Biochemistry Involving Carbon-Fluorine Bonds
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Biochemistry Involving Carbon-Fluorine Bonds
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Biochemistry Involving Carbon-Fluorine Bonds R o b e r t F i l l e r , EDITOR
Illinois Institute of Technology
A symposium sponsored by the Divisions of Fluorine and Biological Chemistry at the 170th Meeting of the American Chemical Society, Chicago, Ill., Aug. 26, 1975.
28
ACS SYMPOSIUM SERIES
AMERICAN CHEMICAL WASHINGTON, D. C.
SOCIETY 1976
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Library of Congress
Data
Biochemistry involving carbon-fluorine fonds. (ACS symposium series; 28, ISSN 0097-6156) Includes bibliographical references and index. 1. Organofluorine compounds—Physiological effect— Congresses. I. Filler, Robert, 1923. II. American Chemical Society. Division of Fluorine. III. American Chemical Society. Division of Biological Chemistry. IV. Series: American Chemical Society. ACS symposium series; 28. QP981.F55B56 ISBN 0-8412-0335-0
Copyright ©
612'.0157 ACSMC8 28 1-215
76-13037 (1976)
1976
American Chemical Society A l l Rights Reserved.
N o part of this book may
be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E UNITED STATES O F AMERICA
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
ACS Symposium Series R o b e r t F. G o u l d ,
Series
Editor
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
FOREWORD The ACS SYMPOSIU
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.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
PREFACE Τη recent years there has been increasing interest and research activity in the biochemistry and medicinal applications of compounds con taining the carbon-fluorine bond. The medicinal chemistry of fluorinated organic compounds was last discussed at a symposium held at the national ACS meeting in Washington, D.C. in September 1971. Simultaneously a Ciba Foundation symposium on the biochemistry and biological activities of carbon—fluorine compound and incisive discussions a symposium, promi nent contributors to the field participated, were published in 1972 by Elsevier. A survey of fluorinated compounds of medicinal interest was presented in the December 1974 issue of Chemical Technology. The biochemical aspects of C - F chemistry have developed rapidly since the pioneer studies in the early 1940s by Sir Rudolph Peters, who elucidated the mechanism of the toxic action offluoroacetateby invoking the concept of 'lethal synthesis." Special mention should also be made of the elegant studies since the late 1950s by Charles Heidelberger and his colleagues on the tumor-inhibitory effects of nucleotides of fluorinted pyrimidines. Important advances in the biochemistry of organofluorine compounds continue unabated and a symposium on this subject, cosponsored by the Divisions of Fluorine and Biological Chemistry, was held at the Chicago ACS meeting in August 1975. This volume includes all ten presentations and discussions at that symposium. The subjects are of broad interest, ranging from fluorocarboxylic acids as enzymatic and metabolic probes to the use of perfluorocarbons as "artificial blood." We hope that these reports will further stimulate those already active in the field and create a sense of excitement and enlightenment to the curious. To the newcomer we say "Welcome—don't be afraid of fluorine compounds—they're lots of fun." Finally, I wish to express my appreciation to all of the contributors for their patience and unstinting cooperation in making this book possible. Illinois Institute of Technology Chicago, Ill. January 1976
ROBERT FILLER
ix In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1 Fluorocarboxylic Acids as Enzymatic and Metabolic Probes E R N E S T KUN University of California, San Francisco, Department of Pharmacology, and the Cardiovascular Research Institute, San Francisco, Calif. 94143
The development of site specific reagents necessarily depends on the knowledge of specific biochemical reactions, which should include the chemical structure of catalysts, substrates, modifiers, and products of enzymatic processes. Very little is known about catalytic sites of most enzymes, therefore comparison of substrates with analogues may be one of the experimental approaches to study this problem. It follows also that extensive information related to analytical enzymology (i.e., catalytic properties of isolated enzymes, enzymatic composition of cells) has to preceed the meaningful application of specific probes in complex systems. This endeavor is conceptually a synthetic one and intends to elucidate biological functions. It is only in relatively rare cases that inhibitors of cellular systems act in a manner predicted from in vitro enzymology. For example, a linear multienzymatic process can be indeed regulated by a rate limiting enzymatic component, susceptible to a specific inhibitor. In this case the biological significance of the linear multienzyme system can be studied with success. If acute inhibition does not cause rapid irreversible changes in cellular economy, sustained inhibition may trigger a variety of compensatory cellular processes, encompassing both intermediary and macromolecular metabolism. Experimental pathology and toxicology may benefit from these studies, since pathophysiology of environmental toxic effects is likely to be traced to specific initiating reactions of inhibitors with specific cellular sites. This experimental subject is at present only in its beginning stages. In contrast to linear systems-distributive, branching,and cyclic multienzymatic processes (1) respond in a far more complex manner to perturbations by a specific inhibitor. Alterations of steady state concentrations of metabolites, and subcellular changes of topographic distribution of intermediates of metabolic pathways will occur. Most of these consequences are unpredictable from idealized metabolic charts (2). Experimental results obtained with relatively uncomplicated fluorocarboxylic acids indicate that major metabolic pathways are apparently not 1 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E
BONDS
regulated by properties of enzymatic components alone but more importantly by a v a i l a b i l i t y of substrates. The role of primary regulatory enzymes in c e l l u l a r systems, problems of molar ratios of substrate to enzymes in biological systems have been reviewed (3) but the majority of biochemical texts have not incorporated these advances at present time. The choice of F substitution of H or OH groups in well known carboxylic acid substrates to obtain specific inhibitors was based on straight forward chemical considerations (4). The present paper is restricted to experimental work related to the mode of action of fluorocarboxy l i c acids, developed in our laboratory. >
F1uoro-dicarboxylic acids A series of chemical and enzymological experiments (5,6,7, 8,9,10,11,12,13,14,15,16,17,18,19) with mono and difluoro oxalacetates, fluoroglutamates mal ate, fluorolactate and mal ate dehydrogenases, transaminases, glutamate and lactate dehydrogenases suggested a reasonably uniform pattern of interaction between fluorodicarboxylic acid substrates with respective enzymatic s i t e s . It is of interest that introduction of one or two F atoms did not change the conformation of the parent molecule to the extent that i t could not be recognized by enzymatic s i t e s . In general^enzyme-fluoro-substrate Michaelis-Menten complexes are readily formed, but in some instances the rates of conversion to products, i . e . , the process of enzymatic catalysis i t s e l f is d r a s t i c a l l y reduced by F-substitution. The molecular reasons for this inhibition by Fsubstitution are as yet unexplored, and constitute a significant problem of mechanistically oriented enzymology, worthy of more detailed investigations. As would be expected F-dicarboxylic acids behave as r e l a t i v e l y uncomplicated l i n e a r l y competitive inhibitors with respect to the non-fluorinated substrate molecule. Kinetic analyses of the effect of fluoro-oxalacetate in bisubstrate systems of mal ate dehydrogenases (19) and lactate dehydrogenase (14) support this conclusion. A summary of experimental results is shown in Table I. Whereas monofluoro oxalacetate is a slowly reacting substrate of MDH, difluoro subs t i t u t i o n converts this substrate homolog to an equally good substrate of MDH to oxalacetate i t s e l f with respect to Vmax,except difluoro-oxalacetate has a Km of 4.0 mM (about 4.10^ higher than Km of oxalacetate). Further exploration of this significant effect of difluoro substitution may shed light on the as yet unknown molecular mechanism of catalysis of MDH. Since the p r i mary purpose of our investigations was to obtain biological probes, the s t a b i l i t y of F-dicarboxylic acids were also i n v e s t i gated in biological systems. Despite the promising in v i t r o properties of e-monofluoro oxalacetatic acid as an i n ï ï ï b i t o r of MDH, i n s t a b i l i t y prevented i t s application in complex systems. As would be expected.monofluoro oxalacetate is susceptible to
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
330 μΜ 710 μΜ
1800 μΜ 640 μΜ
3 F-glutaric
a-F-glutamate(NADP )
a-F-glutamate(NAD )
Liver GDH
Liver GDH
Liver GDH
Liver malic enzyme (decarboxylating)
Kidney MDH
5
6
7
8
9
L(+) 3-fluorolactate
--
300 μΜ
13 μΜ
300 μΜ
330 μΜ
15.0
13.0
transaminative defluorination
45. μΜ
LEGEND TO TABLE I: MDH = malate dehydrogenase; GOT = glutamate oxalacetate amino transferase; GDH = glutamate dehydrogenase; LDH = lactate dehydrogenase; Ma = molecular a c t i v i t y ; (S) = with physiological substrate; (F-S) = with fluoro-analogue
10 Muscle LDH
(-)-erythrofluoromalate
33'-difluoromalate
+
+
3 F-oxalacetic
Liver GOT(mito)
4
33* -F2-oxalacetic
1.0
Liver GOT(mito)
4.0 mM
3
33'-F2-oxalacetic
Kidney MDH(mito)
η
2
1
SUBSTRATE AND INHIBITORY PROPERTIES OF F-CARBOXYLIC ACIDS Ma F-carboxylic acid 1^ Κ* MâXF-S) Kidney MDH(mito) 101 3-F-oxalacetic 0.5 μΜ 0.5 μΜ
Enzyme
No.
4
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
COOH
COOH I ÇH + Ε
I
FCH I c=o I COOH
2
HCNH I COOH ?
COOH
I HC II
C-NH COOH Figure 1.
2
COOH I CH
COOH I FCH I
2
C=0 I COOH
COOH ^ HCH I C=NH I COOH
HCNH
2
COOH
H0 2
COOH I CH I + NH, c=o I COOH 2
Transaminative degradation of monofluoro-oxahcetic acid
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1.
KUN
Fluorocarboxylic
5
Acids as Probes
bivalent metal ion catalyzed decarboxylation to fluoro pyruvate. The kinetics of this reaction has been studied in detail (20). Interaction of monofluoro oxalacetate with glutamate-oxalacetate aminotransferase yields rapid elimination of F~ and Ν Η Λ from a system containing both aspartate and the fluoro-acid (b), repre senting a rare case of elimination reactions characteristic of pyridoxal-phosphate c a t a l y s i s , as well known from the work of Snell and his school. This i s shown in Figure 1. From a com bined use of fluoro-glutarate, a r e l a t i v e l y specific i n h i b i t o r of GDH (11) and of difluoro-oxalacetate, an i n h i b i t o r of GOT (10) the regulation of both transaminative and oxidative pathways of mitochondrial glutamate metabolism was further elucidated (12). Enzymatic reduction of monofluoro-oxalacetate by MDH on a prepar ative scale yielded (-)erythrofluoromalic acid (21) which i s an excellent i n h i b i t o r of malate dehydrogenases in v i t r o . In isolated hepatocytes this i n h i b i t o r acts only on cytosolic MDH isoenzyme because i t doe mitochondrial membrane (cf. 21), therefore i t i s useful as a c e l l u l a r probe of this MDH isoenzyme. Extensive t r i a l s with mitochondria and isolated hepatocytes - as models for the study of complex systems - indicated that only difluoro-oxalacetic and (-)erythrofluoromalic acidsproved useful. Difluoro-oxalacetate, by i n h i b i t i n g GOT,proved to be stable and highly s p e c i f i c in i t s action. Mono fluoro-malate, besides i n h i b i t i n g cytosolic MDH is also an effective activator of the c i t r a t e carrier system of the inner mitochondrial membrane, as shown in Figure 2 (cf. 21). When mitochondria are incubated with c i t r a t e and the exit +
f
.
Cit
I
i 2
F-Mal
ι 4
ι 6
Min.
ι 8
1 10
Molecular Pharmacology
Figure 2.
Spectrophotometric assay of citrate entry into mitochondria
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
6
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
of i s o c i t r a t e is monitored by an externally added NADP + Mg + isocitrate dehydrogenase i s o c i t r a t e detector system in the dual wave length spectrophotometer, rapid i s o c i t r a t e efflux is observed under a wide variety of experimental conditions. This process is greatly stimulated by (-)erythrofluoromalate (see Figure 2) as indicated by the large increase of the rate of extramitochondrial NADPH formation. Fluoromalate appears to act similar to malate (cf. 22) except i t s effect is not complicated by penetration and subsequent metabolism in the matrix - as i t is the case with malate. Consequently (-)erythrofluoromalate is a more specific and useful activator of the c i t r a t e - i s o c i t r a t e translocating membrane system than oxidizable dicarboxylic acids (compare with 22). It is a puzzle why c i t r a t e - i s o c i t r a t e exchange in energized mitochondria proceeds with the observed high efficiency (about 80% of added c i t r a t e appears as i s o c i t r a t e in the extramitochondrial compartment). We présumeras discussed more extensively l a t e r through the inner mitochondrial membrane is a more complex process than a carrier mediated carboxylic acid exchange (22) and may reflect an energy coupled membrane process of as yet unknown physiological significance. In v i t r o chemical and enzymatic studies thus far provided fluoro malate and difluoro-oxalacetate as candidates useful as probes in c e l l u l a r systems. The discovery of a valuable technique, that of isolation of hepatocytes by Berry and Friend (23), gave significant impetus to further studies. Gluconeogenesis from lactate was c h a r a c t e r i s t i c a l l y inhibited by difluoro-oxalacetate and to a lesser extent by (-)erythrofluoromalate,whereas glucose formation from pyruvate was more sensitive to fluoromalate than to difluoro-oxalacetate (24) as shown in Table II and Figure 3. Combination of both fluoro-dicarboxylic acids +
Tabl e II
Effects of fluoro-dicarboxylic acids on rates of gluconeogenesis in intact isolated liver cells Cells (125—225 mg wet weight), obtained from the livers of animals faeted 18 h, were incubated for 40 min at 37 °C. The initial substrate concentration was 10 mM and inhibitor concentration 2.5 m M . The values given are means ± S . E . with the number of observations in parenthesis. Rates of glucose formation have been calculated after subtraction of the average rate observed in the absence of added substrate. °· ± - ° ° n m o l x g ^ X m i n " (32 observations) T l l i 8
w
a
s
Substrate added
0 8
0
3
. ..... . . . Inhibitor added
1
_. . . Glucose formed μπιοί x g" x miu 1
L-Lactate L-Lactate L-Lactate L-Lactate Pyruvate Pyruvate Pyruvate Pyruvate
European Journal of Biochemistry
Fructose Fructose Fructose
0.45 Fluoromalate 0.34 Difluorooxaloacetate 0.24 Fluoromalate, Difluorooxaloacetate 0.11 — 0.51 Fluoromalate 0.21 Difluorooxaloacetate 0.38 Fluoromalate, 0.22 Difluorooxaloacetate 2.82 Fluoromalate 2.81 Difluorooxaloacetate 2.Si
Apparent i n h l b l t l o n -1
·/·
± 0.02 (20) ± 0.06 (4) ± 0.04 (4)
24 56
± 0 . 0 5 (3) ± 0.02 (20) ± 0.04 (5) ± 0.08 (4) ± 0 . 0 7 (4)
76 — 59 25 57
± 0.11 (14) (2) -(2)
— —
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2+
1.
KUN
Fluorocarboxylic
0
Acids
1
as Probes
7
J I • . I 2 3 4 5 Inhibitor concentration (mM) European Journal of Biochemistry
Figure 3. on rate of cells from pyruvate (·) or foctate (O). Each point is the average of two experiments. Conditions were the same as given in the legend of Table 1. , with difluorooxaloacetate; , with fluoromalate.
i n h i b i t gluconeogenesis from both precursors by 76-80% without s i g n i f i c a n t l y altering c e l l u l a r processes other than those i n v o l ved in the transfer of reducing equivalents through the inner mitochondrial membrane (25, 26). In preliminary studies we found no noticable toxic effects of either fluoro carboxylic acids when injected intravenously (20 mg/100 g body weight) into mice. Marked changes in glutamate and aspartate concentrations in the l i v e r indicated that both fluoro carboxylic acids actually penetrated l i v e r parenchyma c e l l s . It would appear reasonable to undertake more extensive in vivo studies with both fluoro carboxy l i c aci-ds as a model for a possible experimental chemotherapeut i c approach to metabolic disorders l i k e diabetes, characterized by abnormally large gluconeogenesis. Studies with fluorocitrate a.)Structure of the inhibitory isomer. In contrast to fluorodicarboxylic acids of apparently low or undetectable acute toxicity,the most important fluorotricarboxylic acid: monofluoroc i t r i c acid, because of i t s remarkable t o x i c i t y , plays a h i s t o r ical significance in the f i e l d of biochemical lesions, an area established by S i r Rudolph Peters (cf. 27). In a series of classical experiments Peters and his school established that the site of action of fluorocitrate is localized at an i n i t i a l step of c i t r a t e metabolism (28). The enzymatic s i t e of action of fluorocitrate was proposed to be mitochondrial aconitase (27).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
This mechanism appears to be incompatible with the results of Guarrierra-Bobyleva and Buffa (29), who found that after in vivo administration of toxic doses of fluorocitrate,only the metabolism of c i t r a t e was inhibited in mitochondria whereas c i s - a c o n i tate - which is also a substrate of aconitase - is oxidized nor mally. Because of the apparent uncertainties surrounding both the chemistry of the toxic isomer of f l u o r o c i t r i c acid and i t s subcellular mode of action this problem was reinvestigated. As i l l u s t r a t e d in Figure4 (cf. 30) the four possible isomers of monofluorocitric acid are formed either from fluoroacetyl-CoA and oxalacetic acid or from monofluoro-oxalacetic acid and acetyl CoA. It was shown (31,32) that the toxic isomer i s formed only by enzymatic condensation of fluoro acetyl CoA with oxalacetate whereas a l l other isomers had no s i g n i f i c a n t inhibitory effect on aconitase. Because of the r e l a t i v e l y weak inhibitory effects of fluorocitrate (Κ · * 50-80 μΜ) the d i s t i n c t i o n between "inhibi tory" and "non-inhibitory would wish for. Besides i n i t i a l velocity kinetic analyses, we, as well as others,observed a time dependent, anomalous i n h i b i t i o n of aconitase a c t i v i t y , but only in the i s o c i t r a t e d hydrogenase coupled test system, containing either Mg or Mn?+ (see l a t e r for more details ). Elucidation of the chemical structure of the toxic fluorocitrate isomer was successful despite the d i f f i c u l t i e s encountered in the enzymology of aconitase. Synthesis and r e s o l ution of isomers was accomplished in 1969 (33) and i t was shown that the electrophoretically separated erythro isomers (Figure 5 and Figure 6) contained the toxic species, which was further re solved and i d e n t i f i e d as (-)erythrofluorocitric a c i d , correctly defined as: 1R: 2Rl-fluoro-2-hydroxy-l,2,3-propane tricarboxylic acid. Crystallographic analysis of rubidium ammonium hydrogen fluorocitrate dihydrate (34) lent further support to our deduction, based o r i g i n a l l y on NMR and pk analyses of electrophoretically resolved diastereoisomers (31,32). Since the c i t r a t e condensing enzyme plays a key role in the biosynthesis of (-)erythrofluorocitric acid from fluoroacetic acid some kinetic characteristics of this enzyme were also determined with F-acetvl CoA as substrate. Results are shown in Table I I I , η
2+
TABLE
3
Summary of Kinetic Properties of Citrate Synthase from Pig Heart" Con stant K
Acetyl-CoA Fluoroacetyl-CoA F l uo roace ty 1-Co A Acetyl-CoA Fluoroacetyl-CoA
m
K K
m x
Kinetic constant
Substrate or inhibitor 25 μλί
23 μλί 2.2 μλί 2.77 (/xmolcs D P N H / m g / m i n ) 0.0084.5 (pinoles D P N H / m g / m i n )
v ' mil D P N , malate and malate dehydrogenase served as asourceof oxalacetate, and the reaction was followed by appearance of D P N H . β
'The Citric Acid Cycle"
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
KUN
Fluorocarboxylic
Acids
as Probes
F of mulot (•)-Mixture
Corbons derived from oxoloocetote or< fluoro-oxaloacetote
_ \
Λ
Λ
f COOH | H-C-H 1
|HOOC-C-OH
Carbons derived Γ from ocetote or < fluoroocetote L
H-C-F 2 1 C
0
0
H
(•)- Mixture
~\ COOH1 | H-C-F 2 1
HOOC-C-OH
H-C-H 1
1
C
COO" Newman projections " O O C ^ L OH olong C - 2 . C - 3 axis T A J (ionized forms) F-V^
C
O
coo" H O ^ L COO" JXj
1
F-C-H 2
HOOC-i-OH F-C-H
H
COOH 1
COOH 1 H-C-H
HOOC-fj-OH
2
H-Ç-H
COOH1
COOH
coo"
COO"
F ^ C >
Configurational P r e f i x * fi/S
System
(2S.3R)-
Racemates E «tensions of carbohydrote-omino acid rules ( C O O H group Ignored)
(2R.3S)-
(2R.3R)-
(2RS.3SR)-
L
0$Lg-
« 9" L
Racemates
erythro-L $
t
$
erythro-0 -
t
threo-Oj-
$
erythro-D L $
U Og0 L .LgC^-
^s^gLg"
Racemates
(2S.3Sh
(2RS.3RS)-
threo-L,-
fhreo-O L -
$
e
f
Provisional Assignments Weaker acids (Component A) H
Stronger octds (Component 8 )
I
From fluoroocetyl-CoA
From fluoro-/ oxaloacetate
«-
Not formed enzymaticolly è
enanttomers
"The Citric Acid Cycle"
Figure 4.
The isomers of monofluorocitric acid *
* T h e t e r m i n o l o g y o f L. D* a n d erythro D„ originates f r o m Η . B . V i c k c r y [ A Suggested N e w N o m e n c l a t u r e f o r the Isomers o f Isocitric A c i d , " / . Biol. Chan. 237, 1739 (1962)1. F o r c l a r i f i c a t i o n o f RS nomenclature, the reader is referred t o : C a h n , R . S., Ingold, C , a n d P r e l o g , V . : Angew. Client. Intern. Ed. Engl. 3 S 5 ( 5 ) , 511 ( 1 9 6 6 ) , a n d C a h n , R . S., 7. Chem. Educ. 1 1 6 ( 4 1 ) , 508 ( 1 9 6 4 ) . M
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E
0
0
0
n
C
ο
Y
ο οο ο 0000 ο
ο ο 0 0 0 00
U χ
CL
I
Ο
·— —
—·
ο
ν
LJL
2 3 4 5 6
Journal of Biological Chemistry
Figure 5. Electrophoresis of fluorocitric acid from enzymatic and chemical syntheses. CS: chemically synthesized fluorocitric acid; P: picric acid marker; fluorocitric acids I and II (see text) (32).
6£
5Ό
I 1 l I
1
CNF I I I t
40
3.0
CHF C H 1
2
I
I I I I i 1 I » 1
CH 1 1 I I t I 1 1 1
2.0
ΙΌ
2
1
W i 1
1
1 I I 1
I
C H , I 1 t 1
I
0 ppm ( δ )
I I 1 I
1
I 1 1 I
t
1 I I I
Journal of Biological Chemistry
Figure 6.
Nuclear magnetic resonance spectrum of triethyl fluorocitrate (32)
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1
1 1
BONDS
1.
KUN
Fluorocarboxylic
Acids
11
as Probes
(cf. 30). Whereas Km for acetyl CoA and i t s F-analog is the same, Vmax in the presence of F-acetyl CoA is about l/300th of V measured in the presence of the physiological substrate. Fluoroacetyl CoA acts also as an inhibitor of the condensation of acetyl CoA with oxalacetate. mx
b.)Mode of action of (-)erythrofluorocitrate. A molecular mechanism of fluorocitrate t o x i c i t y has to account for the i r r e versible cessation of c e l l function in certain s p e c i f i c c e l l s of the nervous system, known to be the probable anatomical organ s i t e of this poison (28). Whereas the question of organ s p e c i f i c i t y in the manifestation of pharmacological responses belongs to the domain of physiology, universality of metabolic enzymes in most organs s t i l l provides acceptable in v i t r o models for the study of biochemical toxicology. Consequently i f i n h i b i t i o n of aconitase is the mode of action of f l u o r o c i t r a t e , irreversible inhibition of this enzyme should b tions of the i n h i b i t o r becaus most probably present in vivo (compare with slow rates of fluoroc i t r a t e biosynthesis cf. 30). Even i f these requirements are met there is some uncertainty why aconitase i n h i b i t i o n should prove f a t a l , when inhibitors of other c i t r i c acid cycle enzymes causes r e l a t i v e l y small t o x i c i t y : eg. malonate is not a powerful poison (cf. 1), or as stated e a r l i e r , inhibition of malate dehydrogenase in vivo e l i c i t s no detectable toxic effects. This question is of particular significance, since the existence of cytoplasmic and mitochondrial isoenzymes of aconitase (35) make i t uncertain why the operation of the tricarboxylate cycle could not proceed undisturbed i f only one, i . e . , the mitochondrial isoenzyme^were inhibited (compare with 29). Because of the h i s t o r i c a l l y developed notion (36) identifying fluorocitrate t o x i c i t y with aconitase i n h i b i t i o n , the present evidence related to this question w i l l be examined in d e t a i l . Notorious problems of enzyme i n s t a b i l i t y at advanced stages of purification are well known to those concerned with the enzymology of aconitase. In a l l but one instance millimolar concentrations of ascorbate, cysteine, and F e are required to demonstrate f u l l aconitase a c t i v i t y in v i t r o . More recent studies concerned with the mechanism of aconitase action were performed almost entirely in this highly a r t i f i c i a l in v i t r o environment (37,38). This enzyme had a molecular weight of 68,000. Gawron et a l . (39,40) while unable to reproduce the i s o lation procedure of Villafranca and Mildvan (37,38) obtained an enzyme (mol. weight = 66,000) which had higher s p e c i f i c a c t i v i t y than reported by e a r l i e r workers. It appears therefore doubtful that previous in v i t r o studies (37,38) are entirely valid even within the framework of cuvette enzymology. Non-competitive i n hibition of heart aconitase (37) by transaconitate with c i t r a t e or i s o c i t r a t e as substrates (41) could not be reproduced (42)» therefore the explanatory mechanism of Villafranca (41) postulating two isomeric forms of aconitase appears to be without firm 2+
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
12
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
experimental foundation. One of the - until recently - unrecog nized complications of in v i t r o enzymology of aconitase l i e s in the frequently used NADP^ dependent i s o c i t r a t e dehydrogenase assay system i t s e l f . As demonstrated in 1974 (43)^various pre parations of aconitase are susceptible to time dependent i n a c t i vation by bivalent cations e g . , Mg or Mn^ , which are necessary constituents of the coupled assay system. Unawareness of this complication can result in double reciprocal plots of reaction rates, which can simulate competitive or non-competitive or mixed types of inhibitions by fluorocitrate with highly variable apparent Κ·,· values calculated from primary plots. We have also been misled by these types of experimental observations and pos tulated e a r l i e r a time dependent inactivation of aconitase by f l u o r o c i t r a t e , presumably by fluoro-aconitate or i t s defluorinated product (cf. 30). The very low Ki values for fluorocitrate reported by Brand et a l . (44), assaying aconitase a c t i v i t i e s of crude mitochondrial extract are apparently due to the inactivation of aconitase by Mg2 , which i s superimposed to the competitive inhibitory effect of fluorocitrate as reported more recently (43). In view of the a r t i f i c i a l i t y of the enzymology of aconitase, we pursued this problem by isolating an electrophoretically homogeneous enzyme which i s , for a f i n i t e period of time, f u l l y active without any a r t i f i c i a l activator whatsoever (43). Purification is shown in Table IV. The molecular weight of this cytoplasmic isoenzyme i s 2+
+
Table IV Purification of cytoplasmic aconitase from pig liwr Purification slq>
Total protein
Total acon itase activ ity at 25°
Specific activitv at 25°'
μηιοΐα/ηηη min/mg protein Step 1 (liver extract) Step 2 Step :> Step I
12,300 5(i0 180 8
1,045 730 500 85
0.134 1.32 2.70 10.0
Molecular Pharmacology
111,000. considerably higher than purified preparations requir ing Fe^+ cysteine for activation. A much less purified mito chondrial isoenzyme has also been isolated under conditions which maintained maximal a c t i v i t y without added activators. From Figure 7 i t i s evident that fluorocitrate acted as a l i n e a r l y competitive reversible i n h i b i t o r . From suitable compu ter models substrate and inhibitory constants were calculated as summarized in Table V. It i s apparent that (-)erythrofluoro%
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1.
