l a r dichroism data of heparin (I), of methacrylated heparin polymer (II) and of the copolymer of methacrylated heparin with butyl methacrylate (III) in water solution are shown on Figure 4, measured with a "Dichrograph-II" JOUAN, P a r i s . In both polymers (II) and ( I I I ) , the (+) and (-) Cotton-effects are increased, when compared with heparin. However, for the polymer (II) the increase of the (-) Cotton-effect is greater (110 %) than the increase of the (+) Cotton-effect (69%). The reversed phenomenon is observed for the copolymer (III) . These changes in the Cotton-effects are due probably to a change in the geometry of the polymerized heparin molecule with methacrylate and/or with butyl methacrylate. This observation should also be taken in consideration to explain the dissociation of the antithrombic and antilipemic a c t i v i t i e s of the heparin polymers. Acknowledgement The authors are grateful to the Hoffmann-La Roche Co., Basle, Switzerland, for testing the heparin polymers . Abstract Heparin methacrylate is polymerized in 1,4-dioxane solution by heating with a z o d i i s o b u t y r o n i t r i l e as cata-­

Heparin polymer

II.

10.5

14.5

11.2

10 m i n .

THROMBIN

TABLE I I

7.5

7.5

9.2

7.3

8.8

180 m i n .

IN SECOND

60 m i n .

TIME

30

1 7

methacrylate

Roche

+ Quick s

T

method

using

4.3

4.6

4.3

0 min.

THROMBIN

4.7

4.9

5.5

16 E / m l t h r o m b i n

6.4

14.0

8.9

0 min.

20

1 2

20

120 m i n .

ACTIVITY 20 m i n .

ANTILIPEMIC

solution

120 m i n .

IN SECOND

20 m i n .

TIME

180 m i n .

OF 2 mg/kg

8

1 8 2

60 m i n .

1 2

1 mg/kg

ACTIVITY

OF

10 m i n .

ANTILIPEMIC

INJECTION

A C T I V I T Y AND THROMBIN TIME AFTER INTRAVENOUS I N J E C T I O N HEPARIN OR HEPARIN POLYMER IN RABBITS

Copolymer of h e p a r i n methacrylate with butyl methacrylate

Heparin

COMPOUNDS

I.

III.

methacrylate

Roche

ANTILIPEMIC

I

A C T I V I T Y AND THROMBIN TIME AFTER INTRAVENOUS HEPARIN OR HEPARIN POLYMER IN RATS

Copolymer of h e p a r i n methacrylate with butyl methacrylate

Heparin polymer

II.

III.

Heparin

I.

COMPOUNDS

ANTILIPEMIC

TABLE

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

H

>

C/3 r

>

d H M

Hi

w ο Κ

>

ο

MESTER E T A L .

Heparin

Derivatives

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

+ 110%

ηm

Figure 4. Circular dichroism data in water (0.75 mg/mL) of heparin (I), methacryhted heparin polymer (II), and co-poly­ mer of methacryhted heparin with butyl methacrylate (III)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch007

120

carbohydrate sulfates

l y s t . Depending on the degree of polymerization, solu­ ble of gelatinous heparin preparations of high molecu­ lar weight are obtained, and separated on a column of Sephadex G-200 from unchanged heparin. A higher degree of polymerization of heparin methacrylate with alkyl methacrylates or v i n y l derivatives resulted in l i p o s o luble heparin preparations. Copolymerization with such r e t i c u l a n t s as divinylbenzene or Ν,N'-methylene-bisacrylamide changed completely the geometry of the molecule. C i r c u l a r dichroïsm measurements were used to follow the structural change of heparin methacrylate polymer and of i t s copolymer with butyl methacrylate. Some of the high molecular weight heparin preparations show a d i s s o c i a ­ tion between the anticoagulant and antilipemic a c t i v i ­ ties of heparin : the antithrombic a c t i v i t y decreased, while the antilipemic a c t i v i t y increased considerably or remained unchanged. Insoluble heparin preparations can be used for coating of surfaces. Literature cited 1. Foster A . B . and Huggard A.J., Adv.Carbohydr.Chem., 1955, 10, 335, Acad.Press, New-York. 2. Velluz L., Plotka C. and Nominé G . , Compt.Rend.Acad. Sci.Fr., 1958, 247, 2203. 3. Velluz L., Nominé G. and Mathieu J., Bull.Soc.Chim. Biol., 1959, 41, 415. 4. Nominé G . , Bucourt R. and Bertin D . , Bull.Soc.Chim. Fr., 1961, 561. 5. Adams S . S . , Heathcote B.V. and Macey P.E., J.Pharm. Pharmacol., 1961, 13, 240. 6. Inch T . D . , Ph.D.Thesis, Birmingham , 1963 . 7. Jacques L.B., Kavanagh L . W . , Mazurek M. and P e r l i n A . S . , Biochem.Biophys.Res.Comm., 1966, 24, 447. 8. P e r l i n A . S . , Ng Ying Kin N . M . K . , Bhattacharjee S.S. and Johnson L.F., Can.J.Chem., 1972, 50, 2437. 9. Jeanloz R.W., in "The Carbohydrates-Chemistry and Biochemistry" 2B, p.589, Ed. by Pigman W., Horton D. and Herp Α . , Acad.Press, New-York and London (1970). 10. Patat F. and E l i a s H., Naturwiss, 1959, 46, 322. 11. Laurent T.C., Arch.Biochem.Biophys., 1961, 92, 224. 12. Kiss J., in "Heparin-Chemistry and C l i n i c a l Usage" p . 3 , Ed.by Kakkar V . V . and Thomas D . P . , Acad.Press, New-York and London , 1976 . 13. Horner A . A . in "Heparin-Chemistry and C l i n i c a l Usa­ ge" p.37, Ed.by Kakkar V . V . and Thomas D . P . , Acad. Press, New-York and London, 1976 . 14. Stone A.L., in "Meth.Carbohydr.Chem.", 7, p.120, Ed.by Whistler R . L . and BeMiller J.N., Acad.Press, New-York and London , 1976 . Received February 6, 1978.

8 Enzymatic Formation and Hydrolysis of Polysaccharide Sulfates K A L Y A N K. DE, K A Z U H I K O Y A M A M O T O , and R O Y L . W H I S T L E R

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Department of Biochemistry, Purdue University, Lafayette, I N 47907

Naturally occuring polysaccharide sulfate esters are widely distributed. These high molecular weight anionic polymers possess rheological and complexing properties that have drawn the attention of industrial interests and lead to extensive commerciall i z a t i o n of several of the polysaccharides. This, in turn, has influenced fundamental and application research to bring about a better understanding of the behavior of hydrocolloids. The next step i s a rational design and development of polysaccharide sulfates to more perfectly serve practical needs. The great bulk of natural occurring polysaccharide sulfates are found in seaweeds where they serve structural functions and possibly act as ion exchange agents and natural absorbents to hold large quantities of water for proper functioning of the sea plant. While normally water soluble, the polysaccharides are retarded from escape into the enveloping sea by the matrix character of the plant c e l l w a l l . Man has extracted these polymers and made use of their highly viscous nature and their gel forming properties. In addition, use i s made of their unique characteristic of combining with protein to produce complexes such as the useful one between carrageenan and the protein in chocolate to maintain suspension uniformity in chocolate milk. Other sulfated polysaccharides are widely distributed in animal tissues where they again serve a water holding use, provide emolliency, l u b r i c i t y and complexing characteristics. Heparin serves a special function in the control of blood coagulation. It can be expected that sulfated, and perhaps phosphorylated polysaccharides, w i l l develop greater pharmaceutical and industrial applications. Applications would be f a c i l i t a t e d by finding techniques by which sulfate-groups could be inserted into and removed from polysaccharides by expectedly low cost and surely the more specific means provided by enzymes. The present review is a summary of the existing knowledge of enzymes that transfer sulfate to ester positions in polysaccharides and of those 0-8412-0426-8/78/47-077-121$06.75/0 ©

1978 A m e r i c a n C h e m i c a l Society

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

122

CARBOHYDRATE SULFATES

enzymes t h a t c a t a l y z e s u l f a t e e s t e r h y d r o l y s i s . Although l i t t l e exact i n f o r m a t i o n i s p r e s e n t l y a v a i l a b l e , the e x c e l l e n t work so f a r done points to the l i k e l i h o o d of r a p i d f u r t h e r development. With proper a p p l i c a t i o n of e v o l v i n g i n f o r m a t i o n , p r a c t i c a l use may w e l l be made of s u l f a t e e s t e r forming and h y d r o l y z i n g enzymes. In the enzymatic formation of s u l f a t e e s t e r s , the s u l f a t e group i s t r a n s f e r r e d from a donor s u b s t r a t e to a polysaccharide acceptor. Only three donor substrates have been i d e n t i f i e d . Ascorbate 2 - 0 - s u l f a t e and adenine 3'-0^-phosphate-5'-0-phosphos u l f a t e are two n a t u r a l donors and p-nitrophenyl s u l f a t e i s a s y n t h e t i c donor. A d d i t i o n a l s y n t h e t i c donors may soon be designated. S u l f a t e t r a n s f e r from j j - n i t r o p h e n y l s u l f a t e and from ascorbate 2 - 0 - s u l f a t e r e q u i r e the presence of ATP as a c o f a c t o r and, hence, adenine 3'-0-phosphate-5'-0-phosphosulfate (PAPS) may be the u n i v e r s a l designate intermediate and s o l e donor f o r a l l s u l f a t e t r a n s f e r a s e s . When other donors are present they presumably i n t e r a c t with ATP through the c a t a l y t i c i n f l u e n c e of two a d d i t i o n a l enzymes to provide PAPS. S u l f a t a s e s capable of h y d r o l y z i n g the h a l f - e s t e r s u l f a t e l i n k a g e to carbohydrates have been observed s i n c e 1931. The group o f esterases t h a t hydrolyze s u l f a t e linkages i n a v a r i e t y of simple sugar s u l f a t e s are termed g l y c o s u l f a t a s e s (sugar s u l f a t e s u l f o h y d r o l a s e s Ε C. 3.1.6.3). S u l f a t a s e s capable of h y d r o l y z i n g s u l f a t e linkages i n polysaccharide s u l f a t e s are named a f t e r t h e i r s u b s t r a t e , as f o r example, chondrosulfatases hydrolyze c h o n d r o i t i n s u l f a t e s , and c e l l u l o s e s u l f a t a s e hydrolyzes c e l l u l o s e s u l f a t e s . The s u l f a t a s e s may be completely s u b s t r a t e s p e c i f i c even as to l o c a t i o n of the s u l f a t e group, but some are not f u l l y s u b s t r a t e s p e c i f i c since apparently a s i n g l e enzyme can hydrolyze both charonin (charonan) s u l f a t e and c e l l u l o s e s u l f a t e . A l l s u l f a t a s e s are a b s o l u t e l y s p e c i f i c f o r the s u l f a t e group. The enzymes are obtained from a v a r i e t y of sources such as m i c r o b i a l , molluscan and higher animals. None have been observed i n higher p l a n t s . Enzymatic S u l f a t i o n Enzymatic s u l f a t i o n of polysaccharide i s u s u a l l y e f f e c t e d through the t r a n s f e r of s u l f a t e from a d e n i n e - 3 ' - p h o s p h o - 5 ' phosphosulfate (3'-PAPS) i n a r e a c t i o n c a t a l y z e d by s u l f o t r a n s f e r ase [E C. 2.8.2] as shown o r i g i n a l l y by F. D'Abramo and F. Lipmann (1_). The process of s u l f a t i o n w i t h i n o r g a n i c s u l f a t e proceeds i n several s t e p s . F i r s t i s the a c t i v a t i o n of s u l f a t e to form adenine-5'-phosphosulfate. This i s immediately f o l l o w e d by phosphorylation at the 3 ' - p o s i t i o n to produce adenine 3'-phospho5 ' - p h o s p h o s u l f a t e , f o l l o w e d by the t r a n s f e r of s u l f a t e from 3'-PAPS to the a c c e p t o r , c a t a l y z e d by s u l f o t r a n s f e r a s e .

8.

DE E T A L .

Polysaccharide

A c t i v a t i o n s t e p s , SO^

Sulfates

123

+ ATP

AMP-S0=

> AMP-S0 ~ + p p . " 3

+ ATP

»

3 ' - P 0 - A M P - S 0 ~ + ADP 3

3

T r a n s f e r r i n g s t e p , ROH + 3 -P0~-AMP-S0 "" (R-NH ) ,

o

6

3

2

>

R 0 - S 0 " + 3'-P0 -AMP 3

3

(RNH-S0 ')

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

3

S u ! f o t r a n s f e r a s e s are of common occurance i n animal t i s s u e s t h a t c o n t a i n aminoglycans and i n p l a n t t i s s u e s , e s p e c i a l l y a l g a l c e l l s , t h a t are major producers of s u l f a t e d p o l y s a c c h a r i d e s . However, evidence f o r the mechanism of the enzymatic r e a c t i o n and c h a r a c t e r i z a t i o n of the enzyme i s incomplete due to d i f f i c u l ­ t i e s i n o b t a i n i n g homogeneous enzyme p r e p a r a t i o n s . Enzyme preparations from c h i c k embrionic c a r t i l a g e ( l _ - 3 h hen o v i d u c t ( 4 ) , hen uterus ( 5 J , r a b b i t uterus ( 6 J , beef lung ( 7 ) , beef eyes ( 8 ) , r a t b r a i n ( 9 J , mouse l i v e r (1_0) serum (11), mast c e l l tumor Τ]_2-1_4), squid c a r t i l a g e (15), molluscus (ΙβΤΤ and marine gastropod (1_7) have been reported to c a t a l y z e s u l f a t i o n of exogeneous and endogeneous a c c e p t o r s . Occurrence of s u l f o t r a n s f e r a s e and 3'-PAPS as s u l f a t e donors (18) are i n d i c a t e d i n red algae Chondrus c r i s p u s (19), Porphyridium (20, 21_) and i n the brown a l g a e , Fucus (22-25). Experiments w i t h i n c o r p o r a t i o n of r a d i o a c t i v e s u l f a t e i n t o carrageenan of Chondrus (19) and i n t o c a p s u l a r p o l y s a c c h a r i d e of Porphyridium (20) demonstrate that the i n c o r p o r a t i o n i s a remarkably r a p i d process through 3'-PAPS as a s u l f a t e p o o l . In Porphyridium 3'-PAPS was a c t u a l l y i s o l a t e d as a water s o l u b l e intermediate ( 2]_). The r a p i d i t y w i t h which s u l f a t e i n c o r p o r a t i o n takes place seems to f a v o r the idea t h a t the s u l f a t i o n of a preformed polymer occurs. During e a r l y embryogenesis of Fucus, exogeneous r a d i o a c t i v e s u l f a t e can be incorporated i n t o an a c i d - s o l u b l e fucose polymer w i t h i n 10 hr. a f t e r f e r t i l i z a t i o n . Experimental evidence i n d i c a t e s t h a t enzymatic s u l f a t i o n occurs i n the region where fucan i s formed and where i t can immediately act as an acceptor (25J). Although several attempts have been made to o b t a i n from these a l g a e , c e l l - f r e e enzyme system which can t r a n s f e r the s u l f a t e from 3'-PAPS to f r e e sugar, sugar n u c l e o t i d e , or p o l y s a c c h a r i d e , no p o s i t i v e r e s u l t s have y e t been seen. L i t t l e i s known of the s p e c i f i c i t y of s u l f o t r a n s f e r a s e and i t i s not e s t a b l i s h e d whether there i s a s i n g l e n o n - s p e c i f i c s u l f o t r a n s f e r a s e or a number of more s p e c i f i c enzymes. The l a t t e r case becomes more l i k e l y as the enzyme systems are i n v e s t i g a t e d in greater d e t a i l . Results of the e a r l i e r work on the s p e c i f i c i t y of the crude enzyme p r e p a r a t i o n are summarized i n Table I. 9

CARBOHYDRATE SULFATES

124

Table I

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Tissue

Acceptor s p e c i f i c i t y of some crude preparations of mucopolysaccharide s u l p h o t r a n s f e r a s e s P o t e n t i a l Acceptors Utilized Not U t i l i z e d Reference

rabbit skin hen o v i d u c t chick cartilage human leimyosarcoma human mammary carcinoma

dermatan sulphate h e p a r i t i n sulphate c h o n d r o i t i n 4- and 6-sulphates dermatan sulphate dermatan c h o n d r o i t i n 4sulphate and 6-sulphates chondroitin, c h o n d r o i t i n 4and 6 - s u l p h a t e s , dermatan s u l p h a t e , h e p a r i t i n sulphate

26 27 28 29

30

More d e t a i l e d i n v e s t i g a t i o n s on the s p e c i f i c i t y of s u l f o t r a n s f e r a s e have been conducted i n an enzyme system s o l u b i l i z e d from hen o v i d u c t ( 4 J . This c e l l - f r e e system i s capable of c a t a l y z i n g the t r a n s f e r of r a d i o a c t i v e s u l f a t e from 3'-PAPS to a number of aminoglycans. The r e l a t i v e rates of t r a n s f e r are shown i n Table II. Further experiments of the system show t h a t simple o l i g o s a c c h a r i d e s c o n t a i n i n g N-acetyl-D-galactosamine can a l s o a c t as acceptors f o r the s u l f o t r a n s f e r a s e although the o l i g o s a c c h a r i d e s are r a t h e r l e s s e f f i c i e n t than the p o l y s a c c h a r i d e s . Both mono- and d i s u l f a t e d d e r i v a t i v e s of N - a c e t y l - D - g a l a c t o samine residues i n the o l i g o s a c c h a r i d e s are formed i n the r e a c t i o n (31, 32). Measurements of r e a c t i o n v e l o c i t y at d i f f e r e n t c o n c e n t r a t i o n s show that the V and the Michael i s constant (K^) d i f f e r among carbohydrate s u b s t r a t e s . The higher V for c h o n d r o i t i n 6 - s u l f a t e as an acceptor compared to c h o n a r o i t i n 4 - s u l f a t e i s i n agreement w i t h the data shown i n Table II.

8.

DE E T A L .

Polysaccharide

Sulfates

Table

125

II

R e l a t i v e extents of sulphate t r a n s f e r to d i f f e r e n t acceptors by the amino glycan sulphotransferases of hen oviduct and of chick embryo c a r t i l a g e

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Acceptor c h o n d r o i t i n 4-sulphate c h o n d r o i t i n 6-sulphate dermatan sulphate h e p a r i t i n sulphate chondroitin (natural) c h o n d r o i t i n (chemical) hyaluronic acid heparin keratosulphate

Oviduct (4)

C a r t i l a g e (2)

1.00 1.8 0.72 0.45 1.8 0.62 0.00 0.00 0.00

1.00 0.46 0.75 1.3

-

0.13 0.08 0.26 0.25

The s u l f o t r a n s f e r a s e system i n the 105000 χ g supernatant f r a c t i o n prepared from the homogenate of c h i c k embryo c a r t i l a g e [2) can use a g r e a t e r number o f polysaccharide acceptors than the oviduct enzyme (Table I I ) . An endogeneous polysaccharide acceptor occurs i n the same enzyme preparation (33). A f t e r d e s u l f a t i o n of t h i s acceptor w i t h methanolic-hydrcgen c h l o r i d e , i t no longer accepts s u l f a t e . S i m i l a r l y , c h o n d r o i t i n prepared by d e s u l f a t i o n of c h o n d r o i t i n 4 - s u l f a t e does not a c t as an acceptor. The a b i l i t y of s u l f a t e acceptors i s l i t t l e i n f l u e n c e d by t h e i r molecular s i z e but i s s t r o n g l y dependent on the charge on the molecules. Treatment of p r o t e i n - p o l y s a c c h a r i d e complex endogeneous acceptors w i t h proteinase or w i t h a l k a l i increase t h e i r a b i l i t y to accept s u l f a t e . However, there i s a s t r i k i n g change i n the type of s u l f a t i o n t h a t r e s u l t s from removal of p r o t e i n . When the aminoglycan i s combined w i t h p r o t e i n s u l f a t e , e s t e r s are formed predominantly a t a x i a l hydroxyl groups. For example, s u l f a t e groups are introduced a t p o s i t i o n 4 of N - a c e t y l D-galactosamine u n i t s . On removal of p r o t e i n , s u l f a t i o n occurs p r i n c i p a l l y a t e q u a t o r i a l or primary hydroxyl groups. One p o s s i b l e e x p l a n a t i o n f o r t h i s i s t h a t although the p r o t e i n p o r t i o n of the endogeneous p r o t e i n - p o l y s a c c h a r i d e complex i s not necessary f o r s u l f a t e i n c o r p o r a t i o n , i t aids i n determining the s i t e s f o r s u l f a t i o n . A s u l f o t r a n s f e r a s e p u r i f i e d from mouse l i v e r homogenate (10) t r a n s f e r s s u l f a t e from 3'-PAPS only to p o s i t i o n C-6 of an N-acetyl-D-galactosamine residue i n c h o n d r o i t i n s u l f a t e and i t s Michael i s constant can be c l o s e l y c o r r e l a t e d to the degree of sulfation. In the s o l u b l e enzyme systems, only a small amount of s u l f a t e i s incorporated i n t o p o l y s a c c h a r i d e . Most of the c h o n d r o i t i n s u l f a t i n g a c t i v i t y i s found to occur as microsomal enzyme r a t h e r than as s o l u b l e enzyme (34,35J. A s u l f o t r a n s f e r a s e

CARBOHYDRATE

126

i n a microsomal preparation from chick embryo c a r t i l a g e t r a n s f e r s a greater amount of s u l f a t e from 3'-PAPS to endogeneous g l y c o saminoglycan than does the s o l u b l e enzyme {3 36). Incubation of the mcirosomal preparation at pH 6.5 with 3'-PAPS r e s u l t s i n the i n c o r p o r a t i o n of s u l f a t e i n t o endogeneous c h o n d r o i t i n 6 - s u l f a t e (60-70%) and i n t o c h o n d r o i t i n 4 - s u l f a t e (30-40%) w h i l e incubation at pH 7.8 r e s u l t s i n t h e . i n c o r p o r a t i o n of s u l f a t e i n t o c h o n d r o i t i n 6 - s u l f a t e e x c l u s i v e l y (37). Although the s u l f o t r a n s f e r a s e a c t i v i t y of chick embryo c a r t i l a g e i s predominantly associated with membrane, a s i g n i f i c a n t proportion of the enzyme a c t i v i t y i s found i n the 105000 χ g supernatant f r a c t i o n (Golgi p r e p a r a t i o n ) . This preparation contains a c t i v i t y f o r 4- and 6 - s u l f a t i o n of endogeneous p o l y s a c c h a r i d e . However, heat treatment of the preparation causes more r a p i d l o s s of a b i l i t y to s u l f a t e p o s i t i o n C-4 of the polysaccharide than l o s s of a b i l i t y to s u l f a t e p o s i t i o n C-6 (38) ( F i g . 1). This suggests t h a t a s p e c i f i c enzyme i s a c t i v e f o r s u l f a t i o n at each p o s i t i o n . Moreover, by chromatography on Sephadex G-200 the enzyme f o r 6 - s u l f a t i o n can be obtained e s s e n t i a l l y f r e e from the a c t i v i t y f o r 4 - s u l f a t i o n . The enzyme e f f e c t i n g 4 - s u l f a t i o n i s not recovered from chroma­ tography perhaps because of i n a c t i v a t i o n . Thus, w i t h i n the l i m i t e d work on the microsomal preparation and the Golgi p r e p a r a t i o n , the s u l f a t i o n system of chick embryo c a r t i l a g e seems to c o n s i s t of at l e a s t two enzyme species t h a t are h i g h l y s p e c i f i c with regard to the p o s i t i o n s u l f a t e d . An aminoglycan s u l f o t r a n s f e r a s e u t i l i z i n g 3'-PAPS as a s u l f a t e donor and endogeneous p r o t e i n - p o l y s a c c h a r i d e complex as an acceptor has been c h a r a c t e r i z e d (1J5). This enzyme, from squid c a r t i l a g e , shows high s p e c i f i c i t y f o r p o s i t i o n C-6 of N - a c e t y l Q-galactosamine moieties t h a t already bear a s u l f a t e group at p o s i t i o n C-4. The s u l f o t r a n s f e r a s e i s separated from endogeneous p r o t e i n polysaccharide complex and p u r i f i e d some n i n e - f o l d on DEAE-Sephadex A-50. The enzyme i s a c t i v e only w i t h exogeneous acceptors. Although various mono-, d i - , and polysaccharide are s u l f a t e a c c e p t o r s , i t i s e s s e n t i a l t h a t they have a s u l f a t e at p o s i t i o n C-4 of t h e i r j^-acetyl-D-galactosamine r e s i d u e s . The p u r i f i e d enzyme s p e c i f i c a l l y c a t a l y z e s the s u l f a t i o n of l ^ - a c e t y l D-galactosamine 4 - s u l f a t e r e s i d u e s , and produces f^-acetyl-Dgalactosamine 4 , 6 - d i s u l f a t e , whether ^-acetyl-D-galactosamine 4 - s u l f a t e i s i n a p r o t e i n - p o l y s a c c h a r i d e complex or whether i t i s p r o t e i n - f r e e . Most l i k e l y , t h e r e f o r e , the s p e c i f i c i t y of t h i s enzyme does not i n v o l v e the r e c o g n i t i o n of a p a r t i c u l a r s i z e or a p a r t i c u l a r monosaccharide sequence of an aminoglycan acceptor molecule. 9

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

8.

DE E T AL.

Polysaccharide

Sulfates

127

Molecular and Cellular Biochemistry

Figure 1. Heat labilities of two different sulfotransferase activities in the 105,000χ g supernatant of cartilage ho­ mogenate (38)

CARBOHYDRATE SULFATES

128

Another enzyme has been p u r i f i e d from a homogenate of the albumin s e c r e t i n g region of hen oviduct (39-41). The enzyme c a t a l y z e s the t r a n s f e r of s u l f a t e from 3'-PAPS to p o s i t i o n C-6 of the N-acetyl-D-galactosamine 4 - s u l f a t e moiety of u r i d i n e diphosphate-N-acetyl-D-galactosamine 4 - s u l f a t e . Although UDP-N-acetyl-D-galactosamine 4 - s u l f a t e i s the most a c t i v e carbohydrate substrate f o r the enzyme, N-acetyl-D-galactosamine 4 - s u l f a t e and i t s 1-phosphate act as a s u l f a t e acceptor at a comparable r a t e and A ' -glucurc!nido-lN-acetyl-D-galactosamine 4 - s u l f a t e [2-acetamido-2-deoxy-3-0-($-D-gluco-4-enepyranosyluronic a c i d ) - 4 - 0 - s u l f o - D - g a l a c t o s e ] and c h o n d r o i t i n accept s u l f a t e s l o w l y . A more completely p u r i f i e d enzyme has a pH optimum f o r s u l f a t e t r a n s f e r to UDP-N-acetyl-D-galactosamine 4 - s u l f a t e at pH 4.8. The k i n e t i c data f o r s u l f a t e t r a n s f e r to several substrate are summarized i n Table I I I .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

4

5

Table III S p e c i f i c i t y of a sulphotransferase from hen o v i d u c t which can u t i l i z e simple sugars or sugar d e r i v a t i v e s as substrates (39) —

Acceptor

m ( m M )

UDP-N-acetylgalactosamine 4 - s u l f a t e ^-acetylgalactosamine 4-sulfate ^ - a c e t y l g a l a c t o s a m i n e 1-phosphate 4-sulfate Δ -glucuronido-N-acetylgagactosamine 4-sulfate 4

5

y (relative)

0.05 1.4

1.00 0.72

0.13

0.39

2.0

0.05

9

The s u l f o t r a n s f e r a s e s considered so f a r have a l l formed s u l f a t e e s t e r s . However, i n the formation of heparin which contains 0 - s u l f a t e and j f - s u l f a t e , a separate s u l f o t r a n s f e r a s e i s i n v o l v e d . This i s demonstrated by the separate i d e n t i f i c a t i o n of JN- and 0 - s u l f o t r a n s f e r a s e a c t i v i t y i n an enzyme preparation from mouse mastocytoma (14·). Both s u l f o t r a n s f erases can be s o l u b i l i z e d from mastocytoma microsomal f r a c t i o n . The pH optimum f o r the enzymes i s about 7.5. The Michael i s constant f o r 3'-PAPS i s estimated to be 2 χ 10" M f o r the N - s u l f o t r a n s f e r ­ ase and 1 χ 1 0 " f o r the 0 - s u l f o t r a n s f e r a s e . The 0 - s u l f o t r a n s ferase i s more s e n s i t i v e to h e a t - i n a c t i v a t i o n ; 60% of the a c t i v i t y being l o s t a f t e r 1 min. at 50°. Under the same c o n d i t i o n s only 15% of N - s u l f o t r a n s f e r a s e i s l o s t . The ^ - s u l f o ­ t r a n s f e r a s e i s s e l e c t i v e l y i n h i b i t e d by sodium c h l o r i d e , whereas, the 0 - s u l f o t r a n s f e r a s e i s e s s e n t i a l l y u n a f f e c t e d . Work on the acceptor s p e c i f i c i t y of the 0 - s u l f o t r a n s f e r a s e from mouse mastocytoma shows t h a t N - s u l f a t e groups i n acceptor polysaccharides are e s s e n t i a l f o r 0 - s u l f a t i o n . The 0 - s u l f a t e group i s 5

4

8.

DE E T A L .

Polysaccharide

Sulfates

129

p r e f e r e n t i a l l y introduced i n t o the N^-sulfated (and thus p r e v i o u s l y l^-deacetylated) r a t h e r than i n t o N-acetylated regions at heparan s u l f a t e (42,43). Almost a l l work on s u l f o t r a n s f e r a s e has been done on t i s s u e s i n which s u l f a t i o n of aminoglycan occurs. L i t t l e work has been done on the mechanism of s u l f a t i o n of n i t r o g e n - f r e e glycans. However, a s u l f o t r a n s f e r a s e from the mucous gland e x t r a c t s of the marine gastropod, charonia lampus, c a t a l y z e s the t r a n s f e r of s u l f a t e from 3'-PAPS to the glucan p o l y s u l f a t e , charonan s u l f u r i c a c i d (17). Charonan s u l f u r i c a c i d c o n s i s t s of two f r a c t i o n s , one a s u l f u r - p o o r f r a c t i o n w i t h a g l y c o g e n - l i k e s t r u c t u r e and the other a s u l f u r - r i c h f r a c t i o n w i t h a c e l l u l o s e ­ l i k e s t r u c t u r e (44,45). When S i s used i n the donor, more than 95% of the a c t i v i t y i s incorporated to the s u l f u r - r i c h fraction.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

3 2

In a d d i t i o n to 3'-PAPS, other s u l f a t e donors e x i s t . One i s j j - n i t r o p h e n y l s u l f a t e , an e f f e c t i v e s u l f a t e donor i n the s u l f o t r a n s f e r a s e preparation of beef cornea e p i t h e l i c a l e x t r a c t . Here, the s u l f a t e group i s t r a n s f e r r e d from j j - n i t r o p h e n y l s u l f a t e to polysaccharide i n the presence of adenosine 3 ' , 5 ' - d i p h o s p h a t e (56). The s u l f a t i o n of polysaccharide seems to occur by p o l y ­ saccharide s u l f o t r a n s f e r a s e coupling w i t h phenol s u l f o t r a n s f e r a s e or phenol s u l f a t a s e which i s a l s o present i n the corneal e x t r a c t . Another donor i s ascorbate 2 - s u l f a t e (47). When r a d i o a c t i v e s u l f a t e i s used, the s u l f a t e i s found incorporated i n t o c h o n d r o i ­ t i n s u l f a t e i n c h i c k embryo c a r t i l a g e epiphyses. Enzymatic D e s u l f a t i o n Glycosulfatasewas observed f i r s t i n the d i g e s t i v e organs of the t r o p i c a l marine mollusc Charonia lampas (48-52). Subsequently, the enzymewas observed (53-63) i n e x t r a c t s of the d i g e s t i v e organs of marine molluscs from B r i t i s h waters, i n the l a r g e p e r i w i n k l e L i t t o r i n a l i t t o r e a and the common limpet P a t e l l a v u l g a t a . I t i s a l s o present i n the mould Trichoderma v i r i d e ( 6 4 ) , and the b a c t e r i a Pseudomonas carrageenovora (65). A s i m i l a r g l y c o s u l f a t a s e i s observed i n the isthmus region of the hen o v i d u c t (95). From the l i v e r of the marine gastropod Charonia lampas ( T r i t o n a l i a s a u l i a e ) there can be obtained a c e l l u l o s e p o l y s u l f a t a s e (66-68) and a chondrosulfatase (79). A porphyran s u l f a t a s e can be prepared from the algae Porphyra u m b i l i c a l i s (69-72). Chondrosulfatase i s observed a l s o i n p u t r i f a c t i v e b a c t e r i a (73-75), Proteus v u l g a r i s (76,77) and Flavobacterium heparinum (78). In a d d i t i o n to occurance i n the l i v e r of (λ lampas, the enzyme i s found i n the l i v e r of squid Ommastrephes s l o a n i

130

CARBOHYDRATE SULFATES

p a c i f i c u s (81-83), i n r a t l i v e r lysosomes (84), i n bovine aorta (85) and the v i s c e r a of P a t e l l a vulgata (80). Two kerato s u l f a t a s e s are present i n £. lampus (86). A heparin s u l f a t a s e has been i s o l a t e d from Flavobacterium heparinum (87). Cerebroside s u l f a t a s e can be i s o l a t e d from the d i g e s t i v e gland of abalone, H a l i o t i s (88) and the lysosomes of pig kidney and from other organs as l i v e r and spleen (89-91).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Glycosulfatases The g l y c o s u l f a t a s e s ( s u g a r - s u l f a t e s u l f o h y d r o l a s e s , E.C.3.1.6.3) capable of h y d r o l y z i n g e s t e r s u l f a t e linkages i n a v a r i e t y of mono-, d i - , and t r i - s u l f a t e s u b s t i t u t e d monosaccharides and d i s a c c h a r i d e s have been p a r t i a l l y p u r i f i e d and i s o l a t e d by a number of d i f f e r e n t groups from a v a r i e t y of d i f f e r e n t sources. Most of the procedures are r a t h e r long and i n v o l v e d . Several are given as examples. Example 1. Washed and macerated v i s c e r e a l organs of L i t t o r i n a ( 5 7 j are placed i n volumes of acetone at 0 ° , f i l t e r e d , macerated i n f r e s h acetone and thoroughly washed i n acetone. The residue i s suspended i n c o l d water, homogenized at pH 9 and adjusted to pH 7 f o r 15 minutes, c e n t r i f u g e d and the c l e a r supernatant d i a l y z e d and the d i a l y z a t e adjusted to pH 2.3 f o r 2 min and readjusted to 4.6, c e n t r i f u g e d , d i a l y z e d , adjusted to pH 6.7, r i b o n u c l e i c a c i d and salmon roe protamine s u l f a t e added to complete p r e c i p i t a t i o n . A f t e r c e n t r i f u g a t i o n the supernatant i s d i a l y z e d , c e n t r i f u g e d and the supernatant adjusted to pH 8, c e n t r i f u g e d , d i a l y z e d and at pH 5.7 t r e a t e d w i t h ammonium s u l f a t e to 55% of s a t u r a t i o n . A f t e r c e n t r i f u g a t i o n the supernatant at pH 4.9 i s 85% saturated w i t h ammonium s u l f a t e . The p r e c i p i t a t e i s removed by c e n t r i f u g a t i o n and d i a l y z e d . The f i n a l product has a high c o n c e n t r a t i o n of g l y c o s u l f a t a s e as w e l l as a high content of a r y l s u l f a t a s e . The p u r i f i c a t i o n procedure r e s u l t s i n the e l i m i n a t i o n of the c h o n d r o i t i n a s e and most of the 3-Ji-acetylglucosaminidase but other s u l f a t a s e enzymes are s t i l l present i n a p p r e c i a b l e amounts. N u c l e i c a c i d c o n c e n t r a t i o n has been c o n s i d e r a b l y decreased and the preparation r e t a i n s a c t i v i t y when stored i n the frozen s t a t e . It is noticed that glycosulfatase a c t i v i t y in L i t t o r i n a v a r i e s c o n s i d e r a b l y w i t h season. R e l a t i v e l y small amounts of the enzyme can be detected i n the w i n t e r months, whereas, organisms c o l l e c t e d i n J u l y provide the most a c t i v e p r e p a r a t i o n . Example 2. Trichoderma v i r i d e (64) i s grown on a medium c o n t a i n i n g the 6 - 0 - s u l f a t e of D-galactose or D-glucose. The

8. DE E T A L .

Polysaccharide

Sulfates

131

washed mycelium i s frozen and ground w i t h alumina a t -15° and e x t r a c t e d with c o l d T r i s s o l u t i o n a t pH 7.9, c e n t r i f u g e d and supernatant s t o r e d .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Example 3. Pseudomonas carrageenovora (65) c e l l s are harvested, and c e n t r i f u g e d . The supernatant i s t r e a t e d w i t h streptomycin s u l f a t e to remove n u c l e i c a c i d s , c e n t r i f u g e d and the supernatant i s t r e a t e d w i t h ammonium s u l f a t e . The p r e c i p i t a t e i s d i s s o l v e d i n water and d i a l y z e d . Example 4. The l i v e r of Charonia lampas (94) i s homogenized w i t h sodium c h l o r i d e , c e n t r i f u g e d , and the supernatant d i a l y z e d at pH 3.6 and c e n t r i f u g e d . The supernatant i s then s u c c e s s i v e l y passed through columns of phosphocellulose, Sephadex G-150 and Concanavalin A-Sepharose when p u r i f i e d g l y c o s u l f a t a s e I i s i s o l a t e d . G l y c o s u l f a t a s e I I , which i s s t i l l unseparated from a r y l s u l f a t a s e a t t h i s stage, i s p u r i f i e d by i s o e l e c t r i c focussing. Example 5. Hen o v i d u c t isthmus (95) i s t r e a t e d w i t h T r i s HC1 a t pH 7.2, homogenized, and c e n t r i f u g e d . The supernatant i s t r e a t e d w i t h ammonium s u l f a t e to 40% s a t u r a t i o n , c e n t r i f u g e d , and the supernatant i s t r e a t e d again w i t h ammonium s u l f a t e t o 65% s a t u r a t i o n , and c e n t r i f u g e d . The p r e c i p i t a t e i s d i s s o l v e d i n T r i s - H C l at pH 7.2 and d i a l y z e d . This i s then a p p l i e d to a column of DEAE-cellulose. The pooled f r a c t i o n i s c o n c e n t r a t e d , d i a l y z e d at pH 4.5 and then a p p l i e d to another column of C M - c e l l u l o s e a t pH 4.5. The pooled f r a c t i o n s are concentrated and d i a l y z e d a t same pH. The enzyme r e t a i n e d 95% and 90% of i t s a c t i v i t y f o r 2 and 9 months, r e s p e c t i v e l y , when stored a t - 2 0 ° . Assay of G l y c o s u l f a t a s e s S u l f a t a s e s c a t a l y z e the r e a c t i o n : R-0-S0 " + H 0 — > 3

2

R-O-H + S 0

2 4

' + H

+

Enzyme assay i s based upon measurement of unchanged d e s u l f a t e d r e s i d u e , i n o r g a n i c s u l f a t e or change o f pH.

substrate,

The e a r l i e r i n v e s t i g a t i o n of g l y c o s u l f a t a s e s has employed assay methods f o r i n d i r e c t determination of unhydrolyzed s u b s t r a t e , by g r a v i m e t r i c estimations of i n o r g a n i c s u l f a t e l i b e r a t e d on a c i d h y d r o l y s i s and the measurement o f l i b e r a t e d i n o r g a n i c s u l f a t e by p o t e n t i o m e t r i c t i t r a t i o n (50) or n e p h e l o m e t r i c a l l y (50). These methods are not capable of a high degree of accuracy unless a p p l i e d t o l a r g e q u a n t i t i e s . Methods f o r the assay o f s u l f a t a s e s based on the t u r b i d i m e t r i c determinat i o n of barium s u l f a t e have been described (96,97). C o l o r i m e t r i c methods of s u l f a t a s e assay a l s o have been used ( 9 8 ) , and a s m a l l - s c a l e v e r s i o n of each o f these methods has been devised (99).

CARBOHYDRATE SULFATES

132

The method of Dodgson and Spencer (54), i n which p r e c i p i t a t e d benzidine s u l f a t e i s estimated c o l o r i m e t r i c a l l y , although i t i s time-consuming and demands very c a r e f u l m a n i p u l a t i o n , has proved useful f o r a number of s u l f u r i c e s t e r s . A g l u c o s e - o x i d a s e p e r o x i d a s e - O - d i a n i s i d i n e method (100) i s a p p l i c a b l e a l s o .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

P r o p e r t i e s of g l y c o s u l f a t a s e s Charonia lampas g l y c o s u l f a t a s e : The e a r l i e s t study of t h i s g l y c o s u l f a t a s e has been made during the years 1931 through 1948 and t h i s work has been p a r t l y reviewed (101). U n f o r t u n a t e l y , the g l y c o s u l f a t a s e used i s not pure. I t shows g r e a t e s t a c t i v i t y a g a i n s t D-glucose 6 - 0 - s u l f a t e and shows a c t i v i t y a l s o a g a i n s t a v a r i e t y of s u l f a t e e s t e r s of monosaccharides and d i s a c c h a r i d e s as w e l l as against adenosine 5 ' - s u l f a t e . Recently p u r i f i e d g l y c o s u l f a t a s e I and II from (λ lampas have been reported as a c t i v e against D-glucose 6 - 0 - s u l f a t e , showing the same K of 25.0 mM, and the same optimum pH of 5.5. m

L i t t o r i n a l i t t o r e a g l y c o s u l f a t a s e : This g l y c o s u l f a t a s e has been e x t e n s i v e l y i n v e s t i g a t e d . The s p e c i f i c i t y of the enzyme i s r e l a t i v e l y low as i t hydrolyzes D-glycose 6 - 0 - s u l f a t e , D-glucose 3 - 0 - s u l f a t e , and D-galactose 6 - 0 - s u l f a t e (Table IV). In common w i t h most other s u l f a t a s e enzymes, g l y c o s u l f a t a s e i s s t r o n g l y i n h i b i t e d by phosphate and pyrophosphate, and to a l e s s extent by f l u o r i d e i o n s . Some increase i n enzyme a c t i v i t y i s observed i n the presence of magnesium and manganous c h l o r i d e s . Table IV Optimum Substrate^ cone χ 10 (M)

Κ χ 10 m

Relative activity

Substrate

Optimum PH

Potassium D-glucose 3-0-sulfate

5.7-5.9

7.0

3.0

1.0

Potassium D-glucose 6-0-sulfate

5.5-5.6

4.0

1.7

10.0

Potassium D-galactose β-0-sulfate

5.2

>8.0

7.2

2.0

2

None of a wide range of polysaccharides of p l a n t or animal o r i g i n i s attacked by the enzyme, nor are the o l i g o s a c c h a r i d e s produced by the a c t i o n of hyaluronidase on c h o n d r o i t i n 4 - s u l f a t e or c h o n d r o i t i n 6 - s u l f a t e (102).

8.

DE E T A L .

Polysaccharide

133

Sulfates

P a t e l l a vulgata g l y c o s u l f a t a s e : This enzyme has a wide s p e c i f i c i t y f o r 6 - 0 - s u l f a t e e s t e r s of D-glucose, D-galactose (Table V ) , and D-mannose, and a l s o f o r 2 - 0 - , 3-0- and 4 - 0 - s u l f a t e e s t e r s of L-fucose. Table V Optimum pH

Optimum substrate cone (M)

Activity (units)

6-0-sulfate

6.0

0.04

3295

D-galactose δ-0-sulfate

6.0

0.04

4035

Substrate

Κ (mM)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

D-glucose 1.86 28.3

The enzyme a c t i v i t y i s s t r o n g l y i n h i b i t e d by phosphate and borate i o n s , w h i l e the e f f e c t s of cyanide, f e r r i c and c u p r i c vary w i t h the s u b s t r a t e . Trichonderma v i r i d e g l y c o s u l f a t a s e : The enzyme i s a simple g l y c o s u l f a t a s e a c t i v e towards the 6 - 0 - s u l f a t e e s t e r s of D-glucose and D-galactose but not towards D-glucose 3 - 0 - s u l f a t e . A c t i v i t y decreases sharply a t e i t h e r s i d e of the optimum temp, of 2 8 ° . The pH f o r maximum a c t i v i t y i s 7.8-7.9. The g l y c o s u l f a t a s e seems to be d i f f e r e n t from analogous molluscan enzymes t h a t a l s o can hydrolyze monosaccharide s u l f a t e e s t e r s . Pseudomonas carrageenovora g l y c o s u l f a t a s e : The enzyme shows a remarkable degree of s p e c i f i c i t y f o r h y d r o l y z i n g neocarrabiose s u l f a t e , 3-0-(3,6-anhydro-a-D-galactopyranose 4 - 0 - s u l f a t e , a degradation product of K-carrageenan, w i t h h y d r o l y s i s leading to the corresponding d i s a c c h a r i d e . The enzyme a l s o acts on D-galactose 6 - 0 - s u l f a t e , but not on D-galactose 4 - Q - s u l f a t e ; a s u r p r i s i n g r e s u l t s i n c e D-galactose 4 - 0 - s u l f a t e i s a u n i t i n the neocarrabiose s u l f a t e .

Neocarrabiose 4 - 0 - s u l f a t e

CARBOHYDRATE SULFATES

134

Enzyme a c t i v i t y i s completely i n h i b i t e d i n 0.1 M a c e t a t e b u f f e r , pH 4.0, and by 25 mM mercuric i o n . P a r t i a l i n h i b i t i o n occurs w i t h 0.1 M a c e t a t e b u f f e r , pH 5.6, and 25 mM phosphate b u f f e r , pH 7.5. There i s no i n h i b i t i o n w i t h 25 mM s u l f a t e and 3 mM ethylene diamine t e t r a a c e t i c a c i d (EDTA). Hen o v i d u c t s u l f a t a s e : This enzyme removes only the s u l f a t e at p o s i t i o n C4 and shows remarkable s p e c i f i c i t y towards UDP-Na c e t y l -Q-gal actosami ne-4-0-sul f a t e , UDP-N-acetyl-D-galactosamine4 , 6 - d i - 0 - s u l f a t e , and N - a c e t y l - D - g a l a c t o s a m i n e - 4 , 6 - d i - 0 - s u l f a t e , but not towards l N - a c e t y l - Q - g a l a c t o s a m i n e - 4 - 0 - s u l f a t e . (Table V I ) .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Table VI Maximum v e l o c i t y V max (units/mg p r o t e i n )

Substrate

1.

UDP-Gal NAc-4-sulfate

Κ m (M)

4,200

4 χ ΙΟ"

4

UDP-Gal NAc-4,6-disulfate

10,500

6 χ ΙΟ"

6

3.

j\[-Acetyl gal a c t o samine 4 - s u l f a t e

0

4.

^-Acetylgalacto­ samine 4 , 6 - d i s u l f a t e

560

2.

0 3 χ 10

The enzyme i s not a c t i v e toward £-nitrophenyl s u l f a t e , D-glucose 6 - 0 - s u l f a t e , D-galactose 3 - 0 - s u l f a t e , D-galactose 6 - 0 - s u l f a t e , or a number of s u l f a t e d polysaccharides of p l a n t and animal o r i g i n . The enzyme i s d i s t i n c t from any of the known a r y l s u l f a t a s e and g l y c o s u l f a t a s e . Polysaccharide

sulfatases

A number of n a t u r a l polysaccharide s u l f a t e s of both p l a n t and animal o r i g i n s are known, f o r example: c h o n d r o i t i n 4 - s u l f a t e , c h o n d r o i t i n 6 - s u l f a t e , dermatan s u l f a t e , shark c h o n d r o i t i n s u l f a t e , keratan s u l f a t e , heparan s u l f a t e , h e p a r i n , porphyran, τ , κ , ι and μ-carrageenan, charonan s u l f a t e and f u c o i d a n . A number of enzymes and enzyme-systems have been i s o l a t e d that d e s u l f a t e some of these s u l f a t e d p o l y s a c c h a r i d e s . C e l l u l o s e p o l y s u l f a t a s e : An enzyme h y d r o l y z i n g s u l f u r i c e s t e r bonds i n c e l l u l o s e p o l y s u l f a t e and charonan s u l f a t e occurs i n l i v e r e x t r a c t of the marine gastropod, Charonia lampas. Since

8.

DE E T A L .

Polysaccharide

Sulfates

135

c h o n d r o i t i n s u l f a t e and amylose p o l y s u l f a t e are s c a r c e l y hydrolyzed by the enzyme p r e p a r a t i o n , t h i s new s u l f a t a s e has been c a l l e d " c e l l u l o s e p o l y s u l f a t a s e " .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

The l i v e r of Charonia lampas (68) i s macerated a t pH 6.0, c e n t r i f u g e d , and to the supernatant i s added a c r i f l a v i n e to 1% c o n c e n t r a t i o n . P r e c i p i t a t e d m a t e r i a l i s separated by c e n t r i f u g a t i o n , ethanol i s added to the supernatant to 70% concentrat i o n , c e n t r i f u g e d , and the p r e c i p i t a t e d i a l y z e d i n water and c e n t r i f u g e d . The supernatant, at pH 5.2, i s p u r i f i e d i n a column of CMC, changing the eluent pH g r a d u a l l y from 5.2 t o 6.0. The appropriate f r a c t i o n s are pooled, and d i a l y z e d . The crude c e l l u l o s e p o l y s u l f a t a s e from C. lampas hydrolyzes a random s u l f a t e d c e l l u l o s e removing most of the s u l f a t e groups w h i l e presumably other enzymes present hydrolyze the c e l l u l o s e to D-glucose and D-glucose s u l f a t e s from which s u l f a t e groups are then hydrolyzed. The p u r i f i e d c e l l u l o s e p o l y s u l f a t a s e hydrolyzes s u l f a t e groups from the polysaccharide without degrading the c h a i n . This enzyme shows remarkable s p e c i f i c i t y towards c e l l u l o s e p o l y s u l f a t e and charonan s u l f a t e by converting these from high s u l f u r content to low s u l f u r content p o l y s a c c h a r i d e s . I t slowly a t t a c k s dextran s u l f a t e , but not amylose s u l f a t e (Table V I I ) . Regarding the e f f e c t of the p o s i t i o n of s u l f a t e bond, Takahashi suggests t h a t c e l l u l o s e p o l y s u l f a t a s e p r e f e r e n t i a l l y a t t a c k s e s t e r bonds a t the C2 and C3 p o s i t i o n s , w h i l e g l u c o s u l f a t a s e a t t a c k s p r e f e r e n t i a l l y C6 p o s i t i o n e s t e r bonds. Porphyran s u l f a t a s e : An enzyme present i n Porphyra e x t r a c t s i s capable of h y d r o l y z i n g porphyran, a complex s u l f a t e d polysaccharide c o n t a i n i n g residues of D - g a l a c t o s e , L - g a l a c t o s e , 6-0-methyl-D-galactose, and 3,6-anhydro-L-galactose. Porphyra seaweed (70) i s washed w i t h water, minced i n t o aqueous sodium carbonate a t pH 8.3, pH adjusted to 6.0-6.5 and c e n t r i f u g e d . To the supernatant i s added a calcium phosphate g e l , s t i r r e d and c e n t r i f u g e d . The gel i s washed a t pH 7.5, c e n t r i f u g e d , and the supernatant t r e a t e d w i t h s o l i d ammonium s u l f a t e to 0.8% s a t u r a t i o n . The p r e c i p i t a t e i s i s o l a t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n water, and d i a l y z e d . The enzyme d e s u l f a t e s porphyran but does not a t t a c k other sulfated polysaccharides. Complete i n h i b i t i o n o f the enzyme occurs on a d d i t i o n of metal binding reagents, thus i n d i c a t i n g t h a t there i s a b i - or t e r v a l e n t c a t i o n e s s e n t i a l to the enzyme. No i n h i b i t i o n i s observed when a mixture of Zn2 or C o ^ ions (1.5 mM) and ethylenediaminetetra a c e t i c a c i d (EDTA) (0.5 mM) i s present. A 50% i n h i b i t i o n occurs i f M n i o n i s s u b s t i t u t e d f o r one of these c a t i o n s , and complete i n h i b i t i o n occurs i f +

2 +

+

136

CARBOHYDRATE SULFATES

Mg ion i s used. Borate i s a powerful a c t i v a t o r producing 60% a c t i v a t i o n at pH 7.6. Since porphyran i s i t s e l f a p o l y e l e c t r o l y t e , i t i s conceivable t h a t c e r t a i n of the a c t i v a t o r s i n f l u e n c e s the r e a c t i o n by i n t e r a c t i o n with the substrate r a t h e r than the enzyme. I t i s known t h a t s a l t s present i n s o l u t i o n w i t h polysaccharide polyanions can a l t e r the c o n f i g u r a t i o n of the polymer, and i t i s p o s s i b l e t h a t some of the c a t i o n a c t i v a t o r s operate by i n c r e a s i n g the time spent by the s u b s t r a t e i n a c o n f i g u r a t i o n f a v o r a b l e f o r the r e a c t i o n .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Chondrosulfatase Chondrosulfatase of Proteus v u l g a r i s : A s t r a i n of Proteus v u l g a r i s (N.C.T.C. 4636) appears to be a p a r t i c u l a r l y potent source of chondrosulfatase (E.C.3.1.6.4). The enzyme i s a s s o c i a t e d w i t h a c h o n d r o i t i n a s e system which can degrade the chain of c h o n d r o i t i n s u l f a t e producing s u l f a t e d o l i g o s a c c h a r i d e s which are subsequently d e s u l f a t e d by chondrosulfatase. Proteus v u l g a r i s (76,77) i s c u l t u r e d , and harvested by c e n t r i f u g i n g . The c e l l s are macerated i n acetone, f i l t e r e d , homogenized at pH 7.0, incubated at 3 7 ° , and c e n t r i f u g e d . The supernatant i s d i a l y z e d , c e n t r i f u g e d , and to the supernatant added sodium s a l t of yeast n u c l e i c a c i d at pH 7.4. The pH i s adjusted to 4.0, the p r e c i p i t a t e removed by c e n t r i f u g i n g and d i s s o l v e d i n aqueous sodium hydroxide at pH 7.4. The s o l u t i o n i s adjusted to pH 6.7 and an aqueous s o l u t i o n of protamine s u l f a t e added, d i a l y z e d and c e n t r i f u g e d . The supernatant i s adjusted to pH 8.0, c e n t r i f u g e d , and the supernatant t r e a t e d w i t h calcium phosphate gel at pH 6.55. The gel-enzyme complex i s separated by c e n t r i f u g a t i o n , washed and t r e a t e d repeatedly w i t h sodium acetate s o l u t i o n at pH 8.0 u n t i l no f u r t h e r p r o t e i n i s e l u t e d from the g e l . The e l u a t e c o n t a i n i n g s u l f a t a s e are combined and d i a l y z e d . Chondrosulfatase, i n the absence of c h o n d r o i t i n a s e , i s v i r t u a l l y i n a c t i v e towards the polymerized form of c h o n d r o i t i n s u l f a t e . However, c h o n d r o i t i n s u l f a t e degraded w i t h t e s t i c u l a r hyaluronidase i s r e a d i l y hydrolyzed. The crude Proteus c o n c e n t r a t e , which i s a c t i v e towards c h o n d r o i t i n s u l f a t e , i s i n a c t i v e towards the s u l f a t e d polysaccharides agar, f u c o i d a n , carrageenan, chondrus o c e l l a t u s mucilage and s u l f a t e d laminaran. Recently, two chondrosulfatases are i s o l a t e d from the e x t r a c t s of Proteus v u l g a r i s (N.C.T.C. 4636). One of these enzymes, chondro 4 - s u l f a t a s e , i s a c t i v e against 2-acetamido-2deoxy-3-0-(3-Q-gluco-4-enepyranosyl-uronic acid)-4-0-sulfo-D-

8. DE E T A L .

Polysaccharide

Sulfates

137

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

g a l a c t o s e , the product from the degradation of c h o n d r o i t i n s u l f a t e A or Β by c h o n d r o i t i n a s e , and i t s saturated analogue, acetylchondrosiη 4 - s u l f a t e , but does not a t t a c k the corresponding 6 - 0 - s u l f a t e isomer, the product from the degradation of c h o n d r o i t i n s u l f a t e C by c h o n d r o i t i n a s e , and i t s saturated analogue, a c e t y l c h o n d r o s i n 6 - s u l f a t e . In c o n t r a s t , chondro 6-sulfatase desulfates disaccharide 6-sulfates andacetyl-D-galactosamine 4 , 6 - d i s u l f a t e a t p o s i t i o n 6, but does not a t t a c k tfie d i s a c c h a r i d e 4 - s u l f a t e isomers.

2-Acetamido-2-deoxy-3-0-(3-Q-gluco-4enepyranosyluronic a c i d ) - 4 - 0 - s u l f o - D galactose

2-Acetami do-2-deoxy-3-0-(3-D-gluco-4enepyranosyluronic a c i d ) - 6 - 0 - s u l f o - Q galactose The enzymes do not a t t a c k polymer c h o n d r o i t i n s u l f a t e s , hexa-, p e n t a - , t e t r a - , or t r i s a c c h a r i d e s derived from c h o n d r o i t i n s u l f a t e s A and C by d i g e s t i o n w i t h crude t e s t i c u l a r hyaluronidase, or a c e t y l g a l a c t o s a m i n e 4- and 6 - s u l f a t e s . Chondrosulfatase of P a t e l l a v u l g a t a : The v i s c e r a of P a t e l l a vulgata (60) i s reported to c o n t a i n a g l y c o s u l f a t a s e and a l s o an enzyme t h a t i s a c t i v e a g a i n s t c h o n d r o i t i n 4 - s u l f a t e . The chondrosulfatase has been p u r i f i e d by s e p a r a t i n g from the g l y c o s u l f a t a s e by f r a c t i o n a t i o n w i t h ammonium s u l f a t e .

138

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Chondrosulfatase from t h i s source i s reported to be capable of d e s u l f a t i n g c h o n d r o i t i n s u l f a t e without p r i o r d e p o l y m e r i z a t i o n . S u l f a t e i s a l s o l i b e r a t e d by the enzyme from a range of c h o n d r o i t i n s u l f a t e s from d i f f e r e n t sources, i n c l u d i n g a commercial preparation b e l i e v e d to be predominately c h o n d r o i t i n 6-sulfate. Chondrosulfatase of Squid l i v e r : The l i v e r e x t r a c t s of the squid (Ommastrephes s l o a n i p a c i f i c u s ) (83) contains chondros u l f a t a s e along w i t h three other enzymes, hyaluronidase (E.C. 3.2.1.35), 3-N-acetyl-hexosaminidase (E.C. 3.2.1.30), and ^-glucuronidase (E.C. 3.2.1.31). Crude enzyme i s prepared from squid l i v e r by e x t r a c t i o n w i t h acetone and then p u r i f i e d by f r a c t i o n a t i o n w i t h ammonium s u l f a t e and by using a column of Sephadex G-100_. The p u r i f i c a t i o n achieved i s about 1 5 - f o l d from the crude enzyme. The p a r t i a l l y p u r i f i e d chondrosulfatase i s stored at - 2 0 ° , without a p p r e c i a b l e l o s s of a c t i v i t y f o r a month. The squid chondrosulfatase a t t a c k s c h o n d r o i t i n 4 - s u l f a t e at g r e a t e r r a t e than i t does c h o n d r o i t i n 4 , 6 - d i s u l f a t e . Chondroitin 6 - s u l f a t e i s a l s o d e s u l f a t e d to a s l i g h t e x t e n t , but keratan s u l f a t e , dermatan s u l f a t e and heparin are not d e s u l f a t e d w i t h t h i s enzyme. A s u l f a t e d t e t r a s a c c h a r i d e and a mixture of s u l f a t e d o l i g o s a c c h a r i d e s , both prepared from c h o n d r o i t i n 4 - s u l f a t e by t e s t i c u l a r hyaluronidase d i g e s t i o n , are r e s p e c t i v e l y d e s u l f a t e d to the extent of about 32% and 56% of the s u l f a t e released from c h o n d r o i t i n 4 - s u l f a t e polymer. D e s u l f a t i o n from the s u l f a t e d di s a c c h a r i d e , 2-acetami do-2-deoxy-3-0-($-Q-gluco-4-enopyranosyluronic a c i d ) - 4 - 0 - s u l f o - Q - g a l a c t o s e , i s n e g l i g i b l e . These s t u d i e s show t h a t the squid chondrosulfatase i s capable of d e s u l f a t i n g mainly 4 - s u l f a t e i n c h o n d r o i t i n s u l f a t e s without p r i o r depolymerization. Chondrosulfatase a c t i v i t y i s extremely i n h i b i t e d by c u p r i c s u l f a t e , sodium phosphate, sodium f l u o r i d e , borax and heparin. Chondrosulfatase i s a c t i v a t e d by sodium c h l o r i d e , c u p r i c c h l o r i d e , EDTA, and c y s t e i n e h y d r o c h l o r i d e . The apparent 1^ of t h i s chondrosulfatase i s estimated as 0.49 mM f o r r e a c t i o n s at pH 5.0 and 37° f o r 3 h w i t h c h o n d r o i t i n 4 - s u l f a t e as the s u b s t r a t e . Chondrosulfatase of bovine a o r t a : I t has been known (103, 104) t h a t r a t s d e s u l f a t e c h o n d r o i t i n s u l f a t e i n v i v o and i t has been claimed (105) t h a t chondrosulfatase can be detected h i s t o c h e m i c a l l y i n human sweat glands, but attempts have been unsuccessful (56) to prepare i t from mammalian t i s s u e , e x t r a c t s w i t h chondrosulfatase a c t i v i t y . On the other hand, crude preparations from p i g kidney are known to l i b e r a t e s u l f a t e from chondroitin sulfate.

8.

DE E T A L .

Polysaccharide

Sulfates

139

Bovine a r t e r i a l t i s s u e (aorta) contains a chondrosulfatase (85). The enzyme i s p u r i f i e d by ammonium s u l f a t e f r a c t i o n a t i o n , p r e c i p i t a t i o n a t pH 4.6, and by gel f i l t r a t i o n . An 85 f o l d p u r i f i c a t i o n i s achieved.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

The enzyme i s a c t i v e against c h o n d r o i t i n 4 - s u l f a t e , but i t does not a t t a c k c h o n d r o i t i n 6 - s u l f a t e , dermatan s u l f a t e , and keratan s u l f a t e . In acetate b u f f e r , the pH optimum i s 4.4, and Km i s 0.735 mM. K e r a t o s u l f a t a s e : Two forms o f k e r a t o s u l f a t a s e (86) (I and I I ) are i s o l a t e d from the marine gastropod, charonia lampas. Both forms r e l e a s e a l l the s u l f a t e from kerato s u l f a t e s and n e i t h e r appears i d e n t i c a l w i t h g l y c o s u l f a t a s e or c h o n d r o s u l f a t a s e , both of which are a l s o present i n charonia lampas. The crude l i v e r e x t r a c t i s p u r i f i e d by f r a c t i o n a t i o n w i t h ammonium s u l f a t e and then by successive column chromatography of CM-Sephadex C-50 and DEAE-Sephadex A-50. K e r a t o s u l f a t a s e preparations I and II are stored a t -20° i n the presence of 0.1 M sodium c h l o r i d e without a p p r e c i a b l e l o s s of a c t i v i t y f o r several weeks. Keratosulfatases desulfate keratopolysulfate, keratosulfate and h o r a t i n s u l f a t e , a s u l f a t e d polysaccharide composed of L-fucose, g-mannose, D-glucose, D - g a l a c t o s e , D-glucosamine and D-galactosamine, i s o l a t e d from the l i v e r of £. lampas. K e r a t o s u l f a t a s e I does not l i b e r a t e s u l f a t e from h o r a t i n s u l f a t e (Table V I I I ) . Table V I I I Enzyme preparation

Substrate

Incubation time

SO^ libérât

(h) 1. 2. 3.

I II I II I II

Keratopolysulfate Same , Kerator S]sulfate Same Horatin s u l f a t e Same 7

D

96 96 96 76 48 48

(%) 80 100 95 100 < 3 100

I t a l s o appears that k e r a t o s u l f a t a s e f i r s t l i b e r a t e s s u l f a t e from k e r a t o s u l f a t e s and the d e s u l f a t e d polymer i s then degraded to D-galactose and N-acetyl-D-glucosamine by the a c t i o n of 3-D-galactosidase and 3-u-acetyl-D-glucosaminidase, enzymes a l s o present i n the p a r t i a l l y p u r i f i e d k e r a t o s u l f a t a s e .

140

CARBOHYDRATE SULFATES

Potassium dihydrogen phosphate, sodium f l u o r i d e and £-chloromercuribenzoate i n h i b i t both fkeratosulfatases I and as they do most other known £. lampus s u l f a t a s e s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

The optimum pH values of k e r a t o s u l f a t a s e s I and II 4.5 and 6.0, r e s p e c t i v e l y .

II,

are

Heparin degrading multienzyme system: No s i n g l e enzyme has been i s o l a t e d which can d e s u l f a t e heparin or h e p a r i t i n s u l f a t e s . However, f i v e enzymes from Flavobacterium heparinum (87), capable of degrading heparin to i t s b a s i c c o n s t i t u e n t s , have been p u r i f i e d and c h a r a c t e r i z e d . The p u r i f i e d heparinase degrades heparin to t r i s u l f a t e d d i s a c c h a r i d e . This i s d e s u l f a t e d f u r t h e r by a d i s a c c h a r i d e s u l f a t a s e y i e l d i n g the d i s u l f a t e d d i s a c c h a r i d e . This product i s a s u b s t r a t e f o r glycuronidase g i v i n g g l u c o s a m i n e - 2 , 6 - d i s u l f a t e and a , 3 - k e t o a c i d . D-glucos a m i n e - 2 , 6 - d i s u l f a t e i s d e s u l f a t e d by a s u l f a t a s e and a sulfamidase y i e l d i n g f r e e glucosamine and i n o r g a n i c s u l f a t e . The crude e x t r a c t from heparinum i s p u r i f i e d by s u c c e s s i v e l y using a column chromatography of Sephadex G-200 and then by agarose gel e l e c t r o p h o r e s i s . The hepa'rinase has no a c t i v i t y upon c h o n d r o i t i n s u l f a t e s A, Β and C or on h e p a r i t i n s u l f a t e s A and B, but degrades h e p a r i t i n s u l f a t e s C and D, y i e l d i n g s u l f a t e d d i s a c c h a r i d e s w i t h the same chromatographic m o b i l i t y as the ones obtained from heparin. The s p e c i f i c i t y of the enzyme, d i s a c c h a r i d e s u l f a t a s e , i s l i m i t e d . The enzyme acts only upon the t r i s u l f a t e d d i s a c c h a r i d e , forming d i s u l f a t e d d i s a c c h a r i d e and i n o r g a n i c s u l f a t e . Heparin, s u l f a t e d hexa- and t e t r a - s a c c h a r i d e s , d i s u l f a t e d d i s a c c h a r i d e , and g l u c o s a m i n e - 2 , 6 - d i s u l f a t e are not substrates f o r t h i s enzyme. Glycuronidase acts only upon the d i s u l f a t e d d i s a c c h a r i d e , y i e l d i n g glucosamine-2,6-disulfate. This enzyme does not a c t upon the t r i s u l f a t e d d i s a c c h a r i d e . Monosaccharide s u l f a t a s e and sulfamidase a c t only upon the s u f l a t e d hexosamine monosaccharides. No a c t i v i t y upon heparin or the d i s a c c h a r i d e s has been d e t e c t e d . The pathway of enzymatic degradation of heparin may proceed as shown.

8.

DE E T A L .

Polysaccharide

Sulfates

141

Heparin

I

heparinase

T r i s u l f a t e d Disaccharide disaccharide sulfatase D i s u l f a t e d Disaccharide + SO^

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

glycuronidase G l u c o s a m i n e - 2 , 6 - d i s u l f a t e + a,3-Keto A c i d monosaccharide sulfatase

sulfamidase Glucosamine-6-sulfate + S0

Glucosamine-N-sulfate

2-

+ so4

4

2-

sulfamidase

monosaccharide sulfatase Glucosamine + SO.

Cerebroside s u l f a t a s e : Presence of a cerebroside s u l f a t a s e has been f i r s t reported i n the d i g e s t i v e gland of the abalone, H a l i o t i s (88). This enzyme, i n a d d i t i o n to h y d r o l y z i n g c e r e b r o s i d e , can a l s o d e s u l f a t e c h o n d r o i t i n s u l f a t e but not p h e n y l s u l f a t e nor glucose 6 - s u l f a t e . Cerebroside s u l f a t a s e has been p u r i f i e d from the lysosomes of p i g kidney (90) and i t s presence has been shown i n other organs, p a r t i c u l a r l y l i v e r and spleen. The p u r i f i c a t i o n i s achieved by high voltage e l e c t r o p h o r e s i s of the crude e x t r a c t . This enzyme d e s u l f a t e s only cerebroside 3 - s u l f a t e s . Cerebroside 6 - s u l f a t e s are not d e s u l f a t e d . D-Galactose 3 - s u l f a t e i s hydrolyzed a t about 20% the r a t e of c e r e b r o s i d e 3 - s u l f a t e but D-galactose 6 - s u l f a t e i s not hydrolyzed. The enzyme a l s o d e s u l f a t e s cerebron and kerasin s u l f a t e s . The optimum pH i s 4.5, and i s 0.1 mM w i t h cerebroside 3 - s u l f a t e s . Enzyme a c t i v i t y i s i n h i b i t e d by s u l f i t e , s u l f a t e , phosphate, and f l u o r i d e w h i l e a c t i v a t e d by hydroxylami ne.

CARBOHYDRATE SULFATES

142

The enzyme has s t r i k i n g resemblance to a r y l s u l f a t a s e A and the enzymes could be i d e n t i c a l (107). Both cerebroside s u l f a t a s e and a r y l s u l f a t a s e A have been i s o l a t e d from human l i v e r (108) and perhaps t h e i r absence could cause metachromatic leucodystrophy i n humans.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Hunter's

Syndrome

Hunter's Syndrome i s a genetic d i s o r d e r a s s o c i a t e d w i t h f a i l u r e to degrade dermatan s u l f a t e and heparan s u l f a t e . Lysosomal storage of these polymers leads to numerous c l i n i c a l problems. F i b r o b l a s t s c u l t u r e d from the s k i n of Hunter's p a t i e n t s do not adequately degrade s u l f a t e d mucopolysaccharides because of a d e f i c i e n c y of a s p e c i f i c p r o t e i n t h a t i s present i n c e l l s e c r e t i o n s , c e l l s , and urine of i n d i v i d u a l s who do not have Hunter's Syndrome (113). Recent s t u d i e s show (109) t h a t a d e f i c i e n c y of a s u l f a t a s e , L - i d u r o n o - s u l f a t e s u l f a t a s e , which s p e c i f i c a l l y cleaves the e s t e r s u l f a t e of i d u r o n i c a c i d , to be r e s p o n s i b l e f o r the d e f e c t i n Hunter's Syndrome. This s u l f a t a s e , when added exogenously to Hunter c e l l s , a c c e l e r a t e s the degradat i o n of s u l f a t e d mucopolysaccharides. Sanfilippo

Syndrome

S a n f i l i p p o Syndrome i s a f a m i l i a l d i s o r d e r of mucopolysaccharide metabolism, t r a n s m i t t e d i n autosomal r e c e s s i v e f a s h i o n . Excessive u r i n a r y e x c r e t i o n of heparan s u l f a t e i s the major d i a g n o s t i c c r i t e r i o n . F i b r o b l a s t s from p a t i e n t s w i t h S a n f i l i p p o Syndrome f a l l i n t o two subgroups, each m a n i f e s t i n g a d e f i c i e n c y of a s p e c i f i c f a c t o r r e q u i r e d f o r normal metabolism of s u l f a t e d mucopolysaccharide. The f a c t o r d e f i c i e n t i n the A subgroup has been i s o l a t e d from normal human u r i n e . The f a c t o r a c c e l e r a t e s degradation of stored mucopolysaccharide i n S a n f i l i p p o A f i b r o b l a s t s . I t has been suggested t h a t S a n f i l i p p o A f a c t o r i s a heparan s u l f a t e s u l f a t a s e (110). When the f a c t o r i s administered exogenously to S a n f i l i p p o f i b r o b l a s t s , a c o r r e c t i o n of the abnormal metabolism occurs. Metachromatic Leucodystrophy

(MLD)

Metachromatic leucodystrophies are a group of human genetic d i s o r d e r s which are c h a r a c t e r i z e d by the d e p o s i t i o n of cerebroside s u l f a t e r i c h granules i n the c e n t r a l and p e r i p h e r a l nervous systems with r e s u l t a n t progressive n e u r o l o g i c a l degeneration. I t has been suggested t h a t the turnover of cerebroside s u l f a t e s to cerebrosides might be blocked as a r e s u l t of d e f i c i e n c y of cerebroside s u l f a t a s e (111, 112). Cerebroside s u l f a t a s e a c t i v i t y i s demonstrated i n normal mammalian t i s s u e s such as kidney and b r a i n , where i n MLD p a t i e n t s the cerebroside s u l f a t e accumulation i s e s p e c i a l l y high.

8.

DE E T A L .

Polysaccharide

Sulfates

143

Biogenesis

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

Recent work has shown t h a t prophyran contains residues of both 3,6-anhydro-L-galactose and L - g a l a c t o s e 6 - s u l f a t e (69, 71). When e x t r a c t s of porphyran c o n t a i n i n g seaweed are incubated w i t h the i s o l a t e d p o l y s a c c h a r i d e , f r e e s u l f a t e i s l i b e r a t e d and s y n t h e s i s of 3,6-anhydro-L-galactosyl residues occurs. Equimolar q u a n t i t i e s of the two products are present i n the m i x t u r e . These r e s u l t s s t r o n g l y i n d i c a t e t h a t L-galactose 6 - s u l f a t e i s an immediate b i o l o g i c a l precursor of 3,6-anhydro-L-galactose, the residue present i n commercial agar. This enzymic h y d r o l y s i s can provide a way of making commercial agar from 6 - s u l f a t e d p o l y ­ saccharides. S t r u c t u r a l work of polysaccharides Enzymic h y d r o l y s i s of polysaccharides can provide i n t e r e s t i n g s t r u c t u r a l i n f o r m a t i o n . Thus, the s u b s t r a t e s p e c i f i c i t y of f i v e enzymes leads to the f o r m u l a t i o n of a pathway f o r the s e q u e n t i a l degradation of heparin (87). L i k e w i s e , the demonstration of the presence of chondroitinase-ABC, chondroitinase-AC, chondro4 - s u l f a t a s e , c h o n d r o - 6 - s u l f a t a s e , and unsaturated d i s a c c h a r i d e glucuronidase provides an enzymatic mechanism f o r the degradation of c h o n d r o i t i n s u l f a t e s (78). Another obvious f e a t u r e of these enzymes i s the use to which they may be put as reagents i n studying the s t r u c t u r e of c h o n d r o i t i n s u l f a t e s as w e l l as the composition of a given mixture of isomeric c h o n d r o i t i n s u l f a t e s . Conclusion Work on enzymatic s u l f a t i o n and d e s u l f a t i o n of polysaccharides i s i n i t s formative stages but has a l r e a d y reached a p o i n t where c e r t a i n b e n e f i c i a l a p p l i c a t i o n s are apparent. C l i n i c a l c o n t r o l of several syndromes may be f a c i l i t a t e d by a p p r o p r i a t e manipulation of enzymes e f f e c t i n g s u l f a t e groups. On a broader b a s i s there i s i n d i c a t i o n of the p o t e n t i a l use of enzymes f o r s u l f a t i o n of at l e a s t c e r t a i n polysaccharides and even a more immediate use of enzymes f o r d e s u l f a t i o n and perhaps d e s u l f a t i o n w i t h r e s u l t a n t i n t r o d u c t i o n of anhydro r i n g s . Consequently, f u r t h e r i n d u s t r i a l examination of these enzyme systems can be expected.

Literature Cited 1. 2. 3. 4.

D'Abramo, F-. and Lipmann, F., Biochim. Biop-ys. Acta, (1957) 25, 211. Meezan, E. and Davidson, Ε. Α., J . Biol. Chem. (1967) 242, 1685. Richmond, M. E., Deluca, S., and Gilvert, J . E., Biochem. (1973) 12, 3898. Suzuki, S., and Strominger, J. L . , J . Biol. Chem. (1960) 235, 257.

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Johnson, A. H., and Baker, J . R., Biochim. Biophys. Acta (1973) 320, 341. Endo, Μ., and Yoshizawa, Z . , J . Biochem. (1976) 79, 293. Foley, T. J . and Baker, J . R., Biochem. J., (1971) 124, 25 p. Wortman, B . , J. Biol. Chem., (1961) 236, 974. Balasubramanian, A. S., and Bachhawat, Β. K., J. Neurochem., (1964) 11, 877. Momburg, M., Stuhlsatz, H. W., Kisters, R., and Greiling, G. Η., Hoppe-Seyler's Z. Physiol. Chem., (1972) 353, 1351. Adams, J. B . , Biochim. Biophys. Acta, (1964) 83, 127. Eisenman, R. Α., Balasubramanian, A. B . , and Marx, W., Arch. Biochem. Biophys. (1967) 119, 387. Balasubramanian, A. B . , Joun, N. S., and Marx, W., Arch. Biochem. Biophys., (1968) 128, 623. Jansson, L . , Hook, M., Wasteson, Α., and Lindahl, U . , Biochem. J., (1975) 149, 49. Habuchi, O., Yamagata, T., and Suzuki, S., J . Biol. Chem. (1971) 246, 7357. Goldberg, I. H., and Delbruch, Α., Fed. Proc. (1959) 19, 235. Yoshida, H., and Egami, F., J . Biochem. (1965) 57, 215. Su, J . C., and Hassid, W. Z . , Biochem. (1962) 1, 474. Loewus, F., Wagner, G., Schiff, J . Α., and Weistrop, J., Plant Physiol. (1971) 48, 373. Ramus, J., and Groves, S. T., J . Cell. B i o l . , (1972) 54, 399. Ramus, J., and Groves, S. T., Plant Physiol. (1974) 53, 434. Bidwell, R. G. S., and Ghosh, N. R., Can. J . Bot. (1963) 41, 209. Qauntrano, R. S., and Crayton, Μ. Α., Devel. Biol. (1973) 30, 29. Crayton, Μ. Α., Wilson, E . , and Quantrano, R. S., Devel. Biol. (1974) 39, 164. Hogsett, W. E . , and Quantrano, R. S., Plant Physiol. (1975) 55, 25. Davidson, E. A. and Riley, J . G., J. Biol. Chem. (1960) 235, 3367. Suzuki, S., Trenn, R. H., and Strominger, J . L . , Biochim. Biophys. Acta (1961) 50, 169. Adams, J. B . , Biochem. J., (1960) 76, 520. Hasegawa, E . , Delbruck, Α., and Lipmann, F., Fed. Proc. (1961) 20, 86. Adams, J. B . , and Meaney, M. F . , Biochim. Biophys. Acta (1961) 54, 592. Suzuki, S., and Strominger, J . L . , J. Biol. Chem. (1960) 235, 267. Suzuki, S., and Strominger, J . L . , J . Biol. Chem. (1960) 235, 274. Meezan, E . , and Davidson, Ε. Α., J . Biol. Chem., (1967) 242, 4956. Deluca, S., and Silvert, J . E . , J. Biol. Chem., (1968) 243, 2725. Silvert, J . E . , and Deluca, S., J . Biol. Chem., (1969) 244, 876.

8. DE ET AL. 36. 37. 38. 39. 40.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.

Polysaccharide Sulfates

145

Richmond, M. E., Deluca, S., and Silvert, J . E., Biochem. (1973) 12, 3904. Deluca, S., Richmond, M. E., and Silvert, J . E., Biochem. (1973) 12, 3911. Kimata, Κ., Okayama, M., Oohira, Α., and Suzuki, S., Mol. Cell Biochem. (1973) 1, 211. Harada, T., Shimizu, S., Nakamishi, Y . , and Suzuki, S., J. Biol. Chem. (1967) 242, 2288. Tsuji, M., Shimizu, S., Nakanishi, Y . , and Suzuki, S., J. Biol. Chem. (1970) 245, 6039. Nakashimi, Y . , Sonohara, H., and Suzuki, S., J . Biol. Chem. (1970) 245, 6046. Hook, M., Lindahl, U . , Hallen, Α., and Backstrom, G., J . Biol. Chem. (1975) 250, 6065. Lindahl, U . , Hook, M., Backstrom, G., Jacobsson, I . , Riesenfeld, J., Malstrom, Α., Roden, L . , and Feingold, D. S. Fed. Proc., (1977) 36, 19. Egami, F., Asahi, T., Takahashi, N . , Suzuki, S., Shibata, S., and Nishizawa, Κ., Bull. Chem. Soc. Japan (1955) 28, 685. Iida, K., J . Biochem., (1963) 43, 181. Wortman, B., J . Biol. Chem. (1961) 236, 974. Hatanaka, H., Yamagata, T., and Egami, F., Proc. Japan Acad. (1974) 50, 747. Soda, T., and Hattori, C., Bull. Chem. Soc., Japan (1931) 6, 258. Soda, T., Bull. Chem. Soc. Japan (1934) 9, 83. Soda, T., J . Fac. Sci. Tokyo Univ. (1936) 3, 150. Soda, T., and Egami, F., Bull. Chem. Soc., Japan (1933) 8, 148. Soda, T., Katsura, T., and Yoda, O., J . Chem. Soc. Japan (1940) 61, 1227. Dodgson, K. S., Lewis, J . I. M., and Spencer, B . , Biochem. J. (1953) 55, 253. Dodgson, K. S., and Spencer, B . , Biochem. J . (1953) 55, 436. Dodgson, K. S., and Spencer, B . , Biochem. J . (1954) 57, 310. Dodgson, K. S., and Lloyd, A. G., Biochem. J . (1961) 78, 319. Dodgson, K. S., Biochem. J . (1961) 78, 324. Lloyd, P. F., Lloyd, K. O., and Owen, O., Biochem. J . (1962) 85, 193. Lloyd, P. F., and Lloyd, K. O., Nature (Lond) (1963) 199, 287. Lloyd, P. F., and Fielder, R. J., Biochem. J . (1967) 105, 33P. Lloyd, P. F., Stuart, C. H., Biochem. J. (1968) 107, 7P. Fielder, R. J., and Lloyd, P. F., Biochem. J . (1968) 109, 14P. Lloyd, P. F., and Forrester, P. F., Biochem. J., (1971) 124, 21P. Yamashina, I . , J . Chem. Soc. Japan (1951) 72, 124. Weigl, J., and Ypahe, W., Can. J . Microbiol. (1966) 12, 874. Takahashi, N . , J. Biochem. Tokyo (1960) 48, 508, 691. Takahashi, N . , and Egami, F., Biochim. Biophys. Acta (1960)

146 68. 69. 70. 71. 72. 73. 74. 75. 76. Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99.

CARBOHYDRATE SULFATES

Takahashi, Ν., and Egami, F., Biochem. J., (1961) 80, 384. Rees, D. Α., Biochem. J . (1961) 78, 25P. Rees, D. Α., Biochem. J . (1961) 80, 449. Peat, S., and Rees, D. Α., Biochem. J . (1961) 79, 7. Peat, S, Turvey, J . R., and Rees, D. Α., J . Chem. Soc. (1961) 1590. Neuberg, C. and Rubin, O., Biochem. Z . , (1914) 67, 82. Neuberg, C., and Hofman, E . , Biochem. Z. (1931) 234, 345. Neuberg, C. and Hofman, E . , Naturwissenschaften (1931) 19, 484. Dodgson, K. S., Lloyd, A. G., and Spencer, B . , Biochem. J . (1957) 65, 131. Dodgson, K. S., and Lloyd, A. G., Biochem. J . (1957) 66, 532. Yamagata, T., Saito, H . , Habuchi, O. and Suzuki, S., J. Biol. Chem. (1968) 243, 1523. Soda, T. and Egami, F . , J . Chem. Soc. Japan (1938) 59, 1202. Lloyd, P. F. and Fielder, R. J., Biochem. J . (1968) 109, 14P. Kawai, Y . , Seno, N . , and Anno, Κ., Anal. Biochem. (1969) 32, 314. Kawai, Y. and Anno, K., Biochim. Biophys. Acta (1971) 242, 428. Atsumi, Κ., Kawai, Y . , Seno, Ν., and Anno, Κ., Biochem. J . (1972) 128; 983. Tudball, N. and Davidson, Ε. Α., Biochim. Biophys. Acta (1969) 171, 113. Held, E . , and Budeecke, E . , Hoppe-Seyler's Ζ. Physiol. Chem. (1967) 348, 1047. Fukuda, M. N . , and Egami, F . , Biochem. J., (1970) 119, 39. Dietrich, C. P., Silva, M. E. and Michelacci, Υ. Μ., J. Biol. Chem. (1973) 248, 6408. Fujino, Y. and Negishi, T., Bull. Agri. Chem. Soc., Japan (1957) 21, 225. Mehl, E. and Jatzkewitz, H., Hoppe-Seyler's Ζ. Physiol. Chem. (1963) 331, 292. Mehl, E. and Jatzkewitz, H . , Hoppe-Seyler's Ζ. Physiol. Chem. (1964) 339, 260. Mehl, E. and Jatzkewitz, H., Biochim. Biophys. Acta (1968) 151, 619. Hatanaka, H., Ogawa, Y. and Egami, F . , J . Biochem. (1975) 77, 353. Hatanaka, H . , Ogawa, Y . , Egami, F., Ishizuka, I. and Nagai, Y . , J. Biochem. (1975) 78, 427. Hatanaka, H. Ogawa, Y . , and Egami, F., J . Biochem. (1976) 79, 27. Tsuji, M., Hamano, M., Nakanishi, Y . , Ishihara, K. and Suzuki, S, J . Biol. Chem. (1974) 249, 879. Dodgson, K. S., Biochem. J . (1961) 78, 312. Rees, D. Α., Biochem. J . (1961) 80, 452. Lloyd, A. G., Biochem. J . (1959) 72, 133. Spencer, B . , Biochem. J . (1960) 75, 453.

8. DE ET AL.

Polysaccharide Sulfates

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch008

100.

147

Huggett, A. St. G. and Nixon, D. Α., Biochem. J . (1957) 66, 12P. 101. Fromageot, C., "The Enzymes", Vol. 1, pt. 1, p. 517, Academic Press, Inc., New York (1950). 102. Lloyd, A. G., Meth. Enzym. (1966) 8, 670. 103. Dziewiatkowski, D. D., J . Biol. Chem. (1956) 223, 239. 104. Dohlman, C. H., Acta Physiol. Scand. (1956) 37, 220. 105. Ohmura, H. and Yasoda, T., Quoted in Biol. Abstr. (1962) 39, 837. 106. Lloyd, A. G., Large, P. J., Davies, M., Olavesen, A. H., and Dodgson, K. S., Biochem. J . (1968) 108, 393. 107. Graham, E. R. B. and Roy, A. B . , Biochim. Biophys. Acta (1973) 329, 88. 108. Mraz, W., Fischer, G., and Jatzkewitz, H . , Hoppe-Seyler's Z.Physiol. Chem. (1976) 357, 201. 109. Sjorberg, I . , Fransson, L. Α., Matalon, R., and Dorfman, A. Biochem. Biophys. Res. Commun. (1973) 54, 1125. 110. Kresse, H. and Neufeld, E. F., J . Biol. Chem. (1972) 247, 2164. 111. Mehl, E. and Jatzkewitz, H., Biochem. Biophys. Res. Commun. (1965) 19, 407. 112. Porter, M. T., Fluharty, A. L., Trammell, J . and Kihara, H. Biochem. Biophys. Res. Commun. (1971) 44, 660. 113. Bach, G., Eisenberg, F., J r . , Cantz, M., and Neufeld, E. F . , Proc. Nat. Acad. S c i . , USA (1973) 70, 2134.

RECEIVED

February 6, 1978.

9 Some Novel Methods and Results in the Sulfation of Polysaccharides K E N N E T H B. G U I S E L E Y

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

Marine Colloids, Inc., Rockland, ME 04841

The methods used in the sulfation of polysaccharides are f a i r l y limited, because the properties of sulfating agents and those of polysaccharides combine to put severe constraints on the synthetic chemist. Thus, in virtually a l l the literature, we find essentially the same techniques - the treatment of a polysaccharide with a Lewis base complex of sulfur trioxide, under anhydrous conditions, usually in the presence of a base such as pyridine. One of the commonest techniques involves the addition of chlorosulfonic acid to pyridine in the cold, followed by introduction of the polysaccharide, and subsequent heating (1-8). The product is usually recovered and purified by alcohol precipitation, dissolution in water, neutralization, dialysis, and alcohol precipitation. The vigor of the sulfating agent is a function of the strength of the Lewis base with which it is combined. Pyridine, being a moderate base, effectively controls the potency of the sulfur trioxide, and the crystalline pyridine-sulfur trioxide complex is readily prepared (9). Triethylamine and trimethylamine form less reactive sulfating reagents (10a), which are so stable, that sulfations in aqueous systems have been reported (4, 11). At the opposite end of the act i v i t y scale are the complexes with dimethylformamide (DMF) and dioxane (10a). These, being much weaker bases, hold the sulfur-trioxide less tightly, permitting reactions at lower temperatures. Virtually a l l polysaccharides have been the object of sulfation reactions, starting with cellulose in 1819 (12). A series of review articles in Industrial and Engineering Chemistry in the 1950's and 1960's, by Gilbert and Jones refer to most of these, and a concise, but thorough, coverage was given the subject in Gilbert's book, "Sulfonation and Related Reactions" (10b). 0-8412-0426-8/78/47-077-148$05.00/0 © 1978 American Chemical Society

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

9.

GuisELEY

Sulfation

of

Polysaccharides

149

Because o f t h e p h y s i o l o g i c a l importance o f h e p a r i n , many attempts have been made over the y e a r s t o produce a s y n t h e t i c h e p a r i n by s u l f a t i o n o f p o l y s a c c h a r i d e s (5-7, 13, 14), and a n t i u l c e r a t i v e m e d i c a t i o n s ( p e p s i n i n h i b i t o r s ) have been s i m i l a r l y p r e p a r e d (1_, 15-17) . D i r e c t s u l f a t i o n o f p o l y s a c c h a r i d e s , as, f o r example, by the p y r i d i n e - c h l o r o s u l f o n i c a c i d method, g e n e r a l l y r e s u l t s i n d e g r a d a t i o n o f t h e polymer, w i t h r e s u l t a n t l o s s o f c o l l o i d a l p r o p e r t i e s . However, employment o f g e n t l e r c o n d i t i o n s o f t e n f a i l s t o e f f e c t enough s u l f a t i o n t o change t h e p r o p e r t i e s o f the p o l y saccharide. Wolfrom and J u l i a n o (_3_) r e c o g n i z e d the need t o r e n d e r p o l y s a c c h a r i d e s more amenable t o s u l f a t i o n through an a c t i v a t i o n p r o c e s s , and Schweiger f u r t h e r e d t h e concept i n a s e r i e s o f p a t e n t s d e s c r i b i n g the s u l f a t i o n o f a l g i n (18), s t a r c h (19), xanthan (20), and c e l l u l o s e (21). In t h e s e c a s e s , t h e a c t i v a t i o n was a c c o m p l i s h e d by t r e a t m e n t o f t h e p o l y s a c c h a r i d e i n a s e r i e s o f s o l v e n t washes which d e h y d r a t e d i t , y e t p r e v e n t e d i t s becoming i n e r t t o s u l f a t i o n , as does oven-drying. The p r i n c i p a l s u b j e c t o f t h i s paper i s a g e n e r a l l y a p p l i c a b l e method o f a c t i v a t i n g p o l y s a c c h a r i d e s , which we d e v e l o p e d a t about the same time. Basically, i t c o n s i s t s o f a two-step p r o c e s s i n v o l v i n g h y d r a t i o n o f t h e gum, f o l l o w e d by d i s t i l l a t i v e s o l v e n t d r y i n g . For i t s h y d r a t i o n , the gum may be t o t a l l y d i s s o l v e d , o r merely s w o l l e n w i t h a l i m i t e d q u a n t i t y o f water. In the d e h y d r a t i o n s t e p , the water and any o t h e r s o l v e n t which may r e a c t i r r e v e r s i b l y w i t h a s u l f a t i n g agent, are removed by d i s t i l l a t i o n i n t h e p r e s e n c e o f a h i g h e r b o i l i n g , water-miscible s o l v e n t . Products of p r e d i c t a b l e degrees o f s u b s t i t u t i o n and h i g h m o l e c u l a r weight can then be p r e p a r e d by c o n v e n t i o n a l methods o f s u l f a tion. Our i n t e r e s t i n s u l f a t i n g p o l y s a c c h a r i d e s was p r i m a r i l y one o f p r o d u c i n g a s y n t h e t i c c a r r a g e e n a n (,22.). Carrageenans a r e g a l a c t a n s u l f a t e s p r e s e n t i n a number o f r e d seaweeds, and which a r e e x t r a c t e d and used f o r t h e i r g e l l i n g , suspending, and v i s c o s i t y - p r o d u c i n g p r o p e r t i e s i n f o o d s , p a r t i c u l a r l y d a i r y p r o d u c t s , and i n such o t h e r d i v e r s e p r o d u c t s as t h e p o p u l a r a i r f r e s h e n e r g e l . A s e m i - s y n t h e t i c gum w i t h p r o p e r t i e s s i m i l a r t o c a r r a g e e n a n c o u l d prove t o be o f g r e a t economic v a l u e i n t h e f a c e o f r i s i n g seaweed c o s t s which put the p r i c e o f carrageenan a t r o u g h l y f o u r times t h a t o f guar and l o c u s t bean gum, and twenty t o t h i r t y times t h a t o f s t a r c h .

CARBOHYDRATE SULFATES

150

Results

and D i s c u s s i o n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

The e f f e c t s o f t i m e , temperature, s u l f a t i n g r e a gent, and p r e c i p i t a t i n g s o l v e n t were d e t e r m i n e d on p o l y s a c c h a r i d e s a c t i v a t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method, a f t e r e a r l i e r e x p e r i m e n t s showed t h a t o t h e r methods o f d r y i n g i n h i b i t e d r e a c t i o n (Table 1 ) . E f f e c t o f Temperature. F o r guar and l o c u s t bean gum s u l f a t e d w i t h DMF:S03, t h e e x t e n t o f s u l f a t i o n was q u i t e comparable a t a g i v e n temperature, whereas o t h e r gums v a r i e d somewhat i n t h e i r tendency toward b e i n g sulfated. R e a c t i o n was found t o be q u i t e r a p i d i n i t i a l l y , as i n d i c a t e d i n F i g . 1, w i t h about 80% o f t h e p o s s i b l e s u b s t i t u t i o n f o r a given temperature o c c u r r i n g i n t h e f i r s t hour. U s i n g t h i s knowledge, i t was p o s s i b l e t o o b t a i n a "product w i t h a d e s i r e d DS, merely by employing t h e c a l c u l a t e d amount o f s u l f a t i n g r e a g e n t and c a r r y i n g o u t t h e r e a c t i o n a t a temperature somewhat above t h e temperature which would produce t h a t DS when an e x c e s s o f s u l f a t i n g r e a g e n t was p r e s e n t . An added advantage i n t h i s t e c h n i q u e was t h e f a c t t h a t t h e r e was no r e s i d u a l s u l f a t i n g r e a g e n t l e f t f o l l o w i n g t h e r e action. E f f e c t o f P r e c i p i t a t i n g S o l v e n t . « A f a c t o r which, r a t h e r s u r p r i s i n g l y , a f f e c t e d t h e DS a c h i e v a b l e under a g i v e n s e t o f s u l f a t i o n c o n d i t i o n s , was t h e p a r t i c u l a r s o l v e n t used f o r t h e i n i t i a l p r e c i p i t a t i o n o f t h e gum. T h i s i s i l l u s t r a t e d i n T a b l e 2. With a l l t h r e e gums t e s t e d , methanol was g e n e r a l l y t h e most e f f i c i e n t a t a c t i v a t i n g , whereas DMF v a r i e d t h e most, b e i n g l e a s t e f f i c i e n t w i t h s t a r c h and most w i t h l o c u s t bean gum. I s o p r o p y l a l c o h o l worked w e l l f o r s t a r c h b u t was unimp r e s s i v e w i t h t h e two galactomannans. Acetone was gene r a l l y poor. A p o s s i b l e explanation i s that the order i s , i n general, r e l a t e d t o the dehydrating a b i l i t y of the s o l v e n t , m o d i f i e d by an a f f i n i t y o f each gum f o r a p a r t i c u l a r solvent. In a l l t h e s e c a s e s , t h e gum was a c t i v a t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method and s u l f a t e d a t 25° f o r 24 h o u r s , u s i n g s u f f i c i e n t 1 M DMF:S0 t o t o t a l l y s u l f a t e t h e gum, i . e . , t o produce a DS o f 3.0. P y r i d i n e was p r e s e n t i n an amount e q u a l t o t w i c e t h e number o f moles o f DMF:SC>3. 3

E f f e c t o f S u l f a t i n g Reagent. U s i n g DMF as a s o l v e n t , and l o c u s t bean gum as t h e s u b s t r a t e , f o u r s u l f a t i n g r e a g e n t s were t e s t e d f o r t h e i r a c t i v i t y a t 70° (for 4 hours). Here, a g a i n , t h e r e were d i f f e r e n c e s i n DS. G e n e r a l l y , t h e y f o l l o w e d t h e o r d e r p r e d i c t e d from

9.

Sulfation

GuiSELEY

of

Polysaccharides

151

T a b l e 1. E f f e c t o f d r y i n g method on degree o f s u b s t i t u t i o n (DS) and v i s c o s i t y (1%, 2 5 ° , B r o o k f i e l d LVF, 6 rpm) f o r l o c u s t bean gum + 1 mole DMF:SO3/mole anhydro sugar, 24 h r s . , room temperature.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

D.S.

25 , cps) 1.0

η

(

Oven D r y i n g , 60°C. ATM.

0. 006

2425

Benzene D i s t i l l a t i o n ,

ATM.

0. 03

3450

Benzene D i s t i l l a t i o n , VAC.

0. 07

1100

DMF D i s t i l l a t i o n , VAC.

0.47

1150

3.0

ρ Sulfation of Guar

2.0

—

D.S. 1.0

70°

0.0

2 3 T I M E , hrs.

Figure 1. Effect of time at different tempera­ tures in the sulfation of guar with DMF:S0 3

T a b l e 2. E f f e c t o f p r e c i p i t a t i n g s o l v e n t on degree o f s u l f a t i o n o f s t a r c h , guar, and l o c u s t bean gum (DMF:S0 , 25°/24 h r s . ) 3

Starch

Guar

LBG

MeOH

1. 51

DMF

1. 67

1. 37

DMF

0. 70

MeOH

1. 08

Acetone

0. 57

Acetone

0. 65

Py

0. 94

DMF

0. 31

i-PrOH

0. 60

Acetone

0. 53

i-PrOH

0. 47

Me OH

1. 65

i-PrOH

CARBOHYDRATE SULFATES

152

t h e s t r e n g t h o f t h e base used t o complex t h e SO3, w i t h t h e e x c e p t i o n o f t r i e t h y l a m i n e , which produced t h e h i g h e s t DS r a t h e r t h a n t h e second l o w e s t as a n t i c i ­ pated: S u l f a t i n g Reagent Et N:S0 DMF:S0 Py:S0 Me N:S0 3

DS 1.35

3

0.99

3

0.88

3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

3

0.

3

39

From a p r a c t i c a l p o i n t o f view, t h e r e i s no advantage i n u s i n g any o f t h e complexes o t h e r t h a n t h a t w i t h DMF, s i n c e i t can be p r e p a r e d and used d i r e c t l y , and i s v e r y efficient. T h i s a s p e c t was i n v e s t i g a t e d no f u r t h e r . A n a l y s i s f o r A c t i v e S u l f a t i n g Reagent. Because o f t h e d e s i r e t o c o n t r o l DS and t o m i n i m i z e polymer degra­ d a t i o n , i t was n e c e s s a r y t o a n a l y z e f o r t h e c o n c e n t r a ­ t i o n o f b o t h a c t i v e s u l f a t i n g r e a g e n t and h y d r o l y z e d DMF:S0 which may be p r e s e n t . P u b l i s h e d methods f o r such an a n a l y s i s u s u a l l y d i r e c t the r e a d e r t o add an e x c e s s of water and t i t r a t e t h e s u l f u r i c a c i d p r o ­ duced ( 2 3 ) . T h i s method f a i l s t o make t h e n e c e s s a r y d i s t i n c t i o n between t h e a c t i v e and t h e h y d r o l y z e d r e a ­ gent. I f , however, one uses anhydrous methanol i n p l a c e o f water i n a second t i t r a t i o n , i t i s p o s s i b l e t o d e t e r m i n e , by d i f f e r e n c e , b o t h t h e amount o f a c t i v e s u l f a t i n g r e a g e n t and t h a t o f s u l f u r i c a c i d . 3

C a l l i n g the r e a c t i o n w i t h water T i t r a t i o n f u r i c a c i d from a l l s o u r c e s i s d e t e r m i n e d : S0

3

+ H 0

+ H S0

4

(2

meq.

H)

H S0

-> H S 0

4

(2

meq.

H)

2

2

Titration S0

3

4

2

2

3

3

H S0 2

4

3

-> H S 0 2

4

+

( 1 meq.

H+)

(2

H)

meq.

Therefore, A =

2 X meq.

S0

3

+ meq.

H S0

4

Β =

meq.

S0

3

+ meq.

H S0

4

meq.

S0

3

A-B and,

=

sul­

+

Β d i s t i n g u i s h e s between t h e two

+ CH OH -> C H O S 0 H

A,

2

2

+

species:

9.

Sulfation

GuiSELEY

2B = 2 X meq. A = 2B-A

=

of

Polysaccharides

153

H S0

4

+ 2 X meq.

SO

meq.

H S0

4

+ 2 X meq.

SO

meq.

H S0

2

2

2

4

To i l l u s t r a t e t h i s , 10 ml. o f DMF:S0 r e a g e n t added to 50 ml. o f water r e q u i r e d 22.65 ml. o f 1 Ν NaOH. A n o t h e r 10-ml. p o r t i o n was added t o 10 ml. o f anhydrous DMF p l u s 2.5 ml. o f anhydrous methanol and 5 ml. anhydrous pyridine. The m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t , then 50 ml. o f water was added, and the s o l u t i o n t i ­ t r a t e d t o a p h e n o l p h t h a l e i n e n d p o i n t w i t h 1 Ν NaOH: 11.90 ml. was required. Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

3

A-B 2B-A

= 22.65 - 11.90

= 10.75

= 23.80 - 22.65 =

1.15

meq. meq.

S0

3

H S0 2

4

E f f e c t of R e s i d u a l Water A f t e r A c t i v a t i o n . This a n a l y t i c a l scheme was put t o use t o d e t e r m i n e how much r e s i d u a l water was p r e s e n t i n an a c t i v a t e d gum, by c a r r y i n g out t h e a n a l y s i s on the r e a c t i o n m i x t u r e a f t e r t h e r e a c t i o n was complete. Guar gum was a c t i v a ­ t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method, f o u r samples b e i n g p r e p a r e d i d e n t i c a l l y except t h a t each was taken t o a d i f f e r e n t s t a t e of d r y n e s s w i t h the DMF. S u l f a t i o n was c a r r i e d out w i t h two moles o f DMF:S0 per mole o f anhydrohexose, and two moles o f p y r i d i n e f o r each mole o f DMF:S0 , employing c o n d i t i o n s o f time and temperature t h a t would n o r m a l l y produce a DS o f about 1.25. When t h e r e a c t i o n was complete, the p r o d u c t was s e p a r a t e d and worked up, and the r e a c t i o n m i x t u r e ana­ l y z e d f o r a c t i v e DMF:S0 and s u l f u r i c a c i d . The v i s ­ c o s i t y o f the p r o d u c t and i t s DS were d e t e r m i n e d and compared w i t h the l e v e l o f s u l f u r i c a c i d found. These r e s u l t s are shown i n T a b l e 3. I t i s quite c l e a r that d i f f e r e n c e s i n v i s c o s i t y a r e not due t o v a r i a t i o n s i n DS, s i n c e t h o s e are v i r t u a l l y i d e n t i c a l , but i t does appear s i g n i f i c a n t t h a t even i n the p r e s e n c e o f an ex­ cess of p y r i d i n e , degradation occurs i n p r o p o r t i o n to the amount of s u l f u r i c a c i d produced. In view o f the f a c t t h a t the s u l f a t e e s t e r o f the polysaccharide s h o u l d be as a c i d i c as the s u l f u r i c a c i d (pyridinium i o n b e i n g the c o u n t e r i o n i n b o t h c a s e s ) , a p o s s i b l e explanation i s that oxidative degradation i s occurring, r a t h e r than h y d r o l y t i c c h a i n c l e a v a g e . (The t i t r a t a b l e a c i d i t y would then be e x p l a i n e d i n terms o f H S 0 plus unreduced H S 0 ) . 3

3

3

2

2

3

4

Other A c t i v a t i o n Methods.