KUN
Fluorocarboxylic
Acids
13
as Probes
Ε Ε 40θ|
//·> —
u
Jj
COO
I
2ΌΟΟ
I
I
OOO
L_
2000
l/S(M) Molecular Pharmacology
Figure 7. Competitive inhibition of cy toplasmic (A) and mitochondrial (B) aconitase of pig liver by (—)-erythrofluorocitrate. Rates of cis-aconitate for mation 240 nm at 25°. plasmic aconitase, and in B, 32 μ% of mitochondrial aconitase, were used per test system (5-cm light path; 3-ml vol ume) at varied concentrations of citrate (abscissa). Curve 1, 500 μΜ. fluorocitrate; 2,100 μΜ fluorocitrate; 3, no fluorocitrate.
Table V Siihslrutt: constants fitr citrate ami fl-ixoritralc and inhihilor constants f<»r (— )-r.rf/thro-Jliiornrilratc determined at μ/Ι 7.5 (10 w.\t Tris-ΙΚΊ) and 25° Isoenzyme
K
Α·
Β tnU
Cytoplasmic Mitochon drial
K,
m
Citrate
-Iso citrate
Citrate
μΜ
μΜ
(Λ;
A
Β
d Isocitrate
(A) μΜ
220
700
17
18
27
20
420
250
17
00
57
35
" A , determined by the cis-aconitatc assay; B , determined by the isocitrate dehydrogenase assay (Μβ-'" concentration varied between 0.1 and 5.3 nm;. Molecular Pharmacology
c i t r a t e behaved as an uncomplicated competitive and reversible i n h i b i t o r , therefore this kinetic property did not satisfy the mechanistic requirements set forth by the well known irreversible and highly potent toxicological effect of f l u o r o c i t r a t e . Although i t i s apparent that the enzyme requiring no a r i f i c i a l activator i s probably closer to the enzyme functioning in the c e l l u l a r environ-
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
14
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E
BONDS
mentjits properties are s t i l l different from aconitase located in isolated mitochondria. It was shown (43) that Mg2 is a potent inactivator of the isolated enzyme. In sharp contrast, lysosome free mitochondria, after accumulating over 200 mM Mg^ in the inner membrane and matrix (46) maintain a f u l l y active intramitochondrial aconitase for several hours at 37°, clearly indicating that in vitro s e n s i t i v i t y to Mg^ of isolated aconi tase has no relationship to the intramitochondrial c a t a l y t i c environment of this enzyme. This fact further stresses the need for extreme caution in projecting cuvette enzymology to b i o l o g i cal systems. Defluorination of fluorocitrate by isolated aconitase (37), has been recently reported by Villafranca and Platus (45) in the presence of an about 100 fold molar excess of fluorocitrate over aconitase. This system also contained 10 mM cysteine and 5 mM Fe^ . It is noteworthy that enzyme preparations requiring no activators f a i l e d to defluorinat lar results were also obtaine Gawron (48) did observe in v i t r o defluorination of f l u o r o c i t r a t e , again in the presence of 5 mM Fe* , 10 mM cysteine, 30 mM ascorbate, 0.126 μ moles of aconitase and 1 mM f l u o r o c i t r a t e . Only 3% of 1.0 mM fluorocitrate was defluorinated and this reversible reaction stopped in 1 to 1.5 minutes. Since inhibition of the enzyme which occurs during this process is reversible (cf. 45), whereas, as shown l a t e r , mitochondrial c i t r a t e u t i l i z a t i o n is inhibited in an irreversible manner by very low concentrations of f l u o r o c i t r a t e , i t seems unlikely that this phenomenon bears on the molecular mechanism of fluorocitrate poisoning. In contrast to kinetic studies with isolated aconitase pre parations, isolated mitochondria respond to fluorocitrate in a manner which seems to bear more directly on toxicology. When the c i t r a t e influx and i s o c i t r a t e efflux (21) of isolated intact mitochondria are measured following preincubation of mitochondria with less than micromolar concentrations of (-)erythrofluoroc i t r a t e , marked and irreversible inhibition of this process is obtained. Isocitrate efflux is inhibited when either c i t r a t e or cis-aconitate are added externally (49). Results are shown in Figure 8 (a & b) and Table VI. When the mitochondrial membrane structure was disrupted by the nonionic detergent Triton X-100 f u l l aconitase a c t i v i t y of mitochondria was obtained even in the presence of low concentrations of fluorocitrate which in the same preparation completely inhibited the flux of tricarboxylic acids into intact mitochondria. It is also apparent (Figure 8b) that activation of i s o c i t r a t e efflux by fluoromalate is also inhibited by preincubation with 2 μΜ f l u o r o c i t r a t e . The absence of i n h i b i tion by 10" to Ι Ο " M fluorocitrate of aconitase of lysed mito chondria agreed with in v i t r o enzyme kinetics (43). Fluoroci trate in intact mitochondria therefore irreversibly inhibited a membrane associated process essential for energy dependent t r i - ' carboxylic acid translocation. Similar experimental results +
+
+
+
+
>
8
6
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1.
KUN
Fluorocarboxylic
Acids
as Probes
15
Mia Biochemical and Biophysical Research Communications
Figure 8a. Rates of isocitrate efflux from cis-aconitate as substrate (1 cis-aconitate C = cis-aconitate added after 4 min preincubation without F-citrate; D = mitochondria preincubated with 5 μΜ F-citrate for 4 min then cis-aconitate addi tion. At arrows, Triton X-100 is added (see Results). y
Abbreviations used in Figures 8a and b: C is = cisaconitate; F-Cit = fluorocitrate; F-Mai = fluoromalate; Cit = citrate; Τ = Triton X-100.
2~
4
6
8
K)
12
Min Biochemical and Biophysical Research Communications
Figure 8b. Rates of isocitrate efflux from citrate (1 mM) as substrate. Upper curve = at 4 min, 1 mM F-malate added. Lower curve = at 2 min, 2 μΜ Fcitrate; at 4 min, 1 mM citrate; at 7 min, 1 mM Fmalate, being added in succession.
were obtained by Brand et a l . (44). Exchange diffusion of c i t r a t e in preloaded mitochondria was not inhibited by added fluorocitrate (44),hence i t was concluded that the anion c a r r i e r i t s e l f was not the target of inhibition by f l u o r o c i t r a t e . It must be noted however, that the essential prerequisite for
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
16
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
Table VI INHIBITION OF ISOCITRATE EFFLUX FROM ADDED CITRATE AND CIS-ACONITATE BY FLUOROCITRATE
% Inhibition M
F-citrate
1.0 X 1 0 "
8
1.25 Χ 1 0 "
Isocitrate
7
7
11
13
Χ 10"
8
31
30
5.0
Χ 10~
8
61
51
1.0 X 1 0 "
7
5.0
7
100
78
Χ 10"
(1 mM) a f t e r
Efflux
cis-aconitate*
2.5
•Added as s u b s t r a t e s (see
8
citrate*
of
1 minute p r e i n c u b a t i o n w i t h
F-citrate
Column 1 ) . Biochemical and Biophysical Research Communications
inhibition of mitochondrial c i t r a t e transfer i s preincubation of mitochondria for 2 to 8 minutes with very low concentrations of fluorocitrate in the absence of c i t r a t e which,if added simul taneously with fluorocitrate at 5 mM concentration prevents i n h i b i t i o n . In preloaded mitochondria c i t r a t e concentration i s 3-4 mM (cf. 44). Itwould be therefore expected that 1(T to 10" M fluorocitrate would not be effective under these conditions. We have reinvestigated this problem in collaboration with Dr. Eva Kirsten ( v i s i t i n g s c i e n t i s t from the University of Berlin)by a different experimental technique. Taking advantage of the extraordinary s t a b i l i t y of lysosome free mitochondria (50) we have followed the time course of ATP synthetase a c t i v i t y of these organells for 40-60 minutes. Since ATP synthetase a c t i v i t y under these conditions i s much faster than substrate permeation into the matrix, the time course of 32p incorporation into ATP, induced by external substrate is a sensitive measure of the rate of substrate translocation. This i s i l l u s t r a t e d in Figure 9. After preincubation of mitochondria for 10 minutes in the presence of unlabel led Pi + ADP to deplete endogenous substrates, P + substrates (either 10 mM c i t r a t e - T r i s , 0.5 mM malate-Tris separately or simul taneously) were added and the rate of ATP synthesis assayed (51) by the radiochemical method. The rate of ATP synthesis reached a plateau in 15 minutes when either substrates were added sep arately, but proceeded at a high rate for 60 minutes when >
6
t
3 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8
1.
KUN
Fluorocarboxylic
Acids
17
as Probes
MINUTES Figure 9. Time course of ATP synthesis by lysosomefree mitochondria (50). After preincubation for 10 min in the presence of 5 mM ADP + 5 m M P<, to deplete endogenous substrates, 32 Ρ (0.5 to 1.0 X JO dpm) and substrates (see text) were added Τ = 3 0 ° . 5
c i t r a t e + malate were added simultaneously. This time course i s characteristic of the kinetics of malate or fluoromalate a c t i v a ted c i t r a t e translocation (Figure 2). When^during preincubation of mitochondrials nanomoles of (-)erythrofluorocitrate per mg mitochondrial protein and 40 mM Mg2+ were present and the reac tion was started by either glutamate (2.5 mM) plus malate (2.5 mM) or c i t r a t e (10 mM) plus malate (0.5 mM)^the rate of ATP synthesis was completely inhibited when c i t r a t e was the permeant substrate but was unaffected when glutamate + malate were the substrates (FigurelO). It i s interesting that Mg2 accumulation (^230 mM into the matrix, cf. 50) caused a 3-fold augmentation of ATP synthetase a c t i v i t y (compare Figures 9 with 10^ thus Mg has no inhibitory effect on aconitase in s i t u . The c r i t i c a l role of the time of preincubation with 50 picomoles fluorocitrate +
2+
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
18
BIOCHEMISTRY
INVOLVING CARBON-FLUORINE BONDS
C 'to
Ο ί
α
Ό)
Ε ο Ε < Ο
MINUTES Figure 10. Conditions were the same as described for Figure 9, except 40 mM Mg * was also present. C = citrate + malate control; A = glutamate + malate control; Ό = glutamate + malate + preincu bation with 50 ρ moles of F-citrate per mg mitochon drial protein; Β = citrate + malate + preincubation with 50 ρ moles of F-citrate per mg mitochondrial protein; Τ = 3 0 ° . 2
per mg mitochondrial protein is i l l u s t r a t e d by the following results. Citrate dependent ATP synthetase a c t i v i t y was inhibited by 52% after 3 minutes preincubation, 63% after 6 minutes preincubation^and 90-100% after 12 minutes preincubation. This time course has direct relationship to the known kinetics of fluorocitrate poisoning, which has a slow onset but is i r r e v e r s i ble. Once c i t r a t e metabolism of mitochondria is inhibited by traces of fluorocitrate no subsequent manipulation can reactivate this specific process. Penetration of other mitochondrial sub strates and their subsequent metabolism i s unaffected under con ditions when c i t r a t e permeation is completely inhibited. No precise molecular interpretation of these results is as yet
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1.
KUN
Fluorocarboxylic
Acids
19
as Probes
possible, however i t seems clear that predictions based on cuvette enzymology of isolated aconitase does not f u l l y account for these phenomena. Technology of enzyme isolation in a l l but one case modifies aconitase and makes i t s c a t a l y t i c function dependent on a r t i f i c i a l activators. The preparation which is f u l l y active without F e + cysteine is inactivated in v i t r o by Mg (or Mn ) yet in the intact mitochondria this inactivation by Mg does not take place, therefore the micro environment of the mitochondrial enzyme has not yet been reproduced in v i t r o . Inhibition of the energy dependent c i t r a t e transport by traces of fluorocitrate in isolated mitochondria exhibits many charact e r i s t i c s which appear to predict the in vivo t o x i c i t y of fluoroc i t r a t e . Further analysis of this system and isolation of inner membrane components of the transport apparatus are necessary for real progress in this f i e l d . It is significant that Peters (cf. 27) i n t u i t i v e l y predicted that the biochemical lesion in fluoroc i t r a t e t o x i c i t y is probabl chemistry. Our recent c i a l l y because no biochemically meaningful argument can be proposed for the c r i t i c a l role of c i t r a t e transfer in mitochondrial and especially in c e l l u l a r metabolism unless an as yet unknown function of this process in the maintenance of energy transduction of the inner mitochondrial membrane is postulated. 2+
2+
2+
2+
Acknowledgement Most of the experimental work was supported by HD-01239, except the experiments concerned with ATP-synthetase (Figures 9 and 10) was sponsored by GM-20552. E. Kun is a research Career Awardee of the USPH. Literature Cited 1. Webb, J.L. "Enzyme and Metabolic Inhibitors," Acad. Press, New York 1963. 2. Dagley, S. and Nicholson, D.E. "An Introduction to Metabolic Pathways," John Wiley & Sons, Inc., New York 1970. 3. Sols, A. and Gancedo, C. "Primary Regulatory Enzymes and Related Proteins," in Biochemical Regulatory Mechanisms in Eukaryotic C e l l s , (Eds. Kun, E. and G r i s o l i a , S.) Wiley Interscience, New York 1972. 4. Kun, E. and Dummel, R.J. "Methods in Enzymology, Vol. XIII," (Eds. Colowick, Kaplan and Lowenstein) Chapt 79, p.623, Acad. Press, New York 1969. 5. Kun, E., Grassetti, D.R., Fanshier, D.W. and Featherstone, R.M. Biochem. Pharmacol. (1958) 158 (207). 6. Kun, E., Fanshier, D.W. and Grassetti, D.R. J . B i o l . Chem. (1960) 235 (416). 7. Kun, E. and Williams-Ashman, H.G. Nature (London) (1962)
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
20
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
194 (376). 8. Kun, E. and Williams-Ashman, H.G. Biochim. Biophys. Acta (1962) 59 (719). 9. Kumler, W.D., Kun, E. and Shoolery, J.N. J. Org. Chem. (1962) 27 (1165). 10. Kun, E., Gottwald, L.K., Fanshier, D.W. and Ayling, J.E. J. Biol. Chem. (1963) 238 (1456). 11. Gottwald, L.K., Ayling, J.E. and Kun, E. J. Biol. Chem.(1964) 239 (435). 12. Kun, E., Ayling, J.E. and Baltimore, B. J. Biol. Chem. (1964) 239 (2896). 13. Kun, E. and Achmatowicz, B. J. Biol. Chem. (1965) 240 (2619). 14. Ayling, J.E. and Kun, E. Mol. Pharmacol. (1965) 1 (255). 15. Gottwald, L.K. and Kun, E. J. Org. Chem. (1965) 30 (877). 16. Cymerman-Craig, J., Dummel, R.J., Kun, E. and Roy, S.K. Biochem. (1965) 4 (2547). 17. Dupourque, D. and Kun 18. Dupourque, D. and Kun, E. "Methods in Enzymology, Vol. XIII," (Eds. Colowick, Kaplan and Lowenstein) Chapt 20, p.116, Acad. Press, New York 1969. 19. Dupourque, D. and Kun, E. Eur. J. Biochem. (1969) 7 (247). 20. Dummel, R.J., Berry, M.N. and Kun, E. Mol. Pharmacol. (1971) 7 (367). 21. Skilleter, D.N., Dummel, R.J. and Kun, E. Mol. Pharmacol. (1972) 8 (139). 22. Chappell, J.B. British Med. Bull. (1968) 24 (150). 23. Berry, M.N. and Friend, D.S. J. Cell. Biol. (1969) 43 (506). 24. Berry, M.N. and Kun, E. Eur. J. Biochem. (1972) 27 (395). 25. Berry, M.N., Kun, E. and Werner, H.V. Eur. J. Biochem. (1973) 33 (407). 26. Berry, M.N., Werner, H.V. and Kun, E. Biochem. J. (1974) 140 (355). 27. Peters, R.A. British Med. Bull. (1969) 25 (223). 28. Pattison, F.L.M. and Peters, R.A. "Handbook of Experimental Pharmacology, Vol. XX," Chapt 8, p.387, Springer Press, New York 1966. 29. Guarriera-Bobyleva, V. and Buffa, P. Biochem. J. (1969) 113 (853). 30. Kun, E. "The Citric Acid Cycle," (Ed. Lowenstein) Chapt 6, p.297, Dekker Publications, New York 1969. 31. Fanshier, D.W., Gottwald, L.K. and Kun, E. J. Biol. Chem. (1962) 237 (3588). 32. Fanshier, D.W., Gottwald, L.K. and Kun, E. J. Biol. Chem. (1964) 239 (425). 33. Dummel, R.J. and Kun, E. J. Biol. Chem. (1969) 244 (2966). 34. Carrell, H.L. and Glusker, J.P. Acta Crystallogr. (1973) 29 (4364). 35. Eanes, R.Z. and Kun, E. Biochim. Biophys. Acta (1971) 227 (204). 36. Morrison, J.F. and Peters, R.A. Biochem. J. (1954) 58 (473).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1.
KUN
Fluorocarboxylic
Acids
as Probes
21
37. Villafranca, J.J. and Mildvan, A.S. J. Biol. Chem. (1971) 246 (772). 38. Villafranca, J.J. and Mildvan, A.S. J. Biol. Chem. (1972) 247 (3454). 39. Gawron, O. Sr., Kennedy, M.C. and Bauer, R.A. Biochem. J. (1974) 143 (717). 40. Gawron, O. Sr., Waheed, Α . , Glaid III, A.J. and Jaklitsch, A. Biochem. J. (1974) 139 (709). 41. Villafranca, J.J. J. Biol. Chem. (1974) 249 (6149). 42. Gawron, O. Sr., manuscript submitted for publication. 43. Eanes, R.Z. and Kun, E. Mol. Pharmacol. (1974) 10 (130). 44. Brand, M.D., Evans, S.M., Mendes-Mourao, J. and Chappell, J.B. Biochem. J. (1973) 134 (217). 45. Villafranca, J.J. and Platus, E. Biochem. Biophys. Res. Comm. (1973) 55 (1197). 46. Kun, E. (unpublished experiments). 47. Peters, R.A. and Shorthouse 48. Gawron, O. Sr. (personal communication). 49. Eanes, R.Z., Skilleter, D.N. and Kun, E. Biochem. Biophys. Res. Comm. (1972) 46 (1619). 50. Kun, E. manuscript submitted to Biochemistry. 51. Sugino, Y. and Miyoshi, Y. J. Biol. Chem. (1964) 239 (2360).
Discussion Professor Kun Q. What sort of methods or approaches are you using for isolating citrate carrier systems? A. For the membrane carrier systems we are in the process of developing techniques of hydrophobic chromatography. Q. On the affinity binding constants? You permit this substrate to remain attached to the carrier you isolated? A. Yes. Q. Chappell suggested that the tricarboxylate carrier is not inhibited by F-citrate. Are you suggesting that F-citrate inhibits energy coupling of the carrier? A. The exchange diffusion carrier of Chappell was tested for inhibition by fluorocitrate (ref. 44) in citrate preloaded mitochondria under conditions (i.e., at 4 mM citrate concen trations), when also in our hands fluorocitrate inhibition is prevented by the simultaneous presence of citrate. As dis-
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
22
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E
BONDS
cussed, preincubation with very low concentrations of fluoroc i t r a t e , in the absence of c i t r a t e are the required conditions to show inhibition of c i t r a t e translocation. Since c i t r a t e translocation in our experiments occurs in energized mitochondria ( i . e . , ATP dependent) i t is possible that Fc i t r a t e inhibits energy coupling. No precise answer to the question is as yet available. Q.
Would you comment on the electrophoretic separation of aconitase.
A.
Yes, we have done that. geneous .
Q.
Did you use a buffer?
A.
Yes, we have describe Pharmacology and I woul (see ref. 43).