A l t e r n a t i v e methods o f

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

154

CARBOHYDRATE

SULFATES

h y d r a t i n g the gums were i n v e s t i g a t e d i n t h e i n t e r e s t of economy - t h e d i s s o l v i n g and r e i s o l a t i o n o f a gum i s v e r y c o s t l y i n view o f t h e energy r e q u i r e m e n t s f o r s o l v e n t r e c o v e r y . In one, t h e gum was b r i e f l y soaked i n aqueous i s o p r o p y l a l c o h o l , then the m i x t u r e was b o i l e d r a p i d l y t o evaporate the a l c o h o l . The r e s u l t i n g wet, s w o l l e n powder was mixed w i t h DMF, broken up, and a l l o w e d t o s t a n d o v e r n i g h t . The water and DMF were r e moved by vacuum d i s t i l l a t i o n as i n t h e p r e c i p i t a t i o n method, and the r e s u l t i n g a c t i v a t e d gum s u l f a t e d . For a g i v e n s u l f a t i o n c o n d i t i o n , the DS and m o l e c u l a r weight (as i n d i c a t e d by v i s c o s i t y ) were dependent on the c o n c e n t r a t i o n o f a l c o h o l i n t h e o r i g i n a l m i x t u r e (Table 4 ) . A t low t i t e r s o f a l c o h o l , low v i s c o s i t i e s suggested incomplete dehydration with r e s u l t i n g degradation. At intermediate t i t e r s , v i s c o s i t i e s increased markedly, a c t u a l l y e x c e e d i n g t h e v i s c o s i t y and DS o f the t y p i c a l DS 1.2 5 guar s u l f a t e s d e s c r i b e d above. F i n a l l y , a t the h i g h e r t i t e r s , t h e DS a g a i n f e l l , p r o b ably a r e s u l t of incomplete a c t i v a t i o n . A v a r i a t i o n o f t h i s p r o c e s s was t h e use o f aqueous DMF t o h y d r a t e t h e gum. T h i s d i d not work w e l l when the o r i g i n a l m i x t u r e p l a c e d on t h e gum was d i s t i l l e d o f f i n vacuo, but i f t h e m i x t u r e were h e a t e d , and the gum t h e n f i l t e r e d o f f and d e h y d r a t e d w i t h f r e s h DMF, s u l f a t i o n w i t h o u t e x c e s s i v e d e g r a d a t i o n was p o s s i b l e . For example, h e a t i n g guar w i t h 3 0% aqueous DMF, filtering, d e h y d r a t i n g , and s u l f a t i n g under c o n d i t i o n s t h a t gave a DS o f 1.25 and a v i s c o s i t y o f about 2000 cps w i t h p r e c i p i t a t e d guar, a DS o f 1.09 and a v i s c o s i t y of 4240 cps were o b t a i n e d , a g a i n somewhat b e t t e r than by t h e p r e c i p i t a t i o n method. A v e r y unexpected r e s u l t came o u t o f a m o d i f i c a t i o n of t h e s e methods: guar was mixed w i t h 60% aqueous i s o p r o p y l a l c o h o l and r e f l u x e d 15 m i n u t e s , then f i l t e r e d o f f and d e h y d r a t e d and s u l f a t e d as d e s c r i b e d above. The p r o d u c t had a DS o f o n l y 0.16, but a v i s c o s i t y o f 29,000 cps a t 1%. (The u n t r e a t e d guar had a v i s c o s i t y of 6600 cps measured by t h e same procedure.) A repeat of t h e experiment r e s u l t e d i n a p r o d u c t w i t h a DS o f 0.20 and a 1% v i s c o s i t y o f 26,000 cps. E f f e c t o f Bases. Because t h e p r i m a r y g o a l o f t h e work was t o make a s y n t h e t i c c a r r a g e e n a n , and s i n c e c a r r a g e e n a n i s used so e x t e n s i v e l y i n f o o d s , an attempt was made t o r e p l a c e p y r i d i n e i n t h e r e a c t i o n m i x t u r e w i t h a l e s s n o x i o u s base. The d i f f i c u l t y was t o f i n d one which would not be so a l k a l i n e as t o r e a c t p r e f e r e n t i a l l y w i t h the DMF:SC>3, n o r so weak as t o f a i l t o p i c k up p r o t o n s as t h e y were l i b e r a t e d (the p o s s i b i l i t y

9.

Sulfation

GuiSELEY

of

Polysaccharides

155

Table 3. E f f e c t of s u l f u r i c acid i n reaction mixture on v i s c o s i t y of sulfated guar. (8.1 g. guar •> 13.1 g. Na guar SO4) . Meq. H S0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

2

4

From D M F i S O o (92 ml. 1.106 M)

From H 0 i n Guar 2

Total

9. 9

10. 0

19.9

2375

1.27

9. 9

14.3

24.2

2175

1.27

9.8

26.8

36. 6

1725

1.22

9. 9

38. 5

48.4

1525

1.24

ηJf (cps) 0

DS

T a b l e 4. E f f e c t o f a l c o h o l t i t e r on DS and v i s c o s i t y o f guar gum h y d r a t e d by a l c o h o l b o i l - o f f (8.1 g. guar + 100 ml. aqueous i s o p r o p y l a l c o h o l ) .

% i-PrOH

DS

η

10

0.70

840

20

0.33

605

30

1.25

850

40

1.26

1450

50

1.46

3400

60

0.88

5700

70

0.21

7300

1 < Q

(cps

CARBOHYDRATE

156

SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

o f o x i d a t i v e d e g r a d a t i o n had n o t been c o n s i d e r e d a t the time). A l t h o u g h none was found t h a t worked as w e l l as p y r i d i n e ( l i k e l y because o f t h e h e t e r o g e n e i t y o f t h e system) , ones t h a t showed promise were sodium and pot a s s i u m a c e t a t e s and d i s o d i u m phosphate. In T a b l e 5 are g i v e n some r e s u l t s . In t h e s e i n s t a n c e s , t h e aqueous DMF method was used except f o r t h e example w i t h p o t a s s i u m a c e t a t e ; t h i s was c a r r i e d o u t w i t h f r e s h l y p r e c i p i t a t e d guar. C o n t r o l o f DS. An i n t e r e s t i n g s i d e l i g h t t o o u r work was d e m o n s t r a t i n g t h e apparent i d e n t i t y o f funoran and s u l f a t e d a g a r o s e . Funoran i s a seaweed p o l y s a c c h a r i d e used i n g r e a t q u a n t i t i e s i n t h e F a r E a s t . Ext r a c t e d from G l o i o p e l t i s f u r c a t a and r e l a t e d s p e c i e s , i t f i n d s use- as a t h i c k e n e r o r g l u e . I t was i d e n t i f i e d as a g a l a c t a n s u l f a t e i n t h e e a r l y 1 9 0 0 ' s , and t h e p r e s e n c e o f 3 , 6 - a n h y d r o - L - g a l a c t o s e was e s t a b l i s h e d i n 1956 by H i r a s e , A r a k i , and I t o ( 2 4 ) . T h i s meant t h a t i n s t e a d o f b e i n g a c a r r a g e e n a n , i t was more l i k e a s u l f a t e d agarose. When agarose was s u l f a t e d t o t h e same e x t e n t as f u n o r a n , and t h e i r i n f r a r e d s p e c t r a compared ( F i g . 2), they were found t o be e s s e n t i a l l y i d e n t i c a l as proposed by S t a n c i o f f and S t a n l e y i n 1968 (2_5) · A d d i t i o n a l p r o o f o f f u n o r a n s s t r u c t u r e was g i v e n by Penman and Rees i n 1 9 7 3 (26J . 1

Conclusion - Properties o f the Products. S u l f a t e d p o l y s a c c h a r i d e s p r e p a r e d i n t h i s work were found t o have one f e a t u r e i n common - t h e y were n o n - g e l l i n g . As such, they were most l i k e lambda-carrageenan, and when t e s t e d i n a v a r i e t y o f a p p l i c a t i o n s , demonstrated an a b i l i t y t o r e p l a c e lambda-, b u t n o t kappa- o r i o t a carrageenan. Of a l l t h e p r o d u c t s made, t h e most comm e r c i a l l y p r o m i s i n g was a guar s u l f a t e h a v i n g a DS o f 2 . 1 9 - i t c o u l d be used t o s t a b i l i z e a c o l d - m i x e d m i l k shake a t o n e - f i f t h t h e l e v e l o f a c a r r a g e e n a n used f o r t h a t purpose. S e v e r a l o f t h e p r o d u c t s made s a t i s f a c t o r y c h o c o l a t e syrups which, when mixed w i t h c o l d m i l k , would t h i c k e n i t and p r e v e n t s e t t l i n g o f t h e cocoa, as i s c h a r a c t e r i s t i c o f lambda-carrageenan. A few o f t h e p r o d u c t s were a l s o s a t i s f a c t o r y as t o o t h p a s t e b i n d e r s a n o t h e r f u n c t i o n n o r m a l l y performed by l_ambda-carrageenan. A number o f f a c t o r s combined t o l e s s e n t h e commerc i a l i n t e r e s t i n t h e s e s e m i - s y n t h e t i c gums, t h e p r i n c i p l e ones b e i n g t h e i n c r e a s e d c o n c e r n over t h e s a f e t y o f f o o d a d d i t i v e s (and t h e r e q u i r i n g o f e x t e n s i v e and c o s t l y animal t e s t i n g ) , and t h e a v a i l a b i l i t y o f l a r g e r q u a n t i t i e s o f seaweeds through t h e s u c c e s s f u l d e v e l o p -

Sulfation

GuisELEY

of

Polysaccharides

T a b l e 5. Replacement o f p y r i d i n e by s a l t s o f weak acids. (Guar gum heated w i t h aqueous DMF, f i l t e r e d d e h y d r a t e d , and s u l f a t e d w i t h excess DMFrSO^).

,

Base

S0 n. 4

n

DS

25 l e 0

, , (c s) P

Na HP0

4

70°/l h r .

0.59

600

Na HP0

4

70°/2 h r s .

0.50

350

NaOAc

50°/l h r .

0. 63

1150

NaOAc

50°/2 h r s .

0. 66

1100

NaOAc

50°/2 h r s .

0. 60

3580

KOAc

70°/4 h r s .

0.77

1130

2

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

Conditions

5.0

6Ό

7.0

MICRONS ao

iq.o

π.ο

13.0

,

,16,

A

A = AGAR0SE

\\

G » G L 0 I 0 P E L T I S POLYSACCHARIDE S » S U L F A T E D AGAROSE

I

2000

1

1

1800

.

1

1600

.

1

1400

.

J

Y\J

1

1200

1 1000

1 800

CM -I Figure 2.

IR spectra of agarose, sulfated agarose (25.5% SO4), and funoran, the polysaccharide from Gloiopeltis furcata (24.7% SO4)

158

CARBOHYDRATE

SULFATES

ment o f seaweed farms i n t h e P h i l i p p i n e s ( 2 7 , 28) . In a d d i t i o n , s i n c e t h e major uses o f carrageenan a r e de­ pendent on i t s g e l l i n g p r o p e r t i e s , f u r t h e r developmen­ t a l work w i t h t h e s e n o n - g e l l i n g d e r i v a t i v e s was n o t warranted. N o n e t h e l e s s , a c o n t r o l l a b l e means o f p r o ­ d u c i n g p o l y s a c c h a r i d e s u l f a t e s was d e v e l o p e d and be­ cause o f i t s g e n e r a l a p p l i c a b i l i t y , i t can r e a d i l y be p u t i n t o p r a c t i c e , u t i l i z i n g v i r t u a l l y any p o l y s a c c h a ­ ride available.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

Experimental Materials. P o l y s a c c h a r i d e s used were o f s t a n d a r d commercial f o o d grade. S u l f u r t r i o x i d e was o b t a i n e d from A l l i e d Chemical Corp. as S u l f a n B. I s o p r o p y l a l ­ c o h o l was t e c h n i c a l grade, 9 9 % . T r i m e t h y l a m i n e - s u l f u r t r i o x i d e was p u r c h a s e d from Hexagon L a b o r a t o r i e s , I n c . , Bronx, N.Y., and used as r e c e i v e d . A l l o t h e r s o l v e n t s and c h e m i c a l s were r e a g e n t grade. DMF:S03 was p r e ­ p a r e d by t h e method o f G a r b r e c h t (23_) . Pyridine-sulfur t r i o x i d e was made by t h e method g i v e n i n I n o r g a n i c Syn­ t h e s e s (9_) , except t h a t S u l f a n Β was used i n p l a c e o f chlorosulfonic acid. In t h i s way, t h e p r o d u c t was f r e e from c h l o r i d e i o n . T r i e t h y l a m i n e - s u l f u r t r i o x i d e was p r e p a r e d as d e s c r i b e d by W h i s t l e r and Spencer ( 2 9 J · A c t i v a t i o n o f P o l y s a c c h a r i d e s . 1. Dissolution/ Reprecipitation. The p o l y s a c c h a r i d e was d i s p e r s e d i n t o r a p i d l y a g i t a t e d water a t room temperature, g e n e r a l l y a t a l e v e l o f 1%. I f n e c e s s a r y , t h e m i x t u r e was heated t o d i s s o l v e t h e p o l y s a c c h a r i d e . F o r guar, i t was n o t n e c e s s a r y ; l o c u s t bean gum, 85°C; s t a r c h and agarose were b o i l e d . The s o l was t h e n poured, i n a s t e a d y stream, i n t o t w i c e i t s volume o f t h e s o l v e n t s e l e c t e d , w i t h a g i t a t i o n p r o v i d e d by a g l a s s s t i r r i n g r o d . The gum was s e p a r a t e d by s t r a i n i n g i t through a p i e c e o f p o l y e s t e r c l o t h suspended on a s c r e e n o r c o l l a n d e r , and f u r t h e r d r i e d by drawing up t h e c o r n e r s o f t h e c l o t h and s q u e e z i n g o u t e x c e s s l i q u i d . The p o l y s a c c h a r i d e was t h e n shredded by hand i n t o a l a r g e necked s t a n d a r d t a p e r round-bottom f l a s k , and c o v e r e d w i t h * anhydrous DMF t o t h e e x t e n t o f about t e n times t h e i n i t i a l weight o f gum. The f l a s k was t h e n a t t a c h e d t o a r o t a t i n g e v a p o r a t o r and t h e s o l v e n t s removed a t 5 - 1 0 mm p r e s ­ sure. The temperature o f t h e b a t h s u r r o u n d i n g t h e f l a s k was a l l o w e d t o i n c r e a s e t o 8 0 ° . D i s t i l l a t i o n was stopped when no v i s i b l e l i q u i d remained i n t h e f l a s k . 2. Aqueous a l c o h o l b o i l - o f f . The p o l y s a c c h a r i d e was mixed w i t h aqueous i s o p r o p y l a l c o h o l i n t h e r a t i o o f 8 . 1 g. gum t o 100 ml l i q u i d , and t h e m i x t u r e q u i c k l y

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

9.

GuiSELEY

Sulfation

of

Polysaccharides

159

brought t o a b o i l i n a s t e a m - j a c k e t e d s t a i n l e s s s t e e l beaker. The a l c o h o l and some o f t h e water were q u i c k l y removed. The r e s u l t i n g damp, s t i c k y gum was mixed with 150 ml. o f DMF, a l l o w e d t o s t a n d o v e r n i g h t , and the vacuum d i s t i l l a t i o n c a r r i e d out as i n 1. 3. Aqueous DMF. The p o l y s a c c h a r i d e was mixed with about 12 p a r t s by weight o f aqueous DMF and h e a t e d i n a b o i l i n g water b a t h f o r 5-10 minutes. The m i x t u r e was s e p a r a t e d by f i l t r a t i o n , and the gum d e h y d r a t e d by v a c uum d i s t i l l a t i o n as i n 1. 4. Aqueous a l c o h o l soak. The p o l y s a c c h a r i d e was mixed w i t h aqueous i s o p r o p y l a l c o h o l and r e f l u x e d f o r 15 minutes o r a l l o w e d t o s t a n d a t room temperature o v e r n i g h t , then s e p a r a t e d by f i l t r a t i o n and d e h y d r a t e d by DMF d i s t i l l a t i o n as i n 1. S u l f a t i o n o f P o l y s a c c h a r i d e s . The a c t i v a t e d p o l y s a c c h a r i d e was s u l f a t e d i n t h e f l a s k i n which i t had been d r i e d down by d i s t i l l a t i o n . An amount o f a p p r o x i mately 1 M DMF:SC>3 ^ded, a c c o r d i n g t o the d e s i r e d r e s u l t or experimental c o n d i t i o n being i n v e s t i g a t e d . For example, f o r 8.1 g. o f s t a r c h , guar, o r l o c u s t bean gum, each o f which has an average o f t h r e e h y d r o x y l groups p e r anhydro sugar, t h e r e i s a t o t a l o f 150 meq. of h y d r o x y l . To p r o v i d e enough s u l f a t i n g agent t o comp l e t e l y s u l f a t e t h e gum t o a DS o f 3.0, 150 ml. o f 1 M DMF:SC>3 would be used; f o r a DS o f 1.0, o n l y 50 ml. o f the r e a g e n t would be added. P y r i d i n e was added a t twice t h e m i l l i e q u i v a l e n t l e v e l o f the s u l f a t i n g reagent. The f l a s k was s t o p p e r e d and p l a c e d on a shaker f o r the des i r e d time. F o r work a t e l e v a t e d t e m p e r a t u r e s , a water b a t h was p o s i t i o n e d beneath the arm o f the shaker and the f l a s k immersed i n i t . When t h e r e a c t i o n time had e l a p s e d , t h e m i x t u r e was q u i c k l y poured through a s i n t e r e d g l a s s f u n n e l on a f i l t e r f l a s k and the gum washed w i t h d r y DMF and/or i s o p r o p y l a l c o h o l t o remove a d h e r i n g u n r e a c t e d s u l f a t ing reagent. I t was then d i s p e r s e d i n water (about 100 p a r t s , based on i n i t i a l w e i g h t ) , and s t i r r e d . D i l u t e (1 N) sodium h y d r o x i d e was added t o m a i n t a i n a pH somewhat over 7 as t h e gum d i s s o l v e d . Usually, i t was h e a t e d toward t h e end o f t h e d i s s o l u t i o n p r o c e s s , then added t o 2 volumes o f i s o p r o p y l a l c o h o l t o r e p r e c i p i t a t e i t . I t was washed 2-3 t i m e s w i t h 85% i s o propyl a l c o h o l s u f f i c i e n t to cover i t , i n order to r e move r e s i d u a l p y r i d i n e . F i n a l l y , i t was squeezed out and d r i e d i n a c i r c u l a t i n g a i r oven a t 60° and ground through a 40-mesh s c r e e n i n a l a b o r a t o r y W i l e y M i l l . w

a

s

a<

A n a l y s i s o f DMF:S03 Reagent.

This i s described i n

CARBOHYDRATE

160

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

the

R e s u l t s and D i s c u s s i o n

SULFATES

section.

A n a l y s i s o f P r o d u c t s . M o i s t u r e was d e t e r m i n e d by d r y i n g a t 70°C and <10 mm. o v e r n i g h t . S u l f a t e was d e t e r m i n e d by d i g e s t i n g a sample o f t h e gum w i t h b o i l i n g c o n c e n t r a t e d n i t r i c a c i d u n t i l a l l o r g a n i c m a t t e r was d e s t r o y e d , then removing e x c e s s n i t r i c a c i d by a d d i t i o n o f s u l f a t e - f r e e f o r m a l i n . Usual proc e d u r e s were f o l l o w e d f o r p r e c i p i t a t i o n w i t h barium c h l o r i d e and w e i g h i n g as barium s u l f a t e . S i n c e the products were i s o l a t e d as sodium s a l t s , DS was c a l c u l a t e d d i r e c t l y from t h e s u l f a t e a n a l y s i s (anhydrous b a s i s ) u s i n g t h e f o r m u l a DS = 162 (%S0 ) 9600 - (102) (%S0 )" 4

m

4

T h i s h o l d s t r u e , w i t h o u t m o d i f i c a t i o n , o n l y f o r sodium s a l t s o f s u l f a t e d gums i n i t i a l l y h a v i n g an average o f t h r e e h y d r o x y l s p e r anhydro sugar u n i t . V i s c o s i t y Measurements. V i s c o s i t i e s were d e t e r m i n e d on 1% s o l u t i o n s o f t h e gums a t 25°C u s i n g a B r o o k f i e l d model LVF o r LVT r o t a t i o n a l v i s c o m e t e r a t 6 rpm, s e l e c t i n g the a p p r o p r i a t e s p i n d l e f o r the v i s c o s i t y being measured. S o l u t i o n s were p r e p a r e d by d i s p e r s i n g t h e gum i n t o 99 times i t s weight o f room temperature d i s t i l l e d water u s i n g good a g i t a t i o n . A l t h o u g h most o f the s u l f a t e d gums were s o l u b l e a t t h i s temperature, t h e m i x t u r e was r o u t i n e l y h e a t e d f o r 15 minutes i n a b o i l i n g water b a t h t o a s s u r e d i s s o l u t i o n o f such m a t e r i a l s as low DS l o c u s t bean gum s u l f a t e , and t o make a l l s o lution preparations similar. A f t e r heating, the s o l was c o o l e d , and water l o s t by e v a p o r a t i o n r e p l a c e d and t h o r o u g h l y admixed p r i o r t o making t h e measurement. M i l k s h a k e T e s t . A d r y b l e n d c o n s i s t i n g o f 20 g. o f sugar, 2 g. o f c o c o a , 0.01 g. a r t i f i c i a l v a n i l l a f l a v o r and 0.5 g. o f c a r r a g e e n a n (or s u l f a t e d p o l y s a c c h a r i d e ) p a s s i n g a 270-mesh s c r e e n , was added t o 8 f l . o z . o f c o l d m i l k and t h e m i x t u r e shaken v i g o r o u s l y f o r 15 seconds, then poured i n t o a 400-ml. B e r z e l i u s beaker. A shake o f h i g h q u a l i t y f i l l e d t h e beaker t o t h e b r i m , r e t a i n i n g t h e volume f o r o v e r 5 minutes, and no s e t t l i n g o f t h e c o c o a o c c u r r e d . T e s t m a t e r i a l s were evaluated against t h i s standard. C o l d Mix C h o c o l a t e M i l k and T o o t h p a s t e T e s t s . These a r e r a t h e r s p e c i a l i z e d t e s t s p e c u l i a r t o t h e i n d u s t r y , and somewhat beyond t h e scope o f t h e s u b j e c t at hand. C o n s e q u e n t l y , t h e y w i l l n o t be i n c l u d e d h e r e .

9.

GUISELEY

Sulfation

of

Polysaccharides

161

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

Abstract In an attempt t o p r e p a r e s u l f a t e d p o l y s a c c h a r i d e s which might have t h e gelling, s u s p e n d i n g , and stabi­ lizing p r o p e r t i e s o f c a r r a g e e n a n , t e c h n i q u e s were de­ v e l o p e d which l e d t o p r o d u c t s h a v i n g c o n t r o l l e d degrees of s u b s t i t u t i o n with r e t e n t i o n o f high molecular weight. B a s i c t o the p r o c e s s was an activation s t e p which i n v o l v e d t h e s e q u e n t i a l h y d r a t i o n - d e h y d r a t i o n o f the p o l y s a c c h a r i d e s . S e v e r a l methods p r o v e d effective, w i t h one resulting in a p r o d u c t h a v i n g f o u r t i m e s t h e viscosity o f t h e starting m a t e r i a l . An analytical method f o r distinguishing between a c t i v e sulfating re­ agent and t h a t which was h y d r o l y z e d , was d e v e l o p e d . S e v e r a l o f t h e p r o d u c t s demonstrated c a r r a g e e n a n - l i k e p r o p e r t i e s , most n o t a b l y , t h o s e o f t h e n o n - g e l l i n g fraction, lambda-carrageenan.

L i t e r a t u r e Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10a. 10b. 11.

12. 13. 14. 15. 16. 17.

Unger, R . , S e i t z , G . , Klockow, M. & Mehrhof, W., U.S. Patent 3,686,164 (1972). Ingelman, B.G.A. & Martensson, Ο . , Swedish Patent 165,090 (1958). Wolfrom, M . L . , & J u l i a n o , B . O . , J. Am. Chem. S o c . , (1960), 82, pp. 2588-2592. Cammarata, P . S . & E i c h , S . , U.S. Patent 3,271,388 (1966). Lee, J., U.S. Patent 2,599,564 (1952). London, E., Theobald, R.S. & Twigg, G . D . , Chem. & I n d . , (1955), pp. 1060-1061. Alburn, H.E., U.S. Patents 2,638,469 & 70, (1953). R i t z e r , H., Austrian Patent, 198,429 (1948). F e r n e l i u s , W.C. "Inorganic Synthesis. I I , " p. 173, McGraw-Hill Book Co., Inc., N.Y. (1946). G i l b e r t , Ε.E.,"Sulfonation and R e l a t e d R e a c t i o n s , " pp. 7-18, I n t e r s c i e n c e P u b l i s h e r s , N.Y. (1965). ibid., pp. 357-359. W h i s t l e r , R.L., G o a t l e y , J . L . & Spencer, W.W., C e r e a l Chem., (1959), 36, pp. 84-90.

Braconnot, Η . , Ann. Chim. Phys., (1819), 12, p. 185. Cook, D.L., E i c h , S. & Cammarata, P . S . , Arch. i n t . Pharmacodyn., (1963), 144, pp. 1-19. Wolfrom, M.L. & Wang, P . Y . , Carbohyd. Res., (1971), 18, pp. 23-27. L'Industrie Biologique, Francaise, French Patent M4431 (1966). M o r i i , E., et al, Japanese Patent 72 46792 (1972). T s u j i , K , , et al, German Offen. 2,546,699 (1976).

162 18. 19. 20. 21. 22. 23. 24. 25.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch009

26. 27. 28. 29.

CARBOHYDRATE SULFATES

Schweiger, R . G . , U.S. Patent 3,349,078 (1967). Schweiger, R . G . , U.S. Patent 3,401,160 (1968). Schweiger, R . G . , U.S. Patent 3,446,796 (1969). Schweiger, R . G . , U.S. Patent 3,624,069 (1971). Guiseley, K . B . , & Whitehouse, P . A . , U.S. Patent 3,720,659 (1973). Garbrecht, W . L . , J. Org. Chem., (1959), 24, pp. 368-372. Hirase, S . , A r a k i , C. & I t o , T., B u l l . Chem. Soc. Japan, (1956), 29, pp. 985-987. Stancioff, D . J . & Stanley, N . F . , Proc. VIth I n t . Seaweed Symp., Santiago de Compostela, Madrid, Spain, (1969), pp. 595-609. Penman, A. & Rees, D . A . , J. Chem. Soc., (1973), pp. 2182-2187. Doty, M . S . , Sea Grant Advisory Report, (1973), UNIHI-SEAGRANT-AR-73-02. Parker, H . S . , Oceans, (1976), pp. 12-19. W h i s t l e r , R . L . & Spencer, W.W., "Methods i n Carbohydrate Chemistry," 4, pp. 297-298, Academic Press, N . Y . , (1964).

RECEIVED

February 6, 1978.

10 Sodium Cellulose Sulfate via Cellulose Nitrite RICHARD G. SCHWEIGER

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

Stauffer Chemical Co., San Jose, C A 95112

It was reported previously that polyhydroxy polymers including cellulose react under certain conditions with Ν2O4 to form nitrite esters (1). The reaction medium must contain a proton acceptor, which may be a highly polar aprotic solvent, such as Ν,Ν-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC). The reaction path is shown in Figure 1. The assymetric isomer of Ν O , nitrosyl nitrate, reacts quantitatively with the cellulosic hydroxyl groups to result in cellulose t r i n i t r i t e ester and an equivalent amount of nitric acid. The nitrite ester has been found to be quite labile and to decompose immediately with a protic solvent, such as water or alcohol, in the presence of an acidic catalyst. It results in regenerated cellulose and nitrous acid or alkyl nitrite depending on the protic solvent used as shown in Figure 2. Analytical data showed that no modification or significant depolymerization occur during this process unless the temperature is excessively high. It was found that, due to the lability of the nitrite groups, cellulose nitrite can be used as a chemical intermediate for the preparation of cellulose derivatives, particularly cellulose sulfate (2). If, to the reaction mixture containing the nitrite ester, SO is added, preferrably in the form of a complex with, for example, DMF to avoid an excessive heat of reaction and degradation, an equivalent number of nitrite groups will be replaced by sulfuric acid ester groups as shown in Figure 3. This results in the formation of a mixed cellulose nitrite sulfate ester and an equivalent amount of N O by the reaction of nitrosyl ion with nitrate ion that was formed previously during nitrosation. The mixed ester is brought into contact with a protic solvent, such as water or alcohol. In the presence of the nitric acid formed during nitrosation, residual nitrite groups are removed immediately as shown in Figure 4 with the formation of cellulose sulfate ester and nitrous acid or alkyl nitrite. The sulfate ester may be precipitated with 2

4

3

2

4

0-8412-0426-8/78/47-077-163$05.00/0 © 1978 American Chemical Society

CARBOHYDRATE SULFATES

164

H C-0H

H C-0N0

2

2

\ Proton + 3 0N0N0

°'

L + 3 HNO3

>

2

Acceptor OH

ΟΝΟ

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

Figure 1.

Formation of cellulose nitrite

H C-0SO H

H C-0S0 H 2

3

N\ONO

2

+ 2

/

\

ROH or HOH

+

3

RONO or HONO

X

H

0

+ 2

> [ \

ΟΝΟ

Figure 2.

Hydrolysis of cellulose nitrite

H C-0S0 H

H C-0N0

2

2

DMF \

• H + NO3 + S 0 +

^\ONO

/

3

>

2~ ^ \

ΟΝΟ

3

N

° > ONO

-N 0 2

4

/ ΟΝΟ

Figure 3.

Sulfation of cellulose nitrite

H C-0N0 2

\ 3 ΟΝΟ

Figure 4.

Hydrolysis of mixed cellulose nitrite sulfate ester

RONO or HONO

10.

SCHWEIGER

Sodium

Cellulose

Sulfate

165

acetone, separated, and n e u t r a l i z e d . However, i t i s p r e f e r r e d to n e u t r a l i z e the complete mixture and p r e c i p i t a t e the sodium c e l l u l o s e s u l f a t e with a l c o h o l . By t h i s method, any D.S. o f up t o about 1.1 i s o b t a i n a b l e simply by c a l c u l a t i n g and using the s t o i c h i o m e t r i c amount o f S 0 . If D.S. values exceeding about 1.1 are t o be o b t a i n e d , lower D.S. n i t r i t e esters have to be used as the i n t e r m e d i a t e , such t h a t the t o t a l D.S., i . e . , the sum of the degree o f n i t r o s a t i o n plus the degree o f s u l f a t i o n , i s about 3. The amount o f Ν 0 f o r producing the n i t r i t e e s t e r i s again c a l c u l a t e d s t o i c h i o m e t r i c a l l y w h i l e that o f S 0 may be the s t o i c h i o m e t r i c amount or a s l i g h t excess. For example, the preparation o f a s u l f a t e e s t e r having a D.S. o f 1.4 requires n i t r o s a t i o n to a D.S. of about 1.6 f o l l o w e d by the a d d i t i o n of a s t o i c h i o m e t r i c q u a n t i t y of DMF-S0 complex t o produce a degree o f s u l f a t i o n o f 1.4 o r of a s l i g h t excess. At the higher degrees o f s u l f a t i o n , a s l i g h t excess o f S 0 i s p r e f e r r e d since t h i s assures a complete r e a c t i o n . Thus, degrees o f s u b s t i t u t i o n o f up t o c l o s e to 2 can be obtained. Although the f a c t t h a t lower D.S. n i t r i t e esters have to be used to o b t a i n degrees o f s u l f a t i o n o f above 1.1 may suggest a d i r e c t s u b s t i t u t i o n o f hydroxyl groups, experimental evidence supports e s t e r exchange s i m i l a r to the s u l f a t i o n o f cellulose t r i n i t r i t e . C e l l u l o s e t r i n i t r i t e , f o r example, can be s u l f a t e d s u c c e s s i v e l y to D.S. values o f 0.7 and 1.1 w i t h about s t o i c h i o m e t r i c amounts o f S 0 . I f , however, a f t e r s u l f a t i o n to D.S. 0.7, the r e s i d u a l n i t r i t e groups were removed by the a d d i t i o n of a s t o i c h i o m e t r i c amount o f methanol, no f u r t h e r s u l f a t i o n o c c u r r e d , even when an amount o f S 0 was added t h a t was s u f f i c i e n t to r a i s e the D.S. to about 2. This new method of d e r i v a t i z i n g c e l l u l o s e i s unique s i n c e i t i s the f i r s t method to u t i l i z e an a c t i v e c e l l u l o s e intermediate and, thus, to permit d e r i v a t i z a t i o n i n a homogeneous r e a c t i o n medium. A l l other methods are heterogeneous r e a c t i o n s using i n s o l u b l e c e l l u l o s e as the s t a r t i n g m a t e r i a l and d i r e c t l y s u b s t i t u t i n g f r e e hydroxyl groups. As a r e s u l t , the s u b s t i tuents are uniformly d i s t r i b u t e d over the molecule, i . e . , a D.S. o f 1 i n d i c a t e s s u b s t a n t i a l l y anhydroglucose monosulfate u n i t s and a D.S. of 2 anhydroglucose d i s u l f a t e u n i t s . In c o n t r a s t , the D.S. o f products of a l l p r i o r methods r e f e r s to an average v a l u e , and the D.S. of t h e i r i n d i v i d u a l u n i t s probably v a r i e s between 0 and 3. This leads t o remarkable d i f f e r ­ ences between products o f p r i o r methods and those of the present method. 1. Water s o l u b l e s u l f a t e e s t e r products are obtained a t a D.S. of as low as about 0.3. Authors o f comparable p r i o r methods s t a t e t h a t a D.S. o f greater than 1.0 i s r e q u i r e d f o r water s o l u b i l i t y ( 3 , 4 ) . 3

2

4

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

3

3

3

3

3

CARBOHYDRATE

166

Table I.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

Metal Mg

2 +

Ca

2 +

Sr

2 +

Ba

2 +

Zn

2 +

Cu

2 +

Co

2 +

Ni

2 +

Fe

2 +

Cd

2 +

H9

2 +

Pb

2 +

Sn

2 +

Al

C o m p a t i b i l i t y with P o l y v a l e n t Metal Ions

Ion

3 +

Ce

3 +

Cr

3 +

Fe

3 +

+ Compatible -

Incompatible

Sodium C e l l u l o s e S u l f a t e D.S. < 1.3 D.S. ;>

+ + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + _

+

-

SULFATES

10.

SCHWEIGER

Sodium

Cellulose

Sulfate

167

2.

Aqueous s o l u t i o n s are p e r f e c t l y c l e a r and do not contain g e l a t i n o u s transparent p a r t i c l e s , such as those o f most commercially a v a i l a b l e c e l l u l o s e products having high v i s c o s i t i e s . D i l u t e s o l u t i o n s , f o r example, can be f i l t e r e d d i r e c t l y through f i n e mi H i pore f i l t e r s without plugging or s u b s t a n t i a l reduction o f the flow r a t e . 3. A l l products, p a r t i c u l a r l y those w i t h a D.S. of below about 1.3, e x h i b i t an unusual c o m p a t i b i l i t y w i t h p o l y v a l e n t metal ions as shown i n Table I. Only i f the D.S. exceeds alj)out 1.3 i s some i n c o m p a t i b i l i t y observed w i t h B a , C e , and F e . S o l u t i o n s of products showing a plus do not form a p r e c i p i t a t e , remain c l e a r , and maintain v i s c o s i t y even when the s o l u t i o n i s saturated w i t h the metal s a l t s i n d i c a t e d . There i s hardly any other water s o l u b l e polymer - p a r t i c u l a r l y a n i o n i c polymer - t h a t e x h i b i t s complete c o m p a t i b i l i t y over such a wide range and i n saturated s a l t s o l u t i o n s . Another great advantage of t h i s method i s the f a c t t h a t no s i g n i f i c a n t depolymerization occurs during s u l f a t i o n v i a n i t r i t e e s t e r i n t e r m e d i a t e . I t has been shown p r e v i o u s l y that n i t r i t e e s t e r formation with Ν 0ι+ does not depolymerize c e l l u l o s e unless the r e a c t i o n mixture i s kept a t room temperature over an extended period o f time (1,2). S i m i l a r l y , no s i g n i f i c a n t depolymerization occurs during the subsequent s u l f a t i o n by e s t e r exchange as i n d i c a t e d by the extremely high v i s c o s i t i e s of the f i n a l products and by the f a c t t h a t i n c r e a s i n g the r e a c t i o n time f o r s u l f a t i o n from 1-2 h r s . to about 20 h r s . does not s i g n i f i c a n t l y reduce the v i s c o s i t y of the products. Table II summarizes the v i s c o s i t i e s o f 1% s o l u t i o n s o f products w i t h varying D.S. prepared from high D.P. cotton l i n t e r s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

2

3

3 +

2

Table I I . App. D.S. 0 0.3 - 0.5 0.6 - 0.9 1.0-1.3 1.4-1.6

V i s c o s i t y and i . r . Spectrum vs. Degree o f S u b s t i t u t i o n V i s c o s i t y , cps 5000 2000 1000 500

-

7000 4000 2000 1000

Frequency (-0H), CM"

1

3400 - 3420 <3430 ^3440 <3450 3460 - 3470

Products having maximum v i s c o s i t i e s of up to about 7000 cps are obtained. No c e l l u l o s e d e r i v a t i v e prepared by p r i o r methods, i r r e s p e c t i v e of the type o f s u b s t i t u e n t i n t r o d u c e d , e x h i b i t s v i s c o s i t i e s o f such magnitude. This i s o f p a r t i c u l a r s i g n i f i c a n c e s i n c e s u l f a t i o n o f polysaccharides g e n e r a l l y causes more serious degradation than most other r e a c t i o n s . Apparently, a f u l l y s u b s t i t u t e d c e l l u l o s e i s c o n s i d e r a b l y more r é s i s t e n t to depolymerization than a p a r t i a l l y s u b s t i t u t e d or u n s u b s t i t u t e d c e l l u l o s e when subjected t o chemical r e a c t i o n s .

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

168

In comparing v i s c o s i t y w i t h D.S., a great v i s c o s i t y increase i s observed i n the lower D.S. range as the D.S. decreases. A c e r t a i n v i s c o s i t y i n c r e a s e , of course, i s expected since the product concentrations i n the s o l u t i o n s are determined by weight and, t h e r e f o r e , the number of actual c e l l u l o s e molecules i n s o l u t i o n increases as the D.S. decreases. However, even when s o l u t i o n s of equal molar concentrations are compared to e l i m i n a t e t h i s e f f e c t remains the v i s c o s i t y increase unexpectedly high. An e x p l a n a t i o n f o r t h i s phenomenon i s hydrogen bond a c t i v i t y . In c e l l u l o s e , there i s i n t e n s i v e i n t e r - m o l e c u l a r hydrogen bond f o r m a t i o n , i n f a c t , to such an extent t h a t the c e l l u l o s e i s water i n s o l u b l e . I t i s , t h e r e f o r e , conceivable t h a t s u b s t i t u t i o n of hydroxyl groups to a low D.S. may reduce hydrogen bond formation s u f f i c i e n t l y to s o l u b i l i z e the c e l l u l o s e but not enough to e l i m i n a t e a c r o s s linking, i . e . , viscosity increasing, effect in solution. Such an e f f e c t would d i m i n i s h and f i n a l l y disappear as the D.S. i n c r e a s e s . Support f o r t h i s assumption i s i n the i . r . data i n Table II. The frequency of the OH peak of products with the lowest degrees of s u b s t i t u t i o n i s c l o s e to t h a t of the h i g h l y bonded hydroxyl groups i n u n s u b s t i t u t e d c e l l u l o s e . There i s a gradual s h i f t towards higher frequencies as the D.S. increases i n d i c a t i n g d i m i n i s h i n g hydrogen bond a c t i v i t y of the hydroxyl groups w i t h i n c r e a s i n g D.S. S i m i l a r thoughts were expressed p r e v i o u s l y w i t h regard to a l g i n s u l f a t e s (5). S i m i l a r to other polysaccharide s u l f a t e s ( 4 - 7 ) , a l l the c e l l u l o s e s u l f a t e products prepared v i a n i t r i t e e s t e r i n t e r mediates are p r o t e i n r e a c t i v e assumingly by the formation of i o n i c bonds between the negative s u l f a t e e s t e r groups and the p o s i t i v e s i t e s i n the p r o t e i n . This r e s u l t s i n v i s c o s i t y increases or p r e c i p i t a t i o n i f both the polysaccharide s u l f a t e and the p r o t e i n are i n s o l u t i o n p r i o r to combining them. Table III shows v i s c o s i t i e s of 0.5% s o l u t i o n s of sodium c e l l u l o s e s u l f a t e at varying D.S. w i t h and without 5% sodium c a s e i n a t e . Table III. App. D.S. 0.5 - 0.6 0.7 - 0.8 1.1-1.2 1.4 - 1.5

Reaction w i t h P r o t e i n V i s c o s i t y , cps With Caseinate Without Caseinate 810 754 1420 2400

690 550 303 226

A l l products show a s i g n i f i c a n t v i s c o s i t y increase over t h a t of the c o n t r o l . The v i s c o s i t y increase becomes greater as the D.S. of the c e l l u l o s e s u l f a t e e s t e r i n c r e a s e s . Combined w i t h the v i s c o s i t y increase i s a change of the flow c h a r a c t e r i s t i c s i n t h a t a s h o r t e r flow i s n o t i c e d and the s o l u t i o n

10.

SCHWEIGER

Sodium

Cellulose

Sulfate

169

appears t o become more coherent and e x h i b i t s a higher degree of p s e u d o p l a s t i c i t y . S i m i l a r r e s u l t s are obtained when, instead o f c a s e i n a t e , soy p r o t e i n i s o l a t e i s used. A number o f methods f o r the s u l f a t i o n o f c e l l u l o s e have been published p r e v i o u s l y , among them r e a c t i o n s with S 0 ( 8 10), c h l o r o s u l f o n i c a c i d (11,12), and s u l f u r i c a c i d w i t h (13-15) and without (16,17) an a l i p h a t i c alcohol i n the r e a c t i o n medium. Most o f these methods, p a r t i c u l a r l y those employing S0 or s u l f u r i c a c i d , s e v e r e l y degrade the c e l l u l o s e r e s u l t i n g i n products o f l i t t l e commercial value. Two other methods have been developed more r e c e n t l y , one by W h i s t l e r employing dimethyl s u l f o x i d e (DMS0)-S0 (4) and another by Schweiger using DMF-S0 complex ( 6 ) . In both methods, c e l l u l o s e i s s u l f a t e d d i r e c t l y under heterogeneous c o n d i t i o n s to r e s u l t i n products having r e l a t i v e l y high v i s c o s i t i e s . So f a r , they have been the only methods w i t h p o t e n t i a l commercial f e a s i b i l i t y . Table IV summarizes some o f the p r i n c i p a l d i f f e r e n c e s r e l a t i n g to the water s o l u b l e products o b t a i n a b l e by the two p r i o r methods and the present method. Both heterogeneous methods are s i m i l a r i n t h a t they r e s u l t i n products w i t h i n a narrow D.S. range. Relatively l a r g e excesses o f s u l f a t i n g agent are r e q u i r e d , and the D.S. cannot be e f f e c t i v e l y c o n t r o l l e d . The products have s i m i l a r maximum v i s c o s i t i e s o f about 200 t o 500 cps and poor compati b i l i t y w i t h a number o f metal ions p a r t i c u l a r l y a t high concentrations. In c o n t r a s t , the homogeneous s u l f a t i o n v i a n i t r i t e e s t e r permits easy c o n t r o l l a b i l i t y o f the D.S. w i t h i n a wide D.S. range - i n c l u d i n g p r e v i o u s l y unaccessible low D.S. ranges - by employing e s s e n t i a l l y s t o i c h i o m e t r i c q u a n t i t i e s of reagent and r e s u l t s i n products e x h i b i t i n g much higher maximum v i s c o s i t i e s and much greater c o m p a t i b i l i t y w i t h metal ions. Another method has been reported most r e c e n t l y by B. P h i l i p p e t a l . employing n i t r o s y l s u l f u r i c a c i d i n DMF (18,19). L i k e the present method, t h i s procedure r e s u l t s i n a mixed c e l l u l o s e n i t r i t e s u l f a t e e s t e r intermediate b u t , i n c o n t r a s t , i s a d i r e c t s u b s t i t u t i o n of c e l l u l o s e i n a heterogeneous system. 3

3

3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

3

Experimental P r e p a r a t i o n o f Sodium C e l l u l o s e S u l f a t e s . Cotton 1 i n t e r pulp (50 g.) i s suspended i n 1.5 1 DMF using a 3 1 three neck round bottom f l a s k equipped w i t h a mechanical s t i r r e r , C a C l tube, and dropping f u n n e l . About 75 g N 0i+ d i s s o l v e d i n 100150 ml DMF i s slowly added over a period o f 30-45 min. and s t i r r i n g continued u n t i l a c l e a r h i g h l y viscous s o l u t i o n i s obtained. A saturated s o l u t i o n o f DMF-S0 complex prepared as described p r e v i o u s l y (6) i s then slowly added t o the 2

2

3

Via N i t r i t e Ester homogeneous

^0.3-^2.0, e a s i l y obtainable >2.0, not o b t a i n a b l e easily control­ lable

not p o s s i b l e

J

<1.2, not o b t a i n a b l e DMF-SO, 3 ^1.2-2.0, d i f f i c u l t heterogeneous ;>2.0, usual D.S.

Obtainable not p o s s i b l e

3

D.S.

Controllability of D.S.

Comparison of D i f f e r e n t Methods

<1.0, not o b t a i n a b l e DMS0-S0 M . 0 - 1 . 5 most d i f f i c u l t heterogeneous ^1.5-1.8, usual D.S.

Method

Table IV.

up to ^7000

200-500

200-500

App. Maximum Viscosity

Compatibility 3+ 2+ Κ Ca Al

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

excess

excess

stoichio­ metric to slight excess

large

large

Sulfating Agent Required

10.

SCHWEIGER

Sodium

Cellulose

Sulfate

171

n i t r i t e e s t e r s o l u t i o n over a s i m i l a r period of time w i t h continuous s t i r r i n g and c o o l i n g i n an i c e bath t o maintain the temperature a t about 10-15°. The amount o f S 0 i s c a l c u l a t e d s t o i c h i o m e t r i c a l l y t o r e s u l t i n the d e s i r e d degree o f s u l f a t i o n . The r e a c t i o n mixture i s then mixed w i t h about 50 ml methanol or water, t r a n s f e r r e d i n t o a large beaker, and n e u t r a l i z e d w i t h an aqueous s o l u t i o n o f N a C 0 . During n e u t r a l i z a t i o n , strong a g i t a t i o n and c o o l i n g are r e q u i r e d . The sodium c e l l u l o s e s u l f a t e i s p r e c i p i t a t e d by the a d d i t i o n o f methanol, washed with 70% aqueous methanol, and d r i e d . The y i e l d i s q u a n t i t a t i v e . C e l l u l o s e n i t r i t e e s t e r s w i t h a D.S. o f below about 3 f o r the preparation of s u l f a t e e s t e r s with a D.S. o f above about 1.1 are obtained s i m i l a r l y by using correspondingly s m a l l e r , s t o i c h i o m e t r i c a l l y c a l c u l a t e d amounts o f H 0i+. The f u r t h e r procedure f o r s u l f a t i o n and n e u t r a l i z a t i o n i s the same as described above. A n a l y t i c a l and Property Determinations. Viscosities were measured with a B r o o k f i e l d Viscometer, LVT Model, a t 20° and 60 rpm. For the i . r . s p e c t r a , f i l m s of the products were used prepared by c a s t i n g a 1-2% s o l u t i o n on a glass p l a t e , drying at room temperature and, subsequently, a t 8 0 ° , and removing the dry f i l m with a razor blade. The D.S. of the sodium c e l l u l o s e s u l f a t e was determined g r a v i m e t r i c a l l y as described p r e v i o u s l y (6) by h y d r o l y z i n g the product i n 10% HC1 a t 100° over n i g h t and p r e c i p i t a t i n g the s u l f u r i c a c i d formed as BaSO^. The c o m p a t i b i l i t y w i t h metal ions was determined by d i r e c t l y d i s s o l v i n g i n c r e a s i n g amounts o f the c r y s t a l l i n e s a l t i n a 1% aqueous s o l u t i o n o f the sodium c e l l u l o s e s u l f a t e u n t i l s a t u r a t e d . Metal s a l t s used were c h l o r i d e s , s u l f a t e s , or a c e t a t e s . I n c o m p a t i b i l i t y was i n d i c a t e d by the formation of a p r e c i p i t a t e combined with a l o s s o f v i s c o s i t y . To demonstrate p r o t e i n r e a c t i v i t y , the v i s c o s i t y o f a s o l u t i o n c o n t a i n i n g both 0.5% sodium c e l l u l o s e s u l f a t e and 5% sodium c a s e i n a t e was compared with t h a t of a 0.5% s o l u t i o n o f the corresponding sodium c e l l u l o s e s u l f a t e . 3

2

3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

2

Summary C e l l u l o s e n i t r i t e prepared as described p r e v i o u s l y was allowed to react with DMF-S0 complex. This r e s u l t e d i n the replacement o f part o f the n i t r i t e e s t e r groups by s u l f a t e e s t e r groups with the formation of mixed c e l l u l o s e n i t r i t e s u l f a t e e s t e r . Residual n i t r i t e groups were removed by the r e a c t i o n with a p r o t i c s o l v e n t , such as a l c o h o l or water, and the c e l l u l o s e s u l f a t e e s t e r was n e u t r a l i z e d t o form sodium c e l l u l o s e s u l f a t e . Both n i t r o s a t i o n and s u l f a t i o n appear to be s t o i c h i o m e t r i c r e a c t i o n s , and the degree o f s u l f a t i o n can be c a l c u l a t e d by the amount o f S 0 used. Water s o l u b l e 3

3

172

CARBOHYDRATE SULFATES

products are thus obtained w i t h i n a D.S. range of about 0.3 to 2. Chemical and p h y s i c a l p r o p e r t i e s of c e l l u l o s e s u l f a t e e s t e r s prepared v i a n i t r i t e e s t e r intermediates d i f f e r s i g n i f i c a n t l y from those o f s i m i l a r products prepared by p r i o r methods.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch010

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Schweiger, R. G., J. Org. Chem. (1976), 41, 90. Schweiger, R. G., Tappi (1974), 57, 86. G u i s e l e y , K. B., U. S. Patent 3,720,659 (1973). W h i s t l e r , R. L., U. S. Patent 3,507,855 (1970). Schweiger, R. G., Carbohydrate Research (1972), 21, 275. Schweiger, R. G., Carbohydrate Research (1972), 21, 219. W h i s t l e r , R. L., " I n d u s t r i a l Gums", 2nd E d . , Academic P r e s s , I n c . , New York and London, 1973, pp. 105-107. Traube, W., B l a s e r , Β., and Grunert, C., Ber. (1928), 61, 745. Traube, W., B l a s e r , Β., and Lindemann, E., Ber. (1932), 65, 603. Meyer, Κ. H., Piroué, R. P., and O d i e r , M. E., Helv. Chim. Acta (1952), 35, 574. Tamba, R., Biochem. Z. (1923), 141, 274. Rabenstein, L., U. S. Patent 2,042,484 (1936). Malm, C. J., and Crane, C. L., U. S. Patent 2,539,451 (1951). Malm, C. J. and Crane, C. L., U. S. Patent 2,675,377 (1954). Frank, G., U. S. Patent 2,559,914 (1951). F e h l i n g , H., Ann. (1845), 53, 135. Kagawa, I., J. Soc. Text. C e l l u l o s e Ind. Jap. (1945), 1, 681. B i s c h o f f , K., Dautzenberg, H., Philipp, B., and Wagenknecht, W., Faserforschung und T e x t i l t e c h n i k (1976), 27, 111. Wagenknecht, W., Philipp, B., and B i s c h o f f , Κ., East Ger. Patent 111,381 (1975).

RECEIVED F e b r u a r y 6,

1978.