It is electrophoretically homo-
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2 Biochemistry and Pharmacology of Ring-Fluorinated Imidazoles KENNETH L. KIRK and LOUIS A. COHEN Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Md. 20014
Medicinal chemists the value of fluorine in the design of analogues of metabolically significant molecules (1, 2). The high electronegativity of fluo rine can effect a marked alteration in electron-density d i s t r i b u tion, pK, conformation, etc.; simultaneously, this atom, by virtue of i t s small van der Waals radius (1.35 A), offers minimal steric interference to binding of the analogue at a specific macromolec ular s i t e . The effective size of fluorine attached to an sp carbon may be considered to fall between those of hydrogen and the hydroxyl group, while fluorine on an sp carbon is probably some what smaller — the result of lone pair overlap with the adjacent pi system (3, 4). Effective size undoubtedly varies, also, with solvation effects and specific lone pair interactions. 3
2
C = C
―
<--> C ― C =
+
Ring-fluorinated analogues of a variety of aromatic and heteroaromatic biomolecules have been synthesized and evaluated as agonists and antagonists of their natural relatives. We realized, some years ago, that the imidazole ring, one of the most ubiquit ous and important of natural heteroaromatic systems, could claim no documented fluoro derivatives, and initiated an effort to fill this gap. After we had exhausted the classical synthetic routes (5, 6), abandonment of the effort seemed inevitable. In other studies, we had found that imidazolediazonium ions, which are un usually stable to heat, are transformed photochemically to highly reactive species, possibly carbonium ions or carbenes (with expul sion of molecular nitrogen) (7). It seemed reasonable that such reactive species might capture fluoride and other poorly nucleo p h i l i c anions. Indeed, the f i r s t ring-fluorinated imidazole was obtained in 1971 by photolysis of ethyl 4-diazoniumimidazole-5carboxylate in 50% aqueous fluoroboric acid (8, 9). Subsequently, a wide variety of both 2- and 4-fluoroimidazoles were prepared for chemical and biological studies, of which many are still in pro gress. 23 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
24
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
S y n t h e t i c Methods The most general procedure f o r the s y n t h e s i s of 4 - f l u o r o imidazoles i s i l l u s t r a t e d i n F i g u r e 1 (10, 11), using aminoimidazole precursors. Unless they have the c a p a b i l i t y f o r resonance overlap with an e l e c t r o n s i n k ( n i t r o , cyano, carboxylate, etc.) at C-5, 4-aminoimidazoles show an i n s t a b i l i t y resembling that of v i n y l a m i n e s , and cannot normally be i s o l a t e d without p a r t i a l decomposition. C a t a l y t i c hydrogénation of 4 - n i t r o i m i d a z o l e s proved u n s a t i s f a c t o r y as a source of n o n s t a b i l i z e d 4-aminoimidazoles; however, z i n c dust r e d u c t i o n of the n i t r o group i n 50% f l u o r o b o r i c a c i d , which proceeded r a p i d l y and q u a n t i t a t i v e l y at low temperat u r e , became the method of choice. Immediately upon completion of r e d u c t i o n , the aminoimidazole i s d i a z o t i z e d w i t h sodium n i t r i t e i n s i t u , and the r e s u l t i n g diazonium i o n i s subjected to p h o t o l y s i s , again i n s i t u . A f t e r n e u t r a l i z a t i o n of the f l u o r o b o r i c a c i d medium, the product i s u s u a l l o v e r a l l y i e l d s (based o Since the other products of p h o t o l y s i s are g e n e r a l l y h i g h l y p o l a r , hydroxylated imidazoles, they are not removed by the e x t r a c t i o n s o l v e n t and do not i n t e r f e r e with p u r i f i c a t i o n . In c o n t r a s t to the 4-amino s e r i e s , 2-aminoimidazoles show the s t a b i l i t y to be expected of a l k y l a t e d guanidines. These compounds are generated by c a t a l y t i c hydrogénation of 2-arylazoimidazoles which, i n t u r n , are obtained by coupling of the imidazole w i t h aryldiazonium i o n (Figure 2 ) . Although such coupling occurs predominantly at C-2, smaller q u a n t i t i e s of the 4- and 2,4-bisa r y l a z o d e r i v a t i v e s are formed (12). I t i s e s s e n t i a l that the d e s i r e d isomer be freed of these contaminants ( u s u a l l y by column chromatography) p r i o r to hydrogénation, s i n c e p u r i f i c a t i o n of the r e s u l t a n t 2-aminoimidazole has proved extremely l a b o r i o u s . The pure 2-aminoimidazole i s then d i a z o t i z e d and the diazonium i o n i s photolyzed i n s i t u (13). This photochemical method has proved i t s e l f of v a l u e , w i t h respect to r a p i d i t y , convenience, and y i e l d , i n the synthesis of r i n g - f l u o r i n a t e d d e r i v a t i v e s of s e n s i t i v e or complex aromatic systems (£.£., a l k a l o i d s (14) and catecholamines (15)), as w e l l as other heteroaromatic systems, such as t h i a z o l e (7) and p y r r o l e (16). Special
Properties
A s i n g l e f l u o r o s u b s t i t u e n t , at C-2 or C-4, reduces the basi c i t y of the imidazole r i n g by 5-6 pK u n i t s and increases i t s a c i d i t y (NH—*N~) by 4-5 u n i t s (17) . The magnitudes of these e f f e c t s are s i g n i f i c a n t l y greater than those of the other halogens and i n d i c a t e that, i n c o n t r a s t to the fluorobenzenes, the i n d u c t i v e e f f e c t of f l u o r i n e on the imidazole r i n g overwhelms any e l e c t r o n - r e l e a s i n g e f f e c t due to resonance. I n c o n s i s t e n c i e s i n the l ^ F nmr s i g n a l s f o r the f l u o r o i m i d a z o l e s i n d i c a t e f u r t h e r that
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
KIRK AND COHEN
/=(
Ring-Fluorinated
0 N
£H CH NHAc 2
2
2
25
Imidazoles
^CH^NHAc
N
HN0
3
Zn 50%HBF -10
4
e
7\
CH2CH2NHAC N^NH
CH CH NHAc 2
2
=
2. h*. -10"
N^NH
Figure 1
ArN + pH 8-9
ArN^
2
.R
ArN ^
N^NH
1
2
NyNH
NaAr
NeAr
Pt.Hî AcOH
Mutt be removed before hydrogenolysn + ArNH
2
N
y
N
^R
H
NM
2
Figure 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
26
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
these compounds cannot be t r e a t e d i n p a r a l l e l w i t h the f l u o r o benzenes (17). The f l u o r i n e atom a t C - 4 , unless a c t i v a t e d by an e l e c t r o n s i n k a t C-5, has shown no evidence of r e a c t i v i t y toward nucleop h i l e s ; i n c o n t r a s t , 2 - f l u o r o i m i d a z o l e s are moderately r e a c t i v e toward displacement, p a r t i c u l a r l y i n the ring-protonated form (13), while the f l u o r o i m i d a z o l e anion i s s i g n i f i c a n t l y more s t a b l e (Figure 3 ) . Thus, c t - N - t r i f l u o r o a c e t y l - 2 - f l u o r o h i s t i d i n e cannot be deacylated by a c i d h y d r o l y s i s without l o s s of the f l u o r i n e atom, but the compound i s r e a d i l y deacylated i n m i l d base. Another consequence of the r e a c t i v i t y of 2 - f l u o r o i m i d a z o l e s i s the f a c i l e condensation to c y c l i c t r i m e r s (Figure 4), which appear t o be l a r g e r i n g , heteroannular aromatic systems. Since imidazole i t s e l f has been found to undergo f a c i l e hydrogen-isotope exchange a t C-2 but not a t C-4 (18), we expected an even more f a c i l e exchang w i t h 4 - f l u o r o i m i d a z o l e s Surprising l y , the l a t t e r compound a wide v a r i e t y of c o n d i t i o n s change r a p i d l y a t C-4 above pH 10, but much more slowly a t n e u t r a l pH. Thus, a route became a v a i l a b l e f o r the s p e c i f i c t r i t i u m l a b e l l i n g of 2 - f l u o r o h i s t i d i n e and 2-fluorohistamine, compounds which subsequently proved t o have extensive biochemical u t i l i t y . The a b i l i t y o f F-2 t o a c t i v a t e H-4 i s q u i t e unique; t o date, no other s u b s t i t u e n t a t C-2, whether more o r l e s s e l e c t r o n e g a t i v e than f l u o r i n e , ^ h a s demonstrated a c a p a b i l i t y of such magnitude. A route t o 2-[ H ] - 4 - f l u o r o h i s t i d i n e was developed r e c e n t l y , based on our observation that these exchange processes a r e subject to strong b u f f e r c a t a l y s i s . B i o l o g i c a l Properties Several d e t a i l e d r e p o r t s o f s t u d i e s w i t h f l u o r o i m i d a z o l e s have already been published or a r e i n press; the m a j o r i t y , however, a r e s t i l l i n t h e i r e x p l o r a t o r y stages. The f o l l o w i n g d i s c u s s i o n does not represent a comprehensive l i s t i n g of these studi e s ; r a t h e r , i t presents a sampling, intended t o demonstrate the v a r i e t y and scope o f the b i o l o g i c a l a p p l i c a t i o n s of f l u o r o i m i d a z o l e s and, h o p e f u l l y , t o suggest d i r e c t i o n s f o r f u r t h e r a p p l i c a tion. 2 - F l u o r o h i s t i d i n e . When t r i t i a t e d 2 - f l u o r o - L - h i s t i d i n e i s administered t o mice subcutaneously, the amino a c i d i s r a p i d l y d i s t r i b u t e d throughout the animal. Ten minutes a f t e r a d m i n i s t r a t i o n , the p r i n c i p a l organs ( i n c l u d i n g b r a i n ) show t r i t i u m l e v e l s 2-7 times that o f the blood. A f t e r 72 h r , two-thirds of the o r i g i n a l t r i t i u m count i s s t i l l w i t h i n the animal, and h a l f o f t h i s amount i s found i n i n s o l u b l e p r o t e i n f r a c t i o n s (19). Since we had shown that replacement of the 2 - f l u o r o group by any other s u b s t i t u e n t prevents exchange o f isotope a t C - 4 , and s i n c e a l l the t r i t i u m could be back exchanged from the p r e c i p i t a t e d p r o t e i n
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
KIRK AND COHEN
Ν
Π η F
Ring-Fluorinated
+
R
S
H
27
Imidazoles
•
H^piH
+
R
S
F
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
"
28
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
f r a c t i o n s at pH 11, the analogue must have entered i n t o de novo p r o t e i n synthesis i n place of h i s t i d i n e ; s i n c e the 2-fluorοimid azole moiety i s i n t a c t , covalent attachment of the f l u o r o h i s t i d i n e must have occurred at the amino a c i d s i d e chain. Such i n c o r p o r a t i o n i n t o the mouse i s blocked by cycloheximide and, i n i s o l a t e d l i v e r ribosomes, by actinomycin, both drugs being i n h i b i t o r s of protein synthesis. Studies with r a t p i n e a l gland have s u p p l i e d f u r t h e r evidence f o r the i n c o r p o r a t i o n of 2 - f l u o r o h i s t i d i n e i n t o newly synthesized p r o t e i n (20). H i s t i d i n e and i t s 2 - f l u o r o analo gue are u t i l i z e d c o m p e t i t i v e l y , s i n c e a d m i n i s t r a t i o n of an excess of h i s t i d i n e reduces f l u o r o h i s t i d i n e i n c o r p o r a t i o n . Presumably, the h i s t i d i n e analogue i s incorporated i n t o a l l newly synthesized pro t e i n without d i s c r i m i n a t i o n ; y e t , i t i s conceivable that c e r t a i n f l u o r i n e - c o n t a i n i n g enzymes may r e t a i n a c t i v i t y . S i n g l e doses of 2 - f l u o r o h i s t i d i n e (up to 250 mg per k i l o ) give no evidence of t o x i c i t y , organ degeneration or r e t a r d a t i o n of growth of the mouse over a 30-day p e r i o d . ^ Concentrations of 2 - f l u o r o h i s t i d i n b a c t e r i o s t a t i c ; i n h i b i t i o n of growth of E. c o l i (wild) i s essen t i a l l y complete i n 3 hr at 37°, but the i n h i b i t i o n i s reversed by a d d i t i o n of h i s t i d i n e (20 yg/ml) (21). During the course of the i n h i b i t i o n , the mass of the c u l t u r e increases about t h r e e f o l d , but the number of v i a b l e c e l l s increases about n i n e f o l d . These data suggest that the b a c t e r i a , which contain two to three copies of t h e i r chromosome during the normal growth phase, are unable to c a r r y out f u r t h e r chromosomal r e p l i c a t i o n i n the presence of the drug but are able to undergo c e l l u l a r d i v i s i o n . As shown i n Table I, 2 - f l u o r o h i s t i d i n e i s the most e f f e c t i v e b a c t e r i o s t a t i c agent of the s e v e r a l f l u o r o h e t e r o c y c l e s t e s t e d to date. The preceding s t u d i e s suggest that 2 - f l u o r o h i s t i d i n e may be u s e f u l wherever p r o t e i n synthesis i n a l i e n organisms or c e l l s occurs more r a p i d l y than i n the host s p e c i e s . Thus, the amino a c i d shows a n t i v i r a l behavior i n various i n f e c t e d c e l l c u l t u r e s (Table I I ) , at concentrations s i g n i f i c a n t l y below those needed to produce any v i s i b l e e f f e c t on the host c e l l s (22). To our know ledge, t h i s i s the f i r s t amino a c i d analogue to show a n t i v i r a l p r o p e r t i e s . Although i t s mechanism of a c t i o n has yet to be e l u c i dated, b i o s y n t h e s i s of ' f a l s e ' phosphorylases or v i r u s - c o a t p r o t e i n s are l o g i c a l p o s s i b i l i t i e s . Small peptides c o n t a i n i n g f l u o r i n e are more r e a d i l y obtained by t o t a l synthesis than by b i o s y n t h e s i s . Thus, t h y r o t r o p i n - r e l e a s i n g f a c t o r (TRF) and l u t e i n i z i n g hormone-releasing f a c t o r (LHRF) have been synthesized with 2- and 4 - f l u o r o h i s t i d i n e as r e p l a c e ments f o r the s i n g l e h i s t i d i n e residues (23). While the 4 - f l u o r o analogues are i n a c t i v e , those c o n t a i n i n g 2 - f l u o r o h i s t i d i n e show 20-30% r e l e a s i n g a c t i v i t y . In view of the marked l o s s i n b a s i c i t y
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
KIRK AND COHEN
Table I .
Ring-Fluorinated
29
Imidazoles
E f f e c t of F l u o r o Compounds on E. c o l l
O.D. Compound added
(wild)
a f t e r 18 h r a t 37° (Klett units)
None 2-Fluorohistidine 4-Fluorohistidine 4-Fluoroimidazole 2-Fluorourocanic a c i d 2-Fluoro-4-hydroxyethylthiazole Same + thiamine (2 yg/ml)
375 8 375 370 370 63 270
In minimal glucos s u l t s were obtaine itor.
Table I I .
A n t i v i r a l A c t i v i t i e s of
Fluorohistidines
Minimal i n h i b i t o r y Cell culture
Virus
d
VSV HSV-1 Vaccinia VSV Coxs. B4 Polio I Measles Coxs. B4
Required
d
PRK PRK PRK HeLa HeLa HeLa VERO VERO
2-Fluorohistidine
30 30 30 30 30 30 10 30
4-Fluoro- , histidine
100 >100 >100 >100 >100 >100 >100 >100
e
cone.
(pg/ml)
a
Ribavirin
C
_
-10 10 30 7 30
to reduce v i r u s - i n d u c e d c y t o p a t h o g e n i c i t y by 50%.
E s s e n t i a l l y s i m i l a r r e s u l t s were obtained f o r p - f l u o r o phenylalanine. An e s t a b l i s h e d a n t i v i r a l agent, Ι-β-Dribofuranosyl-1,2,4-triazole-3-carboxamide. Abbreviations are i d e n t i f i e d i n r e f . 32. At concentrations of 100 ug/ml, morphological a l t e r a t i o n of the host c e l l s was apparent only a f t e r 3 days. e
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
30
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
p-Glu-His-Pro-NH
2
(TRF)
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH
2
(LHRF)
of the imidazole r i n g f o l l o w i n g i n t r o d u c t i o n of f l u o r i n e , the e x i s t e n c e of any a c t i v i t y i s s u r p r i s i n g , and these r e s u l t s suggest that r e c o g n i t i o n of the peptide by i t s receptor may depend more on o v e r a l l conformation than on imidazole b a s i c i t y . A - F l u o r o h i s t i d i n e . Despite the minimal s t e r i c consequences of f l u o r i n e s u b s t i t u t i o n and the s i m i l a r i t y i n r i n g b a s i c i t i e s of the isomers, 2 - f l u o r o - and A - f l u o r o h i s t i d i n e are r e a d i l y d i f f e r e n t i a t e d by the h i s t i d i n e t-RNA systems. To date, the evidence i n d i c a t e s that A - f l u o r o h i s t i d i n e does not s u b s t i t u t e f o r h i s t i d i n e i n p r o t e i n b i o s y n t h e s i s , nor does t h i s analogue show s i g n i f i c a n t b a c t e r i o s t a t i c or a n t i v i r a l a c t i v i t (Table d II) s t u d i e s suggest that th (17), f l u o r i n e at C-A being , perhaps, u l a r hydrogen bond. Yet, the A - f l u o r o isomer i s a s u b s t r a t e f o r b a c t e r i a l h i s t i d i n e decarboxylase (but not f o r the mammalian enzyme), while both isomers are modest substrates f o r h i s t i d i n e ammonia-lyase (Table I I I ) . The a b i l i t y of A - f l u o r o h i s t i d i n e to f u n c t i o n both as a weak s u b s t r a t e and a strong competitive i n h i b i t o r f o r the l a t t e r enzyme prompted a r e i n v e s t i g a t i o n of i t s mechTable I I I . E f f e c t s of F l u o r o h i s t i d i n e s on H i s t i d i n e Ammonia-Lyase (pH 9, 25°) Κ Compound
or (mM)
m
L-Histidine
2.7
A-F-L-Histidine
1.25
2-F-L-Histidine
170
K. 1
V (uM/mg/min)
30 0.85 1-2
anism of a c t i o n (2A). A n a l y s i s of k i n e t i c data, r e v e r s i b i l i t y , i s o t o p e i n c o r p o r a t i o n , and isotope e f f e c t s demonstrates that the r a t e - l i m i t i n g step f o r t h i s enzyme i s not the breakdown of an intermediate aminoenzyme (as p r e v i o u s l y supposed), but the almost concerted l o s s of a 3-hydrogen atom and the c o v a l e n t l y bound amino group. While the isomers l o s e ammonia at comparable r a t e s , Af l u o r o h i s t i d i n e i s bound much more e f f e c t i v e l y . Urocanase, the enzyme f o l l o w i n g the ammonia-lyase i n the c a t a b o l i c sequence f o r h i s t i d i n e (Figure 5), promotes the h y d r a t i o n rearrangement of urocanic a c i d (25). While n e i t h e r f l u o r o u r o c a n i c a c i d i s s i g n i f i c a n t as a s u b s t r a t e (Table IV), the 2 - f l u o r o isomer
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
KIRK AND COHEN
Ring-F luorinated
31
Imidazoles
Table IV. E f f e c t s of F l u o r o u r o c a n i c A c i d s on Urocanase (pH 7.4, 25°)
7
10 K Compound
Urocanic a c i d
m
or (M)
K
±
V (units/ml)
400
1.2
-
4-F-Urocanic
acid
-
2-F-Urocanic
acid
1
0.008
measurable a c t i v i t y as i n h i b i t o r or substrate.
i s now a very potent i n h i b i t o enzyme capable of d i r e c t removal of f l u o r i n e from the imidazole r i n g . The enzymes which degrade the imidazole r i n g of h i s t i d i n e , e v e n t u a l l y to glutamic a c i d , can a l s o operate on the f l u o r o h i s t i d i n e s , a l b e i t very slowly. Thus f a r , we have found no evidence f o r metabolites of these analogues i n v i v o . As already mentioned, the f l u o r o h i s t i d i n e s can be i n c o r p o r a t e d i n t o a p o l y p e p t i d e sequence v i a t o t a l s y n t h e s i s . Thus, the peptide fragment, r i b o n u c l e a s e - S - ( l - 1 5 ) , has been s y n t h e s i z e d with 4- f l u o r o - L - h i s t i d i n e r e p l a c i n g h i s t i d i n e - 1 2 (27). T h i s f l u o r i n e c o n t a i n i n g peptide (4-F-His-RNase-(l-15)) a s s o c i a t e s w i t h RNase-S(21-124) as w e l l as, and even more s t r o n g l y than, RNase-S-(l-20). A v a r i e t y of c r i t e r i a suggest that the f l u o r o h i s t i d i n e - c o n t a i n i n g aggregate has a three-dimensional s t r u c t u r e very s i m i l a r to that of the complex, RNase-S'. The noncovalent recombination of RNase5- (l-15) or -(1-20) w i t h RNase-S-(21-124) r e s t o r e s p r a c t i c a l l y a l l the enzymatic a c t i v i t y of the o r i g i n a l enzyme, RNase-A; the a n a l ogous complex with 4-F-His-RNase-(l-15) i s t o t a l l y devoid of a c t i v i t y , although i t i s probably s t i l l capable of b i n d i n g s u b s t r a t e . The r e s u l t s provide strong support f o r the proposal that His-12 f u n c t i o n s as a general a c i d - g e n e r a l base c a t a l y s t i n the enzymatic process — a r e d u c t i o n of 5 u n i t s i n the pK of the r i n g being more than ample to remove t h i s c a t a l y t i c c a p a b i l i t y . Fluorohistamines. Two types of r e c e p t o r , termed HI and H2, have been i d e n t i f i e d as mediators of histamine's b i o l o g i c a l a c t i o n s . C h a r a c t e r i z a t i o n of these r e c e p t o r s , and of histamine's b i o l o g i c a l r o l e s , have been hampered by l a c k of agents which s e l e c t i v e l y b i n d one type. Recently, i t has been found that v a r i o u s s u b s t i t u t i o n s at C-2 or C-4 of the histamine r i n g s e l e c t i v e l y decrease b i n d i n g f o r H2 or HI r e c e p t o r s , r e s p e c t i v e l y . Thus, 2phenylhistamine r e t a i n s some 20% of histamine's potency at HI r e c e p t o r s , but i s l e s s than 0.1% as e f f e c t i v e at H2 r e c e p t o r s . In a p r e l i m i n a r y study, 2-fluorohistamine was found (28) to have
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
32
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E BONDS
/CH -CHC00H
CH=CHCOOH
2
NH
Lyase
2
-NH3 Urocanase +H2Q CL
£H CH C00H
H00CCHCH2CH C00 2
H
NH CH= NH Figure 5
-do HO-CHl
H0-CH2
ON
OH
5-FICAR 5-Fluoro-L β-D-ribofuranosylimidazole4-carboxamide
ribavirin 1 -β-D-ribofuranosyîl 2,4-triazoh-3carboxamide y
5-AICAR 5-amino-l -β-Ό-rihofuranosuUmidazole4-carboxamide
Figure 6
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
ΚΓΑΚ AND COHEN
Ring-Fluonnated
Imidazoles
33
equal or greater a f f i n i t y f o r HI r e c e p t o r s ( i n guinea p i g ileum) than the p r e v i o u s l y most e f f e c t i v e HI a g o n i s t , 2-aminoethylthiaz o l e . By analogy, 4-fluorohistamine might act as a s e l e c t i v e agon i s t f o r H2 r e c e p t o r s , a p o s s i b i l i t y now under i n v e s t i g a t i o n . 2-Fluorohistamine was a l s o found to be an e f f e c t i v e s t i m u l a t o r of c y c l i c AMP formation i n b r a i n s l i c e s , which c o n t a i n both HI and H2 receptors (29). Fluoroimidazole Ribosides. R e l a t i v e l y simple imidazole d e r i v a t i v e s occupy key r o l e s as intermediates i n the b i o s y n t h e s i s of the purine n u c l e o s i d e s . A major approach to the design of a n t i v i r a l agents i s based on analogues of Imidazole species which might i n t e r f e r e with the b i o s y n t h e s i s of n u c l e o s i d e s or of n u c l e i c a c i d s (Figure 6 ) . Thus, r i b a v i r i n , a t r i a z o l e analogue of 5-AICAR, commands s e r i o u s a t t e n t i o n as a broad-spectrum a n t i v i r a l agent (30) . We have synthesized 5-FICAR a f l u o r o analogue of 5-AICAR (31) ; t h i s compound show s i m i l a r to that of r i b a v i r i to b l o c k the b i o s y n t h e s i s of both DNA and RNA. R i b a v i r i n has been shown to act by i n h i b i t i n g the enzyme, IMP dehydrogenase (33), and we assume, t e n t a t i v e l y , that 5-FICAR f u n c t i o n s at the same p o i n t i n the b i o s y n t h e t i c pathway. Future
Plans
The r e s u l t s of these and other s t u d i e s i n f l u o r o i m i d a z o l e chemistry and biochemistry have r a i s e d i n t e r e s t i n g questions f o r the f u t u r e . Some aspects of the p h y s i c a l and chemical p r o p e r t i e s of f l u o r o i m i d a z o l e s do not conform to expectations based on data f o r other imidazole systems and suggest, » that some sub s t i t u t e d imidazoles a r e , at b e s t , b o r d e r l i n e aromatic systems. Further understanding may be provided by a study of d i f l u o r o imidazoles: 4 , 5 - d i f l u o r o i m i d a z o l e has been synthesized and i s , indeed, found to have anomalous p r o p e r t i e s ; d e s p i t e a r a t h e r ex t e n s i v e e f f o r t , however, the 2 , 4 - d i f l u o r o isomer has not y e t been prepared. On the b i o l o g i c a l s i d e , we may ask, , why the isomeric f l u o r o h i s t i d i n e s show such marked d i f f e r e n c e s i n b i n d i n g and r e sponse to h i s t i d i n e - s p e c i f i c enzymes; whether the replacement of h i s t i d i n e by f l u o r o h i s t i d i n e i n an enzyme sequence i n v a r i a b l y leads to l o s s of a c t i v i t y ; whether the 2 - f l u o r o i m i d a z o l e s can be made s u f f i c i e n t l y r e a c t i v e to f u n c t i o n as covalent a f f i n i t y l a b e l s f o r receptor s i t e s . H o p e f u l l y , these and other questions w i l l have been answered before the next F l u o r i n e Symposium.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
34
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
Literature Cited (1) Goldman, Peter, Science (1969), 164, 1123. (2) Ciba Foundation, "Carbon-Fluorine Compounds," Elsevier, Amsterdam, 1972. (3) Reference 2, p. 211. (4) Belsham, M. G., Muir, A. R., Kinns, Michael, Phillips, Lawrence, and Twanmoh, Li-Ming, J. Chem. Soc., Perkin Trans. 2 (1974), 119. (5) Pavlath, A. E. and Leffler, A. J., "Aromatic Fluorine Com pounds," Reinhold, New York, 1962. (6) Hudlicky, Miklos, "Organic Fluorine Chemistry," Plenum Press, New York, 1970. (7) Kirk, K. L. and Cohen, L. Α., unpublished observations. (8) Kirk, K. L. and Cohen, L. Α., Symposium on Fluorine in Medic inal Chemistry, 162nd National Meetin f th America Chem ical Society, Washington (9) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1971), 93, 3060. (10) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1973), 95, 4619. (11) Kirk, K. L. and Cohen, L. Α., J. Org. Chem. (1973), 38, 3647. (12) Nagai, Wakatu, Kirk, K. L., and Cohen, L. Α., J_. Org. Chem. (1973),38,1971. (13) Kirk, K. L., Nagai, Wakatu, and Cohen, L. Α., J. Amer. Chem. Soc. (1973),95,8389. (14) Lousberg, R. J. J. Ch. and Weiss, Ulrich, Experientia (1974), 30, 1440. (15) Kirk, K. L., manuscript in preparation. (16) Lowenbach, W. A. and King, M. M., unpublished data. (17) Yeh, H. J. C., Kirk, K. L., Cohen, L. Α., and Cohen, J. S., J. Chem. Soc., Perkin Trans. 2 (1975), 928. (18) Wong, J. L. and Keck, J. H., Jr., J. Org. Chem. (1974), 39 2398, and earlier references cited therein. (19) Kirk, K. L., McNeal, Elizabeth, Cohen, L. Α., and Creveling, C. R., manuscript in preparation. (20) Klein, D. C., Kirk, K. L., Weller, J. L., and Parfitt, A. G., Mol. Pharmacol., in press. (21) Furano, Α. V., Kirk, K. L., and Cohen, L. Α., unpublished data. (22) De Clercq, Erik, Kirk, K. L., and Cohen, L. Α., unpublished data. (23) Monahan, Μ. Α., Vale, Wylie, Kirk, K. L., and Cohen, L. Α., unpublished data. (24) Klee, C. Β., Kirk, K. L., Cohen, L. Α., and McPhie, Peter, J. Biol. Chem. (1975), 250, 5033. (25) Kaeppeli, Franz, and Retey, Janos, Eur. J. Biochem. (1971), 23, 198, and earlier references cited therein. (26) Klee, C. B., Kirk, K. L., and Cohen, L. Α., unpublished data.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2.
KIRK AND COHEN
Ring-Fluorinated
Imidazoles
35
(27) Dunn, Β. Μ., DiBello, Carlo, Kirk, K. L., Cohen, L. Α., and Chaiken, I. M., J. Biol. Chem. (1974), 249, 6295. (28) Dismukes, R. Κ., unpublished data. (29) Dismukes, R. K., Rogers, Michael, and Daly, J. W., Neurochemistry, in press. (30) Khare, G. P., Sidwell, R. W., Witkowski, J. T., Simon, L. N., and Robins, R. K., Antimicrob. Ag. Chemother. (1973), 3, 517. (31) Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Tetrahedron Letters, in press. (32) De Clercq, Erik, Luczak, Miroslav, Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Life Sciences (1975), 17, 187. (33) Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. Κ., and Simon, L. Ν., Proc. Nat. Acad. Sci. USA (1973), 70, 1174. Discussion Q. A.
Has there been a check of monitoring on whether C-F cleavage occurs? I assume you r e f e r to s t a b i l i t y i n b i o l o g i c a l systems. The normal pathway f o r h i s t i d i n e degradation i n v o l v e s conversion to urocanic a c i d , and, u l t i m a t e l y , to glutamic a c i d . As I had i n d i c a t e d , 2 - f l u o r o h i s t i d i n e i s a very poor s u b s t r a t e f o r the f i r s t two enzymes i n t h i s pathway; a t the t h i r d s t e p , f l u o r i d e i o n would be r e l e a s e d , together with innocuous degrada t i o n products. 4 - F l u o r o h i s t i d i n e can be transformed slowly to 4 - f l u o r o u r o c a n i c a c i d , which i s probably a dead end. Except f o r t h i s data, we have found no i n d i c a t i o n s f o r enzymatic removal of f l u o r i n e .
Q.
Have you t e s t e d the b i o l o g i c a l a c t i v i t y of f l u o r o a l k y l i m i d a -
A.
We have not made any C F ^ - s u b s t i t u t e d i m i d a z o l e s .
Q. A.
As f a r as you know, are any of them known? Yes. A s e r i e s of 4 - t r i f l u o r o m e t h y l i m i d a z o l e s have been reported by Baldwin and h i s colleagues a t Merck Sharp & Dohme [ c f . J . Med. Chem. (1975), l g , 895] and 2 - t r i f l u o r o m e t h y l imidazoles by Lombardino and Wiseman [ J . Med. Chem. (1974), 17, 1182.]
Q.
What do you think would be the long-term s t a b i l i t y of the C-F bond i n 2-fluoroimidazoles? Simple compounds, such as 2 - f l u o r o i m i d a z o l e and 2 - f l u o r o - 4 methylimidazole can be kept i n d e f i n i t e l y i n the s o l i d s t a t e at -80°, but t r i m e r i z e i n a month or two a t 0 ° . In c o n t r a s t , 2 - f l u o r o h i s t i d i n e appears to be i n d e f i n i t e l y s t a b l e a t room temperature. D i l u t e s o l u t i o n s of 2 - f l u o r o h i s t i d i n e i n phos phate b u f f e r (pH 7) have been kept at 0° f o r months without evidence of d e t e r i o r a t i o n .
z o l e s - r e p l a c i n g F by
A.
CF3?
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
36
Q. A.
Q. A.
Q.
A.
BIOCHEMISTRY INVOLVING
C A R B O N - F L U O R I N E BONDS
Can you say anything about the a n t i - a r t h r i t i c a c t i v i t y of the fluorohistidines ? T e s t i n g has not yet been done. We are aware of the p o s s i b i l i t i e s i n t h i s d i r e c t i o n and hope to get such a study going soon. I'd likÇgto ask whether j g u ever considered the use of r a d i o active F as a probe? F has a h a l f - l i f j g O f 110 minutes. Yes, we have. Studies on the s y n t h e s i s of F imidazoles were i n i t i a t e d some time ago at the Atomic Energy Commission i n Bucharest. You're acquaintecjgwith the work of A l Wolf and others a t Brookhaven with F fluor©phenylalanine and other b i o l o g i c a l l y i n t e r e s t i n g molecules? ^g Yes, I am. A l Wol p o i n t of p a r t i c u l a the b l o o d - b r a i n b a r r i e r very q u i c k l y , and so there i s c o n s i d e r a b l e i n t e r e s t i n t h i s compound f o r b r a i n s c i n t i g r a p h y .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3 2-Fluoro-L-Histidine: A Histidine Analog Which Inhibits Enzyme Induction DAVID C. KLEIN Section on Physiological Controls, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014 KENNETH L. KIRK Section on Biochemical Mechanisms of Arthritis, Metabolism, and Institutes of Health, Bethesda
f Chemistry Nationa Institut
Amino acid analogs have a long and interesting history (1,2) and have been extremely valuable to biochemists, pharmacologists, and clinicians as analytical tools and therapeutic agents. Some of the best established and most useful of these compounds are para-fluorophenylalanine, para-chlorophenylalanine, and alpha-methyldopa. The total number of such compounds which have been synthesized with the hopes of producing potent and specific tools probably is in the thousands. Potential Actions of Amino Acid Analogs The stratagem usually employed with an amino acid analog is to have i t enter cells and substitute for the parent compound (1,2); the structural modification characterizing the analog precludes the normal function or metabolic fate of the parent compound. Substitution of the analog for the parent compound could involve substitution in a metabolic pathway resulting in an altered metabolite. Additionally, a metabolic pathway could be inhibited by an amino acid analog by several mechanisms, including competitive or noncompetitive inhibition of a specific enzyme or false feedback. Lastly, substitution for the parent compound in the synthesis of proteins could result in altered proteins. Such replacement of an amino acid analog for the parent compound in the primary structure of a protein could alter a protein through a number of modes of action, including alteration of the secondary, tertiary, or quarternary structure, alteration of the active site of an enzyme, and alteration of the regulatory site of an enzyme. Role of Histidine in Enzymes Histidine is one of the most important amino acids involved in the catalytic action of enzymes (3,40. It functions via the imidazole group, which can act as a general acid, general base, 37 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
38
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
or n u c l e o p h i l e . This c a t a l y t i c r o l e represents the g e n e r a l l y recognized f u n c t i o n of h i s t i d i n e i n enzymes (5). Some of the enzymes i n which h i s t i d i n e i s a t the a c t i v e s i t e i n c l u d e RNAse, glucose-6-phosphatase, and chymotrypsin. Recently, evidence has appeared i n d i c a t i n g h i s t i d i n e may have a f u r t h e r r o l e i n p r o t e i n s (6). I t i s now g e n e r a l l y accepted that the a c t i v i t i e s of many enzymes are regulated v i a covalent chemical m o d i f i c a t i o n of enzyme p r o t e i n (5_ ]_>Q) » i n c l u d i n g phos phorylation. S p e c i f i c p r o t e i n kinases c a t a l y z e phosphorylations, and s p e c i f i c phosphatases c a t a l y z e dephosphorylation. As a r e s u l t , the a c t i v i t i e s of enzymes can be turned on and turned o f f q u i c k l y . The phosphorylation of p r o t e i n s occurs v i a covalent phosphorylation of s p e c i f i c amino a c i d s , with the r e s u l t i n g formation of both a c i d - s t a b l e and a c i d - l a b i l e phosphate bonds. The a c i d - s t a b l e bonds appear to be formed with the hydroxy groups of s e r i n e and threonine, whereas the a c i d - l a b i l e bonds appear to be formed with the imidazol The importance o obvious; i t i s a l s o p o s s i b l e that the imidazole group may be important i n the r e g u l a t i o n of enzyme a c t i o n . The a v a i l a b i l i t y of a h i s t i d i n e analog i d e n t i c a l i n s i z e and shape to h i s t i d i n e but l a c k i n g the f u n c t i o n a l c h a r a c t e r i s t i c s imparted by the imidazole group would be of general i n t e r e s t . 9
2-Fluoro-L-Histidine:
Stratagem
2 - F l u o r o - L - h i s t i d i n e was synthesized p r i m a r i l y t o provide an analog of h i s t i d i n e which would have the same s i z e and shape as h i s t i d i n e , but which would be devoid of the normal a c t i o n imparted by the imidazole group (9). (For a more d e t a i l e d d i s c u s s i o n see L. Cohen, t h i s Symposium Series.) Fluorine i s about the same s i z e as the hydrogen i t r e p l a c e s i n h i s t i d i n e
NH
2
ι
CHsCHCOOH
2-FLUORO-L-HISTIDINE (SYNTHESIS: Kirk ETAL., J. Am. Chem. Soc.,55,8389 (1973))
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN AND KIRK
39
2-Fluoro-L-Histidine
and thus w i l l not introduce major s t e r i c m o d i f i c a t i o n s of h i s t i dine. F l u o r i n e - s u b s t i t u t i o n , because of the strong e l e c t r o n withdrawing e f f e c t of f l u o r i n e , w i l l s u b s t a n t i a l l y a l t e r the pK of the imidazole group. The r e s u l t i n g compound w i l l be essent i a l l y i n a c t i v e , as compared to the parent compound, as a c a t a l y s t i n enzymes. T h i s e f f e c t of f l u o r i n e s u b s t i t u t i o n i s demonstrated i n ^ Table I. In t h i s experiment, the degree of t r a n s f e r of a [ C ] a c e t y l group from [ C ] a c e t y l coenzyme A to the aromatic^amine tryptamine was determined by measuring the amount of N-[ C]acetyltryptamine formed. Imidazole c a t a l y z e d t h i s t r a n s f e r , whereas 2 - f l u o r o i m i d a z o l e d i d not. Likewise, on a t h e o r e t i c a l b a s i s , f l u o r i n e s u b s t i t u t i o n would preclude the phosphorylation of imidazole. Thus, i f 2 - f l u o r o - L - h i s t i d i n e were incorporated by c e l l s i n t o p r o t e i n s i n p l a c e of h i s t i d i n e these p r o t e i n s could not be phosphorylated at a h i s t i d i n residue i f th histidin were p a r t i c i p a t i n g i n th 2-fluoro-L-histidine-containin Pineal N-Acetyltransferase:
Regulation
We have had a long standing i n t e r e s t i n an enzyme i n the p i n e a l gland, N - a c e t y l t r a n s f e r a s e (10). T h i s enzyme c a t a l y z e s the formation of N-acetylated d e r i v a t i v e s of a number of aromatic amines (10,11,12), such as N-acetyltryptamine and N - a c e t y l s e r o t o nin (N-acetyl-5-hydroxytryptamine). I t i s p h y s i o l o g i c a l l y important because the d a i l y 50-100 f o l d change i n the a c t i v i t y of t h i s enzyme r e g u l a t e s l a r g e changes i n the c o n c e n t r a t i o n of serot o n i n i n the p i n e a l gland and l a r g e changes i n the production of N - a c e t y l s e r o t o n i n and melatonin (5-methoxy-N-acetyltryptamine), the p u t a t i v e hormone of the p i n e a l gland. As i s true of the a c e t y l t r a n s f e r r e a c t i o n presented above, i t i s thought that N - a c e t y l t r a n s f e r a s e molecules i n general act through the imidazole group of h i s t i d i n e by a t t a c k on the t h i o a c e t y l group of a c e t y l CoA with the r e s u l t i n g formation of the unstable i m i d a z o l e - a c e t y l bond and the subsequent t r a n s f e r of the a c e t y l group to an acceptor amine (3). A f u r t h e r r o l e of imidaz o l e i n N - a c e t y l t r a n s f e r a s e molecules might be to accept phosphate groups, although d i r e c t evidence f o r t h i s does not e x i s t . The i n crease i n enzyme a c t i v i t y which occurs on a d a i l y b a s i s appears to depend not only upon p r o t e i n s y n t h e s i s but a l s o , i n yet an undefined way, upon the a c t i o n of adenosine 3 ,5 -monophosphate ( c y c l i c AMP). I t i s not c l e a r , however, whether c y c l i c AMP a c t i vates new enzyme molecules, as they are being synthesized, stimul a t e s the formation of new enzyme molecules, or has both a c t i o n s . We f e e l there i s some evidence to suggest that enzyme a c t i v i t y may be regulated by an a c t i v a t i o n - i n a c t i v a t i o n mechanism. T h i s i s p r i m a r i l y because N - a c e t y l t r a n s f e r a s e a c t i v i t y can be r a p i d l y destroyed, probably via enzyme i n a c t i v a t i o n (13,14). I f an a c t i v a t i o n - i n a c t i v a t i o n mechanism p l a y s a r o l e i n the r e g u l a t i o n f
f
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
40
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Table I Comparison of the c a t a l y t i c a c t i v i t y of imidazole and 2 - f l u o r o imidazole i n an a c e t y l t r a n s f e r r e a c t i o n . 14 N-[ C]Acetyltryptamine formed at 37°C (nanomoles/60 min incubation)
Reaction a d d i t i o n s ( f i n a l concentrations) 14
[ C ] A c e t y l - C o A (0.5 tryptamine (10 mM)
Λ
mM), 0.14
14
[ C ] A c e t y l - C o A (0.5 mM) , tryptamine (10 mM), imidazole (100 mM)
Λ
13.4
14
[ C ] A c e t y l - C o A (0.5 mM) tryptamine (10 mM), 2 - f l u o r o i m i d a z o l e (10
X H
Tubes were prepared by adding 25 y l volumes of 2 mM [ C ] acetyl-CoA (S.A. = 1 yCi/ymole), 40 mM tryptamine, 400 mM imid a z o l e , and 400 mM 2 - f l u o r i m i d a z o l e (9), as i n d i c a t e d above. A l l chemicals were d i s s o l v e d i n .1 M sodium phosphate b u f f e r , pH 6.9. B u f f e r was added to the r e a c t i o n tubes to b r i n g the f i n a l volume to 100 y l . Data i s given as the mean of 3 determinations which were w i t h i n 10% of the mean. -, *The i d e n t i f i c a t i o n of the N-[ C]-acetyltryptamine e x t r a c t e d i n t o chloroform was confirmed by two dimensional TLC [chloroform, methanol, g l a c i a l a c e t i c a c i d (90:10:1) followed by e t h y l ace t a t e ] ; 88.7% of the r a d i o a c t i v i t y a p p l i e d to a pre-coated s i l i c a g e l p l a t e , F-254 (Brinkman Instruments Co.) was i s o g r a p h i c with s y n t h e t i c N-acetyltryptamine. Chlorof2£m e x t r a c t i o n of a tryptamine-free r e a c t i o n c o n t a i n ing 0.5 mM [ C]acetyl-CoA and 100 mM imidazole y i e l d e d a small amount^gf r a d i o a c t i v i t y equivalent to l e s s than 0.02 nanomoles of N-[ C]acetyltryptamine. Chloroform e x t r a c t i o n of a tryptamineTfree r e a c t i o n c o n t a i n ing 100 mM 2 - f l u o r o i m i d a z o l e and 0.5 mM [ C]acetyl-CoA y i e l d e d no r a d i o a c t i v i t y .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN AND KIRK
2-Fluoro-L-Histicline
41
of N - a c e t y l t r a n s f e r a s e , i t may i n v o l v e c y c l i c AMP. As pointed out above, i t i s known that the a c t i v i t i e s of other enzymes are regulated by c y c l i c AMP (7). Furthermore, the a c t i v i t i e s of some of these enzymes a r e c l o s e l y c o r r e l a t e d with t h e i r phosphorylation state. In view of our s p e c u l a t i o n that h i s t i d i n e phosphorylation may be i n v o l v e d i n the r e g u l a t i o n of the a c t i v i t y of N - a c e t y l t r a n s f e r a s e and that h i s t i d i n e might be at the a c t i v e s i t e of Na c e t y l t r a n s f e r a s e , the study of the e f f e c t s of analogs of h i s t i dine on the a c t i v i t y of N - a c e t y l t r a n s f e r a s e became of p a r t i c u l a r interest. The E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Induction of P i n e a l N - A c e t y l t r a n s f e r a s e A c t i v i t y in vivo. A v a l u a b l e t e s t of the p r a c t i c a l use of any amino a c i d anal o g i s to determine i f i t w i l l a c t i n an i n t a c t normal animal without producing sever i n i t i a l experimental e f f o r t a c t i v i t y of N - a c e t y l t r a n s f e r a s e i n the p i n e a l gland can be stimul a t e d by catecholamines a c t i n g d i r e c t l y on the c e l l s c o n t a i n i n g t h i s enzyme (11). Predict-ably, the a c t i v i t y of t h i s enzyme can a l s o be stimulated i n the i n t a c t animal by the i n j e c t i o n of these and s i m i l a r compounds, i n c l u d i n g i s o p r o t e r e n o l (15). We found that s t i m u l a t i o n of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l was blocked by i n j e c t i o n s of 2 - f l u o r o - L - h i s t i d i n e (16). We d i d not, however, detect any acute adverse s i d e e f f e c t s of t h i s compound, nor d i d we f i n d that the i n j e c t i o n of a s i m i l a r dose of h i s t i d i n e could b l o c k the i n d u c t i o n of the enzyme. Although t h i s experiment showed that 2 - f l u o r o - L - h i s t i d i n e could act in v i v o , i t d i d not i n d i c a t e whether i t was a c t i n g d i r e c t l y on the p i n e a l gland, nor d i d i t provide any other i n f o r m a t i o n regarding the mode of a c t i o n of t h i s compound. In Vitro Studies on the Induction of P i n e a l N - A c e t y l t r a n s f e r a s e Activity. E a r l y i n our s t u d i e s on p i n e a l N - a c e t y l t r a n s f e r a s e we d i s covered that we could t r e a t p i n e a l glands i n organ c u l t u r e w i t h adrenergic agents, i n c l u d i n g a d r e n a l i n and i s o p r o t e r e n o l , and i n t h i s way i n c r e a s e the a c t i v i t y of N - a c e t y l t r a n s f e r a s e (11). T h i s provided an e x c e l l e n t model system f o r f u r t h e r s t u d i e s with 2fluoro-L-histidine. E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Adrenergic Induction of N - A c e t y l t r a n s f e r a s e A c t i v i t y . When p i n e a l glands are t r e a t e d with 2 - f l u o r o - L - h i s t i d i n e the adrenergic s t i m u l a t i o n of N - a c e t y l t r a n s f erase a c t i v i t y i s s u b s t a n t i a l l y reduced (Table I I I ) . T h i s b l o c k i n g e f f e c t of 2 - f l u o r o - L - h i s t i d i n e was seen w i t h glands which were removed from animals and t r e a t e d immediately with i s o p r o t e r e n o l and 2 - f l u o r o - L - h i s t i d i n e , or with glands which had
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
42
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Table I I In vivo i n h i b i t i o n by 2-F-HIS of the isoproterenol-induced i n crease i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y .
Exp
Group
Treatment
1
A
Isoproterenol
Β
Isoproterenol, 2-F-HIS
2
C
Control
A
Isoprotereno
D
Isoproterenol, HIS
N-Acetyltransferase A c t i v i t y (nmole/gland/hour) 12.6
+1.42
3.0
+
.58
0.2
+
.011
8.33 + 1.05
Animals (100-gram male Sprague-Dawley Rats) were deprived of food overnight. Groups Β and D were i n j e c t e d subcutaneously i n the lower back r e g i o n with a s o l u t i o n of h i s t i d i n e (HIS) or a suspension o f 2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) (250 mg/kg; 25mg/0.25 ml s a l i n e ) a t 9:00 a.m. Groups A, B, and D were i n j e c t e d sub cutaneously i n the nape of the neck with i s o p r o t e r e n o l (20 mg/kg, 2.0 mg/0.1 ml s a l i n e ) at 10:00 a.m. Groups Β and D r e c e i v e d d u p l i c a t e 2-F-HIS or HIS i n j e c t i o n s at 10:30 a.m. A l l animals were k i l l e d a t noon and t h e i r p i n e a l glands were removed r a p i d l y . Values are based on 4 glands; data are presented as the mean + S.E. (From K l e i n e t a l . , 16).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN AND KIRK
43
2~Fluoro-L-Histidine
been c h r o n i c a l l y denervated (16). Chronic denervation removes a l l n e u r a l elements from the gland; the observation that 2 - f l u o r o L - h i s t i d i n e acted i n denervated glands i n d i c a t e d that i t was a c t i n g on p i n e a l c e l l s d i r e c t l y . E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the C y c l i c AMP Induction of N - A c e t y l t r a n s f e r a s e . Catecholamines s t i m u l a t e the a c t i v i t y of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y by f i r s t s t i m u l a t i n g the production of c y c l i c AMP (10). One p o s s i b l e mechanism of a c t i o n of 2 - f l u o r o - L - h i s t i d i n e was i n h i b i t i o n of the a c t i o n of c y c l i c AMP. We evaluated t h i s by determining whether 2 - f l u o r o - L - h i s t i dine could block the s t i m u l a t i o n of N - a c e t g l t r g n s f e r a s e a c t i v i t y as caused by a d e r i v a t i v e of c y c l i c AMP, Ν ,2' d i b u t y r y l adeno s i n e 3 ,5 -monophosphate ( d i b u t y r y l c y c l i c AMP). The s t i m u l a t i o n of N - a c e t y l t r a n s f e r a s e a c t i v i t y caused by t h i s compound was block ed by 2 - f l u o r o - L - h i s t i d i n e (Table I I I ) T h i s i n d i c a t e d that 2f l u o r o - L - h i s t i d i n e was probabl sequence of events whic and f o l l o w s both the i n t e r a c t i o n of catecholamines w i t h a receptor and the production of c y c l i c AMP. f
f
S p e c i f i c i t y of 2 - F l u o r o - L - H i s t i d i n e
I n h i b i t i o n of Enzyme
Induction
Our f i n d i n g that the i n d u c t i o n of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y was blocked by 2 - f l u o r o - L - h i s t i d i n e r a i s e d the question of whether the i n h i b i t o r y e f f e c t s of t h i s compound were s p e c i f i c to t h i s enzyme, or whether the i n d u c t i o n of other enzymes could be i n h i b i t e d . To examine t h i s important question we studied the s t e r o i d i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e i n f e t a l l i v e r , the benz[a]anthracene i n d u c t i o n of a r y l hydrocarbon hydroxylase a c t i v i t y i n l i v e r c e l l s , and the spontaneous i n c r e a s e of o r n i t h i n e decarboxylase a c t i v i t y i n both mouse mammary t i s s u e and the p i n e a l gland. In a l l cases the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e were examined i n in vitro systems (16). S t e r o i d Induction of L i v e r Tyrosine Amino Transferase. The a c t i v i t y of t y r o s i n e amino t r a n s f e r a s e i n l i v e r can be stimulated by treatment with a low concentration of the s t e r o i d dexamethasone (17). We determined that the i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e a c t i v i t y i n f e t a l l i v e r expiants by s t e r o i d treatment was blocked by 2 - f l u o r o - L - h i s t i d i n e (Table IV, 16). Benz[a]anthracene Induction of L i v e r A r y l Hydrocarbon Hydroxylase A c t i v i t y . The a c t i v i t y of l i v e r a r y l hydrocarbon hydroxylase, a membrane bound enzyme, can be induced by low con c e n t r a t i o n s of a number of compounds (18), i n c l u d i n g benz[a]anthracene. Using a c e l l c u l t u r e system, i t was found that the i n d u c t i o n of the a c t i v i t y of t h i s enzyme was s u b s t a n t i a l l y blocked by 2 - f l u o r o - L - h i s t i d i n e (Table V, 16).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
1
Experiment
1.6
18.8
-HIS, 2-F-HIS, (0-11 hours); I s o p r o t e r e n o l (3-11 hours) -HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours) -HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)
None
SCGX
SCGX
2.5
10.4
-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)
+ 0.52 *
+ 3.00
+ 0.09 *
+ 2.11
3.2 + 0.56 *
16.4 + 2.74
None
-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)
None
4.9 + 0.31 *
-HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)
HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)
None
10.5 + 1.5.
0.2 + 0.03
N-Acetyltransferase A c t i v i t y (nmole/gland/hour)
None
HIS (0-11 hours); I s o p r o t e r e n o l (3-11 h r s )
None
(0-11 hours)
HIS
Treatment i n Organ C u l t u r e
None
Surgical Pretreatment
I n h i b i t i o n by 2-F-HIS of the drug-Induced increase i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y i n organ culture
Table I I I
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
-HIS (0-11 hours); d i b u t y r y l c y c l i c AMP (3-11 h r s ) -HIS, +2-F-HIS (0-11 hours); d i b u t y r y l c y c l i c AMP (3-11 hrs)
None
Treatment i n Organ C u l t u r e
None
Surgical Pretreatment
2.6 + 0.64*
22.7 + 3.43
N-Acetyltransferase A c t i v i t y (nmole/gland/hour)
Superior c e r v i c a l ganglionectomy (SCGX) was performed 14 days p r i o r t o organ c u l t u r e . P i n e a l glands were removed between 10:00 and 12:00 a.m. and placed i n t o c u l t u r e . The c o n c e n t r a t i o n o f 2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) was 3 mM; and, when present, the c o n c e n t r a t i o n of h i s t i d i n e (HIS) was 0.1 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 M HC1 t o a f i n a l c o n c e n t r a t i o n of 10 yM. D i b u t y r y l c y c l i c AMP was added i n 5 y l of c u l t u r e medium t o a f i n a l c o n c e n t r a t i o n of 1 mM. Each value i s based on 4 glands. Data a r e given as the mean + S.E. * S t a t i s t i c a l l y l e s s than glands t r e a t e d with e i t h e r i s o p r o t e r e n o l or d i b u t y r y l c y c l i c AMP Ρ < .01. S t a t i s t i c a l a n a l y s i s was performed using Student's " t " t e s t . (From K l e i n et a i . , 16).
3
Experiment
46
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Table IV The e f f e c t of 2-F-HIS on the dexamethasone i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e a c t i v i t y i n f e t a l r a t l i v e r expiants i n c u l t u r e Treatment i n c u l t u r e (hours) (0-3) (3-11)
Tyrosine amino t r a n s f e r a s e a c t i v i t y (nmole/mg protein/min)
Control
Control
Control
Dexamethasone
2-F-HIS
2-F-HIS
2-F-HIS
2-F-HIS + dexamethasone
6.5 54.2 6.6
10.4
A f t e r a 24-hour i n c u b a t i o n period with 1 mM h i s t i d i n e , the t i s s u e was t r a n s f e r r e d to f r e s h medium c o n t a i n i n g 0.1 mM h i s t i d i n e f o r the i n d i c a t e d treatments. The concentration of 2 - f l u o r o - L h i s t i d i n e (2-F-HIS) was 3 mM. Dexamethasone treatment was i n i t i a t e d by adding 5 y l of c u l t u r e medium c o n t a i n i n g dexamethasone r e s u l t i n g i n a f i n a l medium concentration of 5 yM. Each value i s the average of the means of d u p l i c a t e determinations performed on d u p l i c a t e c u l t u r e s . The d u p l i c a t e means d i d not d i f f e r by more than 3 nanomoles/mg protein/minute. (From K l e i n e t a l . , 16).
Spontaneous Increase of O r n i t h i n e Decarboxylase A c t i v i t y . O r n i t h i n e decarboxylase i s involved i n the s y n t h e s i s of p o l y amines, which appear to play a r o l e i n the process o f c e l l d i v i s i o n and gene r e g u l a t i o n (19). The a c t i v i t y of t h i s enzyme i s g e n e r a l l y high under two circumstances: f o l l o w i n g traumatic treatment o f t i s s u e , such as during regeneration of l i v e r or immediately a f t e r t i s s u e i s placed i n t o c u l t u r e , and during normal growth of t i s s u e . The a c t i v i t y of o r n i t h i n e decarboxylase increases i n both mouse mammary t i s s u e expiants (20) and i n p i n e a l glands (unpublished r e s u l t s , D. C. K l e i n and T. Oka) soon a f t e r these t i s s u e s are placed i n t o c u l t u r e . We examined the e f f e c t of 2f l u o r o - L - h i s t i d i n e on the spontaneous increase of o r n i t h i n e decarboxylase a c t i v i t y i n these systems. I t was found that 2f l u o r o - L - h i s t i d i n e d i d not block the spontaneous increase of t h i s enzyme i n the mouse mammary t i s s u e (Table VI) but d i d block i t i n the p i n e a l gland (Table V I I ) . The l a c k of an e f f e c t of 2 - f l u o r o - L - h i s t i d i n e on the increase of o r n i t h i n e decarboxylase i n the mouse mammary t i s s u e may be due
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN AND
KTRK
47
2-Fluoro-L-Histidine
Table V The e f f e c t of 2-F-HIS on the benz[a]anthracene i n d u c t i o n of a r y l hydrocarbon hydroxylase i n l i v e r hepatoma (Hepa-1) c e l l s A r y l hydrocarbon hydroxylase hydroxylase a c t i v i t y (pmoles/ ιmg protein/min)
Treatment i n C u l t u r e (hours) (0-3)
(3-11)
Control
Control
Control
Benz[a]anthracene
2-F-HIS
2-F-HIS
2-F-HIS
2-F-HIS + benz[a]anthracen
19 159 6.2
C e l l s were obtained from a stock maintained i n c u l t u r e and t r a n s f e r r e d to 15 X 60 mm Falcon dishes c o n t a i n i n g 3 ml of Waymouth MAB medium with 10% f e t a l c a l f serum f o r 72 hours u n t i l 75-80% confluency was achieved. The medium was then replaced with 2 ml of BGJ c o n t a i n i n g 0.1 mM h i s t i d i n e f o r the treatments d e t a i l e d above. The c o n c e n t r a t i o n of 2 - f l u o r o - L - h i s t i d i n e (2-FHIS) was 3 mM. Benz[a]anthracene was added i n a concentrated s o l u t i o n r e s u l t i n g i n a f i n a l c o n c e n t r a t i o n of 13 μΜ. Data are presented as the means of d u p l i c a t e determinations (18) performed on d u p l i c a t e c u l t u r e s . The i n d i v i d u a l values d i d more than 5%. (From K l e i n et al., 16).