11 Reaction of Starch with the Chlorosulfonic AcidFormamide Reagent F. S C H I E R B A U M and K. K Ö R D E L

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

Akademie der Wissenschaften der D D R , Forschungszentrum fur Molekularbiologie und Medizin, Zentralinstitut für Ernährung, Potsdam-Rehbrücke, German Demokratic Republic

Reactions of s t a r c h polysaccharides with s u l f a t i n g agents have been c a r r i e d out (1) f o r making heparinoids with blood a n t i coagulating p r o p e r t i e s (2-4), f o r obtaining products having a n t i u l c e r act i o n by i n h i b i t i n g pepsin (5-7), to produce t h i c k e ning agents f o r foods (8) and a u x i l i a r y substances i n various techniques (9,10) and, g e n e r a l l y to subs t i t u t e for n a t u r a l l y occuring polysaccharide gums (11-13). The methods used f o r introducing s u l f u r i c acid groups i n t o the molecules of s t a r c h vary widely. There are (1) r e a c t i o n s i n homogeneous nonaqeous or aqueous systems, (2) reactions i n heterogeneous liquid systems, and (3) r e a c t i o n in dry s t a t e . A summar i z i n g survey on the most important methods and r e a gents used so far is given i n Table 1. The p r o p e r t i e s of the r e s u l t i n g starch esters depend l a r g e l y on these methods and reagents, and it is shown that r e actions i n homogeneous systems give products with low, medium and high degrees of s u b s t i t i o n (3,6,1417). I t is necessary, however, to p r e - g e l a t i n i z e the starch thus l i m i t i n g the starch concentration by its v i s c o s i t y . Reactions in heterogeneous systems on the other hand which may be performed at higher concent r a t i o n s lead to low degrees of s u b s t i t u t i o n (11,13,18-20). The lowest degrees of s u b s t i t u t i o n are obtained by r e a c t i o n s in a dry state (8,21,22). The system S O 3 - t r i a l k y l a m i n e i s f r e q u e n t l y used and well studied as a reactant i n aqueous a l k a l i n e (6,19, 17,20,23,24) and nonaqeous solvents ( p y r i d i n e , DMF, formamide, dimethylsulfoxide) (6,9,11,15,16,25) using l i q u i d or gaseous s u l f u r t r i o x i d e . Another r e a c t i v e system, c h l o r o s u l f o n i c a c i d (4,6,14,26-28) and formamide (4,14,28,29), seems to be of partial 0-8412-0426-8/78/47-077-173$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

174

CARBOHYDRATE SULFATES

i n t e r e s t with respect to costs and a v a i l a b i l i t y οί' chemicals i n the case of upscaling to an i n d u s t r i a l l e v e l f o r making starch s u l f a t e s as p r o t e i n comp^xing agents (30)· But there i s poor knowledge on the influence of the r e a c t i o n conditions on the proper­ t i e s of the end products. The purpose of the present a r t i c l e i s to present information on a c o r r e l a t i o n between p r i n c i p a l reac­ t i o n conditions and the fundamental properties of the r e s u l t i n g esters regarding degree of s u b s t i t u t i o n ( D S ) , degradation and contamination by decomposition reactants. The c h l o r o s u l f o n i c acid-formamide system has been chosen because of i t s several advantages as com­ pared with other r e a c t i v e systems that have been i n ­ vestigated i n numerous preliminary experiments. Formamide has various functions and acts ciS a swelling and s o l u b i l i z i n g agent f o r s t a r c h granules, a com­ plexing agent with c h l o r o s u l f o n i c a c i d , a b u f f e r i n g agent during the r e a c t i o n , and as a solvent f o r the r e a c t i o n products. Also, i t may be destroyed i n suf­ f i c i e n t concentrations of a l k a l i . The main b e n e f i t , however, i s i n the s i m p l i f i c a t i o n of the i n i t i a l r e ­ a c t i o n steps i . e . , the complex may be formed i n the r e a c t i o n v e s s e l by adding c h l o r o s u l f o n i c acid to formamide ( c a l c u l a t e d amounts f o r complex formation and f o r d i s s o l v i n g the starch)immediately followed by the a d d i t i o n of the dry s t a r c h . Under these conditions, the s t a r c h i s suspended at ambient temperature up to concentrations of 30 % of the amount of formamide used f o r d i s s o l v i n g the s t a r c h . There i s no problem with the f l u i d i t y of t h i s system i f i t i s heated slowly up to the r e a c t i o n temperature d e s i r e d . At a s u f f i c i e n t l y high temperature, the s t a r c h granules begin to d i s s o l v e slowly i n the r e a c t i v e system. Con­ sequently, the r e a c t i o n may be c a r r i e d out a l t e r n a ­ t i v e l y i n heterogenous or homogeneous system (30)· Results C ο e f f i c i ent s of de t erminat i o n . For 16 e f f e c t v a r i a b l e s out of 20 which have been i n v e s t i g a t e d at a r e a c t i o n temperature of bO °C ( l e v e l s ) , c o r r e l a t i o n s by variance between e f f e c t v a r i a b l e s and process v a r i a b l e s have been e s t a b l i s h e d . Only the f o l l o w i n g items are not relevant i n mathematical sense : £*] , c a l c u l a t e d on the basis of anhydroglucose unit ( A G U ) and of pure starch s u l f a t e , R „ c a l c u l a t e d on the basis AGU, and inorganic s u l f a t e ( % S^). At a r e ­ a c t i o n temperature of 70 C ( l e v e l / * ), 12 e f f e c t 9

11.

SCHIERBAUM AND KÔRDEL

Starch and Chlorosulfonic

Acid-Formamide

175

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

v a r i a b l e s out oi' 2 0 are c o r r e l a t e d with process v a r i ables. S i m i l a r l y ^ p r o p e r t i e s without s a t i s f a c t o r y corr e l a t i o n are |V} , inorganic s u l f u r , R g , c a l c u l a t e d on the basis of pure and crude starch s u l f a t e , the o p t i c a l density of s o l u t i o n s , and the y i e l d of pure s t a r c h s u l f a t e , the l a t t e r being e v i d e n t l y more dependent on p u r i f i c a t i o n than r e a c t i o n c o n d i t i o n s . F i s h e r Tggt. The combination of four independent process v a r i a b l e s by assuming a s i n g l e e f f e c t and double and t r i p l e i n t e r a c t i o n s r e s u l t s i n 1 6 explanatory v a r i a b l e s . Twelve of these v a r i a b l e s show a s i g n i f i c a n t influence on e f f e c t v a r i a b l e s . They are summarized i n Table 2 and are v a l i d f o r the two r e a c t i o n temperatures used. Independent from the general a c t i o n of the temperature, which i s of predominant influence on the q u a l i t y of e f f e c t v a r i ables, Table 2 gives a sequence of v a l i d i t y f o r the explanatory v a r i a b l e s involved i n the process. The concentration of water (w), which i s shown 6 7 times to be s i g n i f i c a n t i n s i n g l e and m u l t i p l e a c t i o n and the concentration of S O 3 ( r ) , s i g n i f i c a n t i n 6 4 cases of action are most e s s e n t i a l from t h i s point of view. The concentration of s t a r c h i n formamide ( s ) with 1 9 s i g n i f i c a n t actions and f i n a l l y the time of r e a c t i o n ( t ) with only 7 cases of s i g n i f i c a n c e are l e s s important. The r e a c t i o n time, however, may be of greater importance i f the r e a c t i o n i s c a r r i e d out at a temperature of below 5 0 ° C . analysis and i n t e r p r e t a t i o n of eqations of r e gression. In the f o l l o w i n g only a few c o r r e l a t i o n s between process v a r i a b l e s and e f f e c t v a r i a b l e s may be regarded, s e l e c t i n g those with the greatest importance f o r the process to be studied: S u l f u r , bound i n ester groups, S·.,.; Highly e s t e r i f i e d products containing more than 1 6 % S J J , ( D S > 1 , 4 ) may be obtained only at a temperature of 7 0 ° C and a SO3/AGU r a t i o of 4 : 1 . The water content of s t a r c h , when i n creased from b % to 2 0 % acts i n favor of higher degrees of e s t e r i f i c a t i o n ( f i g . 1 ) . At a SO3/AGU r a t i o of 2 : 1 , however, the degree of e s t e r i f i c a t i o n i s low and appears to decrease with i n c r e a s i n g water content. Very low degrees of e s t e r i f i c a t i o n ( < 1 0 % D S < 0 , 7 ) are also obtained at bO ° C . At both SO3/AGU r a t i o s , the degree of e s t e r i f i c a t i o n increases with i n c r e a s i n g water content, the increase being more pronounced at the 4 : 1 than the 2 : 1 r a t i o ( f i g . 1 ) . F i g . 2 shows that, at 7 0 ° C , the degree of este9

9

Non flu id,

dry,

Aqueous, alkaline, homogene­ ous, heterogeneous inhibitors)

(^swelling

soin.

dit. alkali

bases

organ. N'contatning

homogeneous

heterogeneous

Solvent

Non aqueous,

System

organ.

[amides)

[urea

\

SO 3

salts

sulfite

or

sulfate

acidic

LioM

α min es

3

S0

oleum,

tri alky Ι­

bases

acid,

-

-

chlorosulfonic

3

SO Donor

-300

65

- 65

20

-100

hours

-several

1h

0,22

max.

0,01 -1J

1h -2*th

days

0,1 - 2,6

15min

Degree of Esterificatior, DS

-several

Time

0

Temp. °C

and reagents, used for preparing starch sulfates literature)

Complexing Agent *

from

methods

N'contatning

(taken

survey on preferred

of

Summarizing

Characteristics

Tab. 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

-4

SINGLE EFFECT w Γ

· · * · · ·

Γ r W w r 8

t

(w

F

-

-

TIME OF ACTION)

3

5

MOL. RATIO AGU/S0 , β

5

2

-

3 1 1

4

4

-

7

10

-

7 3

>

-

F

4

10

17 12 6

>

WATER CONTENT OF THE STARCH, r

8 · W · Γ t · 8 · Γ

TRIPLE INTERACTION

w s 8 t t t

DOUBLE INTERACTION

t

8

EFFECT

5 1

3 1 1 1

1

4

2 1

4

1

^ 3

£

12 1

21 9 6 2 2 1

25 19 12 1

ACTIONS

STARCH CONCENTRATION IN FORMAMIDE

F

F I S H E R - T E S T FOR PROVING SIGNIFICANCE BETWEEN EXPLANATORY VARIABLES AND VARIABLES IN THE REACTION OF THE CHLOROSULFONIC ACID - FORMAMIDE SYSTEM WITH STARCH

TYPE OF ACTION

TAB. 2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

CARBOHYDRATE SULFATES

5

10 WATER

15

20wCVoJ

CONTENT

Figure 1. Lines of regression for the influence of the water content (w) of the starch on esterification (% S ) (v= S0 /AGU ratio) E

3

15

20 STARCH

25 s(%> CONTENT

Figure 2. Lines of regression for the influence of the concentration of the starch in formamide (s) on esterification (% S ) (w = water content, r = SO,/ AGU ratio) E

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

11.

scHiERBAUM A N D KORDEL

Starch and Chlorosulfonic

Acid-Formamide

179

r i f i c a t i o n decreases s l i g h t l y with i n c r e a s i n g conc e n t r a t i o n oi' starch i n a l l cases. The l i m i t i n g v i s c o s i t y number may be considered as a general measure f o r molecular degradation without using exact values f o r the molecular weight. Generally, s o l u t i o n s with excess e l e c t r o l y t e are used to eliminate p o l y e l e c t r o l y t e behavior. V i s c o s i t y measurements show a pronounced decrease of [»jj takes place with i n c r e a s i n g v/ater content ( f i g . 3 ) . A comparision of the values of [%] i n d i c a t e s that degradation u s u a l l y takes place under conditions which produce high degrees of ester i f i c a t i o n (high temperature and high SO3/AGU r a t i o ) and, i n v e r s e l y , t h a t s l i g h t l y degraded esters may be obtained under conditions f a v o r i n g low degrees of e s t e r i f i c a t i o n (lower temperature and lower SO3/AGU ratio). The high molecular weight p o r t i o n as determined i n the excluded volume through g e l permeation chromatography on SEPHADEX G 200 may be i n t e r p r e t e d s i m i l a r l y . There i s a strong tendency to decrease with i n c r e a s i n g water content at a SO3/AGU r a t i o of 4:1, both with 15 % and 25 % s, and a tendency to increase with an SO3/AGU r a t i o of 2:1 ( f i g . 4 ) . In t h i s case, however, the d i f f e r e n t p o l y e l e c t r o l y t e behavior of the v a r i o u s l y s u b s t i t u t e d polysaccharide esters could not be overcome by the solvent and t h e r e f o r e , a few r e s u l t s are i n c o n s i s t a n t with these tendencies. The apparent v i s c o s i t y ty) of 5 % aqueous s o l u t i o n s i s h i g h l y i n f l u e n c e d by the water content of the s t a r c h at 70 °C and decreases at a high SO3/AGU r a t i o ( f i g . 5 ) , The same tendencies have been observed at the higher v i s c o s i t y l e v e l at 50 °C. It i s s u r p r i s i n g , that high r a t i o s of SO3/AGU as w e l l as high concentrations of s t a r c h give r i s e t o higher v i s c o s i t i e s of the e s t e r . I t i s p o s s i b l e that the h y d r o l y s i s which i s accelerated by the higher water content of the s t a r c h may be reduced by higher amounts of formamide. I t i s , t h e r e f o r e , recommended to c a r r y out the r e a c t i o n at an SO3/AGU r a t i o of 4:1 and a s t a r c h concentration of 25 % at 70 °C i n order to obtain s t a r c h esters with higher v i s c o s i ties. With respect to the y i e l d of s t a r c h s u l f a t e , which i s s i g n i f i c a n t l y c o r r e l a t e d with process v a r i ables only at 50 °C, the same tendency i s observed as i n the dependency o f S-g on process v a r i a b l e s (see f i g . 1 ) . The tendency of the y i e l d value to i n crease with i n c r e a s i n g water content and i n c r e a s i n g SO3/AGU r a t i o , i s due t o a more complete e s t e r i f i c a t i o n . Thus, high degrees of e s t e r i f i c a t i o n c o i n c i d e

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

180

Figure 3. Lines of regression for the influence of the water content (w) of the starch on limiting viscosity number [η] (r = S0 / AGU ratio")

10 |_

3

nor) 5

10 WATER

CONTENT

Figure 4. Lines of regression for the influ­ ence of the water content (w) of the starch on the high molecular weight portion as excluded by gel permeation chromatography (SEPHA­ DEX G 200) (τ = SO /AGU ratio, s = starch concentration) 3

15

20wf%)

11.

scHiERBAUM AND KÔRDEL

Starch and

Chlorosulfonic

Acid-Formamide

181

with high y i e l d s . The amount of formamide necessary f o r producing 1 g s t a r c h s u l f a t e i s shown i n f i g . 6 and appears to decrease with i n c r e a s i n g s t a r c h concentration ( s ) . An i n c r e a s i n g water content, p r e f e r a b l y with a SCh/ AGU r a t i o of 2:1, leads to a s l i g h t decrease of tne formamide amount a l s o . The amount of c h l o r o s u l f o n i c a c i d necessary f o r 1 g ester decreases only s l i g h t l y with i n c r e a s i n g water content ( f i g . 7 ) ; The reason probably i s the increase of S as w e l l as of the y i e l d . Of course, the higher l e v e l r e f e r s to a SO3/AGU r a t i o of 4:1. The nitrogen content of the e s t e r s , the p r i n c i pal contamination, r e s u l t s from formamide bound by the s u l f a t e group. Therefore, the n i t r o g e n content e x h i b i t s the same tendencies i n dependence from process v a r i a o l e s as the degree of e s t e r i f i c a t i o n , i . e . , the higher the degree of e s t e r i f i c a t i o n , the higher the contamination with formamide. Independent of t h i s , the y i e l d of ammonium s u l f a t e as a byproduct r e s u l t i n g from decomposition of formamide i s highest at 70 °C and an SO3/AGU r a t i o of 4:1 and i s i n c r e a sing considerably with the water content.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

E

Discussion The mutual actions of the explanatory v a r i a b l e s w i t h i n the given l e v e l of numerical values chosen f o r t h i s process are summarized i n Table 3 with r e spect to high degrees of e s t e r i f i c a t i o n , high y i e l d values, low molecular d e s t r u c t i o n , and low s p e c i f i c amount of raw m a t e r i a l s . Among the explanatory var i a b l e s a f f e c t i n g the process, the temperature has the greatest i n f l u e n c e on the e f f e c t v a r i a b l e s . Therefore high degrees of e s t e r i f i c a t i o n w i l l r e s u l t only at the higher temperature l e v e l . At t h i s l e v e l the r e a c t i o n proceeds completely i n the homogeneous system, the s t a r c h being d i s s o l v e d i n the reagent. At the 50 °C l e v e l , on the other hand, the r e a c t i o n takes place i n a predominantly heterogeneous system, rendering some s t a r c h soluble and r e a c t i n g to a higher degree and l e a v i n g another part of the s t a r c h undissolved and unreacted. Consequently these r e a c t i o n products have a non-uniform d i s t r i b u t i o n ofi substituents and a wide rang of molecular degradation* The water content or the s t a r c h has twofold e f f e c t , (a) : i t r e s u l t s i n high degrees of e s t e r i f i c a t i o n and a high e f f i c i e n c y regarding raw m a t e r i a l s , and (b) i t leads t o - pronounced h y d r o l y t i c degradation of molecules. The increase i n r e a c t i v i t y i s due to a

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

182

s = 25% 5

10 WATER

15

20wC%)

CONTENT

Figure 5. Lines of regression for the influence of the water content of the starch on apparent viscosity (5% sol) (r = SO /AGU ratio, s = starch concentration) s

Figure 6. Lines of regression for the influence of the starch concentration on the amount of formamide needed for producing 1 g of starch sulfate (w = water content of the starch)

scHiERBAUM AND KÔRDEL

Starch and Chlorosulfonic

Acid-Formamide

5 (9)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

51

5

10 WATER

15

20w(%)

CONTENT

Figure 7. Lines of regression for the influence of the water content of the starch on the amount of chlorosulfonic acid needed for producing 1 g of starch sulfate (r = AGU/SO ratio) 3

183

70

TEMPERATURE °C

: 1

25

4

STARCH CONCENTRATION %

3

S0 /AGU RATIO

20

WATER CONTENT %

E

S

HIGH

: 1

50

-

4

20

HIGH VIELD VALUE 5

50

-

2 : 1

V

HIGH

4

G

50

25

: 1

5

N

LOW

p

Q

50,70

2:1

20

50,70

25

2:1

20

LOW AMOUNTS OF FORMAMIDE CHLOROSULFONIC ACID

EFFECT OF PROCESS VARIABLES ON THE STARCH CHLOROSULFONIC ACID - FORMAMIDE SYSTEM WITH RESPECT TO HIGH DEGREES OF ESTERIFICATION ( S ) AND YIELD VALUES, SMALL MOLECULAR DESTRUCTION (HIGH f * J VALUES) AND BY - PRODUCTS ( N ) , AND LOW AMOUNTS OF RAW MATERIAL

EXPLANATORY VARIABLES

TAB. 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

C/3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

11.

scHiERBAUM AND KÔRDEL

Starch and Chlorosulfonic

Acid-Formamide

185

pronounced swelling of the s t a r c h that contains r e l a t i v e l y large amounts of water, but the amount of water that causes swelling and increases reactivityw i l l also cause an increased h y d r o l y t i c a c t i v i t y and r e s u l t i n cleavage of g l y c o s i d i c bonds. When the s u l f u r t r i o x i d e content, i . e . , the SO3/AGU r a t i o , that i s h i g h l y s i g n i f i a n t i n a l l actions observed i s increased, higher degrees of e s t e r i f i c a t i o n , high y i e l d s and higher molecular d e s t r u c t i o n are observed. With respect t o the e f f i c i e n c y , i t must be kept i n mind, however, that the amount of raw materials may be increased a l s o . The concentration of s t a r c h i n the formamide may be increased up to 30 ;5, but concentrations of s t a r c h between 15 % and 25 % are l e s s s i g n i f i c a n t than other explanatory v a r i a b l e s . I n creasing amounts of s t a r c h lead to a s l i g h t decrease i n degree of e s t e r i f i c a t i o n but also reduce the amount of formamide and by-products. As to the r e a c t i o n time between 15 and 30 min, i t has been pointed out already that the i n s i g n i f i c a n t i n f l u e n c e on e f f e c t v a r i a b l e s may be due to the small d i f f e rence between the temperatures used. I t can theref o r e be stated that the r e a c t i o n times are long enough f o r the e s t e r i f i c a t i o n s at 70 °C, but not f o r those at 50 °C. Conclusions The mutual actions described above lead to some d i f f i c u l t i e s with respect t o the s e l e c t i o n of proper conditions f o r producing starch s u l f a t e s with predetermined p r o p e r t i e s . I t must be r e a l i z e d , however, that the l i n e a r regression adopted i n the study of t h i s process g e n e r a l l y allows the predetermination of tendencies q u a l i t i v e l y , but does not produce q u a n t i t a t i v e r e s u l t s . A p r e d i c t i o n may be allowable only i f the steps i n the system are w i t h i n concrete l i m i t s and rather narrow. Within these r e s t r i c t i o n s , the s e l e c t i o n of r e a c t i o n conditions f o r the preper a t i o n of s t a r c h s u l f a t e s c o n s i s t s of a compromise oetween process v a r i a b l e s being e i t h e r p o s i t i v e or negative i n t h e i r a c t i o n . As an example f o r u t i l i z i n g the tendencies e l u c i d a t e d i n t h i s study, Table 4 shows the recommended process v a r i a b l e s f o r producing s t a r c h s u l f a t e s with a high degree of e s t e r i f i c a t i o n . Experimental General procedure. The c h l o r o s u l f o n i c a c i d - f o r mamide complex was formed by adding dropwise c h l o r o -

15 % 70 C 15 MIN

CONCENTRATION CP STARCH TEMPERATURE TIME CP REACTION U

\ : 1

12 %

WATER CONTENT OF STARCH SO^/AGU RATIO

PROCESS CONDITIONS PROPOSED

r

7

t~ APPARENT VISCOSITY SPECIFIC CONSUMPTION: FORMAMIDE CHLOROSULFONIC ACID

ESTER CONTENT CONTAMINATION G,5

%Ν

5 . 3 g/1* STARCH SULFATE, 2.4 f / l g STARCH SULF ATP.

2 - 11 cP ( 5 % SOL.)

3 0 ml g" 1

5 -

14 - 16 % S

RESULTING PROPERTIES OF RAW STARCH SULFATE

PROPOSED PROCESS CONDITIONS FOR PREPARING STARCH SULFATES WITH A HIGH DEGREE OF ESTERIFICATION AS DERIVED FROM INTERPRETATION OF THE EQUATIONS OF REGRESSION

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

Η M

>

X

w ο

>

ο

00

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

11.

scmERBAUM AND KÔRDEL

Starch and Chlorosulfonic

Acid-Formamide

187

s u l f o n i c acid t o formamide i n a round bottom f l a s k that was cooled t o maintain the temperature at below 40 ° C . The complex formed was d i l u t e d by a d d i t i o n of the amount of formamide required f o r d i s s o l v i n g the dry s t a r c h . Potato s t a r c h with a known moisture content was added t o the complex s o l u t i o n at 20 ° C with vigorous s t i r r i n g , the r e a c t i o n v e s s e l being immersed i n a temperature c o n t r o l l e d bath. The temperature of the bath was r a i s e d t o the desired value within 30 min and the mixture maintained at t h i s temperature f o r the desired r e a c t i o n time and immediatel y poured with vigorous a g i t a t i o n i n t o excess methan o l (1.5 1) « T^e p r e c i p i t a t e formed was f i l t e r e d o f f by s u c t i o n , washed with 100 ml methanol, and f i n a l l y d r i e d by i n f r a r e d r a d i a t i o n i n a stream of cold a i r . Reaction v a r i a b l e s . The general procedure des c r i b e d above was performed with the f o l l o w i n g var i a t i o n of r e a c t i o n conditions: 1. 2. 3· 4· 5·

r e a c t i o n temperature, 50 ° C and 70 ° C ( < * , / * ) time of r e a c t i o n at given temperature, 15 and 30 min ( t ) concentration of dry starch, 15, 20 and 25 % ( r e l a t e d t o formamide) ( s ) moisture content of potato s t a r c h , 5 and 20 % (w? molar r a t i o of SO3 t o AGU. 2:1 and 4:1 ( r ) (AGU anhydro glucose u n i t )

The s p e c i a l conditions, applied i n 48 r e s u l t i n g preparations are summarized i n Table 5· A n a l y t i c a l . The a n a l y t i c a l procedure used f o r c h a r a c t e r i z i n g r e a c t i o n products r e l a t e t o molecular as w e l l as to f u n c t i o n a l properties of the crude r e a c t i o n products. Further p u r i f i c a t i o n as may be necessary f o r p r a c t i c a l a p p l i c a t i o n of the esters has not been a p p l i e d . I t should be noted, hov/ever, that most of the ammonium s u l f a t e , the main by-product, p r e c i p i t a t e s l a t e r than the s t a r c h s u l f a t e . Therefore a r e l a t i v e l y pure starch s u l f a t e may be obtained by f i l t e r i n g i t o f f immediately a f t e r p r e c i p i t a t i o n . T h e f o l l o w i n g a n a l y t i c a l methods have been applied during the course of the complete program of i n v e s t i g a t i o n . (a)

T o t a l s u l f u r content, S Q %,by t o t a l h y d r o l y s i s with HCi, p r e c i p i t a t i o n with BaCl2 and gravimet r i c determination as BaSO^.; s u l f u r , bound i n ester groups, % c a l c u l a t e d by S 3 = S Q - S ^ ; s u l f u r i n anorganic compounds, %, by complexo9

EXPERIMENTAL CONDITIONS FOR ESTERIFICATION

CHLOROSULFONIC ACID

43,1 86,2 86,2 86,2

2 : 1

2 : 1

4 : 1

4 : 1

4 : 1 2

SAMPLE AMOUNTS OF STARCH: 31,6 g (5 % Η 0 ) ,

43,1 43,1

2 : 1

3

S0 /AGU

186,7

20 25

153,4 123,4 2

15

203,4

37,5 g (20 % H 0 )

25

20

15

106,7

136,7

OP STARCH

CONCENTRATION

OF POTATO STARCH BY MEANS OF THE

FORMAMIDE

CHLOROSULFONIC ACID - FORMAMIDE REAGENT

MOL. RATIO

TAB. 5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

11.

scHiERBAUM AND KÔRDEL

Starch and

Chlorosulfonic

Acid-Formamide

189

metric t i t r a t i o n of the aqueous s o l u t i o n with CHELAPLEX

(b (c (d (e (±-

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

(g (h (i (3 (k

#

L i m i t i n g v i s c o s i t y number [η\ , i n 0,33 M NaCl. T o t a l nitrogen content, N Q % by steam d i s t i l ­ l a t i o n of the a l k a l i n e s o l u t i o n and t i t r a t i o n according to the KJELDAHL semi-micro method. Reduction value, c a l c u l a t e d as glucose, R Q %, according to W i l l s t a e t t e r - Schudel. O p t i c a l r o t a t i o n value,/Vj , of 1 % s o l u t i o n s . Dynamic v i s c o s i t y , 1 (cP), of 5 % s o l u t i o n s i n the HOPPL^R Viscosimeter. Coldwater s o l u b i l i t y , derived from the r e f r a c ­ t i v e index η of a 10 % d i s p e r s i o n . pH-value of 10 % s o l u t i o n . T u r b i d i t y of 1 % solution,8 weeks at 4 C; cuvettes 1 cm, transmittance at 650 nm. Reaction with i o d i n e , maximum aborbancejl with 1 0 " % iodine. Gel permeation chromatography, GPC, on Sephadex G 200, measuring the high molecular weight por­ t i o n ( r e s u l t i n g from the excluded volume). 9

m

a

x

The r e s u l t s f o r k } and [*] r e f e r to pure starch s u l ­ f a t e and to AGU, r e s p e c t i v e l y and those f o r R Q to crude and pure starch s u l f a t e and to AGU, r e s p e c t i v e ­ l y . The y i e l d values were c a l c u l a t e d with reference to pure s t a r c h s u l f a t e and ammonium s u l f a t e . Evaluations. The above r e s u l t s derived from determinations and c a l c u l a t i o n s lead to 20 e f f e c t v a r i a b l e s at two l e v e l s each
CARBOHYDRATE SULFATES

190

s i g n i f i c a n c e between independent and e f f e c t v a r i a b l e s within the l i m i t s F > 10, F > 5, F > 3. indi­ cates that there i s no proof f o r s i g n i f i c a n c e between v a r i a b l e s . Equations of r e g r e s s i o n were c a l c u l a t e d f o r the evident c o r r e l a t i o n s between v a r i a b l e s y and independent v a r i a b l e s χ according to y = a^-b^K^x^^ ) J + b [ ^ 2

(a absolute value, K orthogonal f a c t o r , b r e g r e s s i o n c o e f f i c i e n t , χ mean values between l e v e l s of the i n ­ dependent v a r i a b l e s ) The c o r r e l a t i o n s between process and e f f e c t v a r i a b l e s ( p r o p e r t i e s ) as shown by the equation of regression were p l o t t e d and explained under the as­ pects of i n t e r a c t i o n between process v a r i a b l e s and r e s u l t i n g p r o p e r t i e s as w e l l as between r e a c t i o n va­ r i a b l e s and y i e l d values of the f i n a l products.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

Q

Summary Preparative and a n a l y t i c a l studies on a labora­ tory scale have been c a r r i e d out concerning the be­ havior of the s t a r c h - c h l o r o s u l f o n i c acid-formamide system. The r e s u l t s have been evaluated with respect to the i n f l u e n c e of the explanatory (process) v a r i ­ ables on e f f e c t v a r i a b l e s . The c a l c u l a t i o n was per­ formed by means of a theory of adjustment using or­ thogonal polynomials. The explanatory v a r i a b l e s exhibit a s i g n i f i c a n t influence on. e f f e c t v a r i a b l e s i n s i n g l e e f f e c t s and double and t r i p l e i n t e r a c t i o n s . In a d d i t i o n to the temperature, the concentration of water i n starch and the SO3/AGU r a t i o are e s s e n t i a l . Less important i s the concentration of starch and the l e a s t impor­ tant the time of r e a c t i o n w i t h i n the l i m i t of 15 to 30 min. High degrees of e s t e r i f i c a t i o n (DS > 1) and y i e l d s are obtained by i n c r e a s i n g amounts of water and at a high SO^/AGU r a t i o , simultaneously leading to a higher extent of molecular d e s t r u c t i o n and f o r ­ mation of contaminants and by-products. L i t e r a t u r e Cited 1. 2. 3·

Radley, J. Α., Starch Production Technology, p. 558-561, Applied Science, Publ., London, (1976). Astrup, Τ. and J. Galsmar, Acta P h y s i o l . Scand. (1944), 8, 361. Husemann, E., A. von K a u l l a and R. Kappesser,

11. SCHIERBAUM AND KÖRDEL

4. 5. 6. 7. 8. 9.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

10. 11. 12.

Starch and Chlorosulfonic Acid-Formamide 191

Z. Naturforschung, (1946), 1, 584. Pulver, R., Chernotherapia, (1961), 3, 388. B i a n c h i , R. G. and D. L. Cook, Gastroenterology, (1964), 47, 409. Cammarata, P. S. and S t . E i c h , U.S. Pat. 3 271 388, (1966). Placer, Z. and Z. Roubal, Cesk. G a s t r o e n t e r o l . Vyziva, (1960), 14, 422. Kerr, R. w. and F. C. Cleveland, U.S. Pat. 2 961 444, (1960); U.S. Pat. 3 021 222, (1962). Wurzburg, O. B., M. W. Rutenberg and R. J. Ross, U.S. Pat. 2 786 833, (1957). Schmandke, H., R. Maune, P. Schierbaum and K. Kördel, DDR Pat. 111 792, (1975). Whistler, R. L. and W. W. Spencer, Arch. Biochem. Biophys., (1960), 87, 137. Unrau, D. G., D i s s e r t a t i o n , Purdue U n i v e r s i t y , (1968).

13. Arch. 14.

W h i s t l e r , R. L., D. G. Unrau and G. R u f f i n i , Biochem. Biophys., (1968), 126, 647. Husemann, E., R. Werner, "Methoden der organischen Chemie", 4th. Ed., V o l . XIV/2, p. 868, Thieme-V rl., S t u t t g a r t , (1963). Whistler, R. L. and W. W. Spencer, "Methods i n Carbohydrate Chemistry", V o l . IV, p. 297-298, Academic Press, New York and London, (1964). Guiseley, Κ. B. and P. A. Whitehous, U.S. Pat. 3 720 659, (1973). Kruger, L. H. and O. B. Wurzburg, BRD Ausleges c h r i f t 1 908 353, (1975). Traube, W., German Pat. 490 887, (1927). W h i s t l e r , R. L., A. H. King, G. R u f f i n i and F. A. Lucas, Arch. Biochem. Biophys., (1967), 121, 358. Kerr, R. W., E. F. P a s c h a l l and Η. W. Minkema, U.S. Pat. 2 849 326, (1955); U.S. Pat. 2 967 178, (1957). Martin, J. and O. B. Wurzburg, U.S. Pat. 2 857 377; (1956). Minkema, W. H., U.S. Pat. 2 868 780, (1957). Whistler, R. L., J. L. Goatley and W. W. Spencer, Cereal Chem. (1959), 36, 84· Smith, H. E., C. R. Russel and C. Rist, Cereal Chem., (1962), 39; (1963), 40, 282. Schweiger, R. G., Carbohydrate Res., (1972), 21, 275. Tamba, R., Biochem. Z., (1923), 141, 274. Petracek, P. J. and M. D. Draper, U.S. Pat. 3 017 407, (1962). N i t t a , Υ.,Μ. Yukikata and Y. Nawata, Japan. Pat. e

15. 16.

17. 18. 19. 20. 21. 22. 23· 24· 25. 26. 27. 28.

192

29. 30.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch011

31.

CARBOHYDRATE SULFATES

16 700, (1958). Wander, Α., Swiss Pat. 305 572, (1955). Kördel, K. and F. Schierbaum, DDR Pat. 117 078, (1975). Richter, M., S. Augustat and F. Schierbaum, "Ausgewälte Methoden der Stärkechemie", Fachbuch-Verlag L e i p z i g , (1969)·

Acknowledgement We are g r e a t l y indebted to Dr. Friebe and Dr. Anger (Dept. "Methodik und Théorie") f o r the mathematical ( s t a t i s t i c a l ) part of the study, f o r the GPC and iodine t e s t s and f o r some h e l p f u l advice. We are f u r t h e r indebted to Miss B. Konig and Miss B. Kretschmer f o r s k i l f u l l y performing many of the i n vestigations. RECEIVED F e b r u a r y 6,

1978.

12 S u l f o n i c Acid a n d S u l f o m e t h y l - C o n t a i n i n g G r a f t C o - P o l y m e r s of X a n t h a n Gum I. W. COTTRELL, J. L. SHIM, and G. H. BEST Kelco Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

R. A. EMPEY Kelco Division of Merck & Co., Inc.,P . O.Box 998, Okmulgee, OK 74447 Xanthan gum i s a h i g h m o l e c u l a r weight p o l y saccharide produced in a pure culture fermentation process by the microorganism Xanthomonas c a m p e s t r i s [1]. The structure of the polysaccharide is shown in Figure 1. As detailed in this figure, each repeating u n i t c o n t a i n s f i v e sugar u n i t s c o n s i s t i n g of two g l u c o s e u n i t s , two mannose u n i t s , and one g l u c u r o n i c acid unit. The main c h a i n of xanthan gum i s built up of ß-D-glucose u n i t s l i n k e d through the 1- and 4positions; i.e., the c h e m i c a l s t r u c t u r e of the main chain is identical to the c h e m i c a l s t r u c t u r e of cellulose. The s i d e c h a i n comprises the two mannose u n i t s and the g l u c u r o n i c a c i d u n i t . The t e r m i n a l ß - D - m a n n o s e unit is linked glycosidically to the 4 - p o s i t i o n of β-D-glucuronic a c i d , which i n t u r n i s l i n k e d glycosidically to the 2 - p o s i t i o n of ot-D-mannose. T h i s three– sugar s i d e c h a i n i s l i n k e d to the 3 - p o s i t i o n of every o t h e r g l u c o s e r e s i d u e i n the main c h a i n . A l s o , about h a l f of the t e r m i n a l D-mannose r e s i d u e s c a r r y a pyru­ vic a c i d r e s i d u e ketalically l i n k e d to the 4- and 6positions. The n o n t e r m i n a l D-mannose u n i t c o n t a i n s an a c e t y l group at the 6 - p o s i t i o n [2]. Xanthan gum was c o m m e r c i a l i z e d by K e l c o Company, now K e l c o D i v i s i o n of Merck & C o . , I n c . , i n 1964 and has found widespread u t i l i t y i n food and i n d u s t r i a l a p p l i c a t i o n s because of i t s unique p r o p e r t i e s . Attempts have been made to a l t e r the p r o p e r t i e s of xanthan gum by c h e m i c a l d e r i v a t i z a t i o n and m o d i f i c a t i o n . These d e r i v a t i v e s and m o d i f i c a t i o n s i n c l u d e d e a c e t y l a t e d xanthan gum [_3] , carboxymethy 1 e t h e r s [4.1 , c a t i o n i c d e r i v a t i v e s Γ 5 ,6] , p r o p y l e n e g l y c o l e s t e r s L Z L h y d r o x y a l k y l e t h e r s [8] , s u l f a t e s [9.] , and g r a f t c o ­ polymers [10]. However, w i t h the e x c e p t i o n of d e a c e t y l a t e d xanthan gum, none of these d e r i v a t i v e s have been c o m m e r c i a l i z e d to d a t e .

0-8412-0426-8/78/47-077-193$05.00/0 © 1978 American Chemical Society

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

Figure 1. Structure of xanthan gum

12.

COTTRELL E T A L .

Xanthan

Gum

195

T h i s paper d e s c r i b e s the p r e p a r a t i o n of g r a f t c o p o l y m e r s o f x a n t h a n gum containing sulfonic acid g r o u p s a t t a c h e d by e i t h e r g r a f t c o p o l y m e r i z i n g acrylam i d e and 2 - a c r y l a m i d o - 2 - m e t h y l p r o p a n e s u l f o n i c a c i d (AMPSA) o n t o x a n t h a n gum, o r by sulfomethylating xanthan gum-polyacrylamide. T h e s e r e a c t i o n s a r e shown schematically in Figure 2.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

Results

and

Discussion

The s u l f o n i c a c i d and sulfomethy1-containing g r a f t c o p o l y m e r was p r e p a r e d by g r a f t i n g acrylamide (AM) and 2 - a c r y l a m i d o - 2 - m e t h y l p r o p a n e s u l f o n i c a c i d (AMPSA) o n t o x a n t h a n gum i n an a q u e o u s medium w i t h a eerie s a l t present (Figure 2). The r a t i o o f a c r y l a m i d e t o AMPSA was a d j u s t e d to give g r a f t polymers with v a r y i n g degrees of a n i o n i c c h a r a c t e r . A l s o , the r a t i o o f t o t a l monomer t o x a n t h a n gum was v a r i e d t o g i v e polymers with a range of v i s c o s i t i e s (Table I ) . Prel i m i n a r y s c r e e n i n g o f the p r o d u c t p o l y m e r from example 5 i n T a b l e I ( P o l y m e r I) h a s r e v e a l e d t h a t i t i s p s e u d o p l a s t i c , i s s t a b l e b e t w e e n pH 4^12, and e x h i b i t s a s y n e r g i s t i c v i s c o s i t y i n c r e a s e w i t h guar gum. E f f e c t o f pH and t e m p e r a t u r e on v i s c o s i t y and the shear-stress/shear-rate r e l a t i o n s h i p a r e shown i n F i g u r e s 3, 4, and 5, r e s p e c t i v e l y . These polymers a l s o p o s s e s s g o o d s a l t s t a b i l i t y , w h i c h makes them s u i t a b l e f o r o i l f i e l d u s e s , s u c h as f l o o d i n g and drilling. I n a d d i t i o n , t h e y c a n be u s e d i n o t h e r a p p l i c a t i o n s where t h i c k e n e d w a t e r i s d e s i r a b l e , such as t e x t i l e p r i n t i n g p a s t e t h i c k e n i n g , s l u r r y explosive f o r m u l a t i o n s , and c o a t i n g t e c h n o l o g y . The a f o r e m e n t i o n e d p o l y m e r was a l s o p r e p a r e d by i n t r o d u c i n g s u l f o n i c a c i d g r o u p s i n t o x a n t h a n gum by c o m b i n i n g g r a f t p o l y m e r i z a t i o n and c h e m i c a l modification techniques. F i r s t , polyacrylamide was grafted o n t o x a n t h a n gum a t 1:1 r a t i o by w e i g h t , t h e n t h e a m i d e f u n c t i o n was sulfomethylated with formaldehyde and s o d i u m m e t a b i s u l f i t e ( F i g u r e 2) [11] . P r e l i m i n a r y s c r e e n i n g of t h i s a n i o n i c polymer (Polymer II) has shown t h a t i t i s p s e u d o p l a s t i c ( F i g u r e 5 ) , has good high-temperature s t a b i l i t y ( F i g u r e 4 ) , and i s s t a b l e b e t w e e n pH 6 and 11 ( F i g u r e 3 ) . The p o l y m e r a l s o exh i b i t s a s y n e r g i s t i c v i s c o s i t y e f f e c t with guar gum. P o s s i b l e a p p l i c a t i o n s o f t h i s p o l y m e r a r e u s e as a d i s p e r s a n t and s t a b i l i z e r i n s l u r r i e s ; i n p e t r o l e u m recovery ( d r i l l i n g and f l o o d i n g ) , as an a n t i - s t a t i c f i l m f o r m e r ; and p o s s i b l y i n t e x t i l e s .

CARBOHYDRATE

196

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

1)

2

)

XANTHAN

GUM-POLY(ACRYLAMIDE-CO-AMPSA)

SULFOMETHYLATED

Figure 2.

Z N : AM

(1:1)

GRAFT

COPOLYMER

Polymerization reaction scheme

SULFATES

7.5 12 .5 16.7

42. 5 37.5 33. 3 25 16.7 12. 5 0

26 . 4 25.7 26.2 27.1 28.1 26.5 23.4

1.9

3.1

4.2

6.2

8.4

9.3

12. 5

10. 6

9. 3

8. 4

6.2

4.2

3.1

4

5

6

7

8

9

0

22

11

19 .2

4.2

2.1

3

10

11

22

18. 9

2.1

4.2

2

50

37. 5

33. 3

25

9

25

19.1

1.7

4.6

Weight (gm) copolymer

Content (%) 2-acrylamidoContent (%) 2-methylpropane Monomer sulfonic acid

340 200 250 230 300 290

350

250 250 300 300

50 50 50 50

340 350

50

250

480 500

66

50

480 520

66

50

480

Viscosity of Copolymer (1% KC1 solution)

520

V i s c o s i t y of Copolymer (distilled water)

66

Content (%) Xanthan gum

o f AMPSA and A c r y l a m i d e onto Xanthan Gum

1

Example

Acrylic Monomer Weight (gm)

of Graft Polymerization

Weight (gm) 2-acrylamido2-methylpropane Sulfonic Acid

Results

TABLE I

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

CARBOHYDRATE SULFATES

198

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

(CPS)

lOr

il

,

.

,

,

.

,

1

2

4

6

8

10

12

14

Ρ Η Figure 3.

Viscosity vs. pH for xanthan gum and both polymers

Figure 4.

Viscosity vs. temperature for xanthan gum and both polymers

COTTRELL E T A L .

Xanthan

Gum

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

10,

0.1

PO

SHEAR RATE

(sec-) 0.01

0.001

Ίο

-4-

loo

1000

SHEAR STRESS (dynes/cm ) 2

Figure 5. Low shear rate rheological properties of xanthan gum and both polymers. (These data were obtained using the Wells-Micro Brookfield Viscometer and the relaxation technique of Patton (12).)

CARBOHYDRATE

200

SULFATES

Experimental

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

Xanthan

Gum-Poly(aerylamide-co-AMPSA)

The s a m p l e s w e r e p r e p a r e d i n a 1000 m l r e a c t i o n v e s s e l equipped w i t h a n i t r o g e n i n l e t tube, a thermo­ m e t e r , a n d an a d d i t i o n f u n n e l . For the f i r s t three samples ( s e e T a b l e 1 ) , 500 ml o f a f e r m e n t a t i o n b e e r and 6.3 g o f t o t a l monomer ( a c r y l i c p l u s 2 - a e r y l a m i d o 2-methylpropane s u l f o n i c a c i d ) were combined. Examples 4-9 u s e d 500 ml f e r m e n t a t i o n b e e r a n d 12.5 g t o t a l monomer. E x a m p l e 10 c o m b i n e d 12.5 g o f 2 - a c r y l a m i d o 2-methylpropane s u l f o n i c a c i d a n d 500 ml o f f e r m e n t a ­ t i o n beer. The monomer was d i s s o l v e d i n 50 ml w a t e r . The f e r m e n t a t i o n b e e r c o n t a i n e d 2.50 w e i g h t p e r c e n t o f X a n t h o m o n a s c a m p e s t r i s c o l l o i d , as d e t e r m i n e d by i s o ­ p r o p y l a l c o h o l p r e c i p i t a t i o n o f c o l l o i d from a sample of the f e r m e n t a t i o n beer. The r e a c t i o n m i x t u r e was t h e n h e a t e d t o 7 5 ° C , w h i l e t h e r e a c t i o n v e s s e l was p u r g e d w i t h n i t r o g e n g a s f o r one h o u r . Thereafter, the r e a c t i o n v e s s e l was c o o l e d t o room t e m p e r a t u r e a n d 20 ml o f a c a t a l y s t s o l u t i o n d i l u t e d w i t h 50 ml o f w a t e r was a d d e d o v e r a p e r i o d o f 15 m i n u t e s w h i l e t h e c o n t e n t s were s t i r r e d . The c a t a l y s t s o l u t i o n was com­ p o s e d o f 0.1 Ν e e r i e ammonium n i t r a t e d i s s o l v e d i n 1 Ν n i t r i c acid. T h i s s o l u t i o n was made by d i s s o l v i n g 54.8 g o f e e r i e ammonium n i t r a t e i n 1 Ν n i t r i c a c i d and d i l u t i n g t o make 1 l i t e r o f s o l u t i o n . Following the a d d i t i o n o f the c a t a l y s t , s t i r r i n g o f the r e a c t i o n m i x t u r e was c o n t i n u e d f o r two h o u r s a t a r e a c t i o n t e m p e r a t u r e o f 20 t o 2 5 ° C , a f t e r w h i c h 2 g o f p - m e t h o x y p h e n o l d i s s o l v e d i n 50 ml o f i s o p r o p y l a l c o h o l w e r e added. T h e p r o d u c t was p r e c i p i t a t e d by t h e a d d i t i o n o f e x c e s s i s o p r o p y l a l c o h o l a n d was t h e n c o l l e c t e d a n d d r i e d a t 4 5 - 5 0 ° C f o r two h o u r s . The f i n a l w e i g h t s o f t h e p r o d u c t s a r e shown i n T a b l e I . V i s c o s i t y o f t h e c o p o l y m e r s was d e t e r m i n e d a t 1% by w e i g h t s o l u t i o n i n d i s t i l l e d w a t e r , u s i n g a B r o o k f i e l d L V F v i s c o m e t e r a t 60 rpm, No. 2 s p i n d l e . F o r p u r p o s e s o f c o m p a r i s o n , Xanthomonas c a m p e s t r i s c o l l o i d , w h i c h was o b t a i n e d d i r e c t l y f r o m t h e f e r m e n t a ­ t i o n b e e r , h a d a v i s c o s i t y a t a c o n c e n t r a t i o n o f 1% by w e i g h t i n d i s t i l l e d w a t e r o f 840 cps. Sulfomethylated Polymer I I

ZN: AM

(1:1) G r a f t

Copolymer

-

A g r a f t c o p o l y m e r i s p r e p a r e d by r e a c t i n g e q u a l p a r t s b y w e i g h t o f x a n t h a n gum a n d a c r y l a m i d e a c c o r d ­ i n g t o t h e p r o c e d u r e g i v e n i n E x a m p l e 1 o f U. S. p a t e n t

12.

COTTRELL ET AL.

201

Xanthan Gum

3,708,446. The r e s u l t i n g copolymer (1.44 g) i s d i s s o l v e d i n 100 ml d i s t i l l e d water i n a t h r e e neck, round bottom f l a s k (250 ml) f i t t e d w i t h a thermometer, s t i r r e r , and r e f l u x condenser. To t h i s s o l u t i o n i s added 0.95 g N a S 0 (0.005 m o l e ) , 0.80 g 50% NaOH (0.01 m o l e ) , and 0.81 g 37% CH2O (0.01 m o l e ) . The s o l u t i o n i s heated to 5 0 ° C and s t i r r e d at t h i s tempera­ ture for 3 hours. At the end of t h i s p e r i o d , the mixture i s c o o l e d to room temperature ( ^ 2 2 ° C ) , and the r e a c t i o n p r o d u c t i s r e c o v e r e d by a d d i t i o n of the s o l u ­ t i o n to 2 volumes of i s o p r o p a n o l . The p r o d u c t i s d r i e d i n vacuo at 4 0 ° C and m i l l e d through a 60 mesh (U.S. standard) s c r e e n . The r e s u l t s of t h i s experiment are shown i n T a b l e I I . Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

2

2

5

Table I I . The R e s u l t s of S u l f o m e t h y l a t i o n Gum-Acrylamide G r a f t Copolymer.

Y i e l d (g) T h e o r e t i c a l y i e l d (g) Y i e l d (%) Amide Groups Reacted (%) V i s c o s i t y (cps), 1% KC1 V i s c o s i t y (cps), 1%

of Xanthan

2.13 2.58 82.60 60.53 90 87 B r o o k f i e l d LVF Viscometer, 6 0 rpm

Abstract : Two n o v e l g r a f t copolymers of xanthan gum have been p r e p a r e d by g r a f t c o p o l y m e r i z i n g 2 - a c r y l a mido-2-methylpropane s u l f o n i c a c i d and a c r y l a m i d e onto xanthan gum and by g r a f t p o l y m e r i z i n g a c r y l a m i d e onto xanthan gum f o l l o w e d by s u l f o m e t h y l a t i o n . The p r e p a r a tion and p r o p e r t i e s of these two polymers are d i s c u s s e d. Literature 1. 2. 3. 4. 5. 6. 7. 8. 9.

Cited

Jeanes, A. R . , Pittsley, J. Ε . , and S e n t i , F . R . , J. A p p l . Polymer Sci. (1961), 5, 519-526. J a n s s o n , P. E., Keene, L., and L i n d b e r g , B., Carbohydrate Res. (1975), 45, 275-282. J e a n e s , A . R. and S l o n e k e r , J. H., U. S. Patent 3, 000, 790 (1961). Schweiger, R. G., U . S . P a t . 3,236,831 (1966). Schweiger, R. G., U . S . P a t . 3,244,695 (1966). Schweiger, R. G., U . S . P a t . 3,376,282 (1968). Schweiger, R. G., U . S . P a t . 3,256,271 (1966). Schweiger, R. G., U . S . P a t . 3,349,077 (1967). Schweiger, R. G., U . S . P a t . 3,446,796 (1969).

CARBOHYDRATE SULFATES

202 10. 11. 12.

P e t t i t t , D. J., U.S. Pat. 3,708,446 (1973). S c h i l l e r e t al., Ind. & Eng. Chem., p. 2132-2137 (1956). Patton, T. C., J. o f P a i n t Technology, (19 66), 38 (502), 656.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch012

RECEIVED F e b r u a r y 6,

1978.