American Chemical Society Library 1155
16th
St.
N.
W.
Washington, D. C. 20036 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BIOCHEMISTRY
48
INVOLVING C A R B O N - F L U O R I N E
BONDS
Table VI E f f e c t o f 2-F-HIS on the spontaneous increase of o r n i t h i n e decarboxylase a c t i v i t y i n midpregnant mouse mammary expiants.
Treatment Not incubated
Ornithine ^ c a r b o x y l a s e a c t i v i t y (picomoles of C 0^ produced/mg t i s s u e / h r ) 5.0
Incubated (0-3 hours) Control
41.2
+ HIS (3 mM)
33.6
+ 2-F-HIS (3 mM)
33.
Midpregnancy mouse (CH~/Hen) mammary expiants were used (20) ( 2 - f l u o r o - L - h i s t i d i n e , 2-F-HIS; h i s t i d i n e , HIS). The c u l t u r e medium used was M 199 ([HIS] - 0.175 mM). Data a r e presented as the mean enzyme a c t i v i t y i n two c u l t u r e s . Enzyme a c t i v i t y i n each c u l t u r e i s based on d u p l i c a t e determinations, which a r e w i t h i n 1% of the mean. (From K l e i n et a i . , 16).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 4.37 + 0.84
2-F-HIS (0-11 hrs) + I s o p r o t e r e n o l (3-11 hrs)
P i n e a l organ c u l t u r e s were prepared j s d e s c r i b e d i n Table I I . Each datum i s presented as the mean (+ S.E.) of 4 to 6 determinations. For the determination of o r n i t h i n e decarboxylase a c t i v i t y , i n d i v i d u a l glands were sonicated i n 100 y l of the assay b u f f e r (20) Immediately a f t e r being removed from c u l t u r e . The homogenate was stored at -20C f o r 24 hours, thawed, and t r a n s f e r r e d t o a r e a c t i o n v i a l f o r enzyme assay. The f i n a l volume of the r e a c t i o n was 125 y l and c o n t a i n e d , p y r i d o x a l phos phate (40 yM), d i t h i o t h r e i t o l (5 mM) , EDTA (4 mM) , T r i s - H C l (50 mM), and DL-[1-C ] o r n i t h i n e (20 yM, s p e c i f i c a c t i v i t y = 43 yCi/ymole). * S i g n i f l e a n t l y lower than incubated glands not t r e a t e d with 2F-HIS, ρ > 0.01. (From K l e i n e t a l . , 16).
13.24 + 4.17
C o n t r o l (0-3 hrs) + I s o p r o t e r e n o l (3-11 hrs)
34.52 + 9.5*
6.38 + 0.86
2-F-HIS (0-11 hrs)
.02
67.45+6.47
0.15 +
Ornithine^decarboxylase a c t i v i t y (picomoles of C 0^ produced/gland/hr)
C o n t r o l (0-11 hrs)
Incubated
Not incubated
Treatment
N-Acetyltransferase activity (nmols/gland/hr)
The e f f e c t of 2-F-HIS on the spontaneous i n c r e a s e of o r n i t h i n e decarboxylase a c t i v i t y i n p i n e a l glands.
Table VII
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
50
to the higher c o n c e n t r a t i o n of h i s t i d i n e i n the c u l t u r e medium. T h i s would block the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e , as discussed below. A l t e r n a t i v e l y , mammary t i s s u e may contain more h i s t i d i n e or destroy 2 - f l u o r o - L - h i s t i d i n e more q u i c k l y . General Metabolic
E f f e c t s of
2-Fluoro-L-Histidine
The observation that 2 - f l u o r o - L - h i s t i d i n e blocked the induct i o n of s e v e r a l enzymes r a i s e d the p o s s i b i l i t y that t h i s compound, although not apparently t o x i c to the i n t a c t animal, was i n f a c t h i g h l y t o x i c to t i s s u e i n general. To i n v e s t i g a t e t h i s p o s s i b i l i t y we examined the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e on s e v e r a l metabolic parameters (16). We found that a f t e r 24 hours of treatment w i t h 2 - f l u o r o - L h i s t i d i n e , RNA s y n t h e s i s was not decreased (Table V I I I ) . T h i s was remarkable i n view of th dependency of i t upon enzym s y n t h e s i s a small i n h i b i t i o due to 2 - f l u o r o - L - h i s t i d i n e was apparent immediately. T h i s i n h i b i t o r y e f f e c t d i d not, however, i n c r e a s e during the next 24 hours of treatment. E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on Hydroxyindole-O-Methyltransferase A c t i v i t y . Another t o p i c of i n t e r e s t was the e f f e c t of 2 - f l u o r o - L - h i s t i d i n e on the steady-state l e v e l s of a t h i r d enzyme i n the p i n e a l gland, hydroxyindole-O-methyl-transferase (10). I t i s g e n e r a l l y thought that the steady s t a t e l e v e l of an enzyme depends upon both the ongoing production and d e s t r u c t i o n of enzyme molecules. Thus, even though a l a r g e increase i n hydroxyindole-O-methyltransferase a c t i v i t y could not be s t u d i e d , as i n the case of the enzymes examined above, i t seemed probable that the steady-state l e v e l s of t h i s enzyme d i d i n f a c t r e f l e c t enzyme production at a r a t e equal to that of enzyme degradation. The e f f e c t of a 24-hour treatment w i t h 2 - f l u o r o - L - h i s t i d i n e on the a c t i v i t y of t h i s enzyme, which converts N - a c e t y l s e r o t o n i n to melatonin, was examined. 2 - F l u o r o - L - h i s t i d i n e d i d not a l t e r the a c t i v i t y of t h i s enzyme (Table IX). E f f e c t of 2 - F l u o r o - L - H i s t i d i n e on the I n c o r p o r a t i o n of Histidine into Protein. The l a c k of a n o n s p e c i f i c e f f e c t of 2f l u o r o - h i s t i d i n e on s e v e r a l of the metabolic parameters examined l e d us to suspect that t h i s amino a c i d analog may be a c t i n g on a s p e c i f i c mechanism, which was i n v o l v e d i n the i n d u c t i o n of a l l the enzymes we examined. Whereas i t d i d not seem that p r o t e i n s y n t h e s i s was h a l t e d by 2 - f l u o r o - L - h i s t i d i n e , i t d i d seem p o s s i b l e that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by v i r t u e of being i n c o r porated i n t o p r o t e i n i n p l a c e of h i s t i d i n e . We approached t h i s hypothesis by f i r s t determining i f the i n h i b i t i o n of enzyme i n d u c t i o n by 2 - f l u o r o - L - h i s t i d i n e was accompanied by an i n h i b i t i o n of the i n c o r p o r a t i o n of h i s t i d i n e .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN
AND
KIRK
51
2-Fluoro-L-Histidine
Table V I I I The e f f e c t of long term 2-F-HIS treatment i n organ c u l t u r e on the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o macromolecules i n p i n e a l glands incubated w i t h [ C]LEU and [ H ] u r i d i n e f o r a three hour period. R a d i o a c t i v e precursor i n TCA , insoluble material [ C]LEU [ H]Uridine (nanomoles/gland) (picomoles/gland) 14
Treatment i n Organ Culture
J
C o n t r o l (0-3 hours)
0.34
+
0.041
1.04
+
.098
2-F-HIS (0-3 hours)
0.27
+
0.031
0.87
+
.167
0.28
+
0.018*
1.30
+
.122
C o n t r o l (0-24 hours) 2-F-HIS (0-27 hours)
P i n e a l glands were removed between 10:00 and 12:00 A.M. and placed i n t o organ c u l t u r e . The c u l t u r e medium contained no HIS. The concentration-of 2-F-HIS was 3 mM. [ H ] U r i d i n e (3μΜ, S.A.= 21yCi/mole) and [ C]LEU (0.38 mM,S.A.= 8.62 yCi/ymole) were present f o r only the f i n a l three hours of the i n c u b a t i o n p e r i o d . Values, which are based on 4 glands, are computed from the S.A. of the r a d i o a c t i v e p r e c u r s o r s and the r a d i o a c t i v i t y pgr gland p r e c i p i t a t e . Data are presented as the means + S.E. Statisti c a l l y l e s s than the value f o r c o n t r o l glands incubated f o r 27 hours (p < 0.01) but not s i g n i f i c a n t l y l e s s than [ C]LEU i n c o r p o r a t i o n i n t o glands treated f o r 3 hours with 2-F-HIS. (From K l e i n et a l . , 16).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
52
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
Table
IX
Lack of an e f f e c t of long-term treatment with 2-F-HIS on a c t i v i t y of hydroxyindole-O-methyltransferase.
Treatment i n Organ Culture Not
incubated
the
Hydroxyindole-O-methyltransferase activity(picomoles/gland/hour) 69.2
+
11.1
C o n t r o l (0-24
hours)
109.2
+
19.1
2-F-HIS (0-24
hours)
132.8
+
8.4
C o n t r o l (0-48
hours)
88.8
+
6.0
2-F-HIS (0-48
hours
P i n e a l glands were removed between 10:00 and 12:00 A.M. and placed i n t o organ c u l t u r e i n medium c o n t a i n i n g 0.1 mM HIS. Each value i s based on 4 glands and i s presented as the mean (+ S.E.). The concentration of 2-F-HIS was 3 mM (from K l e i n et a l . , 16).
14 IJ^was found that i n the presence of 7 μΜ [ C ] h i s t i d i n e that [ C ] h i s t i d i n e could be incorporated i n t o p r o t e i n . More i n t e r e s t i n g , however, was the f i n d i n g that i n the presence of 2 - f l u o r o L - h i s t i d i n e t h i s i n c o r p o r a t i o n was blocked (Table X). The e f f e c t s of^glevated e x t r a c e l l u l a r concentrations of h i s t i d i n e on both [ C ] h i s t i d i n e i n c o r p o r a t i o n and i n d u c t i o n of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l were examined i n the presence of 2 - f l u o r o - L - h i s t i d i n e (Figure 1). I t was found that the i n h i b i t o r y e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e could be overcome by higher concentrations of h i s t i d i n e . This observation pointed to the explanation that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by d i r e c t l y competing with h i s t i d i n e as a substrate i n p r o t e i n synthesis. 3 I n c o r p o r a t i o n of [ Η]2-Fluoro-L-Histidine i n t o P r o t e i n A d i r e c t method of determining whether 2 - f l u o r o - L - h i s t i d i n e was a c t u a l l y being incorporated i n t o p r o t e i n was to incubate glands with r a d i o l a b e l e d 2 - f l u o r o - L - h i s t i d i n e and recover the l a b e l l e d amino a c i d from p r o t e i n . We have synthesized [ H]2f l u o r o - L - h i e t i d i n e and have found that i t i s incorporated i n t o p r o t e i n and that t h i s i n c o r p o r a t i o n can be blocked by c y c l o h e x i mide (an i n h i b i t o r of p r o t e i n synthesis) and by high concentra t i o n s of h i s t i d i n e . In a d d i t i o n we have been able to enzymatic a l l y d i g e s t l a b e l l e d p r o t e i n and recover a r a d i o a c t i v e compound which has the same chromatographic and e l e c t r o p h o r e t i c c h a r a c t e r -
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3.
KLEIN
AND
KIRK
53
2-Fluoro-L-Histidine
i s t i c s as does a u t h e n t i c ^ 2 - f l u o r o - L - h i s t i d i n e . This r a d i o l a b e l l e d compound apparently i s [ H ] 2 - f l u o r o - L - h i s t i d i n e (21). T h i s set of observations provide necessary support f o r the hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s incorporated i n t o newly synthesized p r o t e i n . A l t e r n a t i v e Mechanisms of A c t i o n of
2-Fluoro-L-Histidine
Although the above evidence i s necessary to accept the hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s incorporated i n t o p r o t e i n , t h i s f i n d i n g might be c o i n c i d e n t a l and not causative. We have thus examined other p o s s i b l e modes o f a c t i o n of 2 - f l u o r o - L - h i s t i d i n e . F i r s t l y , i t seemed p o s s i b l e that 2 - f l u o r o - L - h i s t i d i n e was a c t i v e only a f t e r decarboxylation to 2-fluorohistamine. This, however, was proven improbabl becaus 2-fluorohistamin itself was without e f f e c t on th f e r a s e a c t i v i t y (16). Secondly f l u o r o - L - h i s t i d i n e to assays of N - a c e t y l t r a n s f e r a s e d i d not i n h i b i t enzyme a c t i v i t y , e l i m i n a t i n g the p o s s i b i l i t y that 2-fluoro-Lh i s t i d i n e was a competitive or noncompetitive i n h i b i t o r of enzyme a c t i v i t y (16). T h i r d l y , we thought that 2 - f l u o r o - L - h i s t i d i n e might have induced the production o f an i n h i b i t o r of N - a c e t y l t r a n s f e r a s e a c t i v i t y . We examined t h i s p o s s i b i l i t y by adding
Molecular Pharmacology
Figure 1. The effect of histidine (HIS) on N-acetyltransf erase activity and [ C]HIS incorporation in pineal glands treated with isoproterenol and 2-fluoro-L-histidine (2-FHIS). Data was collected from 4 experiments; a different concentration of HIS was used in each. Percent inhibition is calculated from the following: 100 [isoproterenol value—(isoproterenol + 2-F-HIS value)] isoproterenol value. In each experiment a set of 4 glands was treated with 10 Μ isoproterenol and a second set of 4 glands was treated with 10 Μ isoproterenol and 3 mM 2-F-HIS (16). 14
μ
μ
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 1.17
1.65
1.43
2-F-HIS (0-11 hours)
Control (0-3); Isoproterenol (3-11 hours)
2-F-HIS (0-11 hours); Isoproterenol (3-11 hours) + .075
+ .076
+ .158
+ .071
~ [ H]LEU
-,
+ .019
C]HIS
+ .022
.126 + .005'
.41
.117 + .018*
.30
[
3.68
14.3
0.18
0.34
+
+
+
+
.548
.034
.034
.068
N-Acetyltransferase A c t i v i t y (nmole/gland/hour)
P i n e a l glands were removed between 10:00 and 12:00 and were placed into,organ c u l t u r e . The c u l t u r e medium contained 0.38 mM [ H]LEU (S.A. = 51.2 yCi/umole) and 1 mM [ C]HIS (S.A. = 24 yCi/ymole). The c o n c e n t r a t i o n of 2-F-HIS was 3 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 mM HCl to a f i n a l c o n c e n t r a t i o n of 10 yM. At the end of the experiment glands were sonicated i n 100 y l of 2 mM p e n i c i l l a m i n e i n 0.01 M sodium phosphate b u f f e r , pH 6.9, at 4°C. T h i s treatment preserves enzyme a c t i v i t y d u r i n g handling. A 50 y l sample of each s o n i c a t e was used f o r enzyme assay; a 25 y l sample was used f o r p r e c i p i t a i o n of TCA i n s o l u b l e m a t e r i a l . Values, which are based on 4-5 glands, are computed from the S. A. of the r a d i o a c t i v g amino a c i d and the r a d i o a c t i v i t y per gland p r e c i p i t a t e . ^ Data are presented as the mean + S.E. S i g n i f i c a n t l y l e s s than c o n t r o l value (P < .01). S i g n i f i c a n t l y l e s s than group t r e a t e d with i s o p r o t e r e n o l alone (P < .01) (From K l e i n et a l . , 16).
1.25
Control (0-11 hours)
Treatment i n Organ Culture
R a d i o a c t i v i t y i n TCA-insoluble m a t e r i a l (nmoles of r a d i o a c t i v e amino acid/gland)
3 E f f e c t of 2-F-HIS on r a d i o a c t i v i t y incorporated i n t o p r o t e i n of glands incubated with [ H]LEU and r\:]HIS.
Table Χ
3.
KLEIN
A N D KIRK
2-Fluoro-L-Histidine
55
homogenates of glands t r e a t e d with i s o p r o t e r e n o l and 2 - f l u o r o - L h i s t i d i n e to homogenates of glands t r e a t e d only with i s o p r o t e r e n o l (16). The r e s u l t i n g enzyme a c t i v i t y was the sum of the a c t i v i t i e s o f each homogenate, i n d i c a t i n g that an i n h i b i t o r probably was not present i n the i s o p r o t e r e n o l + 2 - f l u o r o - L - h i s t i d i n e homo genate. F i n a l l y , we added 2 - f l u o r o - L - h i s t i d i n e to glands which had already been treated with i s o p r o t e r e n o l f o r 3 hours. I f 2f l u o r o - L - h i s t i d i n e were i n a c t i v a t i n g newly formed enzyme mole c u l e s , i t would have caused a r a p i d i n a c t i v a t i o n of enzyme a c t i v ity. T h i s , however, was not observed (16). Conclusions The r e s u l t s o f our s t u d i e s i n d i c a t e that 2 - f l u o r o - L - h i s t i d i n e c e r t a i n l y i s not an a c u t e l y t o x i c compound but that i t can i n h i b i t the i n d u c t i o n of s e v e r a l without b l o c k i n RNA synthe s i s , without i n h i b i t i n out a l t e r i n g the a c t i v i t One explanation of these r e s u l t s i s that 2 - f l u o r o - L - h i s t i d i n e i s incorporated i n t o enzyme p r o t e i n , and that i n c o r p o r a t i o n r e s u l t s i n the s u b s t i t u t i o n of 2 - f l u o r o - L - h i s t i d i n e f o r h i s t i d i n e i n the primary s t r u c t u r e o f p r o t e i n s . The s u b s t i t u t i o n could block the i n d u c t i o n of enzyme a c t i v i t y by causing changes i n the s t r u c t u r e of the enzyme, by producing n o n - f u n c t i o n a l a c t i v e s i t e s i n the enzymes, or by b l o c k i n g phosphorylation o f h i s t i d i n e at a r e g u l a t o r y s i t e on the enzyme. We have not, however, shown whether i n c o r p o r a t i o n of 2f l u o r o - L - h i s t i d i n e i n t o complete enzyme p r o t e i n a c t u a l l y occurs. T h i s i s one of our f u t u r e goals. We may f i n d , a l t e r n a t i v e l y , that 2 - f l u o r o - L - h i s t i d i n e blocks the production o f complete molecules of any p r o t e i n i n which i t i s incorporated. Net i n c o r p o r t a t i o n of r a d i o l a b e l l e d l e u c i n e might appear n e a r l y normal because an increased number of p r o t e i n fragments were synthesized. I t a l s o has not been shown whether the phosphoryl a t i o n of enzyme p r o t e i n i s blocked by the s u b s t i t u t i o n of h i s t i dine with 2 - f l u o r o - L - h i s t i d i n e . T h i s and other f a s c i n a t i n g problems regarding the a c t i o n of 2 - f l u o r o - L - h i s t i d i n e are present l y being studied. The s o l u t i o n s of t h i s problem w i l l not only provide us with information regarding the a c t i o n of 2 - f l u o r o - L h i s t i d i n e , but w i l l a l s o provide b a s i c information about b i o l o g i c a l processes i n general.
LITERATURE CITED 1. Shive, W. and Skinner, C. G. "Metabolic Inhibitors, A Comprehensive Treastise" pp. 1-60, Academic Press, New York (1963). 2. Fowden, L., Lewis, D., and Tristram, Η., Adv. Enz. (1968) 29, 89-163. 3. Jencks, W. P. "Catalysis in Chemistry and Enzymology,"
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
56
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
pp 163-168, McGraw Hill, New York (1969). 4. Kirsch, J., Ann. Rev. Biochem. (1973) 42, 205-234. 5. Lehninger, "Biochemistry" pp 217-248, Worth Publishers, Inc., New York (1975). 6. Chen, C. C., Smith, D. L., Bruegger, B. B., Halpern, R. Μ., and Smith, R. Α., Biochemistry (1974) 13, 3785-3790. 7. Segal, H. L., Science (1973) 180, 25-32. 8. Langan, Τ. Α., Proc. Nat. Acad. Sci. USA (1968) 64, 12671271. 9. Kirk, K. L., Nagai, W. and Cohen, L. Α., J. Am. Chem. Soc., (1973) 95, 8389-8392. 10. Klein, D. C. "The Neurosciences: Third Study Program" pp. 509-519, MIT Press, Cambridge, Mass. (1974). 11. Klein, D. C. and Weller, J. L., J. Pharmacol. Exp. Ther. 186, 516-527. 12. Deguchi, T., J. Neuroche 13. Klein, D. C. and Weller 14. Binkley, S., Klein, D. C. and Weller, J. L., J. Neurochem. (1976) (in press). 15. Axelrod, J., Science (1974) 184, 1341-1348. 16. Klein, D. C., Kirk, K. L., Weller, J. L., Oka, T., Parfitt, Α., and Owens, I. S., Molec. Pharmacol. (1976) (in press). 17. Kenny, F. T., "Mammalian Protein Metabolism" pp 131-145, Academic Press, New York (1970). 18. Gielen, J. E., and Nebert, D. W. J. Biol. Chem. (1972) 247, 7591-7801. 19. Russel, D. Η., "Polyamines in Normal and Neoplastic Growth" Raven Press, New York (1973). 20. Oka, T. and Perry, J., J. Biol. Chem. (1976) (in press). 21. Klein, D. C., Kirk, K. L. and Weller, J. L. (manuscript in preparation).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
4 Thymidylate Synthetase: Interaction with 5-Fluoro and 5-Trifluoromethyl-2'-Deoxyuridylic A c i d DANIEL V. SANTI, ALFONSO L. POGOLOTTI, THOMAS L. JAMES, YUSUKE WATAYA, KATHRYN M. IVANETICH, and STELLA S. M. LAM Department of Biochemistry and Biophysics, and Department of Pharmaceutical Chemistry, University of California, San Francisco, Calif. 94143
Thymidylate synthetas of 2'-deoxyuridylate (dUMP) to thymidylate (TMP) with the concomitant conversion of 5, 10-methylene tetrahydrofolate (CH FAH ) to 7, 8-dihydrofolate (FAH ) as depicted in F i gure 1 (for a recent review, see reference 1). In this process the hydrogen at C-6 of FAH is directly transferred to the methyl group of TMP (2). One of our objectives over the past few years has been to establish the mechanism of catalysis of thymidylate synthetase. Extensive investigations of chemical counterparts (3-6) have in dicated that the reaction is initiated by attack of a nucleophile at the 6-position of dUMP and that many, if not all, reactions along the pathway are facilitated by analogous nucleophilic catalysis. The proposed mechanism of this enzyme, as derived from investigations of chemical models is illustrated in Figure 2. It is proposed that the reaction is initiated by attack of a nucleophilic group of the enzyme to the 6-position of dUMP. In this manner, the 5-position of dUMP could be made sufficient ly nucleophilic (viz I, Figure 2) to react with CH FAH or an equivalent reactive species of formaldehyde. Thus, the initial condensation product between dUMP and CH F A H is now generally accepted (1, 7) to be one which is covalently bound to the enzyme and saturated across the 5, 6-double bond of dUMP (II). Proton abstraction from II would give the intermediate 2
4
2
4
2
2
4
4
enolate III. As with the chemical models, III should readily undergo a β-elimination to produce the highly reactive exocyclic methylene intermediate IV and FAH , bound to the enzyme in close proximity. Intermolecular hydride transfer from FAH to IV would yield dTMP, FAH , and the native enzyme. It should be emphasized that all of the aforementioned reactions and inter mediates have direct chemical counterparts, and are in complete accord with all available biochemical data. With the availability of a stable enzyme from an amethop terin resistant strain of Lactobacillus casei (8, 9) and facile methods for its purification (9-11), we undertook studies which 4
4
2
57 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
58
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
Figure 2. Suggested sequence for the thymidylate synthetase reaction. All pyrimidine structures have a l-(5-phospho-2'-deoxyribosyl) substituent and R = CH NHC H COGlu. t
6
k
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
4.
SANTi E T A L .
Thymidylate
59
Synthetase
might p r o v i d e d i r e c t s u p p o r t f o r p r o p o s a l s w h i c h w e r e b a s e d on the a f o r e m e n t i o n e d n o n e n z y m i c m o d e l s . A l t h o u g h a n u m b e r of d i r e c t i o n s h a v e b e e n p u r s u e d t o w a r d s t h i s o b j e c t i v e , the f o l l o w i n g s u m m a r i z e s o u r i n v e s t i g a t i o n s of the i n t e r a c t i o n of t h y m i d y l a t e synthetase w i t h 5 - f l u o r o - 2 ' - d e o x y u r i d y l a t e ( F d U M P ) and 5 - t r i f l u o r o m e t h y l - 2 - d e o x y u r i d y l a t e ( C R d U M P ) . Studies of these two f l u o r i n a t e d n u c l e o t i d e s have p r o v i d e d c o n v i n c i n g e v i d e n c e for the m e c h a n i s m of this e n z y m e ; i n a d d i t i o n , they h a v e p r o v i d e d i n s i g h t into how these d r u g s act, and how the r e a c t i v i t y of f l u o r i n a t e d m o l e c u l e s m i g h t be u t i l i z e d i n the d e s i g n of e n z y m e i n h i b i t o r s . We e m p h a s i z e that a n u m b e r of other l a b o r a t o r i e s have been engaged i n s i m i l a r i n v e s t i g a t i o n s , and r e g r e t that s p a c e does not p e r m i t c o m p l e t e c i t a t i o n of a l l the e x c e l l e n t s t u d i e s p e r f o r m e d i n this a r e a . f
5 - F l u o r o - 2 ' -deoxyuridylat s o m e t i m e ( 12, 13) tha of t h y m i d y l a t e s y n t h e t a s e , but the n a t u r e of i n h i b i t i o n has b e e n the topic of c o n s i d e r a b l e c o n t r o v e r s y (_1_4). S i n c e the 6 - p o s i t i o n of 1 - s u b s t i t u t e d 5 - f l u o r o u r a c i l s i s quite s u s c e p t i b l e t o w a r d n u c l e o p h i l i c attack ( 15- 17), we s u s p e c t e d that F d U M P m i g h t e x e r t its i n h i b i t o r y effect by r e a c t i o n w i t h the p r o p o s e d n u c l e o p h i l i c c a t a l y s t of t h y m i d y l a t e s y n t h e t a s e . Studies f r o m this (18, 19) and o t h e r (20, 21 ) l a b o r a t o r i e s h a v e s i n c e d e m o n s t r a t e d this to be the c a s e . A s i m p l i f i e d d e p i c t i o n of the i n t e r a c t i o n s of F d U M P and C H ^ F A H ^ is g i v e n below i n F i g u r e 3.
E-FdUMP
Ε
E-FdUMP-CH FAH '4 2
-
ι—ι
1
^E.FdUMP.CH FAH
Figure 3
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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U s i n g the i s o t o p e t r a p p i n g m e t h o d (2_2), we have found that the two l i g a n d s , F d U M P and C H ^ F A H ^ , i n t e r a c t w i t h the e n z y m e i n a r a n d o m f a s h i o n , a n d that f o r m a t i o n of the i n i t i a l t e r n a r y c o m p l e x ( E ^ F d U M P ^ C H ^ F A H ^ ) i s at l e a s t p a r t i a l l y r a t e d e t e r mining. F r o m e q u i l i b r i u m b i n d i n g techniques we h a v e a s c e r tained that the d i s s o c i a t i o n constantes of both b i n a r y c o m p l e x e s (K^ a n d K ^ ) a r e a p p r o x i m a t e l y 10" M . T h e f i r s t t e r n a r y c o m plex which is f o r m e d with F d U M P , C H ^ F A H . and enzyme is d e p i c t e d as E - F d U M P ' C H ^ F A H ^ a n d does not i n v o l v e c o v a l e n t bonds. Interestingly, analogous r e v e r s i b l e t e r n a r y complexes m a y be f o r m e d u s i n g a n a l o g s of the c o f a c t o r . T h r o u g h s t u d i e s of the i n t e r a c t i o n of F d U M P and a n a l o g s of C H ^ F A H ^ we h a v e a s c e r t a i n e d that t h e r e i s a s t r i k i n g s y n e r g i s m i n b i n d i n g of l i g a n d s to this p r o t e i n . T h a t i s , the a f f i n i t y of e i t h e r l i g a n d f o r the cognate b i n a r y c o m p l e x i s c a . two o r d e r s of m a g n i t u d e g r e a t e r than the a f f i n i t y f o r the f r e e e n z y m e With many a n a l o g s of C H ^ F A H ^ , thi that i t m a y be p h y s i c a l l T h e s e t e r n a r y c o m p l e x e s show i n t e r e s t i n g u l t r a v i o l e t d i f f e r e n c e s p e c t r a w h i c h m a y be u s e d f o r t h e i r q u a n t i t a t i o n a n d characterization. Shown i n F i g u r e 4 a r e d i f f e r e n c e s p e c t r a o b tained w i t h C H 2 F A H ^ a n d 5, 8 - d e a z a f o l i c a c i d ; s i m i l a r d i f f e r e n c e s p e c t r a h a v e a l s o b e e n o b t a i n e d w i t h a n u m b e r of other a n a l o g s of f o l i c a c i d . C h a r a c t e r i s t i c of these d i f f e r e n c e s p e c t r a i s a peak at c a . 330 n m a n d , u s u a l l y , a t r o u g h at c a . 290 n m . A l t h o u g h the e x a c t r e a s o n f o r the s p e c t r a l changes w h i c h o c c u r upon f o r m a t i o n of the r e v e r s i b l e t e r n a r y c o m p l e x e s i s y e t u n k n o w n , we s u g g e s t that they r e s u l t f r o m p e r t u r b a t i o n s of the p a m i n o b e n z o y l g l u t a m a t e m o i e t y of the c o f a c t o r a n a l o g s w h i c h r e sult f r o m t h e i r e n v i r o n m e n t w i t h i n the t e r n a r y c o m p l e x . T h i s p e r t u r b a t i o n i s b e l i e v e d to be a m a n i f e s t a t i o n of a c o n f o r m a t i o n a l change w h i c h i s r e l a t e d to the a f o r e m e n t i o n e d s y n e r g i s m i n b i n d i n g of the two l i g a n d s . T h e d i f f e r e n c e s p e c t r u m of the E » F d U M P C H F A H c o m p l e x ( F i g u r e 4) i s s i m i l a r to those o b s e r v e d f o r the c o f a c t o r a n a l o g s , s u g g e s t i n g that s i m i l a r c h a n ges i n e n v i r o n m e n t o c c u r w i t h the n a t u r a l c o f a c t o r , C H ^ F A H . . T h e r e i s one s t r i k i n g d i f f e r e n c e i n that t h e r e i s a l o s s of d i f f e r e n t i a l a b s o r b a n c e at 269 n m i n the c o m p l e x f o r m e d w i t h C H ^ F A H ^ (18, 1 9 ) w h i c h w e h a v e not o b s e r v e d i n c o m p l e x e s f o r m e d w i t h c o f a c t o r a n a l o g s ; the r e a s o n f o r this w i l l b e c o m e a p p a r e n t i n the e n s u i n g d i s c u s s i o n . e
2
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The complex f o r m e d with thymidylate synthetase, F d U M P , and the n a t u r a l c o f a c t o r C H J A H . , h a s b e e n e x t e n s i v e l y i n v e s t i g a t e d . T h i s c o m p l e x i s e x t r e m e l y tight a n d m a y r e a d i l y be i s o l a t e d b y a v a r i e t y of t e c h n i q u e s (18, 1 9 , 21, 23, 24). Using r a d i o a c t i v e F d U M P and C H , F A H ^ , the e n z y m e h a s b e e n t i t r a t e d a n d shown to p o s s e s s two F d U M P a n d two c o f a c t o r b i n d i n g s i t e s p e r m o l e ( 1 9 ) . T h e s t o i c h i o m e t r y of b i n d i n g h a s b e e n v e r i f i e d i n a n u m b e r of l a b o r a t o r i e s b y a v a r i e t y of m e thods ( 11, 25, 26). T h i s i s i n a c c o r d w i t h the e a r l i e r f i n d i n g
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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SANTi E T A L .
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that t h y m i d y l a t e s y n t h e t a s e f r o m L . c a s e i has two a p p a r e n t l y i d e n t i c a l subunits of M W 35, 000 e a c h (9, 27). Studies of the r a t e of a s s o c i a t i o n of F d U M P w i t h the E - C H - , F A H . c o m p l e x (k ) and its d i s s o c i a t i o n (k ) have 2 4 ^ on off ' v
7
x
E-CH^AH^+f îl]FdUMP
Ε-Cr^FArJ·
r r
[hi]FdUMP
off a l l o w e d us t o ^ c ^ l c u l a t e the d i s s o c i a t i o n c o n s t a n t of the c o m p l e x to be c a . 10 M . T h i s p r o v i d e s a n e x p l a n a t i o n f o r the d i s c r e p a n c i e s i n R e v a l u e s r e p o r t e d f o r F d U M P in the l i t e r a t u r e ; n a m e l y , p r e v i o u s e x p e r i m e n t s w e r e u s i n g c o n c e n t r a t i o n s of e n z y m e h i g h e r than the and w e r e i n c o n c e n t r a t i o n r a n g e s w h e r e F d U M P was b e h a v i n g as a s t o i c h i o m e t r i c i n h i b i t o r . The kinetically determined i s a p p r o x i m a t e l y 10 - f o l d l o w e r than that f o r the b i n a r y c o m p l e x ; i n e f f e c t the p r e s e n c e of the c o f a c t o r i n c r e a s e s the 10 k c a l / m o l i n b i n d i n g t u r e of the i n t e r a c t i o n of F d U M P and t h y m i d y l a t e s y n t h e t a s e i n v o l v e s changes w h i c h o c c u r w i t h i n the bound t e r n a r y c o m p l e x . S e v e r a l l i n e s of e v i d e n c e d e m o n s t r a t e r a t h e r c o n c l u s i v e l y that a c o v a l e n t bond i s f o r m e d b e t w e e n F d U M P and t h y m i d y l a t e s y n t h e t a s e w i t h i n the c o m p l e x , (a) T h e E - ^ l F d U M P ^ C r L F A H ^ c o m p l e x m a y be t r e a t e d w i t h a n u m b e r of p r o t e i n d é n a t u r a n t s ( u r e a , g u a n i d i n e h y d r o c h l o r i d e , e t c . ) without a p p a r e n t l o s s of p r o t e i n - b o u n d r a d i o a c t i v i t y . W i t h few e x c e p t i o n s , s u c h t r e a t m e n t i s s u f f i c i e n t to d i s r u p t n o n c o v a l e n t i n t e r a c tions b e t w e e n low m o l e c u l a r w e i g h t l i g a n d s and t h e i r p r o t e i n receptors, (b) U p o n f o r m a t i o n of the c o m p l e x , t h e r e i s a d e c r e a s e of a b s o r b a n c e at 269 n m w h i c h c o r r e s p o n d s to s t o i c h i o m e t r i c l o s s of the p y r i m i d i n e c h r o m o p h o r e of F d U M P . This res u l t s t r o n g l y s u g g e s t s that the 5, 6 - d o u b l e bond of the p y r i m i dine is s a t u r a t e d i n the bound c o m p l e x , (c) T h e r a t e of d i s s o c i a t i o n of [6- H ] F d U M P f r o m the c o m p l e x shows a s e c o n d a r y t r i t i u m i s o t o p e effect ( k ^ / k ^ ) of 1.23. T h i s would c o r r e s p o n d to k £ j / k j 3 1. 15 and c l e a r l y α errions traite s that the 6 - c a r b o n of the h e t e r o c y c l e u n d e r g o e s sp to sp r e h y b r i d i z a t i o n d u r i n g the p r o c e s s as r e q u i r e d i f the 5, 6 - d o u b l e bond of F d U M P i s s a t u r a t e d i n the c o m p l e x , (d) P r o t e o l y t i c d i g e s t i o n of the c o m p l e x y i e l d s a p e p t i d e w h i c h i s c o v a l e n t l y bound to both F d U M P and C H ^ F A H ^ . T h e u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a of this p e p t i d e a r e c h a r a c t e r i s t i c of 5 - a l k y l t e t r a h y d r o f o l a t e s a n d , as w i t h the n a t i v e c o m p l e x , t h e r e i s no e v i d e n c e of u l t r a v i o l e t a b s o r p t i o n of the F d U M P c h r o m o p h o r e . k
=
F r o m t h e s e l i n e s of e v i d e n c e , together w i t h i n f o r m a t i o n g a t h e r e d f r o m m o d e l c h e m i c a l c o u n t e r p a r t s , the s t r u c t u r e of the e n z y m e ' F d U M P ' C H ^ F A H ^ c o m p l e x i s c u r r e n t l y b e l i e v e d to be as d e p i c t e d i n F i g u r e 5. H e r e , a n u c l e o p h i l e of the e n z y m e has a d d e d to the 6 - p o s i t i o n of F d U M P , and the 5 - p o s i t i o n of the p y r i m i d i n e i s c o u p l e d to the 5 - p o s i t i o n of F A H . v i a the m e t h y l e n e
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Figure 4. Dashed line: Ultraviolet difference spectra of FdUMP, CH FAH and thymidylate synthetase vs. CHgFAHi and thymidylate synthetase. Solid line: FdUMP, 5,8-deazofohte and thymidylate synthetase vs. enzyme and 5,8-deazafolate. t
Figure 5.
h
Structure of the FdUMP · CH · F A t f j thymidylate synthetase ternary complex where X represents a nucleophile of one of the enzyme amino acids t
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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g r o u p of the c o f a c t o r . A s i m i l a r structure was proposed f r o m e v i d e n c e o b t a i n e d i n d e p e n d e n t l y i n another l a b o r a t o r y (20). R e f e r r i n g to F i g u r e 5, it i s noted that the a s s i g n e d s t r u c ture f o r the E F d U M P * C H > F A r J c o m p l e x i s a n a l o g o u s to one of the p r o p o s e d s t e a d y state i n t e r m e d i a t e s of the n o r m a l e n z y m i c r e a c t i o n ( v i z II, F i g u r e 2). T h e y d i f f e r i n that II p o s s e s s e s a p r o t o n at the 5 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a b s t r a c ted i n a s u b s e q u e n t s t e p , w h e r e a s the E ' F d U M P - C H ^ F A H ^ c o m p l e x ( F i g u r e 5) p o s s e s s e s a s t a b l e f l u o r i n e at the c o r r e s p o n d i n g position. T h u s , i t a p p e a r s that F d U M P behaves as a ' q u a s i substrate f o r this r e a c t i o n . T h a t i s , it e n t e r s into the c a t a l y t i c r e a c t i o n as d e p i c t e d f o r the s u b s t r a t e d U M P i n F i g u r e 2 up to the p o i n t w h e r e a n i n t e r m e d i a t e i s f o r m e d w h i c h c a n p r o c e e d no f u r t h e r ; i n effect, a c o m p l e x i s t r a p p e d w h i c h r e s e m b l e s a steady state i n t e r m e d i a t e ( v i z II, F i g u r e 2) of the n o r m a l catalytic reaction. e
1
11
We have r e c e n t l y obtaine a n FdUMP* C r L F A H . * peptid t i o n of the F O U M P M C H ^ A H ^ • t h y m i c j v l a t e s y n t h e t a s e c o m p l e x . A s shown i n F i g u r e 6, the 94 M H z F s p e c t r u m c o n s i s t s o f a doublet of t r i p l e t s l o c a t e d 87. 2 p p m u p f i e l d of the e x t e r n a l r e f e r e n c e / t r i f l u o r o a c e t i c a c i d . O u r c u r r e n t i n t e r p r e t a t i o n of this s p e c t r u m i s as f o l l o w s : T h e d o u b l e t i s c a u s e d b y s p l i t t i n g of the F r e s o n a n c e b y the p r o t o n at the 6 - p o s i t i o n of the u r a c i l r i n g (H . ) w i t h a c o u p l i n g constant ^ of 32. 5 H z . E a c h c o m p o n e n t of m e d o u b l e t i s s p l i t f u r t h e r into a t r i p l e t ( i n t e n s i t y r a t i o (1:2:1) c a u s e d b y c o u p l i n g of the f l u o r i n e w i t h the a d j a c e n t m e t h y l e n e p r o t o n s ( H ^ ) of the c o f a c t o r w i t h the m a g n i t u d e of the c o u p l i n g constant b e i n g Λ 9 . 2 H z . T h e i n n e r m o s t l i n e s of the t r i p l e t s o v e r l a p s o the F r e s o n a n c e a p p e a r s to be a quintet w i t h i n t e n s i t y r a t i o 1:2:2:2:1. It h a s b e e n w e l l e s t a b l i s h e d that the t r i g o n a l g e o m e t r y of the c a r b o n y l a t o m s i n u r a c i l d e r i v a t i v e s s a t u r a t e d a c r o s s the 5, 6 - d o u b l e bond r e s u l t s i n a h a l f - c h a i r c o n f o r m a t i o n w i t h s u b stituents o n c a r b o n a t o m s 5 a n d 6 s t a g g e r e d (28-30) as c o m m o n l y fowjid i n c y c l o h e x a n e . The F spectrum, in conjunction with p r e v i o u s l y reported u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a l data (3J.), of the F d U M P » C H ^ F A H . * peptide y i e l d s d e f i n i t i v e e v i d e n c e f o r i t s s t r u c t u r e . T h e d o u b l e t of t r i p l e t s i m p l i e s that the f l u o r i n e - b o n d e d c a r b o n i s f l a n k e d b y C H a n d C H g r o u p s ( i . e. C H C F C P ^ ) . T h e C H , of c o u r s e , o c c u r s at the 6 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a t t a c h e d to the n u c l e o p h i l e of the e n z y m e . T h e m o s t l o g i c a l a s s i g n m e n t f o r the C H - , i s the b r i d g i n g g r o u p between the n u c l e o t i d e and c o f a c t o r a s d e p i c t e d f o r the n a t i v e c o m p l e x i n F i g u r e 5. B a s e d o n the s t a b i l i t y of the F d U M P ' C r ^ F A H . » peptide i n the a b s e n c e o f a n t i o x i d a n t s , we h a v e s u r m i s e d that the C F ^ g r o u p b r i d g e s the n u c l e o t i d e to the 5 - a n d n o t to the 1 0 - n i t r o g e n o f the cofactor. It i s p o s s i b l e to a s s i g n the s t e r e o c h e m i s t r y of the 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Figure 6.
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Fluorine-19 nmr spectrum
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FdUMP
· CH FAH t
k
· peptide
substituents w h i c h have a d d e d a c r o s s the 5, 6 - d o u b l e bond of F d U M P to g i v e the t e r n a r y c o m p l e x by c o m p a r i n g the o b s e r v e d c o u p l i n g constants w i t h those f r o m e x t e n s i v e l y s t u d i e d m o d e l s . A s shown i n F i g u r e 7, the 5 - f l u o r o and 6 - h y d r o g e n of the F d U M P * C H ^ F A H ^ * peptide a r e p r o p o s e d to be i n a t r a n s p s e u d o axial conformation. T h e e n z y m e n u c l e o p h i l e and c o f a c t o r a r e t h e r e f o r e trans_ p s e u d ο e q u a t o r i a l . T h e a d d i t i o n of a n u c l e o p h i l e of t h y m i d y l a t e s y n t h e t a s e to the 6 - p o s i t i o n of d U M P is a p r i m a r y event i n the e n z y m e - c a t a l y z e d r e a c t i o n ( F i g u r e 8). T h e r e s u l t a n t c a r b a n i o n (1) r e a c t s w i t h C H , F A H ^ to p r o d u c e a n i n t e r m e d i a t e w i t h a s t r u c t u r e analogous to tnat of the t e r n a r y c o m p l e x f o r m e d w i t h F d U M P and the c o f a c t o r (2, 3). A b s t r a c t i o n of the 5 - h y d r o g e n f o l l o w e d by a s e r i e s of steps i n v o l v i n g r e d u c t i o n of the one c a r b o n u n i t a n d e l i m i n a t i o n of the n u c l e o p h i l e r e s u l t s i n the o b s e r v e d p r o ducts of the r e a c t i o n . T h e s e steps h a v e b e e n p r e v i o u s l y d e p i c ted i n d e t a i l i n F i g u r e 2. W i t h the l o g i c a l a s s u m p t i o n that the n o r m a l e n z y m e - c a t a l y z e d r e a c t i o n o c c u r s i n a m a n n e r s i m i l a r to the f o r m a t i o n of the t h y m i d y l a t e s y n t h e t a s e « F d U M P · C r | F A H ^ c o m p l e x , s e v e r a l d e t a i l s c o n c e r n i n g the m e c h a n i s m of the n o r m a l e n z y m i c r e a c t i o n m a y be i n f e r r e d . F i r s t , the o v e r a l l s t e r e o c h e m i c a l pathway of the e n z y m e c a t a l y z e d r e a c t i o n m a y be d e d u c e d . T h e a d d i t i o n of the n u c l e o p h i l e and c o f a c t o r a c r o s s the 5, 6 - d o u b l e bond m u s t o c c u r i n a t r a n s f a s h i o n . C o n s e q u e n t l y , the s u b s e q u e n t e l i m i n a t i o n of the 5 - h y d r o g e n and the e n z y m i c n u c l e o p h i l e m u s t o c c u r as a c i s elimination .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
SANTi E T A L .
Thymidyhte
Synthetase
R
Figure 7. Stereochemical projection of the FdUMP · CH FAH · peptide as determined hy its F nmr spectrum; R = 5-phospho-2'deoxyribosyl t
h
19
Figure 8
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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S e c o n d , the s t e r e o c h e m i s t r y c o n c e r n i n g the t r a n s i e n t i n t e r m e d i a t e s shown i n F i g u r e 8 m a y be i n f e r r e d . T h e m e c h a n i s tic d e t a i l s d i s c u s s e d below f o l l o w f r o m the p r i n c i p l e that a g r o u p r e a c t i n g w i t h the π - s y s t e m of the u r a c i l h e t e r o c y c l e ap p r o a c h e s a p p r o x i m a t e l y p e r p e n d i c u l a r to the plane of the r i n g ; by m i c r o s c o p i c r e v e r s i b i l i t y , a s i m i l a r o r i e n t a t i o n i s r e q u i r e d w h e n a g r o u p d e p a r t s to r e f o r m the π - s y s t e m . T h u s , the i n i t i a l attack of the n u c l e o p h i l e of the e n z y m e at the e l e c t r o p h i l i c 6 - c a r b o n of d U M P s h o u l d be p e r p e n d i c u l a r to the plane of the heterocycle. T h e r e s u l t a n t c a r b a n i o n (1) w i l l be d e l ^ c a l i z e d throughout the c a r b o n y l g r o u p s and w i l l be h i g h i n sp c h a r a c ter. T h e a p p r o a c h of C H ^ F A H ^ to the 5 - p o s i t i o n w o u l d ^ p e r p e n d i c u l a r to the plane of the r i n g a n d , b a s e d on the F data p r e s e n t e d a b o v e , t r a n s to the n u c l e o p h i l e attached to the 6 - p o s i t i o n . A s a r e s u l t , as shown i n s t r u c t u r e 2, the c o f a c t o r w o u l d e x i s t i n a p s e u d o a x i a l p o s i t i o n , and the 5 - h y d r o g e n w o u l d be p s e u d o e q u a t o r i a l . F o the p r o t o n f r o m the 5 - p o s i t i o s i t i o n (3) p r i o r to its a b s t r a c t i o n to f o r m the c a r b a n i o n (4). This mechanistic interpretation necessitates a previously u n r e c o g n i z e d c o n f o r m a t i o n a l c h a n g e , w h i c h o c c u r s a f t e r a d d i t i o n of the c o f a c t o r but b e f o r e a b s t r a c t i o n of the 5 - p r o t o n ; and r e s u l t s i n r i n g i n v e r s i o n about the 5 - a n d 6 - p o s i t i o n s of the n u c l e o t i d e 19
i n t e r m e d i a t e s {i.e. 2-*\3). T h e r e s u l t s of F n m r studies d e s c r i b e d h e r e a r e p r e l i m i n a r y ; a d e t a i l e d n m r study of the i n t e r a c t i o n of F d U M P w i t h t h y m i d y l a t e synthetase w i l l be p u b lished elsewhere. 5 - T r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d y l a t e (C ξ d U M P ) . CEdUMP i s a potent i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e . R e y e s aria H e i d e l b e r g e r have r e p o r t e d that u p o n p r e i n c u b a t i o n C E ^ d U M P c a u s e s i r r e v e r s i b l e i n h i b i t i o n of t h y m i d y l a t e synthetase f r o m E h r l i c h A c s i t e s c e l l s (32). B a s e d on the o b s e r v a t i o n that t r i f l u o r o m e t h y l u r a c i l ( C F ~ U ) a c y l a t e s a m i n e s i n aqueous media to give u r a c i l - 5 - c a r b o x a m i d e s (33), it was s u g g e s t e d that the i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase m i g h t r e s u l t f r o m a s i m i l a r a c y l a t i o n of a n a m i n o g r o u p at o r n e a r the a c t i v e site of the e n z y m e as shown i n F i g u r e 9 (32). A q u e s t i o n that a r o s e i s why the tr i f l u o r o m e t h y l g r o u p at the 5 - p o s i t i o n of u r a c i l d e r i v a t i v e s s h o u l d be at a l l s u s c e p t i b l e to these r e a c t i o n s . T h e c a r b o n - f l u o r i n e bond i s quite s t r o n g (34) and a n outstanding c h a r a c t e r i s t i c of m o s t t r i f l u o r o m e t h y l groups is their unusual r e s i s t a n c e toward c h e m i c a l d e g r a d a t i o n . A s r e l e v a n t e x a m p l e s , it is noted that b e n z o t r i f l u o r i d e s , d e r i v a t i v e s of 6 - t r i f l u o r o m e t h y l u r a c i l s (35), d e r i v a t i v e s of 2t r i f l u o r o m e t h y l - 4 - o x o p y r i m i d i n e s (36) and 5 - t r i f l u o r o m e thy Ι ό - a z a u r a c i l (37) a r e quite stable t o w a r d h y d r o l y t i c r e a c t i o n s . In c o n t r a s t , U is r a p i d l y c o n v e r t e d into 5 - c a r b o x y u r a c i l ( C U ) (33 ) i n b a s i c m e d i a a n d , although s o m e w h a t s l o w e r , n u c l e o s i d e s of C F ^ U a r e c o n v e r t e d into the c o r r e s p o n d i n g
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
4.
SANTi E T A L .