13 Sulfated Polysaccharides Metabolized by the Marine Chlorophyceae—A Review ELIZABETH

PERCIVAL

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

Bourne Laboratory, Chemistry Department, Royal Holloway College, Egham Hill, Egham, Surrey, TW20 0EX, England

During the past twenty years my colleagues and I have i n v e s t i g a t e d the polysaccharides o f nine d i f f e r e n t genera o f green s e a weeds. In every case we have been able t o separate a t l e a s t one w a t e r - s o l u b l e p o l y d i s p e r s e heteropolysaccharide s u b s t i t u t e d by h a l f e s t e r s u l f a t e groups. That means not only t h a t each seaweed metabolises molecules c o n t a i n i n g more than a single sugar but t h a t each contains a mixture o f molecules all built up on the same general plan but which differ i n the fine d e t a i l s o f s t r u c t u r e . I should emphasise t h a t the results are only the average o f all the molecules present. Each weed presented its own particular p r o blems, but certain groups o f polysaccharide have emerged. From the Cladophorales and Codiales a s u l f a t e d x y l o g a l a c t o a r a b i n a n , and from the U l v a l e s , A c r o s i p h o n i a l e s and U l o t r i c h a l e s a glucuronoxylorhamnan, sometimes c o n t a i n i n g a small p r o p o r t i o n o f glucose, have been separated. The polysaccharides i n each group appear t o be built up on the same general p l a n , but differ i n the proport i o n s o f the sugars present, and i n the fine d e t a i l s o f s t r u c t u r e . The polysaccharide from the unicellular green a l g a , Acetabulària, a member o f the Dasycladales, resembles the l a t t e r group i n c o n t a i n i n g rhamnose, xylose and g l u c u r o n i c a c i d , but i t s major sugar i s galactose and i t s 4-0-methyl d e r i v a t i v e ( 1 ) . General S t r u c t u r a l

Studies

D e t a i l s o f polysaccharides from the f i r s t group o f seaweeds are given i n Table I. The proportions o f sugars present i n Cladophora and Chaetomorpha polysaccharides d i f f e r i n the s m a l l e r p r o p o r t i o n o f galactose present i n the l a t t e r , and both genera are devoid o f 3,6-anhydrogalactose. Caulerpa polysaccharide appears to be the odd man out i n t h a t i t contains a c o n s i d e r a b l e proport i o n o f mannose residues which d e f i e d f r a c t i o n a t i o n , and t h i s i s true f o r two other species o f Caulerpa we have examined. Nevert h e l e s s a l l four genera c o n t a i n approximately the same p r o p o r t i o n 0-8412-0426-8/78/47-077-203$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

xxxxx xxx xx

xxxxx

xxxxx

xx

L-Arabinose

D-Galactose

D-Xylose

xxxxxx xxx

xxxxx xx

+13°

+46°

+70°

ca.+53°

[a]

D

17.5

17

15

16

+

+

xxx

xx

Caulerpa f i l i f o r m i s

xxxx

Codium f r a g i l e

Sulfate %

3,6-Anhydrogalactose

D-Mannose

Constituent Sugars

Chaetomorpha capillaris

Water-soluble Polysaccharides from

Cladophora rupestris

Table I.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

M

H

>

r

c!

M

> Η

> td g Hi ϋ

ο

to ο

13.

Table I I ,

205

Polysaccharides

Water-Soluble

Polysaccharides

Cladophora rupestris

various

Approx. propor. Sugars Gal Ara Xyl

Cladophora sericea*

Scotland Aug. 1969 A p r i l 1970

Gal Ara Xyl

Cladophora laetevirens

Scotland Sept.1969 A p r i l 1970

Gal Ara Xyl

Rhizoclonium r i p a r i urn

Scotland 1969

Gal Ara Xyl

Chaetomorpha melagonium

Scotland 1969

Gal Ara Xyl

Chaetomorpha 1 inum

Scotland 1969 1970

Gal Ara Xyl

Algal

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

Sulfated

PERCivAL

Key:

Species

Time Place

AgN0 on Papers 3

+++ +++ + ++ ++++ ++ ++ +++++ ++ ++ ++++ ++ ++ ++ +++ + ++ +++ +

GLC Ratio 1.0 1.0 0.4 1.0 1.27 0.80 1.0 1.8 0.9

1.0 1.2 0.45

-

Gal = g a l a c t o s e ; Ara = arabinose; Xyl = xylose

May have been contaminated with C. l a e t e v i r e n s .

Figure 1.

Structural features in Cladophora rupestris (left); after reduction and desulfation (right)

17.5

16

[»]„ -87°

n

-47°

11 17-22 -90°

16

21

-74°

-

17

X

probably contaminated with s t a r c h - t y p e p o l y s a c c h a r i d e [ a ] + 1 5 0 ° .

-31°*

7.8

18

19

Sulphate %

X

XX*

D-Glucuronic A c i d %

xxxx

wormskioldii

xxx

xxx

Urospora penicilliformis

xxxx

XX

D-Glucose

XXX

xxx

XXXXX

Ulva lactuca

D-Xylose

XXXXX

Enteromorpha compressa

xxx

Acrosiphonia central i s

Water-Soluble Polysaccharides from

L-Rhamnose

Constituent Sugars

Table IV.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

*1

M

to ο

13.

PERCivAL

Sulfated

Polysaccharides

207

of h a l f e s t e r s u l f a t e and a l l have a p o s i t i v e s p e c i f i c r o t a t i o n . Examination of the c o n s t i t u e n t s of a number o f d i f f e r e n t seaweeds belonging to the Cladophorales shows (Table II) a wide v a r i a t i o n i n the proportions of the sugars present ( 6 ) . Table III gives the linkages present i n Cladophora r u p e s t r i s polysaccharide and Figure 1 some of the s t r u c t u r a l features Table I I I .

Linkages present i n Cladophora r u p e s t r i s

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

1.3- and 1,6-1 inked g a l a c t o s e ; 1 , 4 - l i n k e d x y l o s e ; 1,4- and t r i p l y l i n k e d arabinose; that have been determined (7) f o r t h i s p o l y s a c c h a r i d e . Although t e n t a t i v e evidence of s i m i l a r linkages i n other genera of t h i s group have been obtained these have not been f i r m l y e s t a b l i s h e d . In c o n t r a s t to the arabinans the glucuronoxylorhamnans a l l have negative s p e c i f i c r o t a t i o n s (Table IV). Furthermore, the proportions o f the sugars i n the d i f f e r e n t species are very s i m i l a r e s p e c i a l l y , i f i t i s remembered t h a t these f i g u r e s are only approximate and are the average o f a l l the molecules present. They are determined on hydrolysates of the p o l y s a c c h a r i d e s , and the a l d o b i o u r o n i c a c i d , 4-0-3-D-glucuronosyl-L-rhamnose, i s i n v a r i a b l y present and not included i n these p r o p o r t i o n s . While t h i s prevents accurate determination of the proportions o f the c o n s t i tuents i t s occurrence i n the hydrolysates from a l l the species examined i s evidence of the o v e r a l l s i m i l a r i t y of t h e i r p o l y s a c c h a r i d e s . A s t r i k i n g d i f f e r e n c e i s the lower p r o p o r t i o n of s u l f a t e i n Acrosiphonia and Urospora w o r m s k i o l d i i . A l l the sugars i n the polysaccharides i n t h i s group are l i n k e d i n the same way (Table V ) . Table V.

Linkages Present i n Glucuronoxylorhamnans

1,3- and 1 , 4 - l i n k e d and end group rhamnose; 1,3- and 1.4- l i n k e d x y l o s e , 1,3-1 inked glucose (when p r e s e n t ) ; 1,4linked glucuronic acid However, the proportions o f the d i f f e r e n t types o f linkages appear to vary i n the d i f f e r e n t s p e c i e s . S t r u c t u r a l features found i n the polysaccharide from Ulva l a c t u c a (13) a r e : GA(Ti4jR; GA(l*3)Xy; GA(l->4)Xy; G(l+3)Xy 2 S GA(l*4)R(l+3)GA(l*3)Xy; R(l*4)Xy(l*3)G/GA. GA = D-glucopyranuronic a c i d ; R = L-rhamnopyranose^O Xy = D-xylopyranose; G = D-glucopyranose; S = S-0-

208

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

The S i t e of Sulphate Groups These polysaccharides e x i s t as mucilages i n the p l a n t and the presence of s u l f a t e groups must play a v i t a l r o l e i n t h e i r c o n f o r ­ mation and p h y s i c a l p r o p e r t i e s . I t i s important, t h e r e f o r e , to determine the s i t e of these groups. This has been achieved i n a v a r i e t y of ways, depending upon the p a r t i c u l a r p o l y s a c c h a r i d e . 1. Cleavage by A l k a l i . A l k a l i w i l l cleave s u l f a t e groups from a polysaccharide i f there i s a f r e e adjacent hydroxyl group trans to the s u l f a t e , t h i s occurs v i a an epoxide r i n g and Walden i n v e r s i o n of the hydroxyl c a r r y i n g the s u l f a t e group. Cleavage of the epoxide r i n g can occur i n two ways and two sugars u s u a l l y r e s u l t . Cleavage a l s o occurs w i t h s u l f a t e on C-6 of the hexose provided the hydroxyl on C-3 i s f r e e . In t h i s event removal of the s u l f a t e r e s u l t s i n the formation of the 3,6-anhydride of the p a r t i c u l a r hexose. In both the U l v a l e s and the Cladophorales t h i s method of r e ­ moval of s u l f a t e has been p a r t i c u l a r l y u s e f u l . The s u l f a t e con­ t e n t i n the rhamnan from U. l a c t u c a was reduced from 14.1 to 12.5% by the a c t i o n of a l k a l i , and the hydrolysate of the r e c o v e r ­ ed polysaccharide was found to contain arabinose (14). This could only have a r i s e n from s u l f a t e d xylose (Figure 2 ) . I t could have come from xylose 2- or 3 - s u l f a t e . However, treatment of the p o l y ­ saccharide with sodium methoxide gave 3-0-methylarabinose which could only have been derived from xylose 2 - s u l f a t e . S i m i l a r l y the treatment of the x y l o g a l a c t o a r a b i n a n from Cladophora r u p e s t r i s w i t h sodium methoxide gave mainly 2-0-methyl-L-xylose and l i t t l e 3-0-methyl-L-arabinose (3) i n d i c a t i n g t h a t the s u l f a t e was i n i t i ­ a l l y l i n k e d to C-3 of arabinose. P a r t i a l h y d r o l y s i s s t u d i e s con­ firmed t h i s r e s u l t . The removal of s u l f a t e w i t h a l k a l i and the formation of 3,6anhydrogalactose was demonstrated i n the s u l f a t e d polysaccharide from Codium f r a g i l e (4). The s u l f a t e decreased from approximately 13% to 97o and the 3,6-anhydrogalactose content increased from 1% to 3% on t h i s treatment (Figure 3). 2. The S i t e of S u l f a t e from Periodate Oxidation S t u d i e s . Treatment of the rhamnan from U. l a c t u c a with dry methanolic hydrogen c h l o r i d e reduced the s u l f a t e to 5% and periodate o x i d a t i o n of the i n i t i a l and the d e s u l f a t e d polysaccharide at 2° i n buffered s o l u t i o n stopped a f t e r 70 hours. I t was found t h a t the d e s u l f a t e d polysaccharide had reduced twice as much periodate as the i n i t i a l polysaccharide (14). P a r a l l e l o x i d a t i o n experiments were c a r r i e d out at room temperature. Again, the d e s u l f a t e d polysaccharide reduced approximately twice as much periodate as the i n i t i a l p o l y ­ saccharide. From t h i s i t can be deduced, t h a t removal of s u l f a t e groups l e d to the formation of a d d i t i o n a l α-glycol groups. I t i s known t h a t , i n buffered s o l u t i o n at low temperature, v i c i n a l c i s hydroxyl groups i n sugars are s e l e c t i v e l y o x i d i s e d by p e r i o d a t e . We were a b l e , t h e r e f o r e , to conclude t h a t d e s u l f a t i o n f u r n i s h e d c i s r a t h e r than trans g l y c o l groupings. The only sugar present i n

13.

PERCivAL

Sulfated

209

Polysaccharides

the polysaccharide i n which c i s - g l y c o l groupings occur i s L-rhamnose at p o s i t i o n 2 and 3. I t may be presumed t h e r e f o r e t h a t the s u l f a t e groups are l i n k e d e i t h e r t o C-2 or C-3 o f rhamnose. I n f r a r e d a n a l y s i s o f the polysaccharide gave a strong peak a t 850 cm"" , c h a r a c t e r i s t i c o f a x i a l s u l f a t e i n galactose or glucose(15). If t h i s i s a p p l i c a b l e to L-rhamnose, the m a j o r i t y o f the s u l f a t e i n t h i s polysaccharide i s l i n k e d to C-2 o f the L-rhamnose i n i t s most s t a b l e 1C conformation. In s i m i l a r periodate o x i d a t i o n experiments on Enteromorpha compressa p o l y s a c c h a r i d e , the r e l a t i v e proportions o f the uncleaved sugars i n the i n i t i a l polysaccharide and i n the d e s u l f a t e d polysaccharide a f t e r o x i d a t i o n are given i n Table V I . Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

1

Table VI.

Enteromorpha S u l f a t e d Polysaccharide Percentage Uncleaved: Glucose:Xylose :Rhamnose

I n i t i a l Polysaccharide

26

16

58

Desulfated Polysaccharide

40

16

44

The amount o f rhamnose and, to a l e s s e r e x t e n t , o f xylose was reduced i n the d e s u l f a t e d p o l y s a c c h a r i d e . From these r e s u l t s i t was c a l c u l a t e d t h a t about 22% o f the rhamnose i n the i n i t i a l p o l y saccharide was cleaved by periodate and about 40% i n the d e s u l f a t e d p o l y s a c c h a r i d e , t h a t i s an increase o f 18% o f f r e e hydroxyl groups i n the rhamnose residues proving t h a t a high p r o p o r t i o n of the s u l f a t e groups are attached to t h i s sugar r e s i d u e . Infrared a n a l y s i s o f the i n i t i a l polysaccharide gave a peak a t 850 c m " which i n galactose i n d i c a t e s a x i a l s u l f a t e . A g a i n , i f t h i s i s a p p l i c a b l e to L-rhamnose which i s i n i t s s t a b l e 1C conformation then p o s i t i o n 2 has an a x i a l h y d r o x y l , and t h i s would t h e r e f o r e , as i n the Ulva p o l y s a c c h a r i d e , be the s i t e of s u l f a t e . 3. The S i t e of S u l f a t e from Methylation R e s u l t s . Methylation before and a f t e r d e s u l f a t i o n and comparison of the two sets of methylated sugars i n the r e s p e c t i v e hydrolysates has not proved a very s a t i s f a c t o r y method f o r determining the s i t e o f the e s t e r s u l f a t e i n these p o l y s a c c h a r i d e s . T h e i r high s u l f a t e content makes complete methylation impossible and so the e f f e c t of the removal o f the s u l f a t e groups cannot be p r o p e r l y assessed. However, with the glucuronoxylorhamnan from Urospora p e n i c i l l i f o r m i s i t was found (11) t h a t the hydrolysate from the methylated d e s u l f a t e d polysaccharide contained considerably more 2 , 4 - d i - 0 - m e t h y l rhamnose than the hydrolysate from the i n i t i a l methylated p o l y saccharide w h i l e the p r o p o r t i o n of both the monomethyl and f r e e rhamnose i n the l a t t e r were reduced to t r a c e amounts. From these r e s u l t s i t was p o s s i b l e to deduce t h a t the 1,3-1 inked rhamnose was monosulfated at C-4 and a l s o d i s u l f a t e d a t C-2 and C-4. The 1

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

210

proportions of the d i f f e r e n t methylxyloses and g l u c u r o n i c acids were the same i n the two hydrolysates i n d i c a t i n g t h a t both of these residues are e s s e n t i a l l y devoid o f s u l f a t e . From the Codiolum, the u n i c e l l u l a r stage i n the l i f e h i s t o r y °f Urospora w o r m s k i o l d i i , a s u l f a t e d mannan i n a d d i t i o n to the glucuronoxylorhamnan, was separated (12). This on methylation and h y d r o l y s i s gave 2 , 3 , 4 , 6 - t e t r a - , 2 , 4 , 6 - t r i , 3,6- and 2,3di-O-methylmannoses i n the r e l a t i v e proportions of 1:13:1.5:1. This mannan i s , t h e r e f o r e , 1 , 4 - l i n k e d , s u l f a t e d and/or branched at C-2 and C-6 and has an average chain length of 16. A f t e r d e s u l f a t i o n with 0.08 M-methanolic hydrogen c h l o r i d e , the s u l f a t e content decreased from 6.5 to 0.6%. The d e s u l f a t e d m a t e r i a l was methylated and hydrolysed and the above methylmannoses were obtained i n the approximate r e l a t i v e proportions of 1:13:0.2:1. The f a c t t h a t the 3,6-di-0-methylmannose had almost disappeared i n the d e s u l f a t e d m a t e r i a l and the q u a n t i t y of 2,3di-0-methylmannose remained v i r t u a l l y unchanged provided evidence t h a t the mannan i s s u l f a t e d on C-2 and branched at C-6. 4. The S i t e of S u l f a t e from P a r t i a l H y d r o l y s i s . This has a l s o been useful f o r the determination of the s i t e of the s u l f a t e groups i n the Chlorophyceaean p o l y s a c c h a r i d e s . For example, h y d r o l y s i s of the Codium polysaccharide with N-sulphuric a c i d f o r one hour at 100° l e d to the separation of galactose 4- and 6-monosulfates (4). These were c h a r a c t e r i s e d as f o l l o w s : Each had a DP o f 1. On h y d r o l y s i s they both gave only g a l a c t o s e . The molar r a t i o s of e s t e r s u l f a t e to galactose were 1.15:1.0 f o r the 4 - s u l f a t e and 1.04:1.0 f o r the 6 - s u l f a t e . Methylation gave r e s p e c t i v e l y 2,3,6- and 2 , 3 , 4 - t r i - 0 - m e t h y l g a l a c t o s e s p r o v i d i n g unequivocal proof of the s i t e of e s t e r s u l f a t e groups as galactose 4- and galactose 6 - s u l f a t e . S i m i l a r h y d r o l y s i s o f the x y l o g a l a c t o a r a b i n a n from Cladophora r u p e s t r i s (3) l e d to the separation of arabinose 3 - s u l f a t e and galactose 6 - s u l f a t e . The l a t t e r was c h a r a c t e r i s e d i n the same way as t h i s d e r i v a t i v e from Codium. The former a f t e r methylation gave 2,4- and 2 , 5 - d i - 0 ^ e t h y l a r a b i n o s e s , showing t h a t C-3 i n v o l v e d i n l i n k a g e to s u l f a t e , thus confirming the r e s u l t s a r r i v e d at by a l k a l i treatment of t h i s p o l y s a c c h a r i d e . Biological

Imp!ications

U n l i k e the g a l a c t a n s , none of these p o l y s a c c h a r i d e s , a f t e r e x t r a c t i o n , gives gels i n aqueous s o l u t i o n , the most t h a t can be s a i d i s t h a t i n some cases the s o l u t i o n s are somewhat v i s c o u s . The l a t e Dr. Haug (16) reported t h a t an aqueous s o l u t i o n of Ulva polysaccharide gave a strong gel i f i t was t r e a t e d w i t h borate and calcium ions at a concentration and a pH found i n seawater. He experimented with a wide v a r i e t y of m e t a l l i c ions found i n the sea, but t h i s was the only combination w i t h which Ulva polysacchar i d e gave a g e l . He p o s t u l a t e d t h a t the C-2 and C-3 in the 1,4l i n k e d rhamnose u n i t s complex with borate and t h a t the Ca ions

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

13.

Sulfated Polysaccharides

PERCivAL

211

2-0-Me-Darabinose

3-0-Me-D xylose

Figure 2.

OH Figure 3.

1

—

Galactose 6-sulfate (left) and 3,6-anhydrogahctose (right)

Ο'

O H

H O

O

B . o ^ — ^ o . Ca Figure 4.

2 +

CI"

212

CARBOHYDRATE SULFATES

form bridges or act as s t a b i l i s i n g influences (Figure 4 ) . When C-2 o f these rhamnose u n i t s are s u l f a t e d complex formation w i t h borate i s prevented, and i t may be t h a t t h i s i s the way i n which Ulva l a c t u c a r e g u l a t e s the extent of complexing w i t h borate and hence, the s t i f f n e s s of the polysaccharide g e l . C a r r y i n g out s i m i l a r s t u d i e s on the Cladophora r u p e s t r i s p o l y s a c c h a r i d e , i t was found t h a t calcium ions alone produced a strong gel at the pH of seawater (17). Literature Cited 1.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch013

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Smestad, B e r i t and Percival, E l i z a b e t h , Carbohyd. Res. (1972) 25, 299. F i s h e r , I . S. and Percival, E l i z a b e t h , J. Chem. Soc. (1957) 2666. H i r s t , S i r Edmund, Mackie, W. and Percival, E l i z a b e t h , J. Chem. Soc. (1965) 2958. Love, J. and Percival, E l i z a b e t h , J. Chem. Soc. (1964) 3338. Mackie, I . M. and Percival, E l i z a b e t h , J. Chem. Soc. (1961) 3010. Percival, E l i z a b e t h and Young, Margaret, Phytochemistry (1971) 1 0 , 807. Bourne, E. J., Johnson, F. G. and Percival, E l i z a b e t h , J. Chem. Soc. (C) 1970, 1561. O ' D o n n e l l , J. J. and Percival, E l i z a b e t h , J. Chem. Soc. (1959) 2168. M c K i n n e l l , J. F. and Percival, E l i z a b e t h , J. Chem. Soc. (1962) 3141. M c K i n n e l l , J. F. and Percival, E l i z a b e t h , J. Chem. Soc. (1962) 2082. Bourne, E. J., Megarry, M. L. and Percival, E l i z a b e t h , J. Carbohyd. Nucleosides, Nucleotides (1974) 1, 235. C a r l b e r g , G. and Percival, E l i z a b e t h , Carbohyd. R e s . , (1977) i n the p r e s s . Haq, Q. N. and Percival, E l i z a b e t h i n "Some Contemporary Studies i n Marine Science" (1966) e d i t e d by Harold Barnes, p. 355. George A l l e n and Unwin Ltd., London. Percival, E l i z a b e t h and Wold, J. Κ., J. Chem. Soc. (1963) 5459. Turvey, J. R., Adv. Carbohyd. Chem. (1965) 20, 183. Haug, Α . , Acta Chem. Scand. Β (1976) 30, 562. Percival, E l i z a b e t h , unpublished work.

RECEIVED February 6,

1978.

14 Sulfated Polysaccharides of the Rhodophyceae— A Review ELIZABETH PERCIVAL

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

Bourne Laboratory, Chemistry Department, Royal Holloway College, Egham Hill, Egham, Surrey, TW20 0EX, England

General S t r u c t u r a l Features The major polysaccharides o f the Rhodophyceae a r e galactans which c a r r y varying proportions o f h a l f e s t e r s u l f a t e l i n k e d t o one or more o f the f r e e hydroxyl groups o f the galactose r e s i d u e s . They are readily e x t r a c t e d from the seaweeds by hot water, and research has shown t h a t they all appear t o c o n s i s t o f chains o f a l t e r n a t i n g u n i t s o f 1,3-linked β-galactose and 1 , 4 - l i n k e d α - g a l a c t o s e , t h a t i s a l t e r n a t i n g A and Β u n i t s (Figure 1 ) . How­ ever, some of the u n i t s may be masked by m o d i f i c a t i o n o r by sub­ stitution. E x t r a c t s from different seaweeds vary i n the amount of D- and o f L - g a l a c t o s e , i n the extent t o which these residues are modified t o the 3,6-anhydrosugar, by the extent and p o s i t i o n of h a l f e s t e r s u l f a t e and methoxyl groups and by s u b s t i t u t i o n with pyruvic a c i d (Figure 2 ) . I t i s c l e a r t h a t there are a l a r g e number o f ways i n which the e x t r a c t s from the different weeds differ from one another. It is these d i f f e r e n c e s i n fine s t r u c ­ ture which determine the conformation o r shape o f the molecules and, hence, the p h y s i c a l p r o p e r t i e s o f the various e x t r a c t s . The members o f the Rhodophyceae can be d i v i d e d i n t o those genera which s y n t h e s i s e agar-type molecules, t h a t i s a l t e r n a t i n g 1,3-1 inked D-galactose residues and 1 , 4 - l i n k e d L - r e s i d u e s , others that metabolise carrageenan-type molecules i n which all the u n i t s are D-galactose and galactans which show a f f i n i t i e s to both agar and carrageenan (Table I ) . Table I.

Seaweeds M e t a b o l i s i n g Polysaccharides o f :

Agar-type

Carrageenan-type

Mixed-type

Gelidium

Chondrus

Porphyra

Gracilaria

Gigartina

Laurencia

Phyllophora

Eucheuma

Bangia

Pterocladia

Furcellaria

Gloiopeltis

0-8412-0426-8/78/47-077-213$05.00/0 © 1978 American Chemical Society

Figure 2.

/

Figure 1.

Β

^Galactose (left) and ^-galactose (right)

Α

Β

(left to right, top to bottom)O-Galactose 6-sulfate, ^-galactose 2,6-disulfate, galactose 4,6-0-1boxylethylidene, a-l,4-linked 3,6-anhydro^-gahctose, and 6-0-methyl-O-galactose

Α

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

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215

In every case, the polysaccharides c o n t a i n a f a m i l y o f molecules which d i f f e r i n the f i n e d e t a i l s o f s t r u c t u r e d e t a i l e d above. It has been p o s s i b l e t o separate the extremes of the s t r u c t u r e s i n some i n s t a n c e s . The separation o f agarose (Figure 3 ) , a n o n - s u l f a t e d g e l l i n g f r a c t i o n from agar, where the chains c o n s i s t o f a l t e r n a t i n g 1,3-1 inked D-galactose residues and 1 , 4 - l i n k e d 3, 6-anhydro-L-galactose, l e a v i n g a g a r o p e c t i n , a mixture of v a r i o u s l y s u l f a t e d molecules, behind i s one example. The p r e c i p i t a t i o n by potassium ions o f kappa-carrageenan (Figure 4) where the 1,3l i n k e d u n i t s c a r r y s u l f a t e groups a t C-4, a l s o a separation o f a g e l l i n g f r a c t i o n from a whole e x t r a c t of Chondrus c r i s p u s o r G i g a r t i n a species i s another example o f t h i s f r a c t i o n a t i o n . Again, i n carrageenan the m a t e r i a l l e f t i n s o l u t i o n , the s o - c a l l e d "lambda"-carrageenan, a n o n - g e l l i n g f r a c t i o n , i s a mixture o f v a r i o u s l y s u b s t i t u t e d molecules with a low 3,6-anhydro content. In recent f u r t h e r f r a c t i o n a t i o n work, Dr. Rees (1) suggests t h a t the term lambda-carrageenan should be r e s t r i c t e d to one f r a c t i o n of t h i s m a t e r i a l (Table I I ) . Methods f o r the Determination of the S i t e of S u l f a t e Groups I t i s important to know the p o s i t i o n o f the s u l f a t e groups on the i n d i v i d u a l galactose residues and t h i s has been determined by (1) removal o f the s u l f a t e by a l k a l i , (2) methylation o f the p o l y s a c c h a r i d e , (3) p a r t i a l a c i d h y d r o l y s i s o f the p o l y s a c c h a r i d e , and (4) i n f r a r e d spectroscopy. 1. A c t i o n o f a l k a l i . - E a r l y studies on model s u l f a t e d monosaccharides revealed t h a t s u l f a t e could be removed on treatment w i t h a l k a l i i f there was an adjacent trans f r e e hydroxyl group on the sugar moiety ( 2 ) , then cleavage occurred w i t h Walden i n v e r s i o n . I f there i s no f r e e trans hydroxyl groups, then the s u l f a t e i s s t a b l e t o a l k a l i . At the same t i m e , any s u l f a t e l i n k e d to C-6 o f the sugar residue w i l l be cleaved by a l k a l i i f the hydroxyl group on C-3 i s f r e e . Walden i n v e r s i o n does not occur i n these circumstances, but a 3,6-anhydro-ring i s formed (Figure 5 ) . t i n s ' l a t e r s t a t e o f a f f a i r s occurs f r e q u e n t l y i n these g a l a c t a n s . This was o f commercial importance during the l a s t war when the supply o f agar from Japan was c u t o f f , and carrageenan, which i s i s o l a t e d from Chondrus c r i s p u s and G i g a r t i n a s p e c i e s , was the source i n Canada and Great B r i t a i n o f m a t e r i a l to r e p l a c e agar. Carrageenan has a lower g e l l i n g strength than agar, but i t was found t h a t treatment with a l k a l i increased the gel s t r e n g t h . Research l a t e r showed t h a t the r e a c t i o n j u s t o u t l i n e d had taken p l a c e , and i t has s i n c e been shown t h a t a l l p o l y saccharides w i t h a high 3,6-anhydrogalactose content give strong gels. I t i s common p r a c t i c e i n Japan a t the present time to increase the gel strength o f e x t r a c t s o f G r a c i l a r i a s p e c i e s , a somewhat low gel strength agar, i n t h i s way. In the p l a n t an enzyme brings about t h i s change so t h a t the seaweed i s able to r e g u l a t e the p r o p o r t i o n o f galactose 6 - s u l f a t e and 3,6-anhydro-

A UNITS

D-Galactose 2-sulphate

3

- A^

UNITS

Β

A

_3 UL

nu

mu

NAME

lambda

D-Galactose 2 , 6 - d i s u l p h a t e

3,6-Anhydro-D-galactose 2-sulphate theta

xi

D-Galactose 2-sulphate

kappa 3,6-Anhydro-D-galactose 3,6-Anhydro-D-galactose 2-sulphate i o t a

D-Galactose 2 , 6 - d i s u l p h a t e

A

_3 1A.

D-Galactose 6-sulphate

Repeating Units of Carrageenans

D-Galactose 4-sulphate

Table II.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

H W

r >

1

cl

>

ο Κ

W

>

1

to h-

14.

PERCIVAL

Sulfated

Polysaccharides

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

Figure 3.

of the

Agarose R=H

Figure 4.

Rhodophyceae

217

or OMe

κ-Carrageenan

+ Na S0 +H 0 2

Figure 5.

4

2

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

218

galactose i n any p o l y s a c c h a r i d e . 2. M e t h y l a t i o n . - Methylation has a l s o been used to d e t e r mine the p o s i t i o n o f s u l f a t e groups. As long ago as 1 9 4 7 , E. G. V. P e r c i v a l and h i s colleague {3)_ found t h a t 2 , 6 - d i - 0 methylgalactose was the major methylated sugar i n the hydrolysate a f t e r methylation of carrageenan from G i g a r t i n a s t e l l a t a . This i n d i c a t e d t h a t C-3 and C-4 were i n v o l v e d i n l i n k a g e e i t h e r to other residues or to s u l f a t e . These authors had p r e v i o u s l y shown t h a t the s u l f a t e was s t a b l e t o a l k a l i and, t h e r e f o r e , i t was not l i n k e d t o C - 3 , and they were able to deduce t h a t the galactose residues were mainly 1,3-1 inked w i t h s u l f a t e on C-4. Confirmat i o n of t h i s conclusion was obtained i n 1966 Ç 4 ) . by the i s o l a t i o n and c h a r a c t e r i s a t i o n of 0-a-3,6-anhydrogalactopyranosyl ( 1 - 3 ) galactose 4 - s u l f a t e from a p a r t i a l enzymic h y d r o l y s a t e . Dolan and Rees (5) used methylation s t u d i e s i n a d i f f e r e n t way t o provide evidence o f the p o s i t i o n o f s u l f a t e groups i n lambda -carrageenans. These authors found t h a t f o u r treatments with 1.5%-methanolic hydrogen c h l o r i d e a t 3 5 ° f o r 48 hours reduced the s u l f a t e content from 31 to 2%, and they recovered the d e s u l f a t e d polysaccharide i n 60% y i e l d . M e t h y l a t i o n o f the o r i g i n a l polysaccharide and the d e s u l f a t e d m a t e r i a l and comparison of the methyl sugars i n the hydrolysates o f the two products allowed assignment of the s u l f a t e groups. From these r e s u l t s , i t was f o l l o w e d that 4?% o f the 1 , 4 - l i n k e d residues are s u l f a t e d a t C-2 and C-6 and a high p r o p o r t i o n of the 1,3-1 inked residues a l s o c a r r y s u l f a t e a t C-2 and, i n c o n t r a s t to kappa-carrageenan, only a small p r o p o r t i o n o f these residues are s u l f a t e d a t C-4. 3. P a r t i a l A c i d H y d r o l y s i s . - A c i d h y d r o l y s i s of these p o l y saccharides r a r e l y gives any s u l f a t e d fragments, the g l y c o s i d i c l i n k s and the s u l f a t e groups having s i m i l a r l a b i l i t y . Painter ( 6 ) , however, found t h a t a u t o h y d r o l y s i s o f the f r e e a c i d form a v a r i e t y o f carrageenans i n a d i a l y s i s sac surrounded by d i s t i l l e d water c o n t a i n i n g a suspension of barium carbonate gave a mixture of galactose and galactose monosulfates. From t h i s mixture he was able t o separate and c h a r a c t e r i s e galactose 2 - , 4 - , and 6-monos u l f a t e s , again confirming the deductions from m e t h y l a t i o n . 4. I n f r a r e d Spectroscopy. - This has proved a useful d i a g n o s t i c t o o l i n the a l l o c a t i o n of the s i t e of s u l f a t e groups. Study on model galactose s u l f a t e s (7)_ has shown t h a t peaks a t 820 cm are c h a r a c t e r i s t i c of s u l f a t e at a primary a l c o h o l i c group, t h a t i s a t C - 6 ; a t 830 c m " o f e q u a t o r i a l s u l f a t e , t h a t i s a t C-2 and a t 850 o f a x i a l s u l f a t e , t h a t i s a t C-4. M

M

α

1

Types o f Carrageenans By means o f these and s i m i l a r s t u d i e s , two major groups o f carrageenans have been recognised ( 8 ) . In the f i r s t , the 1 , 3 Ifnked u n i t s are s u l f a t e d i n the 4 - p o s i t i o n , w h i l e i n the second, the s u l f a t e i s i n p o s i t i o n 2 (Table I I ) . This f i r s t group i s subdivided according t o the nature of the 1 , 4 - l i n k e d u n i t s .

14.

PERCIVAL

Sulfated

Polysaccharides

of the

Rhodophyceae

219

These may be present as galactose 6 - s u l f a t e (mu-carrageenan) o r galactose 2 , 6 - d i s u l f a t e (nu-carrageenan) or as the corresponding 3,6-anhydrides i n kappa- and i o t a - c a r r a g e e n a n . In nature the 3,6-anhydrides o f kappa- and iota-carrageenan are formed by enzymatic e l i m i n a t i o n o f the 6 - s u l f a t e from the mu and nu forms, but the conversion i s not always complete. In some seaweeds, these carrageenan types can be i s o l a t e d i n almost pure form, w h i l e i n others they e x i s t as copolymers. In the second group, the 1 , 4 - l i n k e d u n i t s are s u l f a t e d i n the 2 - p o s i t i o n . In lambda-carrageenan, the 6 - p o s i t i o n i s a l s o s u l f a t e d w h i l e i n theta-carrageenan, i t i s n o t .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

P h y s i c a l P r o p e r t i e s and Conformational E f f e c t s Many o f these e x t r a c t s have the a b i l i t y t o form r e v e r s i b l e gels which l i q u i f y when heated and s e t when c o l d . Some give s t i f f gels i n d i l u t e s o l u t i o n , t h a t i s give gels which r e t a i n a d e f i n i t e shape even though they c o n s i s t of 99.9% water and others have no g e l l i n g p r o p e r t i e s a t a l l . These p r o p e r t i e s depend l a r g e l y on the presence o r absence o f s u l f a t e groups i n c e r t a i n p o s i t i o n s on the sugar r e s i d u e s , and the theory o f change from random c o i l t o double h e l i x conformation i n the s o l gel t r a n s f o r mation, developed by Dr. D. A. Rees and h i s colleagues ( 1 ) , has gone a long way t o e x p l a i n t h i s . X-ray d i f f r a c t i o n s t u d i e s o f o r i e n t a t e d f i b e r s o f kappa- and modified iota-carrageenan i n d i cated two p a r a l l e l 3 - f o l d h e l i c a l c h a i n s , each t w i s t e d around the other and having a r i s e of 26A° per t u r n . The two chains move past each other from r e l a t i v e p o s i t i o n s t h a t are p e r f e c t l y general to form a s p e c i a l arrangement t h a t i s e x a c t l y staggered w i t h i d e n t i c a l groups moved t o h a l f the i n i t i a l spacing (Figure 6 ) . Only the presence o f the 3,6-anhydro-ring allows the sugar r i n g to be so c o n s t r a i n e d as to have three e q u a t o r i a l C-H bonds, an arrangement t h a t increases the f l e x i b i l i t y o f the chain and allows winding and unwinding of the double h e l i x . The conformation i s such t h a t hydrogen bonding takes place between the 0-2 and 0-6 o f galactose residues i n d i f f e r e n t stands o f the same double h e l i x . Thus, every u n s u b s t i t u t e d hydroxyl group i n iota-carrageènan i s engaged i n hydrogen bonding, making the conformation very s t a b l e . On the other hand, a s u l f a t e group on the 0-2 of the 1,31 inked galactose residue as i n lambda-carrageenan w i l l i n h i b i t double h e l i x formation. Even a f t e r a l k a l i treatment t o convert the 1 , 4 - l i n k e d residues i n t o 3,6-anhydrogalactose, t h i s c a r r a geenan w i l l not g e l . Rees by means o f o p t i c a l r o t a t i o n s t u d i e s ( 9 ) , confirmed t h a t when the molecules are i n s o l u t i o n they e x i s t i n random c o i l formation and when the s o l u t i o n i s c o o l e d , the chains l i n k by t h i s double h e l i x formation to give a three dimens i o n a l framework (Figure 7 ) , the i n t e r s t i c e s o f which are occupied by water. Iota-carrageenan i s a copolymer o f D-galactose 4 - s u l f a t e and 3,6-anhydro-D-galactose 2 - s u l f a t e w i t h masking by r e placement o f 1/10th o f the anhydride by D-galactose 2 , 6 - d i s u l f a t e

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

220 CARBOHYDRATE SULFATES

Figure 6. Helical structure

Figure 7.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

14.

PERCIVAL

Sulfated

Polysaccharides

of the

221

Rhodophyceae

and a much s m a l l e r p r o p o r t i o n by D-galactose 6 - s u l f a t e (Figure 8 ) . The masking residues cause k i n k i n g of the h e l i c a l carrageenan c h a i n . Thus, the physical and b i o l o g i c a l f u n c t i o n o f these r e s i dues i s to serve as h e l i x - b r e a k i n g i n t e r r u p t i o n s t h a t cause each chain to enter i n t o double h e l i c a l a s s o c i a t i o n w i t h more than one partner (Figure 7 ) . This i s necessary i f a 3-dimensional network i s to be formed r a t h e r than a c o l l e c t i o n o f i s o l a t e d chain p a i r s . I t i s thought that i n gel formation the aggregation of groups of double h e l i c e s i n t o p a r a l l e l bundles takes place (Figure 7) as a secondary process and adds to the strength o f the g e l . 4 - 0 s u l f a t e groups i n the 1 , 3 - l i n k e d residues and 2 - 0 - s u l f a t e groups in the 3,6-anhydro residues are s i t u a t e d on the o u t s i d e o f the h e l i c e s and can, t h e r e f o r e , be expected to a f f e c t the degree o f aggregation. Thus, the i n c r e a s i n g s u l f a t e i n the s e r i e s : agarose -> f u r c e l l a r a n -> kappa-carrageenan -> iota-carrageenan c o i n c i d e s with i n c r e a s i n g e l a s t i c i t y and decreasing b r i t t l e n e s s o f the g e l s . E f f e c t o f M e t a l l i c Ions I t i s understandable t h a t the nature o f the metal i o n combined w i t h the s u l f a t e groups w i l l i n f l u e n c e the degree o f aggregation of the h e l i c e s . For example, the sodium s a l t of kappacarrageenan gives l i t t l e or only weak g e l l i n g products; whereas, potassium gives a strong g e l . In c o n t r a s t , the gel strength o f iota-carrageenan with potassium i s comparatively weak, whereas, with calcium i t gives a strong e l a s t i c g e l . Commercially, the manufacturer combines d i f f e r e n t e x t r a c t s to give the a p p r o p r i a t e p r o p e r t i e s r e q u i r e d by the buyer. E x t r a c e l l u l a r Polysaccharides from M i c r o s c o p i c Red Algae Recent s t u d i e s on the h i g h l y viscous e x t r a c e l l u l a r p o l y saccharides i s o l a t e d from c u l t u r e d samples of the u n i c e l l u l a r microscopic red a l g a e , Rhodella maculata ( 1 1 ) , Porphyridium cruentum (12-14) and Porphyridium aerugineum (14) reveal complex s u l f a t e d p o l y s a c c h a r i d e s . Not only g a l a c t o s e , but c o n s i d e r a b l e proportions o f x y l o s e , g l u c u r o n i c a c i d and glucose are c o n s t i tuents o f these mucilages. Both Rhodella and F. aerugineum cont a i n a p p r e c i a b l e amounts o f 3-0-methylxylose (11,14) and 3- and 4-0-methylgalactoses are c o n s t i t u e n t s o f both Porphyridium species (14). In a d d i t i o n , F. aerugineum contains as much as 10% o f 2,4d i - 0 - m e t h y l g a l a c t o s e . 2-0-Methylglucuronic a c i d has a l s o been reported f o r F. cruentum polysaccharide (13). In both Porphyridium species the s u l f a t e groups i n the polysaccharides are l a b i l e t o methylation and periodate o x i d a t i o n c o n d i t i o n s , although i n cruentum mucilage they are somewhat more s t a b l e than i n aerugineum. While i n f r a r e d a n a l y s i s o f Rhodella polysaccharide (15) gave peaks at 1200 c m " , 874 c m " and 950 c m " which disappear on d e s u l f a t i o n , the two Porphyridium polysaccha1

1

1

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222 CARBOHYDRATE SULFATES

Figure 8.

14.

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of the

Rhodophyceae

223

r i d e s give the c h a r a c t e r i s t i c peaks f o r galactose s u l f a t e s , t h a t i s broadish bands, a t 820-830 c m " and a sharper band a t 850 c m " (14) i n d i c a t i n g t h a t primary, e q u a t o r i a l and a x i a l s u l f a t e may be present. A l l the s u l f a t e i n F. cruentum polysaccharide i s s t a b l e to a l k a l i whereas i n t h a t from F. aerugineum the s u l f a t e i s decreased from 9% t o 3.7%. I n f r a r e d a n a l y s i s of a s u l f a t e d f r a g ­ ment i s o l a t e d from an autohydrolysate o f the f r e e a c i d form o f F. aerugineum (14) gives a peak a t 830 c m " , i n d i c a t i v e o f equa­ t o r i a l s u l f a t e . This has been c h a r a c t e r i s e d . 1

1

1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

Conclusions Although chemical s t u d i e s have been made on only a m i n o r i t y of known species of red a l g a e , i t appears t h a t the type o f water e x t r a c t a b l e polysaccharides present f o l l o w s the b o t a n i c a l c l a s s i ­ f i c a t i o n i n t o genera. There are d i s t i n c t groups o f polymers which, although having much i n common, d i f f e r i n d e t a i l , the pro­ p o r t i o n and p o s i t i o n of s u l f a t e groups being an important v a r i ­ able. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Rees, D. A. Adv. Carbohyd. Chem. (1969) 24, 267 and refer­ ences c i t e d t h e r e i n . Percival, E. G. V. Quart. Rev. (1949) 3, 369. Dewar, Ε. T. and Percival, E. G. V. J. Chem. Soc. (1947) 1622. W e i g l , J., Turvey, J. R. and Yaphe, W. Proc. V t h . I n t . Seaweed Symp. (1965) H a l i f a x , Nova S c o t i a (Edited E. G. Young and J. L. McLachlan) p. 329. Pergamon P r e s s , Oxford. Dolan, T. C. S. and Rees, D. A. J. Chem. Soc. (1965) 3534. P a i n t e r , T. J. Proc. Vth I n t . Seaweed Symp. (1965) H a l i f a x , Nova S c o t i a (Edited E. G. Young and J. L. McLachlan) p. 305. Pergamon P r e s s , Oxford. Turvey, J. R. Adv. Carbohyd. Chem. (1965) 20, 183 and references c i t e d t h e r e i n . S t a n c i o f f , D. J. and Renn, D. W. i n " P h y s i o l o g i c a l E f f e c t s of Food Carbohydrates" ACS Symposium 15, (1974). Edited by A l l e n e Jeanes and John Hodge p. 282. Rees, D. Α . , S c o t t , W. E. and W i l l i a m s o n , F. B. Nature (Lond.) (1970) 227, 390. Anderson, N. S., Dolan, T. C. S. and Rees, D. A. J. Chem. Soc. (C) (1973) 2173. Sheik Fareed, V. and Percival, E. Carbohyd. Res. (1977) 53, 276. Medcalf, D. G., S c o t t , J. R., Brannon, J. H., Hemerick, G. Α . , Cunningham, R. L., Chessen, J. H. and Shah, J., Carbohyd. Res. (1975) 44, 87. Heaney-Kieras, J. and Chapman, D. J. Carbohyd. Res. (1976) 52, 169.

224 14. 15.

CARBOHYDRATE SULFATES

Percival, E. and F o y l e , R. unpublished work. Evans, L. V. and Callow, M. E., Percival, E. and Sheik Fareed, V. J. Cell. S c i . (1974) 16, 1.

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RECEIVED F e b r u a r y 6,

1978.

15 Sulfated Fucose-Containing Polysaccharides from Brown Algae: Structural Features and Biochemical Implications D A R R E L L G.