Thymidylate
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Figure 9
n u c l e o s i d e s of C U (38-40) and C F g d U R p r o v i d e s C f r o m p y r i m i d i n e s (41). A n u m b e r of other c o m p o u n d s have been r e p o r t e d to h a v e r e a c t i v e C - F g r o u p s (see r e f e r e n c e 42). T h e r e a c t i v i t y of C - F bonds i n m o s t c a s e s has b e e n a t t r i b u t e d to h y p e r c o n j u g a t i v e e f f e c t s (43, 44), h y d r o g e n bonding effects (44, 45), and d i r e c t d i s p l a c e m e n t (S.^2) r e a c t i o n s (46). M o d e l r e a c t i o n s i n v o l v i n g the h y d r o l y s i s o f c o m p o u n d s p o s s e s s i n g C - F bonds w e r e e x a m i n e d in this l a b o t a t o r y i n an a t t e m p t to u n d e r s t a n d the u n d e r l y i n g f e a t a r e s w h i c h r e s u l t e d i n the r e a c t i v i t y of s o m e of these m o l e c u l e s (42). F r o m s u c h s t u d i e s we p r o p o s e d that C - F bond l a b i l i z a t i o n u s u a l l y i n v o l v e s one of s e v e r a l g e n e r a l m e c h a n i s m s , (a) A s d e p i c t e d i n F i g u r e 10a, p r o t o n r e m o v a l i s at an a t o m a to the c a r b o n b e a r i n g the f l u o r i n e a t o m w i t h the r e s u l t a n t negative c h a r g e , e i t h e r i n a s t e p w i s e o r c o n c e r t e d m a n n e r , a i d i n g i n the f o r m a t i o n of an i n t e r m e d i a t e (fluoro) a l k e n e . D e p e n d i n g on the s t a b i l i t y of the a l k e n e , it m a y o r m a y not react with solvent, (b) T h e p r o t o n m a y be s i t u a t e d on a n a t o m s u c h that the n e g a t i v e c h a r g e r e s u l t i n g f r o m the i o n i z a t i o n of the p r o t o n can e x e r t its i n f l u e n c e t h r o u g h an extended π - s y s t e m ( F i g u r e 10b). (c) W h e n the c o m p o u n d i s an a l l y l i c f l u o r i d e i n c a pable of u n d e r g o i n g either of the m e c h a n i s m s d e s c r i b e d a b o v e , it m a y u n d e r g o n u c l e o p h i l i c ( M i c h a e l - t y p e ) attack at the β - c a r bon w i t h a s s i s t a n c e by the d e v e l o p i n g c a r b a n i o n to give an i n t e r m e d i a t e s i m i l a r to those p r e v i o u s l y d e s c r i b e d ( F i g u r e 10c). In any of the a b o v e , t r i f l u o r o m e t h y l g r o u p s give c a r b o x y l i c a c i d s o r d e r i v a t i v e s , d i f l u o r o m e t h y l g r o u p s give a l d e h y d e s or k e t o n e s , and f l u o r o m e t h y l g r o u p s give a l c o h o l s or alkenes. M o s t of the C - F bond c l e a v a g e s thus f a r r e p o r t e d c a n be e x p l a i n e d then i n t e r m s of the a f o r e m e n t i o n e d m e c h a n i s m s ; the a b i l i t y of the c o m p o u n d s to f o r m o l e f i n i c i n t e r m e d i a t e s of the type d e s c r i b e d a p p e a r s n e c e s s a r y f o r s u c h r e a c t i o n s to o c c u r . T h e m e c h a n i s m ( s ) b y w h i c h the o l e f i n i c i n t e r m e d i a t e s a r e t r a n s f o r m e d to p r o d u c t s i s b e l i e v e d to i n v o l v e a l t e r n a t e a d d i t i o n of
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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H
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—c=Q-c—F -*• —c—cic-ί —» N u :
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Figure 10
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yl /
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Figure 11
n u c l e o p h i l e (or solvent) to the i n t e r m e d i a t e , and e l i m i n a t i o n of f l u o r i d e i o n . A p o s s i b l e m e c h a n i s m f o r h y d r o l y s i s of the C F ^ g r o u p is d e p i c t e d i n F i g u r e 11 a n d , as shown, m a y i n v o l v e the i n t e r m e d i a c y of a c y l f l u o r i d e s and k e t e n e s i n the t r a n s f o r m a tion of a t r i f l u o r o m e t h y l g r o u p to a c a r b o x y l a t e f u n c t i o n , a l though these i n t e r m e d i a t e s h a v e , as yet, not been d e t e c t e d .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
4.
SANTi E T A L .
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69
T h e above p r o v i d e d i n s i g h t into the p o s s i b l e m e c h a n i s m s by w h i c h C F ^ d U M P m i g h t a c t as an a c y l a t i n g agent. T o o b tain d i r e c t s u p p o r t i n g e v i d e n c e , the m e c h a n i s m s of h y d r o l y t i c r e a c t i o n s of 5 - t r i f l u o r o m e t h y l u r a c i l a n d its N - a l k y l a t e d d e r i v a t i v e s w e r e e x a m i n e d i n d e t a i l (47). T h e r e s u l t s of these studies a r e s u m m a r i z e d b e l o w , and d e p i c t e d i n F i g u r e s 12-14. A l l r e a c t i o n s a p p e a r to p r o c e e d by f o r m a t i o n of a h i g h l y reactive intermediate having an exocyclic difluoromethylene g r o u p at the 5 - p o s i t i o n w h i c h s u b s e q u e n t l y r e a c t s w i t h w a t e r o r h y d r o x i d e i o n i n a s e r i e s of r a p i d steps to give c o r r e s p o n ding 5 - c a r b o x y u r a c i l s . F o r those d e r i v a t i v e s w h i c h p o s s e s s an i o n i z a b l e p r o t o n at the 1 - p o s i t i o n , the p r e d o m i n a n t m e c h a n i s m i n v o l v e s i o n i z a t i o n to the conjugate b a s e and a s s i s t a n c e by the 1 - a n i o n i n the e x p u l s i o n of f l u o r i d e i o n ( F i g u r e 12). W h e n i o n i z a t i o n at the 1 - p o s i t i o n i s p r e c l u d e d by the p r e s e n c e of a n a l k y l substituent ( F i g u r e 13), a c y l a t i o n r e a c t i o n s p r o c e e d by r a t e d e t e r m i n i n g attac the n e u t r a l o r n e g a t i v e l v i d e the r e a c t i v e i n t e r m e d i a t e . In o r d e r to o b t a i n s u i t a b l e i n t r a m o l e c u l a r m o d e l s , and to v e r i f y the p r i m a r y s i t e of r e a c t i o n of 1 - s u b s t i t u t e d d e r i v a t i v e s , a s e r i e s of 1-(a>-aminoalkyl)trif l u o r o m e t h y l u r a c i l s w e r e p r e p a r e d and t h e i r h y d r o l y s e s e x a m i n e d ( F i g u r e 14). N e i g h b o r i n g g r o u p p a r t i c i p a t i o n was a p p a r e n t w h e r e attack of the a m i n o g r o u p on the 6 - p o s i t i o n of the h e t e r o c y c l e r e s u l t s i n the f o r m a t i o n of f i v e - , s i x - , and s e v e n - m e m b e r e d r i n g s ; i n the c a s e of 1 - ( 3 - a m i n o p r o p y l ) - 5 - t r i f l u o r o m e t h y l u r a c i l , apparent f i r s t - o r d e r constants w e r e m o r e than 10 t i m e s g r e a t e r than s i m p l e 1 - a l k y l d e r i v a t i v e s not possessing a neighboring nucleophile. W i t h r e g a r d to the e n z y m i c r e a c t i o n , the s a l i e n t f e a t u r e of these studies i s that the t r i f l u o r o m e t h y l g r o u p of C F ^ d U M P d e r i v a t i v e s w o u l d o n l y b e h a v e as an a c y l a t i n g agent when a s e c o n d a r y d r i v i n g f o r c e i s f u r n i s h e d by r e a c t i o n s w h i c h o c c u r at other p a r t s of the h e t e r o c y c l e . T h a t i s , it i s n e c e s s a r y that a n u c l e o p h i l e i s a d d e d to the 6 - p o s i t i o n of the h e t e r o c y c l e ; i n this m a n n e r , the n o r m a l l y i n e r t t r i f l u o r o m e t h y l g r o u p w o u l d be c o n v e r t e d into a h i g h l y r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e w h i c h m i g h t then a c y l a t e a n u c l e o p h i l i c g r o u p of the e n z y m e . In a c c o r d w i t h p r o p o s a l s f o r the i n v o l v e m e n t of n u c l e o p h i l i c c a t a l y s i s i n the e n z y m i c r e a c t i o n (viz I, F i g u r e 2), these studies l e d us to p r o p o s e a r e l a t e d m i n i m a l m e c h a n i s m f o r the r e p o r t e d i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase b y CFjdUMP. In the pathway d e p i c t e d i n F i g u r e 15, it was s u g g e s t e d that j u x t a p o s e d w i t h i n the a c t i v e s i t e , a n u c l e o p h i l i c g r o u p of the e n z y m e (:Z) adds to the 6 - p o s i t i o n of C F ~ d U M P , p r o m o t i n g the e x p u l s i o n of f l u o r i d e i o n and the f o r m a t i o n of a r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e s i m i l a r to those e n c o u n t e r e d i n o u r m o d e l s t u d i e s . The reactive interm e d i a t e w o u l d then be t r a p p e d by a n u c l e o p h i l i c g r o u p of
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Figure 13
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
4.
SANTI E T A L .
Thymidylate
Synthetase
Figure 15
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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the e n z y m e to g i v e , a f t e r a n u m b e r of s t e p s , the a c y l a t e d enzyme. Subsequent to c o m p l e t i o n of these m o d e l s t u d i e s , the i n t e r a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase was r e i n v e s tigated i n another l a b o r a t o r y u s i n g the e n z y m e f r o m L . c a s e i (21). T h e s e w o r k e r s o b s e r v e d that C F ^ U M P , C H . F A F L t h y m i d y l a t e synthetase f o r m e d a tight t e r n a r y c o m p l e x w n i c h was i s o l a t a b l e by d i s c g e l e l e c t r o p h o r e s i s u n d e r n o n - d e n a t u r i n g c o n d i t i o n s . H o w e v e r , u n l i k e the F d U M P * C H ^ F A H ^ e n z y m e c o m p l e x , no change i n the d i f f e r e n c e s p e c t r a w a s o b s e r v e d w h e n C F g d U M P was u s e d . F u r t h e r m o r e , g e l e l e c t r o p h o r e s i s i n the p r e s e n c e of a p r o t e i n d é n a t u r a n t r e s u l t e d i n a p p a r e n t d e s t r u c t i o n of the c o m p l e x . A f t e r d e n a t u r a t i o n of the c o m p l e x , the n u c l e o t i d e p r o d u c t was o b s e r v e d to m i g r a t e i d e n t i c a l l y w i t h authentic C F ^ d U M P on D E A E - c e l l u l o s e p a p e r . F r o m these r e s u l t s i t was c o n c l u d e d that C - F bonds of the n u c l e o t i d n u c l e o p h i l e of the e n z y m C F dUMP. R e c e n t r e s u l t s o b t a i n e d i n this l a b o r a t o r y a r e not i n a c c o r d w i t h t h e s e f i n d i n g s . A l t h o u g h the m e c h a n i s m of r e a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase has not b e e n a s c e r t a i n e d at this t i m e , i t a p p e a r s c e r t a i n that C - F bond c l e a v a g e i s c a t a l y z e d by the e n z y m e , p r o b a b l y v i a n u c l e o p h i l i c c a t a l y s i s . O u r p r e l i m i n a r y r e s u l t s w h i c h l e a d to this c o n c l u s i o n are s u m m a r i z e d below. C o n t r a r y to the p r e v i o u s r e p o r t (2 1), we o b s e r v e that s u b t r a c t i o n of the u l t r a v i o l e t s p e c t r u m of e n z y m e a n d C F L F A F L f r o m that of the e n z y m e , C H ^ F A H ^ a n d C F ^ d U M P p r o d u c e s a d i f f e r e n c e s p e c t r a ( F i g u r e 16a) w h i c h i s v e r y s i m i l a r to that o b s e r v e d w i t h the F d U M Ρ · C H ^ F A H ^ · e n z y m e t e r n a r y c o m p l e x ( F i g u r e 4). A s w i t h the t h y m i a y l a t e s y n t h e t a s e ^ F d U M P C H ^ F A H ^ c o m p l e x , t h e r e i s the c h a r a c t e r i s t i c i n c r e a s e of a b s o r b a n c e at 330 n m and a d e c r e a s e at 261 n m ; the l a t t e r is i n a c c o r d w i t h s a t u r a t i o n of the 5, 6 - d o u b l e bond of the n u c l e o t i d e . U p o n a d d i t i o n of s o d i u m d o d e c y l s u l f a t e , the o n l y d i f f e r e n t i a l a b s o r b a n c e i s that c h a r a c t e r i s t i c of a n u c l e o t i d e w i t h λ » 267 n m ; although this i s u p f i e l d f r o m the m a x i m u m of C F ^ d U M P , we have not y e t a s c e r t a i n e d w h e t h e r a n a l t e r a t i o n of s t r u c t u r e i s i n v o l v e d o r whether the s h i f t i s a r t i f a c t u a l . In the a b s e n c e of C H ^ F A H the i n c u b a t i o n of t h y m i d y l a t e s y n t h e t a s e and C F ^ d U M F ^ ( r a t i o 1:6) f o r 20 m i n u t e s at 2 2 ° r e s u l t s i n 89% i n a c t i v a t i o n of the e n z y m e . T h i s i s i n a c c o r d w i t h p r e v i o u s r e p o r t s on the effect of this n u c l e o t i d e on the e n z y m e f r o m E h r l i c h a s c i t e s c e l l s (32). The difference spec t r a of C F g d U M P and e n z y m e vs e n z y m e i s shown i n F i g u r e 16b. T h e m a x i m u m of C F ^ d U M P at 261 n m d e c r e a s e s , and a t r a n sient b r o a d peak a p p e a r s w h i c h has a b s o r b t i o n u p to c a . 340 n m . A f t e r 1 h o u r , the f i n a l s p e c t r u m e x h i b i t s a m a x i m a at 276 n m , r e s e m b l i n g 5 - a c y l d e r i v a t i v e s of d U M P . Paper a
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
n
d
4.
SANTI E T A L .
Thymidylate
73
Synthetase
ι 260
ι
ι 300
ι
ι 340
W A V E L E N G T H Figure 16. Ultraviolet difference spectra, (a) Dashed line: CF dUM? CH FAH and thymidylate synthetase vs. CH FAH and thymidylate synthetase. Solid line: after treatment with sodium dodecyl sulfate, (b) Dashed line: CF dUMP and thymidylate syn thetase vs. thymidylate synthetase after 20 seconds. Solid line: after 1 hr. Broken line: ultraviolet spectrum of CF dUMP. s
t
y
k
s
s
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
t
k
74
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
c h r o m a t o g r a p h y of this r e a c t i o n m i x t u r e shows a s i n g l e spot w h i c h m o v e s s l i g h t l y s l o w e r than the s t a r t i n g m a t e r i a l (C^dUMP). A l t h o u g h this p r o d u c t h a s not yet b e e n i d e n t i f i e d , i t i s not 5 - c a r b o x y - d U M P . W h e n C R d U M P was t r e a t e d w i t h a l i m i t i n g a m o u n t of t h y m i d y l a t e synthetase (50:1) f o r 23 h o u r s at 2 2 ° , we w e r e a b l e to d e t e c t that at l e a s t 0.3 e q u i v a l e n t s of F ~ w e r e r e l e a s e d , d e m o n s t r a t i n g that C - F bonds of the n u c l e o tide w e r e i n d e e d l a b i l i z e d . A l t h o u g h these r e s u l t s a r e too p r e l i m i n a r y to p e r m i t d e f i n i tive i n t e r p r e t a t i o n , c e r t a i n c o n c l u s i o n s m a y be r e a c h e d a n d s p e c u l a t i o n s m a y be f o r w a r d e d . It i s c l e a r that the i n t e r a c t i o n of C I ^ d U M P and t h y m i d y l a t e synthetase i n the a b s e n c e of c o f a c tor m a y r e s u l t i n c l e a v a g e of C - F bonds of the n u c l e o t i d e as w e l l as i n a c t i v a t i o n of the e n z y m e . F r o m the a f o r e m e n t i o n e d m o d e l s t u d i e s , it i s m o s t r e a s o n a b l e to p r o p o s e that a c t i v a t i o n of the C - F bond r e q u i r e s a d d i t i o n of a n u c l e o p h i l e of the e n z y m e tu the 6 - p o s i t i o n of th i n a c t i v a t i o n i s not k n o w n than the r e a c t i o n l e a d i n g to C - F bond c l e a v a g e . It i s a l s o a p p a r e n t that the p r e s e n c e of C H ^ F A H ^ m o d u l a t e s this r e a c t i o n i n s o m e y e t unknown m a n n e r ; tne e n z y m e * C F ^ d U M P C H 2 F A H ^ c o m p l e x h a s u l t r a v i o l e t s p e c t r a l q u a l i t i e s quite s i m i l a r to those of the c o m p l e x f o r m e d w i t h F d U M P , i n d i c a t i n g that the 5, 6 - d o u b l e bond of d U M P i s s a t u r a t e d i n the ternary complex. Further experiments are i n progress which a i m to e l u c i d a t e the m e c h a n i s m of i n t e r a c t i o n of d U M P with thymidylate synthetase. #
Literature Cited 1. Friedkin, Μ., (1973), Advan. Enzymol. 38, 235. 2. Pastore, E. J., and Friedkin, M. (1962), J. Biol. Chem. 237, 3802. 3. Santi, D. V., and Brewer, C. F. (1973), Biochemistry 12, 2416. 4. Pogolotti, A. L., and Santi, D. V. (1974), Biochemistry 13, 456. 5. Santi, D. V., and Brewer, C. F. (1968), J. Amer. Chem. Soc. 90, 6236. 6. Kalman, T.I. (1971), Biochemistry 10, 2567. 7. Benkovic, S. J., and Bullard, W. P. (1973), Prog. Bioorg. Chem. 2, 134. 8. Crusberg, T.C., Leary, R., and Kisliuk, R. L. (1970), J. Biol. Chem. 245, 5292. 9. Dunlap, R. Β., Harding, N. G. L., and Huennekens, F. M. (1971), Biochemistry 10, 88. 10. Leary, R. P., and Kisliuk, R. L. (1971), Prep. Biochem. 1, 47. 11. Galivan, J. H., Maley, G. F., and Maley, F. (1975), Bio chemistry 14, 3338.
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4.
SANTI E T A L .
Thymidylate
Synthetase
75
12. Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb, M. R., and Lichenstein, J. (1958), Proc. Nat. Acad. Sci. U. S. 44, 1004. 13. Heidelberger, C., Kaldor, G., Mukherjee, K. L., and Danenberg, P. B. (1960), Cancer Res. 20, 903. 14. Blakley, R. L. (1969). The Biochemistry of Folic Acid and Related Pteridines, New York, Ν. Υ., American Elsevier. 15. Fox, J. J., Miller, N.C., and Cushley, R.J. (1966), Tetrahedron Lett., 4927 16. Otter, B.A., Falco, Ε. A., and Fox, J.J. (1969), J. Org. Chem. 34, 1390. 17. Reist, E. J., Benitez, Α., and Goodman, L. (1964), J. Org. Chem. 29, 554. 18. Santi, D. V., and McHenry, C.S. (1972), Proc. Nat. Acad. Sci. U.S. 69, 1855. 19. Santi, D. V., McHenry, C.S., and Sommer, H. (1974), Biochemistry 13 20. Langenbach, R. J. C. (1972), Biochem. Biophys. Res. Commun. 48, 1565. 21. Danenberg, P. V., Langenbach, R. J., and Heidelberger, C. (1974), Biochemistry 13, 926. 22. Rose, I. Α., O'Connell, E. L., Litwin, S., and Bar Tana, J. (1974), J. Biol. Chem. 249, 5163. 23. Santi, D. V., McHenry, C.S., and Perriard, E.R. (1974), Biochemistry 13, 467. 24. Aull, J. L., Lyon, J. A., and Dunlap, R. B. (1973), Bio chem. Jour. 19, 210. 25. Sharma, R. Κ., and Kisliuk, R. L. (1973), Fed. Proc., Fed. Amer. Soc. Exp. Biol. 31, 591. 26. Aull, J. L., Lyon, J.A., and Dunlap, R. B. (1974), Arch. Biochem. Biophys. 165, 805. 27. Loeble, R.B., and Dunlap, R. B. (1972), Biochem. Bio phys. Res. Commun. 49, 1671. 28. Rouillier, P., Delmau, J., and Nofre, C. (1966), Bull. Soc. Chim. (France), 3515. 29. Furburg, S., and Janson, L. H. (1968), J. Amer. Chem. Soc. 90, 470. 30. Katritzky, A. R., Nesbit, M.R., Kurtev, B. J., Lyapova, Μ., and Pojarlieff, I. G. (1969), Tetrahedron 25, 3807. 31. Wahba, A. J., and Friedkin, M. (1961), J. Biol. Chem. 236, PC 11. 32. Reyes, P., and Heidelberger, C. (1965), Mol. Pharma col. 1, 14. 33. Heidelberger, C., Parsons, D. G., and Remy, D. C. (1964), J. Med. Chem. 7, 1. 34. Pauling, L. (1960), The Nature of the Chemical Bond. Ithaca, Ν. Υ., Cornell University Press, 85. 35. Giner-Sorolla, Α., and Bendich, A. (1958), J. Amer. Chem. Soc. 80, 5744. 36. Barone, J.A. ( 1963), J. Med. Chem. 6, 39.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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37. Dipple, Α., and Heidelberger, C. (1966), J. Med. Chem. 9, 715. 38. Shen, T. Y., Ruyle, W. V., and Lewis, Η. M. (1965), J. Org. Chem. 30, 835. 39. Khwaja, Τ. A., and Heidelberger, C. (1969), J. Med. Chem. 12, 543. 40. Ryan, K. J., Acton, Ε. Μ., and Goodman, L. (1966), J. Org. Chem. 31, 1181. 41. Heidelberger, C., Boohar, J., and Kampschroer, D. (1965), Cancer Res. 25, 377. 42. Sakai, Τ. Τ., and Santi, D. V. (1973), J. Med, Chem. 1079. 43. Roberts, J. D., Webb, R. L., and McElhill, E. A. (1950), J. Amer. Chem. Soc. 72, 408. 44. Filler, R., and Novar, H. (1960), Chem. Ind. (London), 1273. 45. Jones, R. (1947) 46. Nestler, H. J., an 57, 1117. 47. Santi, D. V., and Sakai, T. T. (1971), Biochemistry 10, 3598.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5 The Effect of Aliphatic Fluorine on Amine Drugs RAYW.FULLERandBRYANB.MOLLOY The Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Ind. 46206
Fluorine is th (Table I), and its presence in a molecule can greatly affect the ionization of acids and bases (2). The withdrawal of electrons toward fluorine on a carbon atom adjacent to a carboxyl carbon or an amino-bearing carbon increases the carboxyl group's ability to re lease a proton and decreases the amine's ability to accept a proton. The carboxylic acid becomes a stronger acid, and the amine becomes a weaker base. For example, the pK of ethylamine is >10 compared to 5.7 forβ,β,β-trifluoroethylamine(3). We have studied some sympathomimetic amine drugs having reduced basicity due to fluorine substituted on the β carbon. The influence of ionization on the ac tivity of sympathomimetic amines has previously been considered (4), but with the exception of compounds having substituents directly on the nitrogen, no such amines having pK values below physiological pH appear to have been available (5). An advantage of the β,βdifluoro compounds is that the substitution is in a position of the molecule that is not a major site of metabolic attack nor a site known to be involved in binding to physiological receptors, and the small size of the fluorine atoms (Table I) results in minimal steric alteration in the molecule. Thus the changes in the pharmacological properties of the amines as a result of β,β-difluoro substitution probably are due to the change in ionization. In solution an amine exists in the following equilibrium, a
a
-H+ RNH3+
^
V
RNH2
+H+
77 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
78
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CARBON-FLUORINE
BONDS
the p o s i t i o n o f t h e e q u i l i b r i u m depending on t h e p K o f t h e amine and t h e pH o f t h e s o l u t i o n . When t h e pH i s e q u a l t o t h e p K , 50% o f t h e drug m o l e c u l e s a r e p r o t o n a t e d ( c a t i o n i c ) and 50% a r e n o n p r o t o n a t e d ( n e u t r a l ) . When t h e pH i s one u n i t below t h e p K , j u s t o v e r 90% o f t h e m o l e c u l e s e x i s t i n t h e c a t i o n i c form, and when the pH i s two u n i t s below t h e p K more t h a n 99% o f t h e m o l e c u l e s a r e charged. I n t h i s paper we compare some p r i m a r y amines w i t h p K v a l u e s w e l l above p h y s i o l o g i c a l pH t o t h e i r 3 , 3 - d i f l u o r o d e r i v a t i v e s h a v i n g p K v a l u e s below p h y s i o l o g i c a l pH. The p a r e n t amines a r e n e a r l y c o m p l e t e l y c a t i o n i c whereas t h e 3 , 3 - d i f l u o r o derivat i v e s e x i s t p r e d o m i n a n t l y as n e u t r a l m o l e c u l e s a t p h y s i o l o g i c a l pH. Presumably as a r e s u l t o f t h i s charge d i f f e r e n c e , t h e p h a r m a c o l o g i c p r o p e r t i e s o f t h e d r u g s — b o t h t h e b i o l o g i c a l f a t e and t h e b i o l o g i c a l actions of the d r u g s — a r a
a
a
a
a
a
TABLE Fluorine
Atom
in
Van
Relation
der radius,
to
Waals
I Other
Halogen
Atoms
1
Electronegativity value
0
A
F
1.
35
4. 0
Cl
1.
80
3. 0
Br
1.95
I
2.
H
1.2
1
Data
from
2.8 15
2.5 2. 1
(1).
Amphetamine The e f f e c t on i o n i z a t i o n o f amphetamine produced by t h e p r e s e n c e o f one o r two f l u o r i n e atoms on t h e 3 carbon i s shown i n T a b l e I I . A s i n g l e f l u o r i n e atom reduces t h e p K v a l u e by more than one pH u n i t . The p K o f t h e monofluoro d e r i v a t i v e o f amphetamine i s s t i l l above p h y s i o l o g i c a l pH, however, so t h a t most o f a
a
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
Aliphatic
AND MOLLOY
79
Fluorine
the monofluoro compound i s i o n i z e d a t p h y s i o l o g i c a l pH. More t h a n 99% o f t h e m o l e c u l e s o f amphetamine e x i s t as c a t i o n s a t p h y s i o l o g i c a l pH. In c o n t r a s t , t h e d i f l u o r o compound has a p K below p h y s i o l o g i c a l pH and thus would e x i s t m a i n l y as a n e u t r a l m o l e c u l e r a t h e r t h a n as a c a t i o n i n t h e body. a
TABLE Effect
of ^ Fluorine Ioni zation
οf
II Substution on Amphetamine Distribution
Compound
at RNH
2
\ \=y
the
of Ion ic pH 7. 4
Forms
RNH
z+
y-CH CHNH
1
2
2
CH
3
(/
\=/
X \=y
y-CHCHNH
1 1
F
CH
8.