MEDCALF

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Department of Chemistry, University of Puget Sound, Tacoma, W A 98416

The cell w a l l of p l a n t cells is an intriguing biochemical system t h a t continues to defy definitive c h a r a c t e r i z a t i o n . Peter Albersheim and his co-workers r e c e n t l y have completed the most comprehensive c h a r a c t e r i z a t i o n of the composition of the wall of t i s s u e c u l t u r e d cells ( 1 , 2 , 3 ) . In a d d i t i o n , Albersheim has suggested a model f o r the structural arrangement of these polymers in the w a l l and in some cases, has suggested specific r o l e s f o r particular p o l y s a c c h a r i d e s (4). N e v e r t h e l e s s , the exact r o l e s of the v a r i o u s f r a c t i o n s is not well understood. Moreover, information on the site and pathways of b i o s y n t h e s i s of w a l l polymers is very imcomplete (5,6). The cell w a l l s of algal cells have a unique composition and a l s o p r e s e n t , in certain systems, a unique o p p o r t u n i t y f o r the study of cell w a l l p o l y s a c c h a r i d e b i o s y n t h e s i s and assembly. The fertilized eggs o f the brown a l g a Fucus appear to be ideal f o r the study of cell w a l l composition, b i o s y n t h e s i s , and c o n t r o l of cell w a l l formation ( 7 , 8 , 9 ) . W a l l - l e s s eggs can be fertilized in a c o n t r o l l e d manner and the development of the wall f o l l o w e d in the synchronously developing, p o p u l a t i o n (10). The composition of the c e l l w a l l i n the brown algae (Phaeophyceae) i s made up of two f i b r i l l a r or s t r u c t u r a l polymers, c e l l u l o s e and a l g i n i c a c i d , and m a t r i x polymers. These m a t r i x polymers are a complex array of f u c o s e - c o n t a i n i n g s u l f a t e d p o l y saccharides (11). Mian and P e r c i v a l (12) concluded t h a t these polymers represented a spectrum from h i g h u r o n i c a c i d low s u l f a t e c o n t a i n i n g polymers to a r e l a t i v e l y pure fucan s u l f a t e . The abundance of s u l f a t e d p o l y s a c c h a r i d e s i n a l l three c l a s s e s of marine a l g a e , Chlorophyceae (green), Rhodophyceae ( r e d ) , Phaeophyceae (brown), w h i l e being e s s e n t i a l l y absent i n land p l a n t s , has i n v i t e d s p e c u l a t i o n as to the p h y s i o l o g i c a l f u n c t i o n of these components. The two most commonly suggested r o l e s are (1) involvement w i t h s e l e c t i v e c a t i o n i c t r a n s f e r i n the s a l i n e medium; (2) p r e v e n t i o n of d e s i c c a t i o n when the p l a n t s are exposed to drying c o n d i t i o n s such as low t i d e s (13). A recent r e p o r t of the presence of a s u l f a t e d p o l y s a c c h a r i d e as a major 0-8412-0426-8/78/47-077-225$05.00/0 © 1978 American Chemical Society

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component i n the c e l l w a l l of a h a l o p h i l i c b a c t e r i a (14) lends support to these suggestions. However, d e f i n i t i v e data on the s p e c i f i c biochemical r o l e of s u l f a t e i n these polymers has been lacking. This r e p o r t w i l l examine the composition of the s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e s from brown algae i n terms of t h e i r s t r u c t u r a l c h a r a c t e r i s t i c s and p o s s i b l e r o l e s i n c e l l development and c e l l w a l l formation and f u n c t i o n .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

P o l y s a c c h a r i d e Composition and S t r u c t u r e The e a r l y work on the chemical composition of the f u c o s e c o n t a i n i n g polymers i n brown algae centered on the m a t e r i a l c a l l e d " f u c o i d a n " , a hygroscopic s u l f a t e d p o l y s a c c h a r i d e f r a c t i o n f i r s t i s o l a t e d by K y l i n i n 1913. This m a t e r i a l seemed to be u b i q u i t o u s i n brown a l g a e , but the most e x t e n s i v e s t u d i e s were done on m a t e r i a l i s o l a t e d from Fucus v e s i c u l o s u s and AscophyHum nodosum by d i l u t e a c i d or water e x t r a c t i o n . Although small amounts of x y l o s e and g a l a c t o s e were always found a s s o c i a t e d w i t h " f u c o i d a n " , i t was g e n e r a l l y considered to be a unique substance. I t s b a s i c s t r u c t u r e was shown by Conchie and P e r c i v a l (15) to be composed p r i m a r i l y o f L-fucopyranose u n i t s l i n k e d a - ( l 2) w i t h s u l f a t e groups p r i m a r i l y l o c a t e d a t p o s i t i o n 4. Small amounts of s i n g l e u n i t branches at C-3 and C-4 a l s o were detected (15,16,17). Larsen, Haug and P a i n t e r (18) showed t h a t crude a l g i n i c a c i d p r e p a r a t i o n s from Ascophyllum nodosum were contaminated by s m a l l amounts o f a f u c o s e - c o n t a i n i n g m a t e r i a l which was not " f u c o i d a n . " This m a t e r i a l was named a s c o p h y l l a n and was composed of fucose (25%), x y l o s e (26%), g l u c u r o n i c a c i d (27%) and 13% s u l f a t e . Traces of g a l a c t o s e and mannose a l s o were found. Data from p a r t i a l base h y d r o l y s i s of the molecule suggested a u r o n i c a c i d "backbone" w i t h branches c o n t a i n i n g x y l o s e and f u c o s e . L a t e r work (19) l e d t o the i s o l a t i o n of a s i g n i f i c a n t q u a n t i t y of 3-0-3-D-xylopyxanosyl-L-fucose from a m i l d a c i d h y d r o l y z a t e of a s c o p h y l l a n . This l e d to the c o n c l u s i o n t h a t both sugars were p a r t of the same branched c h a i n . P e r c i v a l (20,21) a l s o i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d a s u l f a t e d polymer f r a c t i o n from A. nodosum which contained fucose (49%), x y l o s e (10%) and g l u c u r o n i c a c i d (11%). These data i n d i cate t h a t t h i s was a d i f f e r e n t polymer f r a c t i o n from a s c o p h y l l a n . I t was shown to be a h i g h l y branched s t r u c t u r e w i t h end-group and (1-+4)-linked x y l o s e , end group and ( l - * 2 ) - l i n k e d fucose (some u n i t s were s i n g u l a r l y branched at p o s i t i o n 3 or 4 and some doubled branches were a l s o i n d i c a t e d ) and ( l - * 4 ) - l i n k e d g l u c u r o n i c a c i d . 3-0-(3-D-Glucopyranosyluronic a c i d ) - L - f u c o s e was a s i g n i f i cant s t r u c t u r a l e n t i t y and evidence f o r (l->3)-linked fucose was a l s o found. S u l f a t e groups were l o c a t e d on p o s i t i o n 4 of fucose residues. Larsen, e t . a l . (22) confirmed t h a t a s c o p h y l l a n was not the only nonfucoidan fucose polymer i n brown a l g a e . They showed !f

fl

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Fucose-Containing

227

t h a t both F\ v e s i c u l o s u s and A. nodosum contained other p o l y saccharide "complexes" and suggested t h a t perhaps " f u c o i d a n " i t s e l f d i d not e x i s t on these algae i n the n a t i v e s t a t e . More r e c e n t l y , P e r c i v a l s group s t u d i e d the carbohydrates i n s e v e r a l species o f brown algae from d i f f e r e n t f a m i l i e s of p l a n t s (12,23,24). They were unable t o separate the f u c o s e - c o n t a i n i n g f r a c t i o n s i n t o unique e n t i t i e s but d i d accomplish s e p a r a t i o n i n t o a s e r i e s o f f r a c t i o n s ranging from a n e a r l y pure fucan s u l f a t e t o one w i t h high u r o n i c a c i d and low s u l f a t e . These were considered to be a m o r e - o r - l e s s continuous spectrum o f s t r u c t u r e s . A l l species s t u d i e d had e s s e n t i a l l y the same p a t t e r n and the s t r u c t u r a l data suggested the same h i g h l y branched c h a r a c t e r i s t i c s as p r e v i o u s l y r e p o r t e d f o r " f u c o i d a n " from F_. v e s i c u l o s u s (15) and the glucuronoxylofucan from A. nodosum (20,21). One o f the s p e c i e s , Desmarestia a c u l e a t a , was shown t o have a much higher p r o p o r t i o n o f g a l a c t o s e i n i t s " f u c a n s " (24), p a r t i c u l a r l y i n the m a t e r i a l e x t r a c t e d w i t h d i l u t e a l k a l i . This crude e x t r a c t had g a l a c t o s e , fucose, x y l o s e and g l u c u r o n i c a c i d i n the molar p r o p o r t i o n s o f 2:1:0.13:1.7. The g a l a c t o s e was D-galactose and m e t h y l a t i o n s t u d i e s i n d i c a t e d i t was present i n the polymer p r i m a r i l y as end-group and (1-K5)-linked r e s i d u e s . S t i l l other v a r i a t i o n s i n composition have been found i n p a r t i a l l y p u r i f i e d e x t r a c t s from Padina pavonia (25). In l i g h t o f the apparent complexity o f the f u c o s e - c o n t a i n i n g polymers i n brown algae, there i s a need f o r a more d e f i n i t i v e c h a r a c t e r i z a t i o n o f these polymers i n order t o study t h e i r p o s s i b l e r o l e i n c e l l development. Medcalf and Larsen (2(3,27) have r e examined the f u c o s e - c o n t a i n i n g s u l f a t e d p o l y s a c c h a r i d e s from two w e l l - s t u d i e d brown a l g a l s p e c i e s , AscophyHum nodosum and Fucus v e s i c u l o s u s . An attempt was made t o assure minimum degradation during e x t r a c t i o n i n order t o i n c r e a s e the r e l i a b i l i t y of the chemical and b i o l o g i c a l c o r r e l a t i o n s . E x t r a c t i o n o f d r i e d weed w i t h water a t pH 2 o r 0.1M EDTA a t pH 7.5 gave i d e n t i c a l r e s u l t s as shown by both f r e e boundary e l e c t r o p h o r e s i s at pH 2 and pH 7 and c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s a t pH 7.5. Previous work had shown t h a t the f u c o s e - c o n t a i n i n g polymers were a l l r e l a t i v e l y h i g h l y charged w i t h u r o n i c a c i d and/or s u l f a t e groups. Thus, e l e c t r o p h o r e s i s should be a r e l i a b l e technique t o i n d i c a t e the complexity o f the e x t r a c t s and t o f o l l o w the success o f attempts to separate the mixtures i n t o i n d i v i d u a l components. A f r a c t i o n a t i o n procedure developed by Larsen, et_ al^. (18,22) was adapted f o r use i n t h i s study. I t i n v o l v e d f r a c t i o n a l p r e c i p i t a t i o n w i t h ethanol o f polymer s o l u t i o n s c o n t a i n i n g v a r y i n g conc e n t r a t i o n s o f magnesium o r calcium c h l o r i d e . C e l l u l o s e acetate e l e c t r o p h o r e s i s was used t o f o l l o w s m a l l s c a l e t r i a l f r a c t i o n a t i o n s . P r e p a r a t i v e s c a l e f r a c t i o n a t i o n s were done using optimum c o n d i t i o n s and the f r a c t i o n s c h a r a c t e r i z e d by both f r e e boundary (pH 2) and c e l l u l o s e a c e t a t e (pH 7.5) e l e c t r o p h o r e s i s . Isolated f r a c t i o n s were r e f r a c t i o n a t e d u n t i l f r e e boundary e l e c t r o p h o r e s i s i n d i c a t e d a t l e a s t 95% homogeneity. 1

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Polysaccharides

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General f r a c t i o n a t i o n schemes f o r the crude e x t r a c t s from A. nodosum and F. v e s i c u l o s u s are shown i n Figure 1. Figures 2, 3, 4, and 5 show the r e s u l t s of the p u r i f i c a t i o n e f f o r t s as i n d i c a t e d by e l e c t r o p h o r e s i s . Four e l e c t r o p h o r e t i c a l l y pure and d i s t i n c t f r a c t i o n s were i s o l a t e d from A. nodosum (Frac. 1-4, Table I) and two from F. v e s i c u l o s u s (Frac." 1-2, Table I I ) . In each case an a d d i t i o n a l mixed f r a c t i o n was o b t a i n e d . However, the homogenious f r a c t i o n s accounted f o r almost 90% of the crude e x t r a c t s from each s p e c i e s . Therefore, these f r a c t i o n s represent unique p o l y mer molecules present i n these algae which can be e x t r a c t e d w i t h very d i l u t e a c i d . They are probably present as m a t r i x polymers i n the c e l l w a l l . Quatrano (28) has shown t h a t d i l u t e a c i d e x t r a c t s from the c e l l w a l l s of 24 hour embryos of these species have an e l e c t r o p h o r e t i c p a t t e r n very s i m i l a r to those shown here. Tables I and II give the a n a l y t i c a l data f o r these f r a c t i o n s . F r a c t i o n 1 from Ascophyllum was very s i m i l a r i n composition to ascophyllan and was i d e n t i c a l i n e l e c t r o p h o r e t i c m o b i l i t y to an ascophyllan sample i s o l a t e d p r e v i o u s l y (18). H y d r o l y s i s of t h i s f r a c t i o n w i t h d i l u t e base f o l l o w e d by d i a l y s i s and a n a l y s i s f o r fucose, u r o n i c a c i d and unsaturated u r o n i c a c i d gave the r e s u l t s shown i n Table I I I . A s i g n i f i c a n t p o r t i o n of the remaining u r o n i c a c i d and the unsaturated u r o n i c a c i d produced v i a 3 - e l i m i n a t i o n were i n the d i a l y z a b l e f r a c t i o n w h i l e the fucose remained essent i a l l y i n the n o n - d i a l y z a b l e p o r t i o n . These data suggested t h a t the m a j o r i t y of the fucose molecules were i n r e l a t i v e l y large fragments t h a t d i d not c o n t a i n u r o n i c a c i d . This i s c o n s i s t e n t w i t h a molecular s t r u c t u r e having a m a j o r i t y of the u r o n i c a c i d u n i t s i n a main chain to which are attached f u c o s e - c o n t a i n i n g s i d e c h a i n s . While a complete s t r u c t u r a l a n a l y s i s has not been done, these data p l u s the e a r l i e r data of Larsen (19) suggest t h a t a s i g n i f i c a n t p o r t i o n of the ascophyllan s t r u c t u r e can be represented as shown i n Figure 6. The nature of the linkages i n the main u r o n i c a c i d chain and the l i n k a g e of fucose t o xylose have not been determined f o r a s c o p h y l l a n . The s t r u c t u r e i n Figure 6 uses i n f o r m a t i o n obtained by P e r c i v a l (21) f o r these l i n k a g e s . The a s c o p h y l l a n - l i k e polymer i s o l a t e d by Medcalf and Larsen (27) contained mannuronic and g u l u r o n i c a c i d i n a d d i t i o n to g l u c u r o n i c a c i d i n c o n t r a s t to both the data of P e r c i v a l (20, 21) and e a r l i e r work by Larsen, e t . a l . (18,22) where only g l u c u r o n i c a c i d was d e t e c t e d . T h i s o b s e r v a t i o n was confirmed by Larsen (29) on a new Ascophyllum sample. F u r t h e r work i s needed to a s c e r t a i n whether these u r o n i c a c i d s are p a r t of a s i n g l e molecule, or whether there are s e v e r a l types o f a s c o p h y l l a n - l i k e molecules, having the same b a s i c s t r u c t u r e , but d i f f e r i n g i n the r e l a t i v e amounts of each u r o n i c a c i d . F r a c t i o n 1 from Fucus was s i m i l a r to a s c o p h y l l a n i n e l e c t r o p h o r e t i c m o b i l i t y . I t d i f f e r e d somewhat i n composition, p r i m a r i l y i n an i n c r e a s e d fucose content. However, i t s p r o p e r t i e s suggested a s t r u c t u r e c l o s e l y r e l a t e d to t h a t given f o r a s c o p h y l l a n . F r a c t i o n s 2 and 3 from Ascophyllum and F r a c t i o n 2 from Fucus

15.

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Sulfated

Fucose-Containing

Polysaccharides

were complexes which could be e a s i l y hydrolyzed i n very d i l u t e a c i d at 80° t o two new p o l y s a c c h a r i d e s . These polymers could be d i s t i n g u i s h e d e l e c t r o p h o r e t i c a l l y as shown i n F i g u r e 7 f o r the Ascophyllum f r a c t i o n s . The two new polymers were separated by f r a c t i o n a l p r e c i p i t a t i o n w i t h ethanol from a M g C l s o l u t i o n . Compositional data f o r these f r a c t i o n s are shown i n Table IV. The i n s o l u b l e (slower) f r a c t i o n was very s i m i l a r t o ascophyllan i n both e l e c t r o p h o r e t i c m o b i l i t y and composition. However, o n l y mannuronic a c i d could be detected i n s i g n i f i c a n t amounts (27). Base h y d r o l y s i s o f the complex from Ascophyllum gave a p a t t e r n of d i a l y z a b l e and n o n - d i a l y z a b l e fucose, u r o n i c a c i d and unsat­ urated u r o n i c a c i d which was the same as found f o r a s c o p h y l l a n (Table I I I ) . F r a c t i o n 2 from Fucus gave very s i m i l a r e l e c t r o ­ p h o r e t i c r e s u l t s a f t e r d i l u t e a c i d h y d r o l y s i s . The major d i f f e r ­ ence between the "complexes" from the two species seemed t o be that the Fucus f r a c t i o n , on h y d r o l y s i s , gave more o f the f a s t e r moving fucan polymer and l e s s of the a s c o p h y l l a n - l i k e f r a c t i o n . No f r a c t i o n from e i t h e r species corresponding t o " f u c o i d a n " could be detected i n the o r i g i n a l e x t r a c t . I t was concluded t h a t " f u c o i d a n " i s not a n a t i v e polymer i n these s p e c i e s , but i s a product o f the h y d r o l y s i s o f the fucose r i c h "complexes." The two complex f r a c t i o n s from Ascophyllum were shown by a time-course study o f the a c i d h y d r o l y s i s a t 75 t o g i v e a gradual i n c r e a s e i n the m o b i l i t y o f the o r i g i n a l band and a simultaneous gradual i n c r e a s e i n the c o n c e n t r a t i o n o f the slower component (Figure 8 ) . These data were i n t e r p r e t e d t o suggest t h a t the fucan p o r t i o n formed the backbone o f the molecule, and the a s c o p h y l l a n ­ l i k e components were attached as branches by a c i d - l a b i l e l i n k ­ ages. The v a r i o u s complexes d i f f e r from each other only i n the number o f a s c o p h y l l a n - l i k e molecules attached t o the fucan back­ bone. A proposed s t r u c t u r e i s shown i n Figure 9. The fucan i s shown w i t h α - (1 2 ) - l i n k a g e s based on data from Conchie and P e r c i v a l (15) . The nature o f the l i n k a g e between the two p o l y ­ saccharides i n the complex i s not known. Medcalf and Larsen (27) showed i t d i d not i n v o l v e a p e p t i d e b r i d g e . F r a c t i o n 4 from Ascophyllum had a n e u t r a l sugar composition q u i t e d i f f e r e n t from any f r a c t i o n p r e v i o u s l y r e p o r t e d f o r t h i s s p e c i e s . I t contained about equal amounts o f fucose and g a l a c ­ t o s e , some u r o n i c a c i d , and 14% s u l f a t e . Only two g a l a c t o s e r i c h polymers p r e v i o u s l y have been i d e n t i f i e d i n brown algae (24,30) and o n l y P e r c i v a l and Young (24) have p u b l i s h e d s t r u c ­ t u r a l d a t a . T h e i r f r a c t i o n , i s o l a t e d by a l k a l i n e e x t r a c t i o n , was d e s c r i b e d e a r l i e r . Medcalf, Schneider and Barnett (31) r e i s o l a t e d f r a c t i o n 4 i n h i g h l y p u r i f i e d form by repeated f r a c t i o n ­ a t i o n w i t h ethanol from CaCl~ s o l u t i o n s . I t gave a s i n g l e band on e l e c t r o p h o r e s i s and had trie composition shown i n Table V. The u r o n i c a c i d was shown t o be g l u c u r o n i c and only t r a c e s o f n e u t r a l sugars other than g a l a c t o s e and fucose were found i n the p u r i f i e d m a t e r i a l . The g a l a c t o s e was shown by D-galactose o x i ­ dase t o be p r i m a r i l y L - g a l a c t o s e . Only two major methylated 2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

229

Figure 1.

insoluble ( F r a c t . 2) insoluble (Fract.3)

Γ insoluble ( F r a c t . 5)

(0.8*

CTAB

soluble

soluble n e u t r a l glucan 0.5 v o l . ethanol

1 v o l . ethanol

1 v o l . ethanol

soluble (Fract.4)

5 vol. ethanol

2

insoluble i n 0.02 M C a C l )

Fractionation scheme used for the isolation of electrophoretically pure fractions from A. nodosum dilute acid extracts

insoluble ( F r a c t . 1)

1 v o l . ethanol

insoluble (0.75% i n 0.03 M M g C l J I — Ζ

2

(0.5% i n 0.05 M MgCl )

CRUDE EXTRACT

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Figure 1.

soluble ( F r a c t . 3)

CTAB

soluble

soluble ( n e u t r a l glucan)

1 v o l , ethanol

insoluble ( F r a c t . 2)

Fractionation scheme used for the isolation of electrophoretically pure fractions from F'. vesiculosus dilute acid extracts

insoluble ( F r a c t . 1)

0.7 v o l . ethanol

2

insoluble (0.5% i n 0.05 M MgCl )

2

(0.5% i n 0.05 M MgCl )

CRUDE EXTRACT

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

232

CARBOHYDRATE SULFATES

Acid Extract

Fract. 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Fract. 2

Fract. 3

A-

Fract. 4 Figure 2. Ascending pattern at pH = 2 of the original extract and purified fractions from A. nodosum

Fract. 5

Acid Extract J Fract. 1 I Fract. 2 I Fract. 3 Figure 3. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5 of the original extract and purified fractions from A. nodosum

|Fract.4 j Fract. 5

MEDCALF

15.

Sulfated

Fucose-Containing

Polysaccharides

233

Acid Extract

Fract. 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Fract. 2

A y0

Fract. 3

ι

•

Figure 4. Ascending pattern at pH = 2 of the original extract and puri\mmtk fied fractions from F . vesiculosus

j Acid Extract I Fract. 1

Figure 5. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5 of the origi­ nal extract and purified frac­ tions from F . vesiculosus

I Fract. 2 I Fract. 3

TABLE I

MAJOR FRACTIONS FROM A. nodosum Protein .(%)

Uronic Acid (%)

S u l - Fuf a t e cose (%) (%)

Neutral Sugar d i s t r i b u t i o n ^ Fuc X y l Mann Gal Glu (%)

Frac­ tion

Yield (%)

1

31

3.6

26.4

12.8

15

37

29

21

3

11

2

29

2.4

15.8

20.9

33

73

11

10

2

5

3

18

1.8

6.4

25.2

40

81

9

4

2

4

4

11

8.5

7.1

14.7

19

34

14

15

27

10

5

11

3.1

7.4

8.1

36

71

7

4

14

4

a

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where t o t a l area under the f i v e peaks equals 100%.

234

CARBOHYDRATE

SULFATES

TABLE I I , MAJOR FRACTIONS FROM F. v e s i c u l o s u s

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Fraction

Yield (%)

Protein (%)

1

21

6.3

2

66

5.4

3

13

3.0

Uronic Acid (%)

21.9 5.6 13.9

S u l - Fuf a t e cose (%) (%)

Neutral ^ Sugar d i s t r i b u t i o n Fuc X y l Mann Gal Glu (%)

18,.1

50

25.4

48,,1

70

12.7

40,.4

70

4.1

17

4

14

7

4

8

11

7

9

5

9

15

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where t o t a l area under the f i v e peaks equals 100%.

TABLE III* COMPOSITION OF FRACTIONS 1 AND 2 AFTER BASE HYDROLYSIS Total Carbohydrate (mg/ml sample)

Fucose (mg/ml sample)

Uronic Acid (mg/ml sample)

Unsaturated Uronic A c i d (mg/ml sample)

Fraction 1 0.12

0.242

0.0

0.13

0.206

0.042

0.62

0.11

0.153

0.028

Original

0.44

0.22

0.128

0.0

Base-hydrolyzed

0.46

0.23

0.098

0.028

Base-hydrolyzeddialyzed

0.38

0.20

0.067

0.017

Original

0.70

Base-hydrolyzed

0.76

Base-hydrolyzeddialyzed

Fraction 2

Figure 6.

Structural features of ascophyllan

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

236

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Figure 7. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5; (A) ascophyllan; (B) Fraction 2; (C) Fraction 2, hydrolyzed; (D) Fraction 3; (E) Fraction 3, hydrolyzed; (F) hydrolyzed Fraction 2, ethanol-insoluble fraction; (G) hydrolyzed Fraction 2, ethanol-soluble fraction

TABLE IV,FRACTIONS FROM MILD ACID HYDROLYSIS OF A. nodosum COMPLEX

Yield " (%) 3

Fraction

Protein (%)

Uronic Acid (%)

Fucose (%)

Neutral ^ sugar d i s t r i b u t i o n Fuc X y l Mann Gal Glu (%)

2(complex 1)

--

2.4

15.8

33

73

11

10

2

5

Insoluble

32

6.8

24.1

11

28

22

28

6

17

Soluble

68

1.6

4.1

42

77

8

8

1

6

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where the t o t a l area under the f i v e peaks equals 100%.

Figure 8. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5. Mild acid hydrolysis (0.02M HCl, 75°C) of Fraction 2.

MEDCALF

Sulfated

Fucose-Containing

Polysaccharides

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

15.

Figure 9.

Structural features of a fucan "complex

237

CARBOHYDRATE

238

SULFATES

sugars were found a f t e r h y d r o l y s i s o f the u r o n i c a c i d reduced and methylated polymer; 2,3,4-tri-0-methylfucose and 3,6,-di-0-methylg a l a c t o s e . Much smaller amounts o f 2,3,4,6-tetra-0-methylglucose and mono-methylfucose were a l s o detected. These data, along with data on p e r i o d a t e o x i d a t i o n from both the o r i g i n a l and d e s u l f a t e d polymers, suggested the p a r t i a l s t r u c t u r e shown i n Figure 10.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Biochemical Role The e a r l y stages of growth o f f e r t i l i z e d Fucus zygotes are shown i n Figure 11 ( 7 ) . A l o c a l i z e d protuberance or r h i z o i d appears 12-16 hours a f t e r f e r t i l i z a t i o n . T h i s i s subsequently p a r t i t i o n e d from the r e s t o f the c e l l by the f i r s t d i v i s i o n at 20-24 hours. The r h i z o i d c e l l becomes the s i t e o f attachment to the substratum (32) and c o n s i d e r a b l e data has been accumulated to show t h a t t h i s c e l l i s both m o r p h o l o g i c a l l y and chemically d i s t i n c t from the other c e l l o f the t w o - c e l l e d embryo (7,32^33). One o f the d i s t i n g u i s h i n g c h a r a c t e r i s t i c s o f the r h i z o i d c e l l i s i t s accumulation o f a s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e . The p r o p e r t i e s o f t h i s polymer f r a c t i o n and i t s r o l e i n r h i z o i d formation and c e l l d i f f e r e n t i a t i o n have been s t u d i e d e x t e n s i v e l y by Quatrano and co-workers (32,34,35,36) . ^ Both a u d i o r a d i o g r a p h i c techniques with S, and t o l u i d i n e Blue 0 c y t o c h e m i c a l l y , showed that the s u l f a t e d fucan i n i t i a l l y i s observed around the r h i z o i d - h a l f o f the nucleus r a d i a t i n g t o ward the s i t e o f r h i z o i d i n i t i a t i o n , and e v e n t u a l l y becomes l o c a l i z e d i n the r e g i o n o f the c e l l w a l l protuberance. After c e l l d i v i s i o n t h i s m a t e r i a l i s found i n the c e l l w a l l o f the r h i z o i d c e l l (32,37). Quatrano and Crayton (32) showed that s u l f a t i o n o f the preformed polymer began a t about the time o f rhizoid initiation. I t was p o s t u l a t e d that the s y n t h e s i s o f the h i g h l y charged fucan s u l f a t e might be the cause o f p o l a r i t y i n the zygote and lead to r h i z o i d formation. However, some very elegant f u r t h e r work by Crayton, Wilson and Quatrano (34) proved that the establishment o f a p o l a r a x i s and i n i t i a t i o n o f r h i z o i d formation could occur even though s u l f a t i o n o f the fucan polymer was prevented. Very r e c e n t l y , Hogsett and Quatrano (36) i s o l a t e d a p l a n t l e c t i n from R i c i n u s seeds which would b i n d to both s u l f a t e d and d e s u l f a t e d fucans. T h i s l e c t i n was g a l a c t o s e s p e c i f i c , but showed a f f i n i t y f o r the fucans found i n Fucus. These authors a t t r i b u t e t h i s b i n d i n g to g a l a c t o s e r e s i d u e s a s s o c i a t e d with these f r a c t i o n s . By a t t a c h i n g a f l o u r e s c e n t dye to the l e c t i n , they were able to use i t as a cytochemical marker f o r the fucan polymers. The data obtained c l e a r l y showed that the u n s u l f a t e d fucan does not accumulate i n the r h i z o i d r e g i o n . The polymer was a v a i l a b l e but s u l f a t i o n was r e q u i r e d f o r i t s l o c a l i z a t i o n and i n c o r p o r a t i o n i n t o the c e l l w a l l o f the r h i z o i d c e l l . T h i s appears to be the f i r s t c l e a r documentation o f a s p e c i f i c b i o chemical r o l e f o r s u l f a t e groups i n polymers o f brown algae. The a v a i l a b i l i t y o f synchronously developing populations o f

15.

M E D C A L F

Sulfated

Fucose-Containing

239

Polysaccharides

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

ft

Ο

ο

on

< ρ

"ο ρ. "δ Ο

ο "S «+~. Ο co

H—,

8

I Ο Où

S)

CARBOHYDRATE SULFATES

240

TABLE V*GENERAL CHARACTERIZATION OF THE GALACTOFUCAN POLYMER

Molar r a t i o

fa]π

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

S u l f a t e (%)

~

5 4

15

°

Galactose

1.0

Fucose

1.1

Uronic A c i d (%)

8

Xylose

0.1

P r o t e i n (%)

7

Mannose

0.1

Glucose

trace

Experimental Marine Biology Figure 11. Stages in the development of Fucus embryos. (A) 0-12 hr, (B) 12-16 hr, (C) 16-20 hr, (D) 30-36 hr. X600 (7). (Photomicrographs courtesy of G. B. Bouck)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

15.

MEDCALF

Sulfated

Fucose-Containing

241

Polysaccharides

Fucus zygotes from eggs which have no d e t e c t a b l e c e l l w a l l p r o v i d e s a unique system f o r the study o f w a l l formation (8,9). The appearance o f v a r i o u s w a l l f r a c t i o n s can be f o l l o w e d and these data may suggest s p e c i f i c r o l e s f o r i n d i v i d u a l w a l l p o l y s a c c h a r i d e s . Quatrano and Stevens (9) used t h i s system t o f o l l o w w a l l development i n Fucus v e s i c u l o s u s zygotes. C e l l w a l l formation could be observed as e a r l y as 10 t o 15 minutes a f t e r f e r t i l i z a t i o n . However, w a l l b i r e f r i n g e n c e was not observed u n t i l 60 minutes a f t e r f e r t i l i z a t i o n . Complete b i r e f r i n g e n c e ( a l l the p o p u l a t i o n ) d i d not occur f o r f o u r hours. The w a l l was composed of a l g i n a t e and c e l l u l o s e i n about equal amounts a t 30 minutes (Figure 12). Fucans were not detected u n t i l f o u r hours a f t e r f e r t i l i z a t i o n when they represented about 25-30% o f w a l l carbohydrate. While the t o t a l carbohydrate i n the w a l l continued t o i n c r e a s e , p a r t i c u l a r l y during r h i z o i d e l o n g a t i o n (16-24 h o u r s ) , the p r o p o r t i o n o f the major p o l y s a c c h a r i d e groups remained r e l a t i v e l y constant. Embryos a t 24 hours c o n s i s t e d o f 60% a l g i n a t e , 20% c e l l u l o s e and 20% f u c o s e - c o n t a i n i n g polymers. The fucans were composed o f two e l e c t r o p h o r e t i c a l l y d i s t i n c t f r a c t i o n s ; F , a slow moving component, which very l i k e l y was the a s c o p h y l l a n - l i k e F r a c t i o n 1 from Fucus d e s c r i b e d by Medcalf and Larsen (26); and V^> a f a s t e r moving f r a c t i o n having a h i g h e r fucose content than F^. This corresponded t o the complex ( F r a c t i o n s 2 and 3) r e p o r t e d by Medcalf and Larsen (26,27). The two fucan polymers d i d not appear i n developing w a l l s a t the same t i m e . Only F. was detected p r i o r t o 12 hours a f t e r f e r t i l i z a t i o n . By 12 nours, the developing w a l l acquired F which corresponded t o the f r a c t i o n which was s u l f a t e d and a s s o c i a t e d w i t h r h i z o i d f o r m a t i o n . The r o l e these polymers p l a y i n the developing w a l l i s only s p e c u l a t i o n a t t h i s time. However, Quatrano and Stevens (9) have suggested t h a t the appearance o f F^ as the e a r l i e s t m a t r i x component i n the developing w a l l , c o i n cident w i t h the a c q u i s i t i o n o f s t r u c t u r a l i n t e g r i t y and b i r e f r i n gence o f the w a l l , argues f o r i t s b a s i c s t r u c t u r a l r o l e i n w a l l assembly and f u n c t i o n . F which appeared a t the time o f r h i z o i d f o r m a t i o n , and which had t o be s u l f a t e d before i t was i n c o r p o r a t e d (37) may be r e s p o n s i b l e f o r c e l l adhesion. Zygotes i n which s u l f a t i o n o f F was i n h i b i t e d , formed r h i z o i d s but d i d not adhere to the substratum (32). The requirements o f s u l f a t e groups f o r adhesion i s c o n s i s t e n t w i t h data suggesting t h a t the presence o f a n i o n i c groups a t the surface p l a y s an important r o l e i n barnacle adhesion (38). The data on the p o l y s a c c h a r i d e s found i n the primary c e l l w a l l of s u s p e n s i o n - c u l t u r e d sycamore c e l l s has been proposed as a model by which t o compare i n f o r m a t i o n on c e l l w a l l s from other systems (_3). While a l g a l systems have unique f e a t u r e s , such as s t r u c t u r a l polymers other than c e l l u l o s e and high degrees o f s u l f a t i o n , there i s a s i m i l a r i t y o f s t r u c t u r e s which can be noted. Table VI i s an attempt t o show p o s s i b l e r e l a t i o n s h i p s between the d e t a i l e d w a l l i n f o r m a t i o n from sycamore c e l l s (4)and the data 2

242

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

70+

TIME A F T E R F E R T I L I Z A T I O N

(HRS.)

Plant Physiology Figure 12. Changes in the major polysaccharide composition of isolated cell walls from F. vesiculosus at different times after fertilization (9)

TABLE V I . COMPARISON OF CELL WALL POLYSACCHARIDES

% o f T o t a l Carbohydrate Sycamore C e l l s

a

Brown Algae

Structural Polymers

C e l l u l o s e , 27%

C e l l u l o s e , 20% A l g i n i c A c i d , 60%

Pectic

Arabinogalactan, 22% Rhamnogalactan, 18%

Glucuronoxylofucan ( a s c o p h y l l a n - l i k e ) 9% Glucuronogalactofucan, 1%

Xyloglucan, 23%

Fucan complexes, 10%

Polymers

Matrix Polymers

Taken from Albersheim (4) Composite o f data from Medcalf and Larsen (26) and Quatrano and Stevens (!9) .

15.

MEDCALF

Sulfated

Fucose-Containing

Polysaccharides

243

obtained by Medcalf and Larsen (26) and Quatrano and Stevens (9) from brown algae. While the proposed r e l a t i o n s h i p s are over­ s i m p l i f i e d , they do suggest the p o s s i b i l i t y that p l a n t c e l l w a l l s , from whatever the source, i n c l u d i n g algae, have more s i m i l a r i t i e s than may p r e v i o u s l y have been recognized. A l g i n i c a c i d may serve both s t r u c t u r a l and p e c t i c r o l e s in the brown algae and thus could be l i s t e d in both f r a c t i o n s in the t a b l e .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

Summary The complicated nature o f the s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e s from brown algae have been reviewed. For Asco­ phyllum nodosum and Fucus v e s i c u l o s i s , a s e r i e s of e l e c t r o p h o r e t i c a l l y homogeneous f r a c t i o n s have been i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d . These i n c l u d e a xylofucoglucuronan (ascophyllan or a s c o p h y l l a n - l i k e ) , complexes which have a s c o p h y l l a n - l i k e mole­ cules attached t o a fucan backbone, and in Ascophyllum, a novel glucuronogalactofucan. Information on the biochemical r o l e o f major f u c o s e - c o n t a i n i n g polymers in Fucus have been reviewed. Work by Quatrano and co-workers i n d i c a t e d that these polymers have an important r o l e in c e l l wall development. The fucan com­ p l e x e s , when s u l f a t e d , are l o c a l i z e d in the r h i z o i d c e l l and are r e q u i r e d f o r adhesion o f the embryo t o the substratum. Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

Talmadge, K.W., K. Keegstra, W.D. Bauer, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 158. Bauer, W.D., K.W. Talmadge, K. Keegstra, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 174. Keegstra, Κ., K.W. Talmadge, W.D. Bauer, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 188. Albersheim, P., in "Plant Biochemistry, 3rd ed.", J. Bonner and J.E. Varner, eds., P. 225, Academic Press, N.Y., 1976. C h r i s t p e e l s , M.J., Ann. Rev. Plant P h y s i o l . , (1976), 27, 19. Northcote, D.H., in "Plant Carbohydrate Biochemistry", J.B. Pridham, ed., P. 165, Academic Press, N.Y., 1974. Quatrano, R.S., in "Experimental Marine B i o l o g y " , R. M a r i s c a l , ed., P. 303, Academic Press, N.Y., 1974. Novotny, A.M. and M. Forman, P l a n t a , (1975), 122, 67. Quatrano, R.S. and P.T. Stevens, Plant P h y s i o l . , (1976), 58, 224. Ley, A.C. and R.S. Quatrano, B i o l . B u l l . , (1973), 145, 446. Percival, E. and R.M. McDowell, "Chemistry and Enzymology of Marine A l g a l P o l y s a c c h a r i d e s " , p. 176, Academic Press, London, 1967. Mian, A . J . and E. Percival, Carbohydr. Res., (1973), 26, 133.

244

(13)

(14) (15)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38)

CARBOHYDRATE

SULFATES

Percival, E. and R.S. McDowell, "Chemistry and Enzymology of Marine A l g a l P o l y s a c c h a r i d e s , " p. 23, Academic Press, London, 1967. Steber, J. and K.H. S c h l e i f e r , Arch. M i c r o b i o l . , (1975), 105, 173. Conchie, J. and E.G.V. Percival, J. Chem. soc., (1950), 827. Côté, R.H., J. Chem. Soc., (1959), 2248. O ' N e i l l , A.N., J. Am. Chem. soc., (1954), 76, 5074. Larsen, B., A. Haug, T.H. P a i n t e r , Acta Chem. Scand., (1966), 20, 219. Larsen, B. Acta Chem. Scand., (1967), 21, 1395. Percival, E., Carbohydr. Res., (1968), 7, 272. Percival, E., Carbohydr. Res., (1971), 17, 121. Larsen, B., A. Haug, and T.J. P a i n t e r , A c t a . Chem. Scand., (1970), 24, 3339. Mian, A.J. and E. Percival, Carbohydr. Res., (1973), 26, 147. Percival, E. and M. Young, Carbohydr. Res., (1974), 32, 195. Abdel-Fattah, A.F. and M. Endrees, Phytochem., (1977), 16, 939. Medcalf, D.G. and B. Larsen, Carbohydr. Res., in p r e s s . Medcalf, D.G. and B. Larsen, Carbohydr. Res., in p r e s s . Quatrano, R.S., unpublished r e s u l t s ( p r i v a t e communication). Larsen, B., unpublished r e s u l t s ( p r i v a t e communication). Mangel-Din Hussein, M., Phytochem., (1975), 14, 1866. Medcalf, D.G., T.L. Schneider and R.W. Barnett, Carbohydr. Res., in p r e s s . Quatrano, R.S. and M.A. Crayton, Dev. Biol., (1973), 30, 29. J a f f e , L.F., Advan. Morphog., (1968), 7, 295. Crayton, M.A., E. Wilson, and R.S. Quatrano, Dev. Biol., (1974), 39, 164. Hogsett, W.E. and R.S. Quatrano, Plant P h y s i o l . , (1975), 55, 25. Hogsett, W.E. and R.S. Quatrano, unpublished r e s u l t s . F u l c h e r , R.G. and M.E. McCully, Can. J. Bot., (1971), 49, 161. Otness, J.S. and D.G. Medcalf, Compar, Biochem. P h y s i o l . , (1972), 43B, 443.

RECEIVED February 6, 1978.

16 Co-Ion and Counter-Ion Interactions with Sulfonated Polysaccharides MARGARET TOMASULA, NANCY SWANSON, and PAUL ANDER

1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

Department of Chemistry, Seton Hall University, South Orange, NJ 07079

With t h e advent o f t h e Debye-Hückel t h e o r y and its subsequent scrutinization t o e x p e r i m e n t a l testing, a p h y s i c a l p i c t u r e emerged f o r t h e b e h a v i o r o f s i m p l e electrolyte s o l u t i o n s a l o n g w i t h its limiting laws which a r e valid f o r v e r y dilute s o l u t i o n s . T h i s model, a l o n g w i t h its f u r t h e r development by Onsager, and t h e Bjerrum i o n - p a i r concept prove u s e f u l . I t s h o u l d be noted t h a t more r e c e n t t h e o r i e s have extended o u r u n d e r s t a n d i n g o f electrolyte solutions to higher concentrations. With the particularly high electrostatic p o t e n t i a l on linear polyelectrolytes due t o c o n s t r a i n e d charges a l o n g t h e c h a i n , it is p r e s e n t l y b e l i e v e d t h a t a rod-like o r cylindrical model f o r t h e polyion w i t h " i t s i o n i c atmosphere is t h e a p p r o p r i a t e r e p r e s e n t a t i o n f o r t h e linear polyelectrolyte in solution. The most comprehensive modern t h e o r y o f polyelectrolyte b e h a v i o r in s o l u t i o n is by Manning ( 1 ) . H i s line charge model f o r t h e p o l y e l e c trolyte r e s u l t s in limiting laws f o r thermodynamic (2) , mass (2) and electrical t r a n s p o r t (3,4,5.) p r o p e r t i e s . G e n e r a l l y , t h e infinite l i n e charge i s thought t o intera c t w i t h s i m p l e i o n s via two phenomena, by ion-atmosphere interaction and by c o n d e n s a t i o n ( s i t e - b i n d i n g ) o f c o u n t e r i o n s onto t h e p o l y i o n . I f t h e charge d e n s i t y o f the polyelectrolyte, which i s p r o p o r t i o n a l t o t h e dimens i o n l e s s parameter ξ

ξ

=

ΤΈτΈ

(

1

)

where e i s the p r o t o n i c c h a r g e , ε i s t h e d i e l e c t r i c con­ s t a n t o f the medium, k i s t h e Boltzmann c o n s t a n t , Τ i s the a b s o l u t e temperature and b i s t h e average d i s t a n c e between a d j a c e n t charge groups on t h e p o l y e l e c t r o l y t e , i s l e s s than a c r i t i c a l charge d e n s i t y ξ , which i s given ξ = I " , where ζ i s t h e charge o f t h e σ

1

α

1

χ

Author to whom correspondence should be sent. 0-8412-0426-8/78/47-077-245$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

CARBOHYDRATE SULFATES

246

c o u n t e r i o n and s i s the charge o f a s i n g l e c h a r g e - s i t e on the p o l y i o n , no c o n d e n s a t i o n o f c o u n t e r i o n s o c c u r s and b o t h c o u n t e r i o n s and c o i o n s ( i f a s i m p l e s a l t i s p r e s e n t ) i n t e r a c t w i t h charges on the p o l y i o n by DebyeHuckel f o r c e s , i . E . , the p o t e n t i a l a t any p o i n t in the i o n i c atmosphere s u r r o u n d i n g the p o l y i o n i s g i v e n by the Debye-Huckel s c r e e n i n g p o t e n t i a l . If ξ i s greater than ξ f o r a p o l y e l e c t r o l y t e , an i n s t a b i l i t y i s shown t o e x i s t which i s r e l i e v e d by c o n d e n s a t i o n o f c o u n t e r ­ i o n s onto the p o l y i o n so as t o reduce ξ t o i t s e f f e c t i v e value ξ . A l l uncondensed c o u n t e r i o n s and c o i o n s in the i o n i c atmosphere i n t e r a c t w i t h the p o l y i o n by DebyeHlickel f o r c e s . S i n c e | s | = 1 f o r almost e v e r y p o l y ­ e l e c t r o l y t e , then f o r ζ>\ζ \~ , and the f r a c t i o n o f con­ densed (site-bound) c o u n t e r i o n s i s ( 1 - | ζ \ ~ ζ ~ ) . The Manning"model has been t e s t e d e x p e r i m e n t a l l y u s i n g d i f f e r e n t p o l y e l e c t r o l y t e s ( 6 . - 2 J 3 ) . S i n c e the model demands a r o d - l i k e geometry f o r the p o l y i o n , i o n i c p o l y s a c c h a r i d e s have been used because o f t h e i r r e l a ­ t i v e l y s t i f f s t r u c t u r e as compared t o the more f l e x i b l e synthetic polyelectrolytes. For s u l f o n a t e d p o l y s a c ­ c h a r i d e s , a c t i v i t y c o e f f i c i e n t measurements f o r the s i m p l e i o n s gave e x c e l l e n t agreement w i t h Manning's t h e o r e t i c a l r e s u l t s o v e r a l a r g e c o n c e n t r a t i o n range f o r sodium c h o n d r o i t i n s u l f a t e (_12) , the sodium s a l t o f s u l ­ f a t e d p r o t e o g l y c a n from b o v i n e i n t e r v e r t e b r a l d i s c (12^) , d e x t r a n s u l f a t e ( 1 J . , _ 2 2 , 2 _ 3 ) and i o t a , kappa and lambda c a r r a g e e n a n s a l t s o f sodium, p o t a s s i u m and c a l c i u m (ISO . S e l f - d i f f u s i o n measurements o f N a Ca and S r + in the p r e s e n c e o f c h o n d r o i t i n s u l f a t e have e s t a b l i s h e d the v a l i d i t y o f the c o n d e n s a t i o n phenomena p r e d i c t e d by Manning's t h e o r y (10,24) . S i m i l a r s t u d i e s w i t h sodium i o t a c a r r a g e e n a n in aqueous s o l u t i o n s o f NaCl, Na^O^ and Na^FeiCN) i n d i c a t e t h a t w h i l e the Manning t h e o r y c o r r e c t l y p r e d i c t s the s e l f - d i f f u s i o n c o e f f i c i e n t s f o r monovalent C I " i o n , i t c l e a r l y overemphasized the e l e c ­ t r o s t a t i c i n t e r a c t i o n s between the d i v a l e n t SO "", the t e t r a v a l e n t F e ( C N ) g ~ and the p o l y i o n , e x c e p t f o r the good agreement a t low r a t i o s o f p o l y e l e c t r o l y t e t o s i m p l e s a l t c o n c e n t r a t i o n s (6_) . With our r e c e n t emphasis on the i n t e r a c t i o n s of c o i o n s w i t h p o l y e l e c t r o l y t e s , i t was o f i n t e r e s t t o examine the e f f e c t o f the charge d e n s i t y o f s e v e r a l c o i o n s on t h i s i n t e r a c t i o n by p o t e n t i o m e t r i c and s e l f - d i f f u s i o n t e c h n i q u e s . Here we r e p o r t r e s u l t s f o r s i n g l e i o n a c t i v i t y c o e f f i c i e n t s f o r Na , CI", B r " and I " in aqueous s o l u t i o n s o f sodium i o t a c a r r a g e e n a n (NaCarr) and sodium d e x t r a n (NaDS) a t 25°C. The r e s u l t s w i l l be d i s c u s s e d in l i g h t o f modern Manning theory of p o l y e l e c t r o l y t e s o l u t i o n s . α

α

p

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

1

ι

1

+

1

1

+ 2

2

f

2

+

16.

TOMASULA E T A L .

Co-Ion

and Counter-Ion

Interactions

247

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

Experimental Section Materials. Sodium iota-carrageenan, an extraction of the red algae Eueheuma Spinosum was kindly supplied by Marine C o l l o i d s , Inc. I t consists of β-D-galactose4-sulfate and 3,6-anhydro-a-D-galactose-2-sulfate r e s i ­ dues with an average distance between charges on the chain of b = 4.4A. Analysis of the carrageenan (sample Re 7275) by the manufacturer indicated that t h i s sample has a weight average molecular weight of 291,000 and a number average molecular weight of 191,000. No con­ taminating cations or anions were present in the sample; the c r i t e r i a f o r contamination being that the cation or anion i s present in the sample in excess of 0.5%. The sample has an equivalent weight of 255.2 ± O.lg. Chem­ i c a l analysis of the p u r i f i e d iota-carrageenan gave a sulfate-hexose r a t i o of 0.99; therefore, one s u l f a t e group i s assumed per sugar unit. The sample was p u r i ­ f i e d by the manufacturer by washing of the unpurified carrageenan with a 1 Ν NaCl solution in 50% isopropyl alcohol. This procedure was repeated four times. This was followed by four p l a i n alcohol washes to remove r e s i d u a l s a l t . The product was then dried and ground. Solutions were prepared by adding weighed amounts of the carrageenan, which were dried in vacuo at 40°C f o r at l e a s t 24 hours, to a volumetric f l a s k and an aliquot of the appropriate s a l t from a stock s a l t s o l u t i o n . The f l a s k was d i l u t e d to the mark with deionized water. Dried reagent grade NaCl, NaBr, and Nal were used to prepare a l l solutions. Dextran s u l f a t e , a sulfated d e r i v a t i v e of dextran, was kindly supplied by Pharmacia Fine Chemicals, Inc. The sample had a molecular weight of 500,000. Foreign ions were removed from the sample by passing the sample through appropriate ion exchange columns. The equi­ valent weight of the sample was found to be 180.5 ± 0.5 g/equiv., i n d i c a t i n g 2.06 s u l f a t e groups per sugar u n i t . This gives an average spacing between charges on the NaDS sample of b = 2. 5Â (11). The concentration of the NaDS solutions were determined by converting the NaDS to the hydrogen form followed by t i t r a t i o n . Electromotive Force Measurements. The counterion a c t i v i t i e s of the i o n i c polysaccharide solutions with added simple s a l t were determined using the Orion Model #94-11 sodium s e l e c t i v e s o l i d - s t a t e electrode in conjunction with a calomel electrode. Coion a c t i v i t i e s of the i o n i c polysaccharide solutions with added simple s a l t were determined using the appropriate anion select i v e s o l i d state electrode. Orion Model #97-17 chloride

American Chemicaf Society Library

1155 IGth St. N. W. W*htigtoii, D, 6. 20036

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

248

CARBOHYDRATE SULFATES

i o n e l e c t r o d e s , O r i o n Model #94-35 bromide i o n e l e c ­ t r o d e s and O r i o n Model #94-53 i o d i d e e l e c t r o d e s were used t o determine the a p p r o p r i a t e c o i o n a c t i v i t i e s . A l l the above a n i o n e l e c t r o d e s were used in c o n j u n c t i o n w i t h a standard calomel e l e c t r o d e . The e l e c t r o m o t i v e f o r c e was measured u t i l i z i n g a C o r n i n g Model 12 pH meter o r an O r i o n Model 801A pH meter w i t h an a c c u r a c y o f ± 0.1 mv. A l l measurements were c a r r i e d out in a c o n s t a n t temperature water b a t h t h e r m o s t a t e d a t 25.00 ± 0.01°C. A i r was bubbled through a l l s o l u t i o n s . A c o n s t a n t p o t e n t i a l r e a d i n g over a 20 minute p e r i o d was r e g a r d e d as a r e l i a b l e emf. Stable p o t e n t i a l s were o b t a i n e d in 2 minutes f o r d i l u t e p o l y ­ e l e c t r o l y t e s o l u t i o n s w i t h added s a l t and 10 minutes f o r the more v i s c o u s s o l u t i o n s . R e p r o d u c i b i l i t y o f ± 0.1 mv was o b t a i n e d which c o r r e s p o n d s t o an u n c e r ­ t a i n t y o f ± 0.002 a c t i v i t y c o e f f i c i e n t u n i t s . The c a l i ­ b r a t i o n o f e l e c t r o d e s was c a r r i e d out r e p e a t e d l y d u r i n g each run u s i n g s o l u t i o n s of the a p p r o p r i a t e simple s a l t w i t h o u t p o l y e l e c t r o l y t e p r e s e n t . in the c o n c e n t r a t i o n range s t u d i e d , the s l o p e s of the emf in mv v s . l o g a were found t o always g i v e c l o s e t o N e r n s t i a n b e h a v i o r . The d a t a i s r e p o r t e d as Y-j/Y^/ the r a t i o o f the e x p e r i ­ mental i o n a c t i v i t y c o e f f i c i e n t s in the p r e s e n c e o f p o l y e l e c t r o l y t e t o t h a t in the absence of p o l y e l e c t r o ­ lyte . +

R e s u l t s and

Discussion

A p r i n c i p a l aim o f t h i s study was t o examine the e f f e c t o f the charge d e n s i t y o f the c o i o n on i t s i n t e r ­ a c t i o n w i t h the p o l y i o n . Thus the s i n g l e i o n a c t i v i t y c o e f f i c i e n t s f o r C l ~ , Br~ and I ~ i o n s were determined in aqueous s o l u t i o n s c o n t a i n i n g NaCarr and NaDS a t 0.00100 M, 0.00500M and 0.0100M N a C l , NaBr and Nal a t 25°. The r e s u l t s are g i v e n in T a b l e s I and I I f o r NaCarr and NaDS, r e s p e c t i v e l y , where γ / γ i s the r a t i o of the c o i o n a c t i v i t y c o e f f i c i e n t s in the p r e s e n c e o f p o l y ­ e l e c t r o l y t e t o t h a t in the absence o f p o l y e l e c t r o l y t e and X = η / n , where n and n a r e the e q u i v a l e n t con­ c e n t r a t i o n s o f p o l y e l e c t r o l y t e and simple s a l t , r e s p e c ­ tively. The g e n e r a l t r e n d f o r the NaCarr r e s u l t s shows that γ / γ i s f a i r l y c o n s t a n t a t u n i t y f o r X < 1 and d e c r e a s e g r a d u a l l y t o f a i r l y c o n s t a n t v a l u e s f o r X > 4, w i t h the e x c e p t i o n f o r 0.0010Af NaCl and NaBr where the Y / Y r a t i o s are c o n s t a n t a t about 0.9 f o r X < 0.5 p r i o r to t h e i r gradual decrease. The γ / γ ° r a t i o s f o r 0.00100M and 0.00500M NaCl and NaBr are almost i d e n t i c a l a t c o r ­ r e s p o n d i n g simple s a l t c o n c e n t r a t i o n s and X v a l u e s , in­ d i c a t i n g t h a t the C l ~ and B r " i o n s i n t e r a c t w i t h the 2

s

2

2

p

2

s

2

2

2

1 .000 1 .000 0 .976

1 .004 1 .004

1 .00 1 . 60

0 .996

0 .949

0 .966 0 .955 0 .948 0 .948 0 .948

0 .961 0 .957 0 .938 0 .938 0 .938

1 .004 0 .985 0 .965 0 .906 0 .884

2 .00 3 .00 4 .00 6 .00 8 .00

0 .961 0 .942

-0 .981 0 .927

0 .942 0 .925

-0 .882 0 .888

0 .854 0 .822 0 .725 0 .765 0 .773

1 . 92 3 . 20 4 . 00 6 . 00 8 . 00

0. 891

0 . 868

0. 860

0. 863

0. 879

0. 913

0. 880

0. 878

0. 893

0. 863

0 .809

0 .803

0 .800

0 .765

0 .741

1 .96

3 .00

4 .00

6 .00

8 .00

0 .978

0 .981

0 .947

0 .854

1 . 60

0 . 902

0. 926

0 .831

888

1 .48

0 .

0 . 96

0. 922

0. 928

0 .

0 .98

835

1 .000

0 .966

0 .896

0 . 80

0. 958

0. 962

0 .845

1 .004 1 .009 1 .004 0 . 80

0 .70

1 .044 1 .036

1 .012

0 .52

1 .025

0 .985

0 .900

0 . 48

0. 958

0. 962

0 .883

0 .48

1 .047 1 .036 0 .965

0 .28

000

1 .028

1 .

0 .907

0 . 22

1 . 000

1 . 003

0 .894

0 .28

-

1 .012 1 .015

1 .004

0 .12

1 .040

0 .957

0 . 10

1 . 000

χ

ο

ο ο

ο

ο ο

ο ο LO ο ο ο

ο ο .Η ο ο ο

Nal

ο

1 . 003

ο ο LO ο ο

0 .914

ο

ο ο

ο ο ιΗ ο ο ο

0 .080

Ο ο LO Ο ο ο

NaBr

2

R a t i o γ / Υ 2 Dependence on X f o r

ο

ο ο fH Ο Ο

NaCl

The C o i o n A c t i v i t y C o e f f i c i e n t Sodium I o t a Carrageenan,

χ

Table I.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

CD

to

2:

ο

3

ο

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CARBOHYDRATE

250

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

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SULFATES

16.

TOMASULA E T A L .

Co-Ion

and

Counter-Ion

Interactions

251

NaCarr p o l y i o n t o t h e same e x t e n t . A l s o , f o r the 0.00100M NaCl and NaBr s o l u t i o n s , t h e Y / Y r a t i o s are c o n s i s t e n t l y lower a t each X v a l u e when compared t o t h e 0.00500M and 0.0100M s o l u t i o n s , which i s p r o b a b l y due t o l e s s s c r e e n i n g o f t h e charges on t h e p o l y i o n a t t h e lowest simple s a l t c o n c e n t r a t i o n . The Y / Y ° r a t i o s f o r the t h r e e N a l c o n c e n t r a t i o n s c l o s e l y approximate one another f o r each X v a l u e and t h e r a t i o s f o r t h e 0.0100M N a l a r e c l o s e t o t h e r a t i o s f o r O.OlOOAf NaBr and NaCl f o r each X v a l u e . A t t h e two lower s i m p l e s a l t concen­ t r a t i o n s t h e C l ~ and B r ~ i o n s i n t e r a c t w i t h NaCarr t o the same e x t e n t , w h i l e a t t h e h i g h e s t s i m p l e s a l t con­ c e n t r a t i o n t h e C l ~ , B r " and I " i o n s i n t e r a c t t o t h e same extent with t h i s p o l y e l e c t r o l y t e . Except f o r 0.00100M NaCl and NaBr, t h e γ / γ r a t i o s approximate u n i t y a t low X v a l u e s , i n d i c a t i n g t h a t t h e c o i o n i n t e r a c t s in s o l u ­ t i o n as i f t h e p o l y e l e c t r o l y t e were not present. I t s h o u l d be noted t h a t c o i o n a c t i v i t y c o e f f i c i e n t s were a l s o determined f o r NaCarr in 0.000500M simple s a l t s . They were found t o be c l o s e t o t h e v a l u e s found f o r 0.00100M s i m p l e s a l t s and w i l l be d i s c u s s e d below. The Y / y r e s u l t s f o r NaDS a r e l i s t e d in T a b l e I I f o r 0.00100M, 0.00500M and 0.0100M NaCl, NaBr and N a l . With t h e e x c e p t i o n o f 0.00100M N a l , all the Y / Y ° r a t i o s c l o s e l y approximate u n i t y f o r t h e whole X range from one to ten. S i m i l a r r e s u l t s were o b t a i n e d w i t h sodium p o l y ­ phosphate f o r a l l t h r e e simple s a l t c o n c e n t r a t i o n s (_25) . From t h e s e c o i o n a c t i v i t y c o e f f i c i e n t measurements, i t appears t h a t t h e monovalent c o i o n s i n t e r a c t w i t h t h e p o l y i o n only a t v e r y low simple s a l t c o n c e n t r a t i o n s , where t h e Debye-Huckel atmosphere i s l a r g e s t . The Manning t h e o r y f o r p o l y e l e c t r o l y t e s o l u t i o n s g i v e s f o r monovalent c o i o n a c t i v i t y c o e f f i c i e n t s (2) 2

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

2

2

2

2

2

2

γ

2

= exp[-|

Γ ' χ / ί ζ - ' Χ

+ 2)]

(2)

where o n l y Debye-Hiickel i n t e r a c t i o n s w i t h t h e p o l y e l e c ­ t r o l y t e are considered. For a given p o l y e l e c t r o l y t e ζ i s f i x e d , so eqn (2) p r e d i c t s t h a t γ d e c r e a s e s as X i n c r e a s e s u n t i l h i g h v a l u e s o f X cause γ t o l e v e l o f f . S i n c e o n l y 0.00100M NaCl and NaBr s o l u t i o n s o f NaCarr i n d i c a t e c o i o n - p o l y i o n i n t e r a c t i o n over an extended range, t h e s e r e s u l t s w i l l be used t o t e s t eqn ( 2 ) , a l o n g w i t h v a l u e s f o r 0.000500M NaCl and NaBr. F o r 0.000500M N a l c o n t a i n i n g NaCarr i t was found t h a t Y / Y approxi­ mated u n i t y over t h e whole X range. The r e s u l t s f o r 0.000500M and 0.00100M NaCl and NaBr s o l u t i o n s con­ t a i n i n g NaCarr a r e p r e s e n t e d in F i g u r e s 1 and 2, r e s p e c ­ t i v e l y , where b = 4 . 4 Â and ξ = 1.62. I t s h o u l d be noted t h a t t h e s m a l l i o n - s m a l l i o n c o r r e c t i o n o f W e l l s (26) 2

2

2

2

252

CARBOHYDRATE

need n o t be a p p l i e d because t h e r a t i o Υ / Ύ used. A l s o , t h e somewhat d i f f i c u l t e x t r a p o l a t i o n o f t h e d a t a t o z e r o i o n i c s t r e n g t h a t a g i v e n X v a l u e suggests t h a t o n l y t h e lowest s i m p l e s a l t c o n c e n t r a t i o n s be c o r r e l a t e d t o t h e t h e o r y when an i o n i c s t r e n g t h dependence i s p r e ­ sent. The agreement between Manning's p r e d i c t e d v a l u e s and e x p e r i m e n t a l v a l u e s i s q u i t e good over most o f t h e range o f X. Using c l u s t e r expansion theory f o r p o l y ­ e l e c t r o l y t e s o l u t i o n s , Iwasa and Kwak (_27) r e c e n t l y p r e ­ d i c t e d an a p p r e c i a t e d i f f e r e n c e between t h e a c t i v i t y c o e f f i c i e n t s o f c o u n t e r i o n s and c o i o n s . From t h e c o n t r i ­ b u t i o n o f h i g h e r o r d e r c l u s t e r terms t o t h e a c t i v i t y c o e f f i c i e n t o f s m a l l i o n s in p o l y e l e c t r o l y t e s o l u t i o n s , i t was shown t h a t f o r ζ > 1 c o n s i d e r a b l e asymmetry between y and γ v a l u e s , w i t h t h e v a l u e s o f y a p p r o x i ­ mating u n i t y . While t h i s t h e o r y might b e t t e r e x p l a i n our r e s u l t s f o r y , f u r t h e r e v a l u a t i o n awaits near future publications. C h l o r i d e i o n and sodium i o n s e l f - d i f f u s i o n measure­ ments were performed on aqueous s o l u t i o n s o f NaCarr ((5) and NaDS (28) in 0 . 0 0 0 5 0 M , 0 . 0 0 1 0 M , 0 . 0 0 5 0 M and 0 . 0 1 0 M NaCl. The same i o n i c p o l y s a c c h a r i d e samples were used as t h o s e employed in t h e p r e s e n t study. When compared t o t h e c o i o n d i f f u s i o n c o e f f i c i e n t s in t h e absence o f p o l y e l e c t r o l y t e D°, t h e c o i o n d i f f u s i o n c o e f f i c i e n t s D f o r C I " i o n showed t h e same t r e n d f o r NaCarr and NaDS. The D /D° r a t i o s d e c r e a s e d from u n i t y f o r X < 1 and l e v e l e d o f f at approximately # / £ ° = 0 . 9 f o r X < 1 . The p r e d i c t e d v a l u e s from Manning's t h e o r y were c l o s e l y obeyed over t h e whole X range. I t s h o u l d be noted t h a t f o r NaDS, t h e D /D° v a l u e s were below u n i t y f o r a l l NaCl c o n c e n t r a t i o n s , w h i l e t h e γ / γ ? v a l u e s were u n i t y f o r almost e v e r y c o n c e n t r a t i o n . T h i s p r o b a b l y r e f l e c t s t h e g r e a t e r s e n s i t i v i t y o f t h e d i f f u s i o n measurements in m o n i t o r i n g Debye-Huckel atmosphere i n t e r a c t i o n s as com­ pared t o a c t i v i t y c o e f f i c i e n t measurements. It i s dif­ f i c u l t t o d e t e c t t h e e f f e c t o f t h e i n t e r a c t i o n s between c o i o n s and p o l y e l e c t r o l y t e s w i t h a c t i v i t y c o e f f i c i e n t measurements because o f t h e h i g h background l e v e l due to small i o n - s m a l l i o n i n t e r a c t i o n s . Sodium i o n a c t i v i t y c o e f f i c i e n t s Y]sj + were d e t e r ­ mined in aqueous NaCarr s o l u t i o n s c o n t a i n i n g 0 . 0 0 0 5 0 0 M , 0.00100M, 0 . 0 0 5 0 0 M and 0 . 0 1 0 0 M N a C l , NaBr and N a l a t 25°C. The r e s u l t s a r e p r e s e n t e d in F i g u r e s 3 , 4 and 5 , where i t can be seen t h a t f o r each s i m p l e s a l t concen­ t r a t i o n ΎΝ3+/ΎΝΛ+ d e c r e a s e s as X i n c r e a s e s and l e v e l s o f f approximately X > 5 . While t h e e x p e r i m e n t a l p o i n t s are c l o s e in v a l u e f o r X > 2 , t h e s i m p l e s a l t c o n c e n t r a ­ t i o n dependence i s e v i d e n t f o r h i g h e r X v a l u e s , where a t a g i v e n X v a l u e Y ^ a d e c r e a s e s as t h e n o r m a l i t y o f w

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

SULFATES

l

a

s

2

2

2

2

2

2

2

2

2

2

2

a

+

TOMASULA E T A L .

Co-Ion and Counter-Ion

Interactions

m Ο.Ο005 Ν α 0.0010 0.0050 ο 0.0100

•

•

ο

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

• _ι

• 1

1

1

I

Figure 1. The dependence of the Cl' ion activity coefficient ratio on X in NaCl solutions containing NaCarr. The solid line is predicted from Man­ ning's theory.

Figure 2. The dependence of the Br~ ion activity coefficient ratio on X in NaBr solutions containing NaCarr. The solid line is predicted from Mannings theory.

• • • Ο

0.9 0\

0.71

0.0005 Ν 0.0010 0.0050 0.0100

Ο .•\· Ο v

0.5\2

3

χ

Figure S. The dependence of the Na* ion activity coefficient ratio on X in NaCl solutions containing NaCarr. The solid line is predicted from Man­ nings theory.

254

CARBOHYDRATE

SULFATES

simple s a l t decreases. A l s o note that Y + i s f a i r l y independent o f t h e nature o f t h e coion a t a l l concen­ trations. I t i s g r a t i f y i n g t h a t t h e r e s u l t s f o r Yvr + r e p o r t e d b y P a s s , P h i l l i p s a n d W e d l o c k (19) f o r 0.OU50M a n d 0.010M N a C l a r e in e x c e l l e n t a g r e e m e n t w i t h o u r s . Sodium i o n a c t i v i t y c o e f f i c i e n t s were d e t e r m i n e d f o r 0.00100M, 0.00500M a n d 0.0100M N a C l , N a B r a n d N a l s o l u t i o n s c o n t a i n i n g NaDS. The r e s u l t s a r e p r e s e n t e d in F i g u r e s 6, 7 a n d 8, w h e r e i t c a n b e n o t e d t h e e x p e r ­ i m e n t a l v a l u e s o f YNa+/Y^ + a t t h e c o r r e s p o n d i n g X values are p r a c t i c a l l y superimposable forall three simple s a l t s used. T h u s , Yvr + a p p e a r s t o b e i n d e p e n d e n t of the nature o f the monovalent coion. Also, l i t t l e i o n i c s t r e n g t h dependence i s noted a t each X v a l u e . As c o m p a r e d t o YN +/Y]sf + v a l u e s o b t a i n e d f o r N a C a r r , t h e v a l u e s a p p e a r t o b e l o w e r f o r NaDS a t c o r r e s p o n d i n g X values. T h i s i s p r o b a b l y due t o t h e h i g h e r c h a r g e d e n s i t y o f t h e NaDS c h a i n w i t h b = 2.5& a n d ξ = 2 . 8 5 . To c o r r e l a t e t h e e x p e r i m e n t a l d a t a t o t h e p r e d i c t e d v a l u e s from t h e l i n e - c h a r g e model o f Manning, t h e r e ­ s u l t s s h o u l d b e e x t r a p o l a t e d t o z e r o i o n i c s t r e n g t h . We a v o i d e d t h i s b e c a u s e t h e two l o w e s t s i m p l e s a l t c o n c e n ­ t r a t i o n s u s e d f o r N a C a r r a n d f o r NaDS s o l u t i o n s w e r e v e r y c l o s e in YNa+/Yïia+ l u e s f o r e a c h v a l u e o f X. A l s o , e x t r a p o l a t i n g t h e d a t a a t t h e s e v e r y low c o n c e n t r a t i o n s u s e d , i . E . , 0.000500M a n d 0.00100M s a l t , w o u l d give only a small correction. The t h e o r e t i c a l e q u a t i o n f o r t h e c o u n t e r i o n a c t i v i t y coefficient y f o r ξ > 1 i s (2) N a

a

a

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

a

a

a

v

a

1

Y! =

1

(ξ' * 1

+ D

(Χ + D - ' e x p C - i χ

Γ'Χ/ίΓ'Χ

+ 2)]

(3)

where ( ζ " ^ + 1)(Ζ + 1 ) ~ r e p r e s e n t s t h e f r a c t i o n o f t o t a l uncondensed c o u n t e r i o n s . When c o m p a r e d t o t h e experimental p o i n t s f o r NaCarr, the s o l i d t h e o r e t i c a l l i n e s in F i g u r e s 3, 4 a n d 5 i n d i c a t e t h a t e x c e l l e n t agreement i s o b t a i n e d f o r a l l simple s a l t c o n c e n t r a t i o n s b e l o w X = 1 a n d o v e r t h e w h o l e r a n g e o f X f o r t h e two lowest simple s a l t c o n c e n t r a t i o n s employed. Similar f i n d i n g s a r e e v i d e n t f r o m F i g u r e s 6, 7 a n d 8 f o r NaDS a t low X v a l u e s , w i t h s m a l l n e g a t i v e d e v i a t i o n s f r o m t h e theoretical values at the higher X values. W e l l s (11) r e p o r t e d t h a t t h e thermodynamic measurements o b t a i n e d f o r a q u e o u s N a C l s o l u t i o n s c o n t a i n i n g NaDS w e r e in e x ­ c e l l e n t agreement w i t h t h e t h e o r e t i c a l v a l u e s from t h e Manning t h e o r y . T h e NaDS s a m p l e h e u s e d was a l m o s t i d e n t i c a l t o ours w i t h r e s p e c t t o charge d e n s i t y and molecular weight. Upon c o m p a r i n g t h e r e p o r t e d (11) N a C l mean a c t i v i t y c o e f f i c i e n t s t o o u r e x p e r i m e n t a l v a l u e s f o r (γ Y - . _ ) ^ , we f i n d v e r y g o o d a g r e e m e n t o v e r t h e +

r

Co-Ion and Counter-Ion Interactions

TOMASULA E T A L .

Figure 4. The dependence of the Na ion activity coefficient ratio on X in NaBr solutions containing NaCarr. The solid line is predicted from Man­ ning's theory.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

+

Figure 5. The dependence of the Na ion activity coefficient ratio on X in Nal solutions containing NaCarr. The solid line is predicted from Manning's theory. +

I

.

i

l

2

!

L.._

1 . !

4

6

I

1

8

X

O0.010N NaCl • 0.005 3 0.001

-

δ

δ

Figure 6. The dependence of the Na ion activity coefficient ratio on X in NaCl solutions containing NaDS. The solid line is predicted from Manning's theory. +

I

2

1

4

6

Χ

8

10

255

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

CARBOHYDRATE SULFATES

Figure 7. The dependence of the Na ion activity coefficient ratio on X in NaBr solutions containing NaDS. The solid line is predicted from Manning's theory. +

ο0.01 ON N a l

• 0.005

-

Ο 0.001

A

C

~ °\\ 9

-

a» Figure 8. The dependence of the Na ion activity coefficient ratio on X in Nal solutions containing NaDS. The solid line is predicted from Manning's theory.

S

+

1

1

1

I

ι

2

4

6

8

10

Χ

TOMASULA E T A L .

16.

Co-Ion

and Counter-Ion

257

Interactions

same X range, g i v i n g s u b s t a n t i a t i o n t o o u r s i n g l e i o n activity results. The N a i o n s e l f - d i f f u s i o n c o e f f i c i e n t s f o r NaDS in NaCl s o l u t i o n s (2_8) f o l l o w t h e same t r e n d as does Na ^Na+ * ^ i ^ 6. The sharp i n i t i a l d e c l i n e f o l l o w e d by a l e v e l i n g o f f o f ^ + / ^ a ^ "*" ¥ 9 agreement w i t h t h e v a l u e s p r e d i c t e d from M a n n i n g s theory. This i s f u r t h e r evidence o f the v a l i d i t y o f the c o u n t e r i o n c o n d e n s a t i o n c o n c e p t and t h e i o n i c atmosp h e r e - p o l y i o n i n t e r a c t i o n c o n c e p t which a r e paramount in the t h e o r y . +

Y

+//

v

s

X

n

F

u

r

e

+

s

nv

e

r

o o d

a

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch016

1

Acknowledgement. The a s t u t e c o n t r i b u t i o n s o f Dr. M. Kowblansky a r e g r e a t l y a p p r e c i a t e d . T h i s p r o j e c t was s u p p o r t e d by Grant No. GM 21234, awarded by t h e P u b l i c H e a l t h S e r v i c e , DHEW. Abstract. C h l o r i d e , bromide, i o d i d e and sodium ion a c t i v i t y coefficients have been determined a t 25°C for aqueous s o l u t i o n s o f sodium i o t a c a r r a g e e n a n and sodium d e x t r a n s u l f a t e o v e r a l a r g e range o f i o n i c p o l y s a c ­ c h a r i d e c o n c e n t r a t i o n s f o r 0.000500M, 0.00100M, 0.00500M NaCl, NaBr and N a I . The e q u i v a l e n t c o n c e n t r a t i o n r a t i o s o f i o n i c p o l y s a c c h a r i d e t o s i m p l e salt was v a r i e d from 0.10 t o 8.0 for sodium i o t a c a r r a g e e n a n and 1.0 t o 10.0 f o r sodium d e x t r a n s u l f a t e . The e x p e r i m e n t a l r e s u l t s a r e d i s c u s s e d in l i g h t o f t h e monovalent c o i o n and mono­ v a l e n t c o u n t e r i o n i n t e r a c t i o n s w i t h t h e polyelectrolyte a c c o r d i n g t o t h e modern t h e o r y o f p o l y e l e c t r o l y t e s o l u ­ t i o n s by Manning. Literature Cited 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Manning, G. S., Annu.

Rev.

Phys.

Chem.

(1972) 23,

117. Manning, G. S., J. Chem. Phys. (1969) 51, 924, 934. Manning, G. S., J. Phys. Chem. (1975) 79, 262. Devore, D. I . and Manning, G. S., J. Phys. Chem. (1974) 78, 1242. Ross, P. D., Scruggs, R. L. and Manning, G. S., Biopolymers, in press. Kowblansky, Α., Sasso, R., Spagnuola, V. and Ander, P., Macromolecules (1977) 10, 78. Kowblansky, M. and Ander, P., J. Phys. Chem. (1976) 80, 297. D i x l e r , D. S. and Ander, P., J. Phys. Chem. (1973) 77, 2684. Menezes-Affonso, S. and Ander, P., J. Phys. Chem. (1974) 78, 1756. Magdelenat, Η., T u r q , P. and Chemla, Μ., Biopolymers

CARBOHYDRATE SULFATES

258

11. 12. 13. 14. 15.

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

(1974) 1 3 , 1 5 3 5 . W e l l s , J. D., Proc. R. Soc. London, Ser Β (1973) 183, 3 9 9 . P r e s t o n , Β . Ν., Snowden, J. McK. a n d H o u g h t o n , K.T., Biopolymers (1972) 1 1 , 1 6 4 5 . Kwak, J. C. Τ . a n d H a y e s , R. C., J. Phys. Chem. (1975) 7 9 , 2 6 5 . S z y m c z a k , J., H o l y k , P. a n d A n d e r , P., J. Phys. Chem. (1975) 19, 2 6 9 . Kwak, J. C. T. a n d J o h n s o n , A. J., Can. J. Chem. (1975) 5 3 , 7 9 2 . H o l y k , P. a n d S z y m c z a k , J., J. Phys. Chem. (1976) 80, 1 6 2 6 . Tuffile, F. M. a n d A n d e r , P., Maoromolecules (1975) 8, 7 8 9 . K o w b l a n s k y , M. a n d A n d e r , P. J. Phys. Chem., in press. P a s s , G., Phillips, G. O. a n d W e d l o c k , D. J., Macromolecules (1977) 1 0 , 1 9 7 . Kwak, J. C. T . , J. Phys. Chem. (1973) 7 7 , 2 7 9 0 . Kwak, J. C. T., O'Brien, M. C. a n d M a c l e a n , D. Α . , J. Phys. Chem. (1975) 7 9 , 2 3 8 1 . S a t a k e , I . et al., J. Polym. Sci., Phys. Ed. (1972) 10, 2 3 4 3 . N o g u c h i , H., G e k k o , K. a n d M a k i n o , S., Macromole­ cules (1973) 6, 4 3 8 . M a g d e l e n a t , Η . , Turq, P. a n d C h e m l a , Μ . , Biopoly­ mers (1976) 1 5 , 1 7 5 . K o w b l a n s k y , M. a n d A n d e r , P., t o b e published. W e l l s , J. D., Biopolymers (1973) 1 2 , 2 2 3 . I w a s a , K. a n d Kwak, J. C. T . , J. Phys. Chem. (1977) 81, 4 0 8 . G a n g i , G. a n d A n d e r , P., to b e published.

RECEIVED F e b r u a r y 6, 1 9 7 8 .

17 Interaction of Sulfated Polysaccharides with Counter-Ions G. PASS—Department of Chemistry and Applied Chemistry, University of Salford, England G. O. Phillips, and D. J. WEDLOCK—School of Natural Sciences, North Wales Institute, Clwyd, Wales, CH5 4BR, U.K.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

R. G. MORLEY—Dari-Tech Corporation, Atlanta, GA 30301

The i n t e r a c t i o n between i n o r g a n i c counterions and polyanions has been e x t e n s i v e l y s t u d i e d with particular reference to the relative order o f c a t i o n b i n d i n g s t r e n g t h s . The interaction may be t r e a t e d theoretically in terms o f condensation of the cations on to the polyanion under the i n f l u e n c e of the charge density of the polyanion and electrostatic i n t e r a c t i o n of uncondensed counter­ ions with the polyanion1,2. The t h e o r e t i c a l treatment has been a p p l i e d to the i n t e r a c t i o n of counterions with a v a r i e t y of p o l y anions3-11, i n c l u d i n g sulphated p o l y s a c c h a r i d e s , and i s here a p p l i e d to the carrageenans. The interaction o f metal ions with carrageenan is of critical importance in controlling its behaviour as stabiliser in milk products. For this reason we have here i n v e s t i g a t e d the i n t e r a c t i o n o f sodium, potassium and calcium counterions with и-, l- and λ- carrageenan. An e.m.f. method, using i o n selective e l e c t r o d e s , was utilised to yield activity c o e f f i c i e n t data, both in the presence and absence o f added simple electrolyte. We have extended the range of counterions f o r which a r e l a t i v e order o f b i n d i n g strength can be determined using a simple t e c h n i q u e l 2 , in which a c a t i o n i c dye, Methylene Blue, which undergoes a strong metachromatic s p e c t r a l s h i f t , i s d i s p l a c e d by a competing metal counterion in the form o f added simple e l e c t r o ­ l y t e . The concentration o f added simple e l e c t r o l y t e r e q u i r e d f o r complete suppression of dye b i n d i n g , and consequent r e v e r s a l of metachromasia, gives a q u a l i t a t i v e order o f c a t i o n b i n d i n g affinity—. Highly charged sulphated polyanions such as the carrageenans, with t h e i r a s s o c i a t e d counterions, cause é l e c t r o ­ s t r i c t i o n of large amounts of solvent water molecules — . The condensed counterions i n v e s t i g a t e d here, namely calcium, potassium and sodium, can e x i s t in three s t a t e s o f h y d r a t i o n ^ * 16. Changes in the s t a t e o f hydration cause volume changes. This f u l f i l l s the c o n d i t i o n s r e q u i r e d f o r a pressure change to cause p e r t u r b a t i o n of the e q u i l i b r i u m which can be monitored i n d i r e c t l y by u l t r a ­ sound attenuation measurements!?. U l t r a s o n i c waves e f f e c t the p e r t u r b a t i o n on a time s c a l e compatible w i t h the k i n e t i c s of the forward and reverse r e a c t i o n s . in the method, excess absorption 0-8412-0426-8/78/47-077-259$05.75/0 © 1978 American Chemical Society

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260

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o f a p o l y i o n s a l t compared w i t h t h e a b s o r p t i o n o f t h e e q u i v a l e n t c o n c e n t r a t i o n o f the tetramethylammonium s a l t form i s measured. T h i s b u l k y r e f e r e n c e i o n has a low a f f i n i t y f o r t h e p o l y i o n and a l l o w s , t o a r e a s o n a b l e a p p r o x i m a t i o n , a measure o f t h e e x c e s s a b s o r p t i o n , r e s u l t i n g from r e l a x a t i o n a l processes a r i s i n g from t h e c o u n t e r i o n b i n d i n g phenomenon. A b s o r p t i o n s due t o o t h e r p r o c e s s e s s u c h as p o l y m e r m o t i o n s a r e e f f e c t i v e l y c a n c e l l e d o u t . E x c e s s a b s o r p t i o n s so o b t a i n e d can be r e l a t e d t o t h e s q u a r e o f t h e volume change i n v o l e d . in a d d i t i o n t o é l e c t r o s t r i c t i o n o f w a t e r by t h e i n t e n s e e l e c t r i c f i e l d of the s t r o n g l y e l e c t r o l y t i c s u l p h a t e e s t e r groups o f c a r r a g e e n a n , h y d r a t i o n o f c a r b o h y d r a t e s — can a r i s e a l s o f r o m i n t e r a c t i o n o f s o l v e n t w a t e r w i t h t h e p o l a r -OH g r o u p s o f t h e sugar u n i t s . T h i s b e h a v i o u r c a u s e s c o n s i d e r a b l e amounts o f w a t e r t o be bound t o t h e c a r r a g e e n a n — , and i s one o f t h e m a j o r f a c t o r s i n f l u e n c i n g the rheology of carrageenan s o l u t i o n s . This i s a n o t h e r i m p o r t a n t f a c t o r in e s t a b l i s h i n g t h e e f f e c t i v e n e s s o f c a r r a g e e n a n s as s t a b i l i s e r s in f r o z e n m i l k p r o d u c t s , s i n c e t h e r e s u l t i n g reduced d i f f u s i o n u L r a t e of water prevents i c e c r y s t a l g r o w t h and changes in p r o d u c t t e x t u r e . F o r t h i s r e a s o n we have d e v i s e d a d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r i c method f o r m e a s u r i n g t h e n o n - f r e e z i n g ( i . E . bound) w a t e r in a s e r i e s o f s o l u t i o n s o f sodium s a l t s o f κ - a n d λ carrageenan. A l t h o u g h p r o t e i n - p o l y s a c c h a r i d e i n t e r a c t i o n s have g e n e r a l l y been w i d e l y s t u d i e d , m i l k p r o t e i n - s t a b i l i s e r i n t e r a c t i o n s have been n e g l e c t e d — — S u c h i n t e r a c t i o n s a l s o e x e r t a m a j o r i n f l u e n c e on the q u a l i t y o f f r o z e n d a i r y p r o d u c t s . Protein i n s t a b i l i t y , d u r i n g p r o c e s s i n g and s t o r a g e o f s u c h p r o d u c t s , i s undesirable. Whereas c a r b o x y m e t h l c e l l u l o s e , g u a r gum and l o c u s t bean gum t e n d t o c a u s e p r o t e i n d é s t a b i l i s a t i o n , c a r r a g e e n a n can p r e v e n t i t . κ - and λ - C a r r a g e e n a n s r e a c t in m i l k s y s t e m s o n l y w i t h c a s e i n p r o t e i n s and no c o m p l e x e s a r e d e t e c t a b l e in t h e whey. T h i s r e a c t i o n i s dependent upon t h e p r e s e n c e o f c a l c i u m i o n s . The e f f e c t o f s e v e r a l h y d r o c o l ^ r j i d s on t h e s t a b i l i s a t i o n o f O g - c a s e i n and c a s e i n m i c e l l e s ' — » • have been t e s t e d , f o r e x a m p l e , l o c u s t bean gum and g u a r gym o f t h e n e u t r a l h y d r o c o l l o i d s ; gum a r a b i c , C . N . C . , p e c t i n , h y a l u r o n i c a c i d and a l g i n a t e s o f t h e c a r b o x y l a t e p o l y a n i o n s and a g a r o s e , h e p a r i n , c h o n d r o i t i n s u l p h a t e s , c e l l u l o s e s u l p h a t e , f u c o i d a n and c a r r a g e e n a n o f t h e s u l p h a t e d p o l y a n i o n s . Only carrageenan i n d u c e d s i g n i f i c a n t s t a b i l i s a t i o n a g a i n s t p r e c i p i ­ t a t i o n by c a l c i u m i o n s a t pH 6 - 8 , i n d i c a t i n g i t s u n i q u e v a l u e within s t a b i l i s e r mixtures, κ - C a r r a g e e n a n was t w i c e as e f f e c t i v e as λ - c a r r a g e e n a n and c o m p l e t e s t a b i l i s a t i o n was e f f e c t e d a t a r a t i o o f 5:1 c a s e i n : κ - c a r r a g e e n a n and 2*5:1 c a s e i n : λ carrageenan. The complex f o r m e d between c x ^ c a s e i n and κ c a r r a g e e n a n was t h e more s t a b l e and r e s i s t e d t h e d e s r u p t i v e i n f l u e n c e o f c a l c i u m i o n s and h e a t t o a g r e a t e r e x t e n t t h a n t h e c o r r e s p o n d i n g a - c a s e i n / κ - c a s e i n complex. A v a r i e t y of t e c h n i q u e s have been used t o s t u d y s u c h m i l k p r o t e i n - p o l y a n i o n i n t e r a c t i o n s , f o r example l i g h t s c a t t e r i n g — ' p o l y a c r y l a m i d e g e l 2 0

2 4 ,

-

s

17.

PASS E T A L .

Sulfated

Polysaccharides

and

Counter-Ions

261

27 99 e l e c t r o p h o r e s i s — , and T h e o l o g i c a l t e c h n i q u e s ^ . Here we d e s c r i b e a new method u s i n g p u l s e r a d i o l y s i s t o s t u d y t h e i n t e r ­ a c t i o n between α ^ c a s e i n and c a r r a g e e n a n in t h e p r e s e n c e o f calcium ions. The method was p r e v i o u s l y used in o u r l a b o r a t o r y to study o t h e r polyanion - c a t i o n i n t e r a c t i o n s ! ^ . Results

and D i s c u s s i o n .

Measurements w i t h I o n S e l e c t i v e E l e c t r o d e s . V a l u e s o f a , where a i s t h e c a t i o n i c a c t i v i t y o f a p o l y e l e c t r o l y t e o r p o l y e l e c t r o l y t e / s i m p l e s a l t s o l u t i o n were o b t a i n e d u s i n g c a l i b r a t i n g s o l u t i o n s o f t h e a p p r o p r i a t e c h l o r i d e s a l t and s i n g l e c a t i o n i c a c t i v i t y values f o r the c h l o r i d e s a l t s , c a l c u l a t e d from the e x t e n d e d D e b y e - H u c k e l e q u a t i o n f o r aqueous s o l u t i o n s a t 2 5 ° C , w i t h the i o n s i z e parameters o f K i e l l a n d — . Together w i t h values o f m , t h e m o l a l c o n c e n t r a t i o n in e q u i v a l e n t s o f c o u n t e r i o n / k g , t h e s i n g l e i o n a c t i v i t y c o e f f i c i e n t s a r e g i v e n by t h e u s u a l r e l a t i o n s h i p , eq ( 1 ) . +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

+

+

- - S Î (1). + m F o r a l l c a r r a g e e n a n t y p e s , we f i n d ( F i g s . 1, 2) t h a t s i n g l e i o n a c t i v i t y c o e f f i c i e n t s o f t h e m o n o v a l e n t i o n s a l t s in p u r e p o l y e l e c t r o l y t e s o l u t i o n are g r e a t e r f o r N a than f o r K , which i s in a c c o r d w i t h p r e v i o u s measurements on s u l p h a t e d p o l y Y

+

+

+

+

s a c ch a r i de s ÇL-LLZS. .

The a c t i v i t y c o e f f i c i e n t s i n c r e a s e s l i g h t l y w i t h d i l u t i o n f o r κ - carrageenans, but are r e l a t i v e l y constant with d i l u t i o n f o r i - and λ - c a r r a g e e n a n s in m o n o v a l e n t i o n f o r m s . The a c t i v i t y c o e f f i c i e n t v a l u e s a t t h e most d i l u t e c o n c e n t r a t i o n can be r e l a t e d t o t h e l i n e a r c h a r g e p a r a m e t e r g, ( F i g . 3 ] , c a l c u l a t e d from e q ( 2 ) , f o r monovalent-monovalent i o n i n t e r a c t i o n s . 2 ekTb

with e water, k = Boltzmann's constant, Τ = absolute temperature, b = a v e r a g e d i s t a n c e between c h a r g e s . A v a l u e f o r b was o b t a i n e d f r o m measurements o f m o l e c u l a r models in t h e i r most e x t e n d e d f o r m , g i v i n g a mean v a l u e o f 0-445nm f o r t h e l e n g t h o f a s u g a r u n i t in a c a r r a g e e n a n m o l e c u l e , and t a k i n g i n t o a c c o u n t t h e degree o f s u b s t i t u t i o n o f t h e sample. The t h e o r e t i c a l l i m i t i n g v a l u e o f t h e a c t i v i t y c o e f f i c i e n t i s g i v e n by t h e s o l i d l i n e when a p p l y i n g M a n n i n g ' s c o n d e n s a t i o n t r e a t m e n t — as g i v e n b y . e q . (3) f o r u n i v a l e n t c a t i o n s , Q

γ

+

=

BxpC-i3 |z|F,

f q 1

where | z | i s t h e m a g n i t u d e o f t h e c h a r g e on t h e c o u n t e r i o n . O u r r e s u l t s p r o v i d e b e t t e r agreement f o r p o t a s s i u m s a l t s o f t h e carrageenans w i t h the t h e o r e t i c a l l i m i t i n g a c t i v i t y c o e f f i c i e n t , than w i t h t h e sodium s a l t forms. T h i s w o u l d be a n t i c i p a t e d s i n c e t h e n a t u r e o f t h e s e l e c t i v e i n t e r a c t i o n o f t h e p o t a s s i u m and

262

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

0-65V

Figure 1.

Activity coefficient of sodium counterions vs. con­ centration for κ, i, and λ-carrageenan

0-50

Figure 2.

Activity coefficient of potassium counterion vs. con­ centration for κ-, t.-, and λ-carrageenan

17.

PASS E T A L .

Sulfated

Polysaccharides

and

263

Counter-Ions

sodium c o u n t e r i o n s i s e l e c t r o s t a t i c i o n b i n d i n g in a m a i n l y h y d r a t e d i o n f o r m . The s m a l l e r h y d r a t e d c a t i o n ^ J L K , s h o u l d be more s t r o n g l y bound t h a n N a , and s h o u l d c o i n c i d e more n e a r l y w i t h M a n n i n g ' s l i m i t i n g v a l u e o f γ , as i t a p p r o a c h e s t h e p o i n t charge a p p r o x i m a t i o n o f t h i s t h e o r y . The r e l a t i v e c o n s t a n c y o f a c t i v i t y c o e f f i c i e n t s as a f u n c t i o n o f c o n c e n t r a t i o n i n d i c a t e s that these polyanions approximate t o a r i g i d l i n e charge, not c o i l e d , w i t h i n t h e c o n c e n t r a t i o n range i n v e s t i g a t e d . The a c t i v i t y c o e f f i c i e n t s o f t h e c a l c i u m s a l t s in K - a h d X c a r r a g e e n a n a r e shown as a f u n c t i o n o f c o n c e n t r a t i o n in F i g . 4 . A g a i n t h e λ f o r m shows a g r e a t e r c o n s t a n c y w i t h c o n c e n t r a t i o n than t h e κ - form s u p p o r t i n g t h e view t h a t t h i s f r a c t i o n w i t h i t s h i g h e r l i n e a r c h a r g e d e n s i t y , i s more r o d - l i k e . Comparing t h e a c t i v i t y c o e f f i c i e n t s o f t h e N a and C a s a l t s o f κ - and λ - carrageenan ( F i g . 5 ) o v e r a range o f c o n c e n t r a t i o n s , i n d i c a t e s t h a t t h e y a r e in t h e r a t i o o f c a . 2 : 1 , w h i c h i s in good agreement w i t h t h e r e q u i r e m e n t s o f eq ( 3 ) . C a l c i u m and s o d i u m were t h e i o n s used f o r t h e c o m p a r i s o n s i n c e t h e y have a l m o s t t h e same +

+

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+

+

2 +

on

hydrated i o n size^2. I t i s observed t h a t both κ - a n d λ carrageenans e x e r t a c o n s i d e r a b l e c a l c i u m i o n s e q u e s t e r i n g e f f e c t in s o l u t i o n . The l o w e r a c t i v i t y c o e f f i c i e n t f o r t h e K s a l t o f each + c a r r a g e e n a n compared t o t h e Na s a l t i n d i c a t e s e l e c t r o s t a t i c b i n d i n g o f t h e h y d r a t e d i o n , when t h e s m a l l e s t h y d r a t e d i o n w o u l d be most s t r o n g l y b o u n d . T h i s i s in a c c o r d w i t h t h e view t h a t t h e i n t e r a c t i o n o f t h e s u l p h a t e e s t e r group w i t h t h e c a t i o n i s l e s s than t h e i n t e r a c t i o n o f w a t e r o f h y d r a t i o n w i t h t h e c a t i o n — . Consequently i t i s e n e r g e t i c a l l y unfavourable f o r t h e s u l p h a t e e s t e r group t o d i s p l a c e w a t e r f r o m a r o u n d a s o d i u m o r p o t a s s i u m c a t i o n . The r e s u l t s o f o u r u l t r a s o n i c r e l a x a t i o n studies also support t h i s conclusion. Thus t h e s m a l l e s t h y d r a t e d c a t i o n w i l l b i n d most s t r o n g l y , f o r c a t i o n s o f t h e same c h a r g e . The r e v e r s e s i t u a t i o n w o u l d a p p l y w i t h c a r b o x y l i c and p h o s p h a t e d p o l y a n i o n s , w h i c h w o u l d a c c o u n t f o r t h e now w e l l e s t a b l i s h e d r e v e r s a l o f a f f i n i t y sequence when c o m p a r i n g s u l p h a t e w i t h p h o s p h a t e and c a r b o x y l a t e polyanions™ S a t a k e e t a i e - and N o g u c h i e t air?-, o b s e r v e d t h e same t r e n d w i t h s o d i u m and p o t a s s i u m d e x t r a n s u l p h a t e s . We now c o n s i d e r p o l y s l e c t r o l y t e s w i t h added s i m p l e e l e c t r o l ­ ytes. Manning— d e r i v e d an e q u a t i o n f o r s i n g l e c o u n t e r i o n a c t i v i t y c o e f f i c i e n t s o f u n i v a l e n t i o n s in a p o l y e l e c t r o l y t e p l u s added s a l t system f o r ξ > 1 , e q . ( 4 ) . Y = ( Ε X + D (X + D " * exp (4) Where X = n / n i s t h e r a t i o o f t h e number o f e q u i v a l e n t s o f p o l y ­ e l e c t r o l y t e c o u n t e r i o n t o t h e number o f e q u i v a l e n t s o f s i m p l e e l e c t r o l y t e c o u n t e r i o n in u n i t volume o f s o l u t i o n . F i g . 6 shows f o r L - c a r r a g e e n a n t h a t s o l u t i o n s most d i l u t e in s i m p l e s a l t (5 X 10 m), where m i s t h e m o l a l c o n c e n t r a t i o n in eq / k g , g i v e t h e b e s t c o r r e s p o n d e n c e between t h e o r y and p r a c t i c e . Similar +

- 1

+

e

s

264

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

I Q

Figure 3.

05

Activity coefficients of counterions in most dilute solution vs. linear charge parameter

Κ)

K5 concentration

Figure 4.

20 eq/kgxIO

25

2

Activity coefficient of calcium counterion vs. concentration for calcium salts of κ- and λ-carrageenan

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

Figure 5. Activity coefficients of sodium counterions vs. ac­ tivity coefficients of calcium counterions at identical concen­ trations for κ- and k-carrageenans

266

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

ΙΌ,

0·2

Γ

>·θ" Ο

• 2

4

•

•

•

•

6

8

ΙΟ

12

• χ

14

Figure 6. Activity coefficient of common cation in icarrageenan/simple electrolyte mixture, uncorrected for small ion—small ion interactions. Comparison with theo­ retical curve according to Equation 3. NaCl: (#)5 X 10- m; (θ) 1 χ 10' m. KCl; (A) 5 X 10r*m; 1 X 10- m;(O)5 X 10~ m. 3

2

2

2

17.

Sulfated Polysaccharides and Counter-Ions

PASS E T A L .

267

r e s u l t s were o b t a i n e d f o r κ - and λ - c a r r a g e e n a n — . The c o r r e s ­ pondence becomes l e s s s a t i s f a c t o r y as t h e c o n c e n t r a t i o n o f s i m p l e salt increases. T h i s c o u l d be due t o t h e n e g l e c t o f m u t u a l i n t e r ­ a c t i o n s between i o n s o f t h e s i m p l e e l e c t r o l y t e w h i c h i s i n h e r e n t in M a n n i n g ' s a s s u m p t i o n s — . An e m p i r i c a l c o r r e c t i o n o f mean a c t i v i t y c o e f f i c i e n t s has been p r o p o s e d in w h i c h t h e mean a c t i v i t y c o e f f i c i e n t o f t h e i o n s in t h e p u r e s i m p l e s a l t s o l u t i o n i s t a k e n as a measure o f t h e i r n u t u a l i n t e r a c t i o n , t h e t h e p r e s e n c e o f p o l y e l e c t r o l y t e — . I f a s i m i l a r c o r r e c t i o n i s a p p l i e d to the s i n g l e i o n a c t i v i t y c o e f f i c i e n t t h i s may be w r i t t e n in t h e f o r m , eq ( 5 ) .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

1n γ

+

= 1n γ

ρ +

+ 1n y

(5)

C +

where γ i s t h e s i n g l e i o n a c t i v i t y c o e f f i c i e n t o f t h e c a t i o n determined e x p e r i m e n t a l l y , γ i s the s i n g l e i o n a c t i v i t y c o e f f i c i e n t c a l c u l a t e d a c c o r d i n g to Manning, y ° i s the s i n g l e i o n a c t i v i t y c o e f f i c i e n t o f t h e c a t i o n in t h e absence o f p o l y ­ e l e c t r o l y t e and i t s c o u n t e r i o n s . A t low c o n c e n t r a t i o n s o f s i m p l e e l e c t r o l y t e , γ °__^1 and γ = γ in agreement w i t h [ F i g . 6 ) . The c o r r e c t e d a c t i v i t y c o e f f i c i e n t s a r e shown in ( F i g . 7 ] , and p r o v i d e much b e t t e r agreement w i t h t h e v a l u e s c a l c u l a t e d f r o m M a n n i n g ' s theory. A s e l e c t i v e i o n b i n d i n g p r o c e s s i s s t i l l o b s e r v e d , even when t h e r e i s c o n s i d e r a b l e e x c e s s o f s i m p l e e l e c t r o l y t e . This i s f u r t h e r borne o u t by t h e r e s u l t s o b t a i n e d u s i n g t h e M e t h y l e n e B l u e (MB ] dye d i s p l a c e m e n t t e c h n i q u e — . I t was f o u n d t h a t t h e l i m i t i n g s a l t c o n c e n t r a t i o n (LSC) r e q u i r e d t o remove c o m p l e t e l y t h e MB f r o m 2/1 s i t e / dye r a t i o p o l y a n i o n - d y e complexes d e c r e a s e s in t h e o r d e r L i : > N a > K > - C s and Mg>*Ca:>Sr f o r λ - and κ - c a r r a g e e n a n s , where a p p r o p r i a t e , ( T a b l e I ) . For κ - carragee­ nan, n e i t h e r E.m.P. n o r dye d i s p l a c e m e n t measurements were c a r r i e d o u t in t h e p r e s e n c e o f p o t a s s i u m o r c a e s i u m c h l o r i d e b e c a u s e o f t h e t e n d e n c y o f κ - c a r r a g e e n a n t o g e l in t h e p r e s e n c e o f t h e s e e l e c t r o l y t e ' s . Our r e s u l t s , t h e r e f o r e , a g a i n s u p p o r t t h e view t h a t t h e s t r e n g t h o f i o n b i n d i n g i s g r e a t e s t f o r t h e s m a l l e s t h y d r a t e d i o n . The s t r e n g t h o f b i n d i n g i s , f o r b o t h c a r r a g e e n a n f r a c t i o n s , in t h e o r d e r Sr>Ca*>-Mg and C s > R> N a > L i , and s e l e c t i v e i o n b i n d i n g i n t e r a c t i o n s a r e s t i l l m a n i f e s t in systems o f high i o n i c s t r e n g t h . T h i s o b s e r v a t i o n c o u l d have b i o l o g i c a l s i g n i f i c a n c e , p o i n t i n g to i o n i c i n t e r a c t i o n s of g l y c o s a m i n o g l y c a n s o c c u r r i n g in t h e c o n n e c t i v e t i s s u e m a t r i x . Measurements were c a r r i e d o u t u s i n g a c a l c i u m e l e c t r o d e on calcium s a l t s of λ - a n d κ c a r r a g e e n a n in t h e p r e s e n c e o f c a l c i u m c h l o r i d e . Here a u s e f u l p a r a m e t e r i s t h e e m p i r i c a l r u l e o f a d d i t i v i t y o f a c t i v i t i e s — . in summary t h i s s t a t e s t h a t a c o u n t e r i o n a c t i v i t y o f a pure p o l y e l e c t r o l y t e s o l u t i o n , a P , can be added t o a c o u n t e r i o n a c t i v i t y in a s i m p l e s a l t s o l u t i o n , a to give a c a l c u l a t e d a c t i v i t y f o r the mixture a ^ not s i g n i f i c a n t l y d i f f e r e n t in many i n s t a n c e s f r o m t h e o b s e r v e d v a l u e a f o r t h e m i x t u r e . D e v i a t i o n s f r o m t h i s r u l e can be e x p r e s s e d +

ρ

+

+

ρ

+

+

+

+

+

s

ca

+

+

o

+

b

s

C,

carrageenan

2

LSC χ 1 0 " Γ 1

2

LSC χ 1 0 ~ N

Kappa c a r r a g e e n a n

2

LCS χ 1 0 " Γ 1

2

LSC χ 1 0 ~ Ι Ί

Lambda

b

Sr 8.13

14.45 Ca 13.18

Ng 19.95

6.03

8.75

11.50

24.40

Sr

Ca

Ng

Na

8.32

12.20

18.20

Li

Κ

Na

Li

E f f e c t o f i n o r g a n i c i o n s on c a r r a g e e n a n - N e t h y l e n e B l u e i n t e r a c t i o n s . S i t e / D y e r a t i o 2/1. Dye 1 χ 1 0 " n ^

TABLE 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

17.

PASS E T A L .

Aa+

Sulfated

= a„

c

a

l

c

Polysaccharides

-a+ a obs

o b s

X

and

Counter-Ions

269

100

+

A l t h o u g h t h e r u l e i s o n l y e m p i r i c a l , many i n s t a n c e s o f v e r y good agreement have been shown i n c l u d i n g m o n o v a l e n t i o n c a r r a g e e n a n s a l t s p l u s monovalent i o n c h l o r i d e s — . F o r sodium dextran s u l p h a t e and added s o d i u m c h l o r i d e , a l l d e v i a t i o n were w i t h i n 1.5%^. However f o r o u r measurements o f c a l c i u m s a l t s o f λ - and κ - c a r r a g e e n a n p l u s c a l c i u m c h l o r i d e , Aa+ v a l u e s a r e s i g n i f i c a n t ­ ly g r e a t e r [Table I I ) . F o r κ - c a r r a g e e n a n , in d i l u t e s i m p l e s a l t s o l u t i o n , v a l u e s o f A a a r e 26%, g r e a t e r t h a n o b s e r v e d v a l u e s of A a f o r λ - c a r r a g e e n a n . T h i s does n o t c o n f o r m t o t h e g e n e r a l r u l e t h a t a d d i t i v i t y i s more c l o s e l y obeyed by p o l y i o n s w i t h lower degrees o f s u b s t i t u t i o n i . E . κ - carrageenan s h o u l d g i v e b e t t e r agreement. I t i s therefore indicated that extra binding o f c a l c i u m i o n s o c c u r s w i t h κ - c a r r a g e e n a n and p r o b a b l y a s t o i c h i o m e t r y o f one c a l c i u m i o n p e r e s t e r s u l p h a t e u n i t i s b e i n g approached. 32 Lyons a n d K o t i n — o b s e r v e d s i m i l a r b e h a v i o u r w i t h t h e magnesium s a l t s o f DNA w i t h added magnesium c h l o r i d e s , where A a v a l u e s up t o c a . 30% were o b s e r v e d when t h e s i m p l e s a l t was p r e s e n t in low c o n c e n t r a t i o n . I t i s p o s s i b l e t h a t c a l c i u m i o n s m i g h t be b i n d i n g t h u s : +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

+

+

0S0

3

•SA

Ca

+

C1~

C a C1~ + Ca C1 +

3

GSA

3

Here c a l c i u m i o n s a r e r e p r e s e n t e d as s i t e b o u n d , w i t h t h e r e s u l t t h a t t h e c h a r g e on t h e c a r r a g e e n a n i s r e v e r s e d . Viewed f r o m t h e s o l v e n t t h e segment a p p e a r s t o be a p o l y c a t i o n w i t h negative univalent counterions. Such c h a r g e r e v e r s a l phenonenon a r e c o m p a r a t i v e l y common and have been i n v o l v e d t o a c c o u n t f o r t h e r e v e r s a l in t h e s i g n o f e l e c t r o p h o r e t i c m o b i l i t y o f p o l y v i n y l p y r d i n i u m b r o m i d e s in e x c e s s K B r 2 4 . • u r r e s u l t s i n d i c a t e t h a t c a l c i u m κ - c a r r a g e e n a n has a g r e a t e r t e n d a n c y t o undergo s i t e b i n d i n g t h a n c a l c i u m λ - c a r r a g e e ­ nan. These r e s u l t s a r e in agreement w i t h t h e work o f H a n s e n — ' 2 1 ' .25. who f o u n d t h a t t h e s t a b i l i s a t i o n o f 0 5 - c a s e i n by c a r r a g e e ­ nan a g a i n s t p r e c i p i t a t i o n o f c a l c i u m i o n s was g r e a t e r w i t h κ carrageenan than w i t h λ - c a r r a g e e n a n . The i n t e r a c t i o n between c a r r a g e e n a n a n d t h e c a s e i n p r o t e i n s was shown t o be dependent in some measure on t h e p r e s e n c e o f c a l c i u m i o n s , in as much as t h e complex f o r m e d i s a c a l c i u m / c a r r a g e e n a n / otg- c a s e i n c o m p l e x . I f in t h e p r e s e n c e o f e x c e s s c a l c i u m i o n s t h e κ - f r a c t i o n shows an enhanced a b i l i t y t o complex c a l c i u m i o n s f r o m s o l u t i o n , t h e n i t would a i d f o r m a t i o n o f t h e p r o t e i n - c a r r a g e e n a n complex. M o r e o v e r , o u r u l t r a s o n i c r e l a x a t i o n s t u d i e s show an anomalous b e h a v i o u r f o r t h e r e l a t i v e volume changes in c a l c i u m κ - and λ -

χ

10

2

χ

0.025 0.055 0.104 0.143 0.176

Ρ a+ 10

2

χ

0.09 8 0.259 0.512 0.765 0.985

m+

p

,n2 10

χ

0.040 0.087 0.152 0.205 0.258

Ρ a+ n

, 2 10

Cb) C o u n t e r i o n a c t i v i t i e s

0-106 0-252 0.519 0.782 1.023

Ρ m+

in

s

in χ

χ

0.500 0.500 0.500 0.500 0.500

s m+

Ca κ -

0.500 0.500 0.500 0.500 0.500

m+

7

o χ

0.375 0.374 0.374 0.374 0.374

a+

10

?

10

2

χ

0.374 0.374 0.374 0.374 0.374

s a+ 10

2

carrageenan - C a C l

10

2

obsd 0.386 0.406 0.427 0.455 0.482

3

systems

ao b s d 0.384 0.399 0.440 0.469 0.504

systems

2

f o r polyanion + s a l t

II

Ca λ - c a r r a g e e n a n - Ca C l

of calcium ion a c t i v i t i e s

(a3 C o u n t e r i o n a c t i v i t i e s

Additivity

TABLE

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

17.

Sulfated

PASS E T A L .

carrageenan b i n d i n g in carrageenan radiolysis

Polysaccharides

and

Counter-Ions

271

i o n b i n d i n g p r o c e s s e s , b u t can be e x p l a i n e d by s i t e calcium κ - carrageenan. A unique c a l c i u m / κ / a c a s e i n i n t e r a c t i o n was a l s o d e t e c t e d by p u l s e s u p p o r t i n g t h e view advanced h e r e . g

U l t r a s o n i c R e l a x a t i o n M e a s u r e m e n t s . Zana and T o n d r e — ' — have g i v e n t h e t h e o r y and a s s u m p t i o n s u n d e r l y i n g t h i s t e c h n i q u e . We have measured t h e q u a n t i t y Δ α / f as a f u n c t i o n o f f w h e r e , f u l t r a s o n i c - f r e q u e n c y and Δα= u l t r a s o u n d a b s o r p t i o n c o e f f i c i e n t o f p o l y a n i o n s a l t minus t h e u l t r a s o u n d a b s o r p t i o n c o e f f i c i e n t o f an e q u i v a l e n t c o n c e n t r a t i o n o f t h e t e t r a m e t h y l a m m o n i u m CTMA) s a l t form. We a s c r i b e any e x c e s s a b s o r p t i o n o f a p o l y i o n s a l t o v e r i t s TMA s a l t f o r m t o a r e l a x a t i o n o r r e l a x a t i o n s a r i s i n g f r o m t h e c o u n t e r i o n b i n d i n g phenonenon ( F i g s . 8 , ^ 9 ) • From a c o m p a r i s o n o f p l o t s o f p r o p o r t i o n a l v a l u e s o f Δτχ/f v f f o r m o n o v a l e n t and d i v a l e n t i o n s a l t s o f p o l y p h o s p h a t e and c a r b o x y l i c p o l y a n i o n s and c a r r a g e e n a n s , i t i s e v i d e n t t h a t t h e c a r r a g e e n a n s show t h e weakest r e l a x a t i o n s . T h i s a f e a t u r e o f s u l p h a t e d p o l y a n i o n s , f o r e x a m p l e , s o d i u m p o l y ( e t h y l e n e s u l p h o n a t e ) 15. The low f r e q u e n c y a b s o r p t i o n s , b e l o w 4MHz a r e p r o p o r t i o n a l t o c o n c e n t r a t i o n ( F i g s . 10, 1 1 ) . T h i s i s i n d i c a t i o n o f t h e r e l a x a t i o n a r i s i n g by monom o l e c u l a r p r o c e s s e s 1 5 . The low f r e q u e n c y a b s o r p t i o n can be a s c r i b e d t o a r e l a x a t i o n o f the o u t e r s p h e r e / i n n e r sphere equilibrium. Any r e l a x a t i o n can be c h a r a c t e r i s e d by a r e l a x a t i o n a m p l i t u d e , A , and a r e l a x a t i o n f r e q u e n c y , P. As a r e s u l t o f p r a c t i c a l d i f f i c u l t i e s in m e a s u r i n g v a l u e s o f Δ α / f below 1MHz, a c t u a l v a l u e s o f t h e r e l a x a t i o n a m p l i t u d e c a n n o t be c a l c u l a t e d . However, g e n e r a l l y , v a l u e s o f t h e r e l a x a t i o n a l f r e q u e n c y f o r i n n e r s p h e r e complex f o r m a t i o n do n o t show marked dependence on t h e n a t u r e o f t h e c o u n t e r i o n f o r c o u n t e r i o n s o f t h e same v a l e n c y and a l s o show s m a l l e r d i f f e r e n c e s t h a n a n t i c i p a t e d between mono­ v a l e n t and d i v a l e n t c o u n t e r i o n s . The v a l u e s o f Δ α / f a t any p a r t i c u l a r f r e q u e n c y t h e r e f o r e , s h o u l d be in p r o p o r t i o n t o t h e r e l a x a t i o n a m p l i t u d e , g i v e n by e q . ( 6 ) .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

2

2

2

A = AVo

f ( k „ , k ., c. ) . (6) 1 -1 ι Where AVo f o r t h e c o u n t e r i o n b i n d i n g p r o c e s s i s e s s e n t i a l l y due t o t h e volume change f r o m r e l e a s e o f e l e c t r o s t r i c t e d w a t e r by t h e p o l y i o n and w a t e r o f h y d r a t i o n by t h e c o u n t e r i o n . The r e l a x a t i o n t i m e (τ) c h a r a c t e r i s e s t h e p r o c e s s and f ( k ^ , k_^, c-^), i s a f u n c t i o n o f t h e f o r w a r d and r e v e r s e r a t e c o n s t a n t s f o r t h e e q u i l i b r i u m , and t h e c o n c e n t r a t i o n c o f t h e p a r t i c i p a t i n g s p e c i e s . From F i g s . 8, 9 i t can be seen t h a t t h e t o t a l volume change on c o u n t e r i o n b i n d i n g t o b o t h λ - a n d κ - c a r r a g e e n a n , i s in t h e o r d e r C a > k > N a . V a l u e s o f Δ α / f f o r κ - c a r r a g e e n a n in t h e c a l c i u m and p o t a s s i u m s a l t forms a r e g r e a t e r than t h o s e f o r λ - carrageenan a t t h e same c o n c e n t r a t i o n s o f c o u n t e r i o n . I t was f o u n d f o r p o l y ( s t y r e n e s u l p h o n a t e ) (PSS) t h a t v a l u e s o f Δα/f were l e s s t h a n 2

τ

i

2

272

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

CARBOHYDRATE SULFATES

Figure 8.

Plots of ultrasonic absorption Δ α / f for 0.04N aqueous solutions of calcium (Q), potassium and sodium (Φ) salts of κ-carrageenan 2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

PASS E T A L .

Sulfated

Polysaccharides

and

Counter-Ions

frequency M H z Figure 9. Plots of ultrasonic absorption Δ α / Ρ for 0.04N aqueous solutions of calcium (Q), potassium (Θ), and sodium (%) salts of λ-carrageenan

I Ο

2·0

3-0

4 0

equiv/litre χΙΟ'

Figure 10. Variation of ultrasonic absorption Δ α / Ρ at par­ ticular frequencies for sodium salts of κ-carrageenan, as a function of polyanion concentration

274

CARBOHYDRATE SULFATES

f o r p o l y ( e t h y l e n e s u l p h o n a t e ) (PES) — . While the l i n e a r charge p a r a m e t e r o f t h e s e two p o l y i o n s i s v i r t u a l l y t h e same, t h e m i n i ­ mum d i s t a n c e between two c h a r g e s (d) on t h e r o d - l i k e model o f these molecules i s d i f f e r e n t : dpcrg^dp^g. F o r s a m p l e s o f sod^jjm c a r b o x y m e t h y l c e l l u l o s e o f d i f f e r e n t d e g r e e s o f s u b s t i t u t i o n — , Δ α / f i n c r e a s e d w i t h i n c r e a s e in d e g r e e o f s u b s t i t u t i o n , and so r e l a t e s t o t h e d e c r e a s e in minimum d i s t a n c e between c h a r g e d s i t e s . Since the expected t r e n d Δ α / f κ - c a r r a g e e n a n
Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

2

Pulse

2

radiolysis

measurements.

F o r w h o l e a c i d c a s e i n and c a r r a g e e n a n a t pH6.6 a n o n - a d d i t i v e r e l a t i o n s h i p i s o b s e r v e d f o r k^ v a l u e s , ( f i r s t o r d e r r a t e c o n s t a n t s f o r t h e d i s a p p e a r a n c e o f t h e h y d r a t e d e l e c t r o n ) in c a r r a g e e n a n and c a s e i n s o l u t i o n s , compared w i t h m i x t u r e s w i t h i d e n t i c a l ^ g o n c e n trations (k/j° ). Interaction i s , therefore, i n d i c a t e d — . A t y p i c a l e x p e r i m e n t i s shown in F i g . 1 2 . A f t e r repeated e x p e r i m e n t s u s i n g a r a n g e o f s y s t e m s E.g. p u r i f i e d κ - c a r r a g e e n a n , p u r i f i e d λ - c a r r a g e e n a n and G e l c a r i n HMR ( A l g i n C o r p o r a t i o n o f A m e r i c a ) , i t was f o u n d t h a t non r e p r o d u c i b i l i t y was a c o n s t a n t feature. This v a r i a b i l i t y in v a l u e s c o u l d be overcome i f t h e b s

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

Figure 11. Variation of ultrasonic absorption Δ α / f at particular frequencies for calcium salts of κ-carrageenan, as a function of concentration 2

fzasein]- 0 0 3 %

k, = I - 6 2 X I 0

5

b a c 801

Figure 12.

TO I

60I

SOI

4CM

1

3CH

20-I

ΙΟΊ |.-| hlO caseimiccarrageenan ratio

Plot of first-order rate constant for reaction of the hydrated electron with the casein: κ-carrageenan complexes

276

CARBOHYDRATE SULFATES

deoxygenation o f t h e s o l u t i o n was c a r e f u l l y s t a n d a r d i s e d . This mechanical p e r t u r b a t i o n i n f l u e n c e d the p o l y m e r i c systems. Fig.13. shows s i m i l a r b e h a v i o u r o b t a i n e d f o r w h o l e a c i d c a s e i n in t h e p r e s e n c e o f CMC. C u r v e Ca) k/|Obs f o r m i x t u r e , c u r v e (b) = k>| p r o t e i n +k^ p o l y s a c c h a r i d e , and c u r v e ( c ) =k>| p o l y s a c c h a r i d e , applied throughout. A l i n e a r i n c r e a s e in r e a c t i v i t y o f c a s e i n t o w a r d s e ~ w i t h i n c r e a s i n g s o d i u m c h l o r i d e c o n c e n t r a t i o n was f o u n d , b u t s o d i u m c h l o r i d e d i d not i n f l u e n c e the r e a c t i v i t y of the p o l y a n i o n s . The i n t e r a c t i o n s o f c a r r a g e e n a n - c a s e i n and CMC - c a s e i n were u n a f f e c t e d by Ι Ο ' ^ Μ N a C 1 . Two t y p e s o f i n t e r a c t i o n can be i d e n t i f i e d f o l l o w i n g the argon b u b b l i n g d e a e r a t i o n . 'Type A ' o c c u r s a t p r o t e i n : p o l y a n i o n r a t i o s o f 80 : 1 t o 1 : 1, l e a d i n g t o a d e c r e a s e in t h e r e a c t i v i t y o f t h e m i x t u r e t o w a r d s e~ [i.E. k
Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

q

o b s

r o î : e i n

1

a n i o n

1

obs> i rotein 1polyanion) I n i t i a l l y , b a s e d on p r e v i o u s e x p e r i e n c e — ' ~ , ' T y p e A ' i n t e r a c t i o n was c o n c l u d e d t o be e l e c t r o s t a t i c in n a t u r e , due t o i n t e r a c t i o n between n e g a t i v e s i t e s on t h e p o l y a n i o n and p o s i t i v e c e n t r e s on t h e p r o t e i n . ' T y p e B ' was a t t r i b u t e d t o a s t r u c t u r a l change in t h e p r o t e i n , m a k i n g a v a i l a b l e more p o s i t i v e sites for e~ a t t a c k as a r e s u l t o f i n t e r a c t i o n w i t h t h e p o l y ­ anion. L a t e r o u r e x p e r i e n c e i n d i c a t e d t h a t a t pH 6 . 6 , where b o t h p o l y a n i o n and p r o t e i n b e a r an o v e r a l l n e g a t i v e c h a r g e ( a l t h o u g h p o s i t i v e s i t e s a r e s t i l l a v a i l a b l e on t h e p r o t e i n ) e l e c t r o s t a t i c i n t e r a c t i o n s o f w h o l e c a s e i n and ' c a s e i n w i t h c a r r a g e e n a n and CMC do n o t o c c u r . I t i s a l s o d o u b t f u l w h e t h e r s t r u c t u r a l changes in t h e p r o t e i n a r e r e s p o n s i b l e f o r t h e ' T y p e B ' i n t e r a c t i o n . It i s more p r o b a b l e t h a t t h e d e c r e a s e in t h e r e a c t i v i t y o f w h o l e c a s e i n and a ^ c a s e i n t o w a r d s e^q w i t h p r o g r e s s i v e a r g o n b u b b l i n g i s due t o a s u r f a c e phenonmenon. When c a s e i n - c a r r a g e e n a n and c a s e i n - C M C i n t e r a c t i o n s were s t u d i e d using the s t a n d a r d i s e d ' s y r i n g e t e c h n i q u e ' f o r deoxygena­ t i o n — , ' T y p e B' i n t e r a c t i o n s were n o t f o u n d . Type ' A ' i n t e r ­ a c t i o n s o c c u r r e d a t a l l r a t i o s o f p r o t e i n : p o l y a n i o n between 40 : 1 and 1 : 10. The i n t e r a c t i o n was u n a f f e c t e d by 1 0 " M Na C1 i n d i c a t i n g t h a t ' T y p e A ' i n t e r a c t i o n s a r e n o t e l e c t r o s t a t i c in n a t u r e , w h i c h i s a d d i t i o n a l l y s u p p o r t e d by o u r o b s e r v a t i o n t h a t ' T y p e A ' i n t e r a c t i o n s a r e p r e s e n t in s u c c i n y l c a s e i n and CMC m i x t u r e s . We a r e n o t a b l e , t h e r e f o r e , t o d e m o n s t r a t e by t h i s t e c h n i q u e t h a t i n t e r a t i o n between p o s i t i v e s i t e s on t h e p r o t e i n and n e g a t i v e s i t e s on t h e c a r r a g e e n a n o c c u r s . 'Type A ' i n t e r a c t i o n s are observed at a l l c o n c e n t r a t i o n r a t i o s investigated even in c a s e i n g u a r gum m i x t u r e s ( F i g . 14) when components a r e not s i g n i f i c a n t l y c h a r g e d . We n e v e r t h e l e s s f i n d t h a t t h e c t ^ c a s e i n - c a r r a g e e n a n i n t e r ­ a c t i o n i s u n i q u e ( F i g . 15) in t h a t k/j obs ~ k/j c a s e i n + k/j K

K

+

K

P

q

s

1

Sulfated

Polysaccharides

and

Counter-Ions

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

PASS E T A L .

Figure 14. Plot of first-order rate constant for reaction of the hydrated electron with casein: guar gum complexes

277

278

CARBOHYDRATE SULFATES

c a r r a g e e n a n a t t h e p r o t e i n : p o l y a n i o n r a t i o o f 1 : 5. in a d d i t i o n t h e a d m i x t u r e o f 10 -"-ΓΠ C a C l 2 i n d u c e s an i n c r e a s e in r e a c t i v i t y s i m i l a r t o t h e ' T y p e B' i n t e r a c t i o n s o b s e r v e d in o u r i n i t i a l e x p e r i m e n t s , a l t h o u g h k/| o b s j ^ k ^ a ^ c a s e i n + k/j carrageenan. L i n and Hansen2JL have s u g g e s t e d t h a t in t h e presence o f Ca i o n s , a ^ c a s e i n and c a r r a g e e n a n f o r m m i c e l l e s in a s i m i l a r manner t o cig' c a s e i n and κ- c a s e i n . in t h e w h o l e c a s e i n - CMC i n t e r a c t i o n a t pH 6 • 6 i t was shown t h a t c a l c i u m i o n b r i d g e s are not found s i n c e a 'Type A' i n t e r a c t i o n i s observed b o t h in t h e p r e s e n c e and a b s e n c e o f ICT^N C a C ^ T h e r e f o r e we c o n c l u d e t h a t p o l y s a c c h a r i d e s g e n e r a l l y a f f e c t t h e r e a c t i v i t y o f m i l k p r o t e i n s t o w a r d s e ^ q , b u t no e l e c t r o s t a t i c i n t e r a c t i o n can be d e m o n s t r a t e d u s i n g p u l s e r a d i o l y s i s . Since hydrophobic bonding i s u n l i k e l y a s a t i s f a c t o r y e x p l a n a t i o n f o r t h e s e e f f e c t s must be a w a i t e d . The t h e r m o d y n a m i c i n c o m p a t i b i l i t y o f h i g h m o l e c u l a r w e i g h t s p e c i e s s u c h as p o l y a n i o n s and p r o t e i n s i s a w e l l e s t a b l i s h e d phenomenon—. A p a r t i a l cause o f t h i s i s ' v o l y m e e x c l u s i o n ' — w h i c h r e n d e r s t h e s y s t e m l e s s s t a b l e so t h a t i t can be t o t a l l y d e s t a b i l i s e d , r e s u l t i n g in phase s e p a r a t i o n , by s m a l l changes in e n v i r o n m e n t . The p o l y s a c c h a r i d e in s o l u t i o n i s in a h i g h l y hydrated s t a t e . Fig.16 shows t h e r e s u l t s o b t a i n e d w i t h s o d i u m s a l t s o f λ and κc a r r a g e e n a n f o r ' n o n f r e e z i n g ' w a t e r , some­ t i m e s t e r m e d 'bround w a t e r ' , u s i n g d i f f e r e n t i a l s c a n n i n g c a l o r i metry t o d e t e r m i n e t h e e n t h a l p y o f f u s i o n o f t h e c a r r a g e e n a n s o l u t i o n s — . Thus t h e p o l y s a c c h a r i d e o c c u p i e s a c e r t a i n , q u i t e c o n s i d e r a b l e , volume in t h e s o l u t i o n f r o m w h i c h t h e p r o t e i n i s excluded. The e f f e c t i s i n d é p e n d a n t o f pH, i o n i c s t r e n g t h and i s o e l e c t r i c p o i n t of the p r o t e i n , but i n c r e a s e s w i t h the concent r a t i o n o f p o l y s a c c h a r i d e and t h e s i z e o f t h e p r o t e i n . As t h e volume e x c l u d e d i n c r e a s e s , t h e a c t i v i t y o f t h e p r o t e i n s o l u t i o n i n c r e a s e s and t h e p r o t e i n a p p r o a c h e s i t s s o l u b i l i t y l i m i t . Any e f f e c t w h i c h l o w e r s t h e s o l u b i l i t y f u r t h e r , E.g. h e a t i n g o r t h e p r e s e n c e o f c e r t a i n i o n s w i l l t h e n push t h e p r o t e i n o v e r i t s s o l u b i l i t y l i m i t r e s u l t i n g in phase s e p a r a t i o n . I t i s n o t p o s s i b l e t o e x p l a i n o u r r e s u l t s c o m p l e t e l y in t e r m s o f volume e x c l u s i o n , a l t h o u g h i t does u n d o u b t e d l y c o n t r i b u t e t o t h e s e complex i n t e r a c t i o n s w h i c h e x e r t s u c h a v i t a l c o n t r o l l i n g i n f l u e n c e on t h e q u a l i t y o f m i l k p r o d u c t s d u r i n g p r o d u c t i o n and storage. —

+

s

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

1

Experimental E.m.P. p r o c e d u r e s t o g i v e c a t i o n i c b i n d i n g d a t a have been reported p r e v i o u s l y — . An E.I.L. model 1048-4 s o d i u m i o n s e l e c t i v e e l e c t r o d e , a model 1057-2 p o t a s s i u m i o n s e l e c t i v e e l e c t r o d e and on O r i o n 9 2 - 2 0 c a l c i u m i o n s e l e c t i v e e l e c t r o d e were used w i t h an E.I.L. model 7050 mV/pH m e t e r . The dye d i s p l a c e m e n t t e c h n i q u e ! 2 . ' .12. and t h e d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r i c method f o r m e a s u r i n g w a t e r b i n d i n g ^ - h a v e a l s o been d e s c r i b e d .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

17.

PASS E T A L .

Sulfated

Polysaccharides

and Counter-Ions

ι30=1

20I IO--I hi <x casein: k* c a r r a g e e n a n ratio ( ^ w

31

w t

hlO )

Figure 15. Plot of firsUorder rate constant for reaction of the hydrated electron with a l casein: κ-carrageenan complexes s

0·5

IO

1-5

Figure 16. Plot of grams of unfrozen water/gram of carrageenan as a function of weight percentage of carrageenan in solution

279

280

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

U l t r a s o n i c a b s o r p t i o n was measured u s i n g t h e r e s o n a n c e i n t e r f e r o m e t r i c technique of Eggers—. The e l e c t r o n i c s y s t e m was a m o d i f i e d v e r s i o n o f t h e s t a n d a r d sweep l e v e l m e a s u r i n g s e t o f Wandel and G o l t e r m a n n ^ - . P u l s e r a d i o l y s i s was c a r r i e d o u t a t t h e C h r i s t i e H o s p i t a l and H o l t Radium I n s t i t u t e , M a n c h e s t e r 2 0 , U . K . A p u l s e d l i n e a r a c c e l e r a t o r was used t o d e l i v e r y a 50n s e c p u l s e o f a p p r o x i m a t e l y 10NeV e l e c t r o n s t o t h e r a d i a t i o n c e l l . A s p e c i a l deaeration p r o c e d u r e was used t o p r e v e n t s u r f a c e d e n a t u r a t i o n o f p r o t e i n w h i c h can c a u s e e r r o r i n t h e r e s u l t s — . Samples o f λ , l and κ - c a r r a g e e n a n were s u p p l i e d by Copenhagen P e c t i n f a b r i k , Κ - c a r r a g e e n a n used i n t h e p u l s e r a d i o l y s i s e x p e r i m e n t s , was a c o m m e r c i a l sample [ G e l c a r i n HNR) s u p p l i e d by D a r i Tech C o r p , A t l a n t a , G a . We a c k n o w l e d g e t h e a s s i s t a n c e o f D r . E . Wyn-Jones p r o v i d i n g f a c i l i t i e s and i n t e r p r e t i n g t h e u l t r a s o n i c relaxation data.

for

ABSTRACT

Our results show that the interaction of simple ions with the carrageenans can be explained i n terms of electrostatic binding for λ - carrageenan, but some site binding i s evident for и- carrageenan, p a r t i c u l a r l y the calcium salt of и- carrageenan. Whereas there are indications from pulse radiolysis measurements that there i s an interaction between the casein proteins and carrageenans, with a unique interaction between αs casein and carrageenan, there i s no evidence to suggest that these inter­ actions are electrostatic in nature. Volume exclusion provides a p a r t i a l explanation. 1

"Literature Cited" 1. Lifson, S., Katchalsky, Α., J.Poly.Sci. (1954), 13, 43. 2. Manning, G.S., J.Chem.Phys. (1969), 51, 924. 3. Podlas, T . J . , Ander, P., Macromolecules, (1970), 3, 154. 4. Rinaudo Μ., Milas Μ., J.Poly.Sci., Poly.Chem.Ed. (1974), 12, 2073. 5. Podlas T . J . , Ander P., Macromolecules (1969), 2, 432. 6. Satake I . , Fukuda M., J.Poly.Sci., Poly.Phys.Ed. (1972) 10, 2343. 7. Noguchi H., Gekko Κ., Makino S., Macromolecules (1973), 6, 438. 8. Oman S., Die Makromol.Chem. (1974), 175, 2133. 9. Wells J.D., Proc.R.Soc.Lond.B. (1973), 183, 399. 10. Preston B.N., Snowden J.McK., Houghton K.T., Biolpolymers (1972), 11, 1645. 11. Kwak J.C.T., O'Brien M.C., Maclean P.Α., J.Phys.Chem. (1975), 79, 2381.

17.

12. 13. 14. 15. 16. 17. 18.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch017

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

PASS E T A L .

Sulfated

Polysaccharides

and

Counter-Ions

281

Pass G . , Phillips G.O., Wedlock D.J., J.Chem.Soc. (Perkin II), (In p r e s s ) . Jooyandeh F., Moore J.S., Phillips G.O. J. Chem.Soc.(Perkin II) (1974), 1468. Biswas A.B., Kumsah C . A . , Pass G., Phillips G.O., J . S o l u t i o n Chem. (1974), 5, 581. Zana R . , Tondre C. Biophys.Chem. (1974), 1, 367. C. Tondre, R. Zana, J.Phys.Chem. (1971), 75, 3367. Stuehr J., Yeager E., ' P h y s i c a l A c o u s t i c s ' V o l . II, 351, Academic P r e s s , New York, (1965). Kumsah C . , Pass G . , Phillips G . O . , J.Solution Chem. (1976), 5, 799. Shipe W . F . , Roberts W . M . , Blanton L.F., J. Diary Sci. (1973), 46, 169. L i n C.F., Hansen P . M . T . Macromolecules (1970), 3, 269. Franz G.J., U . S . Patent No. 3,407,076 (1968). Asano Y . , I n t e r n a t i o n a l Dairy Congress P r o c . 17th Munich, (1966), 5, 695. Asano Y . , A g r . B i o l . C h e m . (1970), 34, 102. Hansen, P . M . T . , J.Dairy.Sci., (1968), 51, 192. L i n C.F., Hansen P.M.T. J . D a i r y Sci. (1968), 51, 945. Payens T.A.J., J.Dairy Sci. (1972), 55, 141. Grindrod J., Nicherson T.A., J . D a i r y Sci. (1967), 50, 948. K e i l l a n d J., J.Amer.Chem.Soc. (1937), 59, 1675. Pass, G., Phillips G.O., Wedlock D.J., Macromolecules (1977), 10, 197. Bungenburg de Jong, H . G . , 'Colloid Science', Vol.II Elsevier, Amsterdam, (1949). W e l l s , J.D., Biopolymers (1973), 12, 223. Lyons J . W . , K o t i n L., J.Amer.Chem.Soc. (1965), 87, 1670. Podlas T.J., Ander P., Macromolecules (1969), 2, 432. Strauss U . P . , G e r s h f e l f M . L . , S p i e r a Η . , J.Amer.Chem.Soc. (1954), 76, 5909. Zana R . , Tondre C . , Rinando M., Milas M., J.Chim.Phys. P h y s i c o l c h i m . B i o l (1971), 68, 1258. McKinnon Α.Α., Rees D . A . , Williamson F.B., Chem.Comm.(1969), 701. Arnott S . , Scott W . E . , J.Mol.Biol. (1974), 90, 253. Phillips G.O., Power D . M . , Robinson C . , Davies J.V., Biochim. Biophys.Acta (1971), 215, 491. Armand G., PhD T h e s i s , U n i v e r s i t y of S a l f o r d , U . K . (1972). Robinson C., PhD T h e s i s , U n i v e r s i t y of S a l f o r d , U . K . (1971). Morley R . G . , PhD T h e s i s , U n i v e r s i t y of S a l f o r d , U . K . (1972). Koningsveld R., A d v a n . C o l l o i d . I n t e r f a c e . S c i . (1968), 2, 151. Laurent T . C . , 'The Chemical Physiology of Mucopolysaccharides,' Proc.Symp.Milan p.153, Little and Brown, Boston (1968). Eggers F . , A c o u s t i c s (1968), 19, 323. P e t h r i c k R . A . , Wyn-Jones E., U l t r a s o n i c s (1972), 10, 228.

RECEIVED February 6, 1978.

INDEX

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

A Absorption spectral shifts of acridine orange .... 68 spectroscopy 67 ultrasonic 272-275 Acceptor specificity of some crude preparations of mucopolysaccharide sulfotransferases 124 Acetabuhria 203 6-O-Acetylamylose, preparation of 110 Acid alginic 225 amount of chlorosulfonic 181 influence of the water content of the starch on the 183 L-ascorbate, properties of monosulfate esters of 11 L-ascorbic C chemical shifts of 10 in concentrated sulfuric acid 4, 5 4-d 16 monosulfate esters of 1 in sulfuric acid 8 chlorosulfonic 173 guluronic 228 hyaluronic 88 hydrolysis, partial 218 mannuronic 228 residues, periodate, cleavage of D-glucuronic 101 O-sulfates, isomeric chlorogenic 26 and sulfomethyl-containing graft co-polymers of xanthan gum, sulfonic 193 Acridine orange absorption spectral shifts of 68 complexes, critical electrolyte concentrations of polyanion68, 76 complexes, effect of p H on the stoichiometry of polyanion70 effect of protein and lipid on the titration of 79 fluorescence shifts of 68 titration of carrageenan vs 69 Acrosiphonia 207 Acrylamide and 2-acrylamido-2methylpropane sulfonic acid 195 1 3

Acrylamide graft co-polymer, results of sulfomethylation of xanthan gum201 2-Acrylamido-2-methylpropane sulfonic acid, acrylamide and 195 Activation effect of residual water after 153 methods, other 153 of polysaccharides 158 Agarose 217 IR spectra of sulfated 156,157 Alcohol boil-off, aqueous 158 Alcohol soak, aqueous 159 Alditol components from heparin 102 Algae extracellular polysaccharide from microscopic red 221 structural features and biochemical implications, sulfated fucosecontaining polysaccharides from brown 225 unicellular green 203 Algin 149 sulfates 168 Alginic acid 225 Alkali, action of 215 Alkali, cleavage by 208 Allyl ethers 50 2-Amino-2-deoxyamylose, preparation of 108 Amino glycan sulfotransferases of hen oviduct and of chick embryo cartilage, sulfate transfer to different acceptors by the 125 Ammonium sulfate, yield of 181 Amylose, aminated 108 Amylose into a 6-carboxyl, 2-sulfoamino analog, conversion of 106 Analytical and property determinations 171 3,6-Anhydrogalactose 211 3,6-Anhydrosugar 213 Animal tissues 123 Anticoagulant activity 96 Arabinans 207 Ascophyllan 226,236 structural features of 234 Ascophyllum nodosum 226,229-231

285

CARBOHYDRATE SULFATES

286

Carbohydrate chains of hog stomach blood group sulfated gylcoproteins 35 Carbohydrate derivatives, benzylated 50 Carboxyl to sulfate groups present in the same polyanion, estimation of 78 6-Carboxyl, 2-sulfoamino analog, conversion of amylose into a 106 Carrageenan(s) 217,218 vs. acridine orange, titration of 69 activity coefficient of calcium counter-ion vs. calcium salts of 264 analysis of the 247 -casein interactions 276 doping of babyfood with 79 in milk products, estimation of 78 potassium counter-ions vs 262 samples, equivalent weights of 88 /simple electrolyte mixture 266, 272 Β sodium counter-ion vs .262 standards 82 Babyfood with carrageenan, doping of 79 sulfate ester groups of 260 Bases, effect of 154 synthetic 149 Binding /x-Carrageenan effects of simple electrolytes on calcium salts of 272-275 PA-AO 71,72 complexes, « 1 casein279 with p H , variation of polyanion-AO 73 complexes, hydrated electron with with p H , variation of polyanionthe casein275 collagen 86 potassium salts of 272, 273 profiles, time-dependence of polysodium salts of . 272, 273 anion-polyanion interaction on 83 strengths of polyanions 75 Casein -/x-carrageenan complexes, « 1 279 Biochemical implications, sulfated -ti-carrageenan complexes, hydrated fucose-containing polysaccharides electron with the 275 from brown algae: structural -carrageenan interactions 276 features and 225 - C M C complexes 277 Biochemical role 238 -guar gum complexes 277 Biogenesis 143 Caulerpa polysaccharide 203 Biological 238 activities of the polymers 115 Cell, rhizoid implications 210 Cell walls from P. vesiculosus, poly­ saccharide composition of isolated 242 systems, spectrofluorimetric Cellulose 149, 225 methods for estimating and acetate electrophoresis 231-236 studying the interactions of polysulfatase 129,134 polysaccharides in 67 141 Birefringence 241 Cerebroside sulfatase Cerebroside, sulfate ester of a 44 Borohydride-reduced heparin, tetra­ Chaetomorpha polysaccharides 203 saccharide fragment from perioCharge parameter, activity coefficients date-oxidized 102 of counter-ions vs. linear 264 Bovine aorta, chondrosulfatase of 138 131,132 Br" ion activity coefficient ratio 253 Charonia lampas Bromides, polyvinylpyridinium 269 Chick embryo cartilage, sulfate trans­ fer to different acceptors by the C. amino glycan sulfotransferases of hen oviduct and of 125 Calcium 259 Chitosan 103 counter-ion vs. calcium salts of biological properties ofC.-6oxidized carrageenan, activity coefficient M-sulfation 106 of 264 oxidation of 104 salts of /x-carrageenan 272-275 sulfation of 104 Capparaceae 21

Ascophyllum, structural features of the galactose-rich polymer from .. 239 L-Ascorbate acid, properties of monosulfate esters of 11 L-Ascorbate 2-sulfate 1 chronology of 2 synthesis of 4 L-Ascorbate, 5-sulfate 7,15 synthesis of 13 L-Ascorbate 6-sulfate 3,14 chronology of 6 L-Ascorbic acid C chemical shifts of 10 in concentrated sulfuric acid 4, 5 4-d 16 in deuterium oxide, 4-deuterio5 monosulfate esters of 1 in sulfuric acid 8

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

1 3

s

s

287

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

INDEX Chloride(s) ion activity coefficient ratio 253 ion self-diffusion measurements 252 magnesium 269 sodium 269 Chlorogenic acid O-sulfates, isomeric 26 Chlorophyceae (green) 225 Chlorosulfonic acid 173 amount of 181 influence of the water content of the starch on the 183 -formamide reagent, reaction of starch with the 173 -formamide system 175 method, pyridine149 Choline sulfate 24 Chondroitin sulfate 88 Chondrosulfatase 129,136 of bovine aorta 138 of "Patella vulgata 137 of Proteus vulgaris 136 of squid liver 138 Chondrus crispus 215 Chromatography, gel permeation 180 Chromatography, ion-exchange column 9 Cladophora polysaccharides 203 Cladophora rupestris 208-212 linkages present in 207 polysaccharide 207 structural features in 205 Cleavage by alkali 208 Cleavage of D-glucuronic acid resi­ dues, periodate 101 C M C - c a s e i n complexes 277 C M C - c a s e i n interactions 276 Codiolum ..... 210 Codium 210 fragile 208 Coefficient s) activity 265,266,272 of calcium counter-ion vs. calcium salts of carrageenan 264 of counter-ions vs. linear charge parameter 264 of determination 175 ratio Br" ion activity 253 Cl~ ion activity 253 N a ion activity 253-256 Co-ion and counter-ion interactions with sulfonated polysaccharides .. 245 Collagen binding with p H , variation of polyanion86 Collagen complexes, critical electro­ lyte concentrations of some poly­ anion88 Colonic goblet cell glycoprotein, oligo­ saccharide structure of desialated 35 +

Concentration on the amount of form­ amide, influence of the starch .... 182 Concentration of the starch in form­ amide on esterification 178 Connective tissues 29 Conversion of amylose into a 6-carboxyl, sulfoamino analog 106 Co-polymer, results of sulfomethylation of xanthan gum-acrylamide graft 201 Co-polymerization 115 Co-polymers of xanthan gum, sulfonic acid and sulfomethyl-containing graft 193 Counter-ion (s) vs. calcium salts of carrageenan, activity coefficient of calcium .. 264 vs. carrageenan, potassium 262 vs. carrageenan, sodium 262 interaction of sulfated polysac­ charides with 259 interactions with sulfonated poly­ saccharides, co-ion and 245 vs. linear charge parameter, activity coefficients of 264 Cruciferae 21 Cyclization, intramolecular 21

D Dairy products, procedure for the analysis of pigmented polyanionstabilized 81 Debye-Huckel theory 245 Desmarestia aculeata 227 Desulfation, enzymatic 129 4-Deuterio-L-ascorbic acid in deuterium oxide 5 Dextran sulfate 247 Diffusion measurements 252 Disaccharides from carboxyl-reduced heparin 99 Dissolution/reprecipitation 158 method 153 D M F , aqueous 159 D M F : S 0 reagent, analysis of 159 Dye release profiles 85 Stern-Volmer plots of 90, 91 Dyebinding, p H dependence of 74 8

Ε Electrodes, measurements with ion selective 261 Electrolyte (s) concentrations for polyanion-acridine orange complexes 68, 76 concentrations of some polyanioncollagen complexes 88

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

288 Electrolyte(s) (continued) mixture, carrageenan/simple 266 on P A - A O binding, effects of simple 72 on polyanion-AO binding, effect of simple 71 Electromotive force measurements .... 247 Electron with the casein-/x-carrageenan complexes, hydrated 275 Electrophoresis, polyacrylamide gel 260,261 Embryo, two-celled 238 Embryos, stages in the development of Fucus 240 Enteromorpha compressa 209 Enteromorpha-suliated polysaccharide 209 Enzymatic formation and hydrolysis of polysaccharide sulfates 121 Epithelial-sulfated glycoproteins, composition of 32, 33 Equations of regression, analysis and interpretation of 176 Equivalent weights of carrageenan samples 88 Ester(s) of L-ascorbate acid, properties of monosulfate 11 of L-ascorbic acid, monosulfate 1 of a cerebroside, sulfate 44 groups of carrageenan, sulfate 260 groups as potential information regulators in glycoproteins, sulfate 29 heparin methacrylate 113 hydrolysis of mixed cellulose nitrite sulfate 164 nitrite 163 nitrogen content of the 181 Esterification 176 influence of the concentration of the starch in formamide on 178 influence of the water content of the starch on 178 Ethanol-insoluble fraction 236 Ethanol-soluble fraction 236 Ethers, allyl 50

F Fibroblast growth, effect of sulfated glycopeptides on 3T3 Fisher test Flavones Flavonols Fluorescence emission spectroscopy .. Fluorescence shifts of acridine orange Formaldehyde

41 176 25 25 67 68 195

Formamide amount of 181 influence of the starch concentration on the 182 -chlorosulfonic acid reagent, reaction of starch with the 173 -chlorosulfonic acid system 175 on esterification, influence of the concentration of the starch in .. 178 Fourier transform 5 Fucan complex, structural features of a 237 Fucose-containing polysaccharides from brown algae: structural features and biochemical implications, sulfated 225 Fucus 225 embryos, stages in the development of 240 vesiculosus 226,230-232 polysaccharide composition of isolated cell walls from 242 zygotes 241 zygotes 238 Funoran 156 IR spectra of 157

G Galactans 213 Galactose 214 -rich polymer from Ascophyllum, structural features of 239 6-sulfate 211 Gel(s) 219 electrophoresis, polyacrylamide 260 permeation chromatography 180 Gigartina 215 stellata 218 Gloiopeltis furcata 156 Glucosinolates and other naturally occuring O-sulfates 19 D-Glucuronic acid residues, periodate cleavage of 101 Glucuronoxylorhamnans 207 linkages present in 207 Glycolipids ("sulfoglycolipids"), studies on the synthesis of sulfurcontaining 44 Glycopeptides composition of pig duodenal 37-39 in 3T3 fibroblast growth, effect of sulfated 41 by ion exchange, fractionation of duodenal 39 Glycoprotein s) carbohydrate chains of hog stomach blood group sulfated 35

289

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

INDEX Glycoprotein (s) (continued) composition of epithelial-sulfated .32,33 oligosaccharide structure of desialated colonie goblet cell 35 sulfate ester groups as potential informational regulators in 29 sulfated 31 Glycosaminoglycans composition of 30 obtained by chemical modification of polysaccharides, sulfated .... 95 sulfated 29 Glycosulfatase (s) 129,130 assay of 131 Littorina littorea 132 Patella vulgata 133 properties of 132 Pseudomonas carrageenovora 133 Trichonderma viride 133 Goblet cell gylcoprotein, oligosac­ charide structure of desialated colonic 35 Gracilaria 215 Guar, effect of time at different tem­ peratures in the sulfation of 151 Guar gum 195 complexes, casein277 Guluronic acid 228 G u m complexes, casein-guar 277

H Halobacterium cutirubrum 46 Heat labilities of two different sulfo­ transferase activities 127 Helical structure 220 H e n oviduct isthmus 131 specificity of a sulfotransferase from 128 sulfate transfer to different accept­ ors by the amino glycan sulfo­ transferases of chick embryo cartilage and of 125 Heparin 77,95 alditol components from 102 degrading multienzyme system 140 derivatives of high molecular weight 113 disaccharides from carboxylreduced 99 methacrylate ester 113 methacrylate, polymer of 116 residues present in 99 segment, methacry lated 114 a suggested tetrasaccharide repeat­ ing unit 101 by sulfation of polysaccharides, synthetic 149

Heparin (continued) tetrasaccharide fragment from periodate-oxidized, borohydridereduced Hog stomach blood group sulfated glycoproteins, carbohydrate chains of Huckel-Debye theory Hunter's syndrome Hyaluronic acid Hydrolysis of cellulose nitrite of mixed cellulose nitrite sulfate ester partial acid of polysaccharide sulfates, enzy­ matic formation and the site of sulfate from partial Hydroxylamine-O-sulfate moiety

102

35 245 142 88 164 164 218 121 210 20

I Ice cream stabilizers Intramolecular cyclization Iodide quenching of dye, SternVolmer plots for the Ion(s) effect of metallic -exchange column chromatography exchange, fractionation of duodenal glycopeptides by selective electrodes, measurements with IR spectra of agarose vs. degree of substitution, viscosity and of funoran of sulfated agarose IR spectroscopy Isothiocyanates

77 21 90 221 9 39 261 157 166 157 157 218 21

Κ Keratosulfatase

139

L Lactosyl ceramide Leukodystrophy, metachromatic Light scattering Lipid on the titration of acridine orange, effect of protein and Littorina littorea glycosulfatase Lossen rearrangement

44 44 260 79 130 132 22

290

CARBOHYDRATE SULFATES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

M Magnesium chlorides 269 Magnesium salts 269 Manning model 246 Mannuronic acid 228 Marine Chlorophyceae, sulfated poly­ saccharides metabolized by the .. 203 Merosinigrin 22 Metachromatic leukodystrophy (MLD) 44,142 Methacrylate, polymer of heparin 116 Methacrylated heparin segment 114 Methacry lation and polymerization .... 113 Methanol 150 Method, dissolution/precipitation 153 Methylation 218 results, the site of sulfate from 209 Milk products, estimation of carra­ geenan in 78 Milk and toothpaste tests, cold mix chocolate 160 Milkshake test 160 Model, Manning 246 Molecular weight heparin derivatives of 113 portion, high 179 and viscosity 97 Monosulfate esters of L-ascorbate acid, properties of 11 Monosulfate esters of L-ascorbic acid 1 Moringaceae 21 Mucopolysaccharide sulfotransferases, acceptor specificity of some crude preparations of 124 Mycobacterium tuberculosis 46

Ν N a ion activity coefficient ratio 253-256 Neber-type reaction 23 Neocarrabiose 4-O-sulfate 133 Nernstian behavior 248 Nitriles 21 Nitrite, cellulose 163 formation of 164 hydrolysis of 164 sodium cellulose sulfate via 163 sulfate ester, hydrolysis of mixed .... 164 sulfation of 164 Nitrite esters 163 Nitrogen content of the esters 181 +

Ο Oligosaccharide structure of desialated colonic goblet cell glyco­ protein

35

Oligosaccharide synthesis to the syn­ thesis of sulfoglycolipids, applica­ tion of a general method of 48 Organic sulfates of plant origin, other 24 Ostwald viscometer flow times 89 Oxidation procedure for 6-O-tritylamylose alternative 107 Oxidation studies, the site of sulfate from periodate 208

Ρ Padina pavonia 227 Patella vulgata, chondrosulfatase of .. 137 Patella vulgata, glycosulfatase 133 Periodate oxidation studies, the site of sulfate from 208 Periodate-oxidized, borohydridereduced heparin, tetrasaccharide fragment from 102 Persicarin 25 pH dependence of dyebinding 74 titrations of varying 77 variation of polyanion-AO binding with 73 variation of polyanion-collagen binding with 86 for xanthan gum, viscosity vs 198 Phaeophyceae (brown) 225 Phenylglycine 23 Pig duodenal glycopeptides, compo­ sition of 37-39 Pigmented polyanion-stabilized dairy products, procedure for the anal­ ysis of 81 Plant origin, other organic sulfates of 24 sulfonolipid, studies towards the synthesis of the 60 tissues 123 Polyacrylamide gel electrophoresis ... 260 Poly (acrylamide-co-AMPS A ) , xanthan g u m 200 Polyanion(s) -acridine orange complexes, critical electrolyte concentrations for .68,76 -acridine orange complexes, effect of p H on the stoichiometry of .. 70 - A O binding, effect of simple elec­ trolytes on 71 - A O binding with p H , variation of .. 73 binding strengths of 75 -collagen binding with p H , varia­ tion of 86 -collagen complexes, critical elec­ trolyte concentrations of some .. 88 concentration 69

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

INDEX

Polyanion (s) (continued) -containing solutions, procedure for the analysis of estimation of carboxyl to sulfate groups present in the same interaction of protein and -polyanion interaction on binding .. profiles, time-dependence of .... and polycations, interaction of -stabilized dairy products, procedure for the analysis of pigmented Polycarboxylates, analysis of polysulfatesinthe presence of Polycations, interaction of polyanions and Polyelectrolyte complexes, investigation of the structure of model Polymer(s) from Ascophyllum, structural features of the galactose-rich biological activities of the of heparin methacrylate matrix Polymerization, methacrylation and .. Polymerization reaction scheme Polysaccharide (s) activation of in biological systems, spectrofluorimetric methods for estimating and studying the interactions of from brown algae: structural features and biochemical implications, sulfated fucosecontaining Caulerpa Chaetomorpha Cladophora rupestris

co-ion and counter-ion interactions with sulfonated composition of isolated cell walls from P. vesiculosus composition and structure with counter-ions, interaction of sulfated Enteromorpha sulfated interactions, proteinionic metabolized by the marine Chlorophyceae, sulfated from microscopic red algae, extracellular novel methods and resultsinthe sulfation of of the Rhodophyceae, sulfated seaweeds metabolizing

291

80 78 82 83 83 92 81 70 92 246 87 239 115 116 225 113 196 158 67

Polysaccharide (s) (continued) structural work of 143 sulfatases 134 sulfated glycosaminoglycans obtained by chemical modification of 95 sulfates 168 enzymatic formation and hydrolysis of 121 sulfation of 159 synthetic heparin by sulfation of .... 149 Polysulfatase, cellulose 129,134 Polysulfates in the presence of polycarboxylates, analysis of 70 Polyvinylpyridinium bromides 269 Porphyran sulfatase 135 Potassium 259 counter-ions vs. carrageenan 262 salts of /A-carrageenan 272, 273 Products, analysis of 160 Products, properties of the 156 Property (ies) and conformational effects, physical 219 determinations, analytical and 171 of xanthan gum, low shear rate rheological 199 Protein and polyanions, interactions of 82 -polysaccharide interactions 260 reaction with 168 on the titration of acridine orange, effect of lipid and 79 Proteus vulgaris, chondrosulfatase of .. 136 Pseudomonas carrageenovora

225

203 203 203 207

245 242 226 259 209 260 246 203 221 148 213 213

131

glycosulfatase 133 Pyridine-chlorosulfonic acid method .. 149 Q Quenching constants, Stern-Volmer .. Quenching of dye, Stern-Volmer plots for the iodide

88 90

R Radiolysis measurements, pulse 274 Reaction scheme, polymerization 196 Reaction variables 187 Reagent, effect of sulfating 150-152 Regression, analysis and interpretation of equations of 176 Relaxations 271 Reprecipitation/dissolution 158 method 153 Resedaceae

21

Residues present in heparin Rheological techniques

99 261

Rhodophyceae, sulfated polysaccharides of the

213

Rhodophyceae

(red)

225

292

CARBOHYDRATE SULFATES

Rhizoid cell Ricinus seeds Rumex

238 238 26

S S 0 reagent, analysis of D M F : 159 S 0 -trialkylamine 173 Salts of carrageenan activity coefficient of calcium counter-ion vs. calcium 264 of /x-carrageenan calcium 272-275 potassium 272, 273 sodium 272,273 magnesium 269 Sanfilippo syndrome 142 Seaweeds metabolizing polysaccharides 213 Seminolipid, synthesis of 54 Semi-synthetic studies 34 Shear rate rheological properties of xanthan gum, low 199 Sodium 259 cellulose sulfate via cellulose nitrite 163 cellulose sulfates, preparation of .... 169 chloride . 269 counter-ions vs."carrageenan 262 dextran sulfate 269 ion self-diffusion measurements 252 metabisulfite 195 salts of /x-carrageenan 272,273 Solvent, effect of precipitating 150 Spectra, IR of agarose 157 vs. degree of substitution, viscosity and 166 of funoran 157 of sulfated agarose 157 Spectral shifts of acridine orange, absorption 68 Spectrofluorimetric methods for estimating and studying the interactions of polysaccharides in biological systems 67 Spectroscopy absorption 67 fluorescence emission 67 IR 218 Squid liver, chondrosulfatase of 138 Starch 149 on the amount of chlorosulfonic acid, influence of the water content of 183 on apparent viscosity, influence of the water content of 182 with the chlorosulfonic acid-formamide reagent, reaction of 173 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

3

Starch (continued) concentration on the amount of formamide 182 on esterification, influence of the water content of 178 in formamide on esterification, influence of the concentration of 178 on limiting viscosity number, influence of the water content of .... 180 sulfate, yield of 179 Stern-Volmer plots of dye 90, 91 Stern-Volmer quenching constants .... 88 Stoichiometry of polyanion-acridine orange complexes, effect of p H on the 70 Stomach blood group sulfated glycoproteins, carbohydrate chains of hog 35 Structural features and biochemical implications, sulfated fucose-containing polysaccharides from brown algae 225 Structural studies, general 203 Substitution, viscosity and IR spectrum vs. degree of 166 Sucrose, ultracontrifugation in 116 Sulfatase(s) 130 cerebroside 141 polysaccharide 134 porphyran 135 Sulfate(s) algin 168 cellulose 163 via cellulose nitrite, sodium cellulose 163 choline 24 dextran 247 enzymatic formation and hydrolysis of polysaccharide 121 ester, hydrolysis of mixed cellulose nitrite 164 groups of carrageenan, ester 260 methods for the determination of the site of 215 as potential informational regulators in glycoproteins, ester 29 present in the same polyanion, estimation of carboxyl to the site of 208 from methylation results, the site of 209 moiety, hydroxylamine-O20 from partial hydrolysis, the site of .. 210 from periodate oxidation studies, the site of 208 of plant origin, other organic 24 polysaccharide 168

293

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

INDEX Sulfate (s) (continued) preparation of sodium cellulose 169 sodium dextran 269 transfer to different acceptors by the amino glycan sulfotransferases of hen oviduct and chick embryo cartilage 125 triglycosyl ceramide 44 yield of ammonium 181 yield of starch 179 2-Sulfate, L-ascorbate 1 chronology of 2 synthesis of 4 5- Sulfate, L-ascorbate 7,15 synthesis of 13 6- Sulfate, L-ascorbate 3,14 chronology of 6 O-Sulfates, glucosinolates and other naturally occuring 19 O-Sulfates, isomeric chlorogenic acid .. 26 Sulfation of cellulose nitrite 164 enzymatic 122 of guar, effect of time at different temperatures in the 151 of polysaccharides 159 some novel methods and results in the 148 synthetic heparin by 149 2-Sulfoamino analog, conversion of amylose into a 6-carboxyl 106 Sulfoglycolipids application of a general method of oligosaccharide synthesis to the synthesis of 48 previous synthetic studies on 46 studies on the synthesis of sulfurcontaining glycolipids 44 Sulfomethyl-containing graft co-poly­ mers of xanthan gum, sulfonic acid and 193 Sulfomethylation of xanthan g u m acrylamide graft co-polymer, results of 201 Sulfonic acid, acrylamide and 2-acrylamido-2-methylpropane 195 Sulfonolipid, studies towards the synthesis of the plant 60 Sulfotransferase (s) 122 acceptor specificity of some crude preparations of mucopolysac­ charide 124 activities, heat labilities of two different 127 of hen oviduct and of chick embryo cartilage, sulfate transfer to different acceptors by the amino glycan 125 from hen oviduct, specificity of a .... 128

Sulfur-containing glycolipids ("sulfo­ glycolipids"), studies on the syn­ thesis of Sulfuric acid, L-ascorbic acid in concentrated Syndrome, Hunter's Syndrome, Sanfilippo Synthesis of sulfoglycolipids, applica­ tion of a general method of oligo­ saccharide synthesis to the Synthetic studies on sulfoglycolipids, previous

44 8 4,5 142 142

48 46

Τ Temperature(s) effect of in the sulfation of guar, effect of time at different for xanthan gum, viscosity vs Test, Fisher Tetrasaccharide fragment from periodate-oxidized, borohydridereduced heparin Tetrasaccharide repeating unit, heparin: a suggested Theory, Debye-Huckel Thiocyanates /^D-Thioglucopyranosidic linkage Thioglucose (Ζ)-Thiohydroximate arrangement .... Time-dependence of polyanion-polyanion interaction on binding profiles Time at different temperatures in the sulfation of guar, effect of Tissues animal connective plant Titration of acridine orange, effect of protein and lipid on the Titrations of varying pHs Toothpaste tests, cold mix chocolate milk and Tovariaceae Trialkylamine-SOa Trichoderma viride glycosulfatase Triglycosyl ceramide sulfate 6-O-Tritylamylose, alternative oxida­ tion procedure for

150 151 198 176

102 101 245 21 20 23 20

83 151 123 29 123 79 77 160 21 173 130 133 44 107

U Ultracentrifugation in sucrose Ultrasonic absorption

115 116 272-275

294

CARBOHYDRATE SULFATES

Ultrasonic (continued) relaxation measurements waves Ulva lactuca Urospora penicilliformis Urospora wormskioldii

W 271 259 207,212 209 207-210

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ix001

V Vinylglycine Viscometer flow times, Ostwald Viscosity apparent influence of the water content of the starch on apparent and IR spectrum vs. degree of substitution measurements and molecular weight number, influence of the water con­ tent of the starch on limiting .... number, limiting vs. p H for xanthan gum vs. temperature for xanthan gum .... Volmer-Stern plots of dye plots for the iodide quenching of dye quenching constants

23 89 179 182 166 160 97 180 179 198 198

Water after activation, effect of residual Water content of the starch on the amount of chlorosulfonic acid, influence of the apparent viscosity, influence of the esterification, influence of the limiting viscosity number, influence of the

183 182 178 180

X Xanthan gum -acrylamide graft co-polymer, results of sulfomethylation of low shear rate rheological prop­ erties of -poly (acrylamide-co-AMPS A ) .. structure of sulfonic acid and sulfomethylcontaining graft co-polymers of viscosity vs. p H for viscosity vs. temperature for

91 90 88

153

149

201 199 200 194 193 198 198

Ζ Zygotes, Fucus

238-241