35
10
90
73
27
2
y-CF CHNH 2
2
1
6.97
CH
3
The i n t e r a c t i o n o f amphetamine w i t h b i o l o g i c a l macromolecules ought t o be p r o f o u n d l y a f f e c t e d by t h e f l u o r i n e s u b s t i t u t i o n and a t t e n d a n t i o n i c changes, s i n c e t h e t r a n s p o r t and enzymic systems t h a t determine d i s t r i b u t i o n and m e t a b o l i s m o f t h e drug s h o u l d a c t d i f f e r e n t l y on a n e u t r a l u n p r o t o n a t e d amine compared to a c a t i o n . A number o f s t u d i e s on t h e i n t e r a c t i o n o f amphetamine and i t s f l u o r i n a t e d d e r i v a t i v e s w i t h enzymes and o t h e r p r o t e i n s i n v i t r o and o f t h e pharma c o l o g i c c h a r a c t e r i s t i c s o f t h e s e drugs i n v i v o have borne o u t t h e e x p e c t e d a l t e r a t i o n s i n p r o p e r t i e s o f amphetamine r e s u l t i n g from f l u o r i n e s u b s t i t u t i o n . In V i t r o I n t e r a c t i o n s . β,β-Difluoro substitution i n c r e a s e s t h e a c t i v i t y o f amphetamine as an i n v i t r o s u b s t r a t e f o r l u n g N - m e t h y l t r a n s f e r a s e (6) o r l i v e r microsomal deaminase (Ί). On t h e o t h e r H a n d , β , β -
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
80
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E
BONDS
d i f l u o r o s u b s t i t u t i o n d e c r e a s e s t h e a c t i v i t y o f amphet amine as an i n h i b i t o r o f m i t o c h o n d r i a l monoamine o x i dase (Table I I I ) . L i k e w i s e , β , β - d i f l u o r o s u b s t i t u t i o n d i m i n i s h e s t h e b i n d i n g o f amphetamine t o b o v i n e serum albumin (Table I I I ) . Thus r e d u c i n g t h e p K o f amphet amine by β , β - d i f l u o r o s u b s t i t u t i o n can e i t h e r enhance or i n h i b i t i t s i n t e r a c t i o n s with v a r i o u s b i o l o g i c a l macromolecules i n v i t r o . a
TABLE Effect
of &jβ-Difluoro Amphetamine : and Binding
of
III
Substitution on Monoamine Oxidase to Bovine Serum
Two
Properties Inhibition Albumin
Drug
Amphetamine ft,
β-Difluoroamphetamine
3.
23
82
2.
56
45
MAO inhibition was measured with rat liver mito chondrial enzyme and ^C'-tryptamine as substrate. The pl50 value is the negative log of the molar concentra tion of inhibitor producing 50% inhibition. Binding to 4% bovine serum albumin in pH 7.4 sodium phosphate buf fer was measured with 10 micromolar drug concentration; after f i l t r a t i o n through Aminco Centriflo u l t r a f i l t r a tion cones drug levels were assayed colorimetrically with methyl orange.
In V i v o P r o p e r t i e s . The e f f e c t o f β - f l u o r i n e sub s t i t u t i o n on t h e d i s t r i b u t i o n o f amphetamine among v a r i o u s body t i s s u e s a f t e r d r u g a d m i n i s t r a t i o n t o ex p e r i m e n t a l a n i m a l s was s t r i k i n g . F i g u r e 1 shows t h e t i s s u e d i s t r i b u t i o n o f amphetamine, β-fluoroamphetamine, and β , β - d i f l u o r o a m p h e t a m i n e i n r a t s one hour a f t e r t h e drugs were i n j e c t e d i n t r a p e r i t o n e a l l y a t e q u i m o l a r doses. H i g h e s t t i s s u e l e v e l s o f amphetamine were i n l u n g , and l o w e s t l e v e l s were i n t h e f a t , a l l t i s s u e s h a v i n g h i g h e r l e v e l s than b l o o d . The r e l a t i v e d i s t r i b u t i o n o f t h e monofluoro d e r i v a t i v e was s i m i l a r , l e v e l s i n a l l t i s s u e s e x c e p t l u n g b e i n g about t h e same as t h o s e o f amphetamine. T h i s s i m i l a r i t y i s e x p e c t e d s i n c e t h e monofluoro compound, l i k e amphetamine, i s m o s t l y p r o t o n a t e d a t p h y s i o l o g i c a l pH. I n marked con t r a s t was t h e d i s t r i b u t i o n o f t h e d i f l u o r o d e r i v a t i v e .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
Aliphatic
A N D MOLLOY
81
Fluorine
H i g h e s t l e v e l s o f t h i s d r u g were i n f a t , t h e t i s s u e t h a t c o n t a i n e d l o w e s t l e v e l s o f t h e o t h e r two amines. L e v e l s o f t h e d i f l u o r o compound i n o t h e r t i s s u e s were c o n s e q u e n t l y reduced; f o r example, t h e l e v e l s i n b r a i n were o n l y about o n e - f o u r t h t h o s e o f amphetamine. This d i s t r i b u t i o n a t one hour i s s i m i l a r t o t h a t seen a t o t h e r times ( 7 ) , i . e . t h e r e i s l i t t l e r e d i s t r i b u t i o n o f drug and t K e r a t e o f d i s a p p e a r a n c e o f t h e drugs from a l l t i s s u e s i s about t h e same. S i n c e t h e predominant p h a r m a c o l o g i c e f f e c t s o f amphetamine r e s u l t from i t s a c t i o n on t h e b r a i n , we compared r e g i o n a l d i s t r i b u t i o n o f amphetamine and 3 , 3 d i f l u o r o a m p h e t a m i n e i n b r a i n (Table I V ) . The drugs were g i v e n a t doses chosen t o produce comparable whole brain levels. Some d i f f e r e n c e s i n d i s t r i b u t i o n among the v a r i o u s anatomi region noted but thes not as g r e a t as t h v a r i o u s o r g a n s . Th amphetamine i n b r a i n was n o t s i g n i f i c a n t l y a l t e r e d by d i f l u o r o s u b s t i t u t i o n (1) . TABLE Regional Distribution 3, β-Dif'luoroamphetamine
Brain (% of
Total
Region Brain
IV of
Amphetamine in Rat
Drug
Level,
hemispheres (62-67%)
Cerebellum Midbrain Brain
(14-17%) (8-10%)
stem
Hypothalamus
nanomoles/g
Weight) Amphetamine
Cerebral
and Brain
(6-10%) (3-4%)
3> $-Difluoro amphetamine
46
± 4
57
± 2
18
± 6
33
±
54
± 4
92
± 6
65
± 20
71
±
86
±
84
± 6
11
Drugs were injected i . p . (amphetamine at 10 3,3-difluoroamphetamine at 40 mg/kg) 1 hour rats were killed. Mean values and standard 5 rats per group are shown.
mg/kg, before errors
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
3
7
the for
82
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
J u s t as t h e t i s s u e d i s t r i b u t i o n o f amphetamine i s changed by β, β - d i f l u o r o s u b s t i t u t i o n , so a l s o i s t h e metabolism o f t h e drug changed. A l t h o u g h t h e h a l f l i v e s o f amphetamine and β , β - d i f l u o r o a m p h e t a m i n e a r e about t h e same i n r a t s , t h e pathways o f m e t a b o l i s m f o r the two drugs a r e d i f f e r e n t (Table V ) . Amphetamine i s m e t a b o l i z e d i n t h e r a t p r e d o m i n a n t l y by h y d r o x y l a t i o n on t h e p a r a p o s i t i o n o f t h e a r o m a t i c r i n g , whereas β , β - d i f l u o r o a m p h e t a m i n e appears n o t t o be m e t a b o l i z e d by p a r a - h y d r o x y l a t i o n a t a l l (7). Instead the d i f l u o r o compound i s m e t a b o l i z e d r a p i d l y by o x i d a t i v e d e a m i n a t i o n (7). T i s s u e l e v e l s o f amphetamine and i t s h a l f - l i f e a r e i n c r e a s e d by agents l i k e d e s m e t h y l i m i p r a mine t h a t i n h i b i t a r o m a t i c h y d r o x y l a t i o n , whereas t i s sue l e v e l s o f β , β - d i f l u o r o a m p h e t a m i n e a r e i n c r e a s e d by t y p i c a l i n h i b i t o r s o f microsomal enzymes l i k e SKF 525A and DPEA {7)· Inductio by c h r o n i c phénobarbita the r a t e o f removal o f β , β - d i f l u o r o a m p h e t a m i n e from t i s s u e s a f t e r i t i s i n j e c t e d i n t o r a t s b u t has no e f f e c t on t h e r a t e o f removal o f amphetamine (0). β , β - D i f l u o r o s u b s t i t u t i o n increases the a c t i v i t y of amphetamine as a s u b s t r a t e f o r microsomal deaminases, an e f f e c t t h a t can be shown i n v i t r o . Apparently the d i f l u o r o s u b s t i t u t i o n abolisKes the a c t i v i t y of amphetamine as a s u b s t r a t e f o r t h e a r o m a t i c hydroxyl a t i n g system, though t h i s system i s d i f f i c u l t t o study i n v i t r o . The f a i l u r e o f t h e d i f l u o r o compound t o be H y d r o x y l a t e d i l l v i v o may be due t o one o r b o t h of the f o l l o w i n g reasons: (a) i t i s more r e a d i l y deaminated t h a n i s amphetamine, (b) i t may be l e s s r e a d i l y h y d r o x y l a t e d t h a n amphetamine. TABLE Effect of &,$-Difluoro Amphetamine Metabolism
Drug
Half-life in Brain
V Substitution in the
Rat
Major
Metabolic
Amphetamine
1.δ
hrs
Ring
β, $-Difluoro amphetamine
1.5
hrs
Oxidative
on
Route
hydroxylation deamination
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
AND
MOLLOY
Aliphatic
Fluorine
83
3,3-Difluoroamphetamine has been shown t o i n c r e a s e locomotor a c t i v i t y i n mice t o the same e x t e n t as amphetamine (9), though a p p r o x i m a t e l y f o u r t i m e s h i g h e r doses o f the 3 i f l u o r o compound have t o be g i v e n . L i k e w i s e about f o u r times h i g h e r doses o f t h e d i f l u o r o compound a r e r e q u i r e d t o produce e q u i v a l e n t drug l e v e l s i n b r a i n compared t o amphetamine, so t h e i n t r i n s i c a b i l i t y o f t h e d i f l u o r o compound t o cause CNS s t i m u l a t i o n seems about e q u a l t o t h a t o f amphetamine. In mice, t h e h a l f - l i f e o f t h e d i f l u o r o compound (0.3 hours) i s much s h o r t e r than i s t h a t o f amphetamine (0.9 h o u r s ) . A p p a r e n t l y t h i s d i f f e r e n c e i s r e l a t e d t o the f a c t t h a t mice have t h e enzymic machinery t o metabo l i z e amphetamine by o x i d a t i v e d e a m i n a t i o n (10), and s i n c e t h e d i f l u o r o d e r i v a t i v e i s a much b e t t e r s u b s t r a t e f o r d e a m i n a t i o n i t i s m e t a b o l i z e d much more r e a d i l y i n mice t h a of amphetamine and 3 , 3 s i m i l a r r e s u l t s i n mice and i n r a t s . One use t h a t has been made o f 3 , 3 - d i f l u o r o a m p h e t amine i s as a t o o l t o e l u c i d a t e i n t e r r e l a t i o n s h i p s among the v a r i o u s a c t i o n s o f amphetamine. Gessa, C l a y and B r o d i e (11) had s u g g e s t e d t h a t the h y p e r t h e r m i a f o l l o w i n g ampEetamine i n j e c t i o n i n t o r a t s was a d i r e c t consequence o f t h e e l e v a t i o n o f plasma f r e e f a t t y acids. We found t h a t 3 , 3 - d i f l u o r o a m p h e t a m i n e injected at an e q u i m o l a r dose e l e v a t e d f r e e f a t t y a c i d s t o t h e same e x t e n t as amphetamine but produced no h y p e r t h e r m i a (12), showing t h a t t h e s e two e f f e c t s o f amphetamine were c o m p l e t e l y d i s s o c i a b l e by v i r t u e o f d i f l u o r o substitution. Presumably the h y p e r t h e r m i c response t o amphetamine i s due t o an a c t i o n i n b r a i n , hence 3*3d i f l u o r o a m p h e t a m i n e produces h y p e r t h e r m i a o n l y when i t i s i n j e c t e d a t higher doses. Another d i f f e r e n c e between amphetamine and 3,3d i f l u o r o a m p h e t a m i n e i s t h e i n a b i l i t y o f t h e l a t t e r drug to cause d e p l e t i o n o f n o r e p i n e p h r i n e l e v e l s i n b r a i n and h e a r t (Table V I ) . H i g h doses o f amphetamine have l o n g been known t o d e p l e t e n o r e p i n e p h r i n e i n t h e s e t i s s u e s (13). We gave t h e d i f l u o r o compound a t f o u r times the~cTose o f amphetamine t o produce e q u i v a l e n t drug l e v e l s i n b r a i n and h e a r t but s t i l l found no reduction of norepinephrine l e v e l s . The p r e c i s e mechanism(s) by which amphetamine lowers n o r e p i n e p h r i n e l e v e l s i s s t i l l u n c e r t a i n , but h y d r o x y l a t e d m e t a b o l i t e s may p l a y a r o l e i n t h i s e f f e c t . The f a i l u r e o f 3/3d i f l u o r o a m p h e t a m i n e t o lower n o r e p i n e p h r i n e c o u l d t h e n be e x p l a i n e d because i t i s not m e t a b o l i z e d by hydroxylation.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
84
BIOCHEMISTRY
TABLE Inability
of
VI
β,ë-Difluoroamphetamine
Amphetamine-Like
Treatment
INVOLVING C A R B O N - F L U O R I N E BONDS
Depletion
of
to Tissue
Norepinephrine Heart
Group
Cause Norepinephrine
Levels,
vg/g Brain
Control
0.89±.05
dl-Amphetamine 0.1 mmole/kg
0.58±.01
(P<.001)
0.351.01
dl-&,&-Difluoroamphetamine 0.4 mmole/kg
0.91±.06
(ns)
0.41±.03
Drugs were injected killed. Mean values group are shown.
i.p. and
0.40±.01
6 hours standard
before errors
(P<.05)
(ne)
the rats were for 5 rats per
Phenethylamine The i o n i z a t i o n o f phenethylamine i s a f f e c t e d by β - f l u o r i n e s u b s t i t u t i o n i n much t h e same way as t h a t o f amphetamine (Table V I I ) . A s i n g l e f l u o r i n e reduces t h e p K , and a second f l u o r i n e f u r t h e r reduces t h e p K t o below p h y s i o l o g i c a l pH. a
a
TABLE Effect
of Fluorine Ionization of
VII Substitution PhenethyI
on amine
Distribution pK
Compound
at
a
of pH
the
Ionic 7.4
RNH2
CH2CH2NH2
Ο
CHCH 2NH 2
Forms RNH3+
99
9.55
8. 20
13
87
6. 75
82
18
I
F
^~^-CF2CH2NH2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
AND
MOLLOY
Aliphatic
Fluorine
85
Figure 1.
Tissue distribution of amphetamine in rats as affected by fluorine substitution on the β carbon. Drugs were injected i.p. at 0.1 mmole/kg 1 hr before groups of 5 rats were killed. Mean values and standard errors are shown (7).
Figure 2. Effect of β fluorine atoms on the activity of phenethylamine as an enzyme substrate. The phenethulamines were compared at 4 mM with the N-methyltransjerase and at 1 mM with monoamine oxidase.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
86
BIOCHEMISTRY
INVOLVING CARBON-FLUORINE
BONDS
In V i t r o I n t e r a c t i o n s . The e f f e c t o f β - f l u o r i n e s u b s t i t u t i o n on the a c t i v i t y o f phenethylamine as a s u b s t r a t e f o r two enzyme systems i n v i t r o i s shown i n F i g u r e 2. A d d i t i o n o f one and two f l u o r i n e atoms p r o g r e s s i v e l y r e d u c e d t h e a c t i v i t y o f phenethylamine as a s u b s t r a t e f o r m i t o c h o n d r i a l monoamine o x i d a s e b u t p r o g r e s s i v e l y i n c r e a s e d a c t i v i t y as a s u b s t r a t e f o r l u n g N - m e t h y 1 t r a n s f e r a s e . The l a t t e r enzyme p r o b a b l y i s n o t i m p o r t a n t i n phenethylamine metabolism i n v i v o but i l l u s t r a t e s t h a t l o w e r i n g t h e p K o f t h e amine can i n c r e a s e as w e l l as d e c r e a s e i t s a f f i n i t y f o r enzymes. The a b i l i t y o f monofluoro and d i f l u o r o phenethylamines t o i n h i b i t t h e o x i d a t i o n o f 14c-phenethylamine by t h r e e p r e p a r a t i o n s o f monoamine o x i d a s e i s shown i n F i g u r e 3. There was a s u b s t a n t i a l d i f f e r e n c e i n t h e i n h i b i t o r y a c t i v i t y o f t h e two compounds and t h i s d i f f e r e n c e appeared t o r e s u l t e n t i r e l values. P l o t t i n g percen c o n c e n t r a t i o n o f p r o t o n a t e d form o f t h e two i n h i b i t o r s r e v e a l e d t h a t t h e p o i n t s f e l l a l o n g t h e same l i n e ; t h i s f i n d i n g s u g g e s t s t h a t t h e RNH3+ form i s t h e a c t i v e i n h i b i t o r o f monoamine o x i d a s e and t h a t t h i s form o f b o t h compounds has e q u a l a f f i n i t y f o r t h e enzyme. a
In V i v o P r o p e r t i e s . Phenethylamine i t s e l f was v e r y r a p i d l y degraded by monoamine o x i d a s e when i t was i n j e c t e d i n t o a n i m a l s , b u t t h e d i f l u o r o d e r i v a t i v e was l e s s r e a d i l y d e s t r o y e d . I t s l e v e l s were h i g h e r t h a n those o f phenethylamine i n a l l t i s s u e s , and t h e t i s s u e d i s t r i b u t i o n o f t h e d i f l u o r o compound resembled v e r y much t h a t o f β , β - d i f l u o r o a m p h e t a m i n e ( 1 4 ) . Thus d i f l u o r o s u b s t i t u t i o n on phenethylamine l e d t o g r e a t e r b i o l o g i c a l s t a b i l i t y whereas d i f l u o r o s u b s t i t u t i o n on amphetamine d e c r e a s e d b i o l o g i c a l s t a b i l i t y a t l e a s t i n some s p e c i e s . Figure 4 i l l u s t r a t e s t h i s d i f f e r e n c e i n the e f f e c t o f d i f l u o r o s u b s t i t u t i o n on i n v i t r o metabolism o f phenethylamine and amphetamine E y l i v e r homogenates. The metabolism b e i n g measured i s d e a m i n a t i o n i n b o t h c a s e s ; microsomal enzymes a r e r e s p o n s i b l e f o r t h e d e a m i n a t i o n o f amphetamine, whereas m i t o c h o n d r i a l monoamine o x i d a s e i s r e s p o n s i b l e f o r the d e a m i n a t i o n o f p h e n e t h y l a m i n e . β , β - D i f l u o r o a m p h e t a m i n e was more r a p i d l y d e s t r o y e d than amphetamine, b u t β , β - d i f l u o r o p h e n e t h y l a m i n e was l e s s r a p i d l y destroyed than phenethylamine. In r a t s , t h e a d d i t i o n a l metabolic route of para-hydroxylation o p e r a t e s on amphetamine i n v i v o , so i n f a c t amphetamine i s d e s t r o y e d a t about t h e same r a t e as d i f l u o r o a m p h e t amine i n r a t s b u t more s l o w l y i n mice. Phenethylamine
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
Aliphatic
A N D MOLLOY
(aj Soluble MAO (rat]
10
(b) Mitochononal MAO (rat)
100 1000 10 100 Micromolar concentration of inhibitor
/ 10
100
1000
87
Fluorine
1000
(c) Mitochondrial MAO (human)
10
100
/ 10
100
Micromolar concentration of RNH
3
+
1000
1000
/ 10
100
1000
form of inhibitor
Figure 3 Inhibition of three monoamine oxidase preparations by β-monofluorophenethylamine (dots) and bu β,β-difluoro-phenethylamine (half circles). The substrate was phenethylamine (.2 mM). Total inhibitor concentration is shown at the top, and the concentration of the protonated form of the inhibitor (pH 7.4) is shown at the bottom. Monoamine oxidase was solubilized from rat liver mitochondria (a), or in tact liver mitochondria were used as enzyme source (b) and (c).
(a)
lb)
minutes Figure 4. Effect of β,β-difluoro substitution on the in vitro degra dation of (a) amphetamine and (b) phenethylamine by rat liver homogenates. The amines were added at .125 mM concentrations to liver homoge nates.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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INVOLVING C A R B O N - F L U O R I N E BONDS
i s d e s t r o y e d more r a p i d l y t h a n d i f l u o r o p h e n e t h y l a m i n e b o t h i n mice and i n r a t s . In mice, t h e c e n t r a l s t i m u l a n t a c t i v i t y o f 3 , 3 d i f l u o r o p h e n e t h y l a m i n e becomes a p p a r e n t a t lower doses than w i t h phenethylamine i t s e l f , b u t i n mice p r e t r e a t e d w i t h an i n h i b i t o r o f monoamine o x i d a s e t h e c o n v e r s e i s t r u e (14). Two f a c t o r s a r e i n v o l v e d : the greater b i o l o g i c a l s t a b i l i t y o f t h e d i f l u o r o compound and t h e more f a v o r a b l e ( f o r b r a i n ) t i s s u e d i s t r i b u t i o n o f phenethylamine i t s e l f . I n normal mice t h e m e t a b o l i c d i f f e r e n c e s a r e more i m p o r t a n t and so t h e d i f l u o r o compound i s more a c t i v e as a s t i m u l a n t than i s p h e n e t h y l amine. When t h e m e t a b o l i c d i f f e r e n c e s a r e e l i m i n a t e d by i n h i b i t i o n o f t h e enzyme r e s p o n s i b l e (monoamine o x i d a s e ) , then phenethylamine has more s t i m u l a n t a c t i v i t y t h a n t h e d i f l u o r o compound because o f t h e l a t t e r s tendency t b r a i n and o t h e r o r g a n s 1
p-Chloroamphetamine p-Chloroamphetamine i s a drug o f s p e c i a l i n t e r e s t because o f i t s s e l e c t i v e e f f e c t s on s e r o t o n i n neurons in brain. p-Chloroamphetamine l e a d s t o a r a p i d d e c r e a s e i n t h e l e v e l s o f s e r o t o n i n and i t s major metabolite, 5-hydroxyindoleacetic a c i d , i n b r a i n . In the r a t and some o t h e r s p e c i e s , p-chloroamphetamine leads further to c y t o t o x i c d e s t r u c t i o n o f serotonin neurons as i n d i c a t e d by r e m a r k a b l y l o n g - l a s t i n g d e c r e a s e s i n parameters s p e c i f i c a l l y a s s o c i a t e d w i t h s e r o t o n i n neurons i n b r a i n ( t r y p t o p h a n h y d r o x y l a s e , s e r o t o n i n and 5 - h y d r o x y i n d o l e a c e t i c a c i d l e v e l s ; h i g h a f f i n i t y s e r o t o n i n uptake) (15,16) and by d i r e c t h i s t o l o g i c e v i d e n c e ( 1 7 ) . TEïïs i t was o f i n t e r e s t t o study t h e d i f l u o r o d e r i v a t i v e o f p-chloroamphetamine. The r e d u c t i o n o f t h e p K o f p-chloroamphetamine by 3 , 3 - d i f l u o r o s u b s t i t u t i o n i s shown i n T a b l e V I I I . The t i s s u e d i s t r i b u t i o n o f p-chloroamphetamine, which resembles t h a t o f amphetamine i t s e l f , was a l t e r e d by the d i f l u o r o s u b s t i t u t i o n . 3,3-Difluoro-p-chloroamphetamine l o c a l i z e d t o an even g r e a t e r e x t e n t i n f a t than d i d 3 , 3 - d i f l u o r o a m p h e t a m i n e , a p p a r e n t l y t h r o u g h a c o n t r i b u t i o n o f the c h l o r i n e t o the o v e r a l l l i p o p h i l i c i t y o f t h e m o l e c u l e i n a d d i t i o n t o t h e reduced i o n i z a t i o n due t o t h e d i f l u o r o s u b s t i t u t i o n (18). The h a l f - l i f e o f t h e d i f l u o r o d e r i v a t i v e was l e s s t h a n t h a t of p-chloroamphetamine i n r a t b r a i n (IS) , presumably because o f more f a c i l e d e a m i n a t i o n o f t h e d i f l u o r o compound. When h i g h doses o f t h e d i f l u o r o compound were i n j e c t e d t o produce drug l e v e l s i n b r a i n a
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
A N D MOLLOY
Aliphatic TABLE
Effect
of β Ionization
89
Fluorine VIII
^-Difluoro of
Substitution on p-Chloroamphetamine Distribution
Compound
pK
at
a
of pH
the
Ionic 7.4
Forms
RNH2
p-Chloroamphetamine
SK3
ë>,ë>-Difluoro-pChloroamphetamine
6.8
RNH3+
1
99
80
20
e q u i v a l e n t t o those t o n i n and 5 - h y d r o x y i n d o l e a c e t i were d e p l e t e d t o about t h e same e x t e n t as by p - c h l o r o amphetamine a t 6 hours (Table I X ) . However, t h e r e was TABLE Brain Levels
Drug
Serotonin and after Injection or its β,β-Di
IX
5-Hydroxyindoleaoetic Acid of p-Chloroamphetamine fluoro Derivative
Treatment
Serotonin
5HIAA % of
Control
p-Chloroamphetamine (0.1 mmole/kg) 6 24
hrs
46
± 3 *
54
+
2*
hrs
43
± 3 *
50
t
2*
3*
β, ë-Difluorop-chloroamphetamine (0.4 mmole/kg) 6
hrs
44
±
1*
56
±
24
hrs
97
t
6
95
t
^Significant
drug
effect,
P<.01
(5
rats/group).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
2
90
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
a d i f f e r e n c e i n d u r a t i o n o f the a c t i o n of the two compounds. With the d i f l u o r o d e r i v a t i v e , b r a i n 5 - h y d r o x y i n d o l e l e v e l s had r e t u r n e d t o normal w i t h i n 24 hours (Table I X ) . On the o t h e r hand, p-chloroamphetamine lowers b r a i n 5 - h y d r o x y i n d o l e s not o n l y 24 hours (Table IX) but f o r s e v e r a l months (15,.16) a f t e r a single injection. T h i s l o n g - l a s t i n g efïect appears t o be due t o a t o x i c a c t i o n o f p-chloroamphetamine on s e r o t o n i n neurons (17). Thus the d i f l u o r o compound has the same s h o r t - t e r m e f f e c t as p-chloroamphetamine on b r a i n s e r o t o n i n but l a c k s the n e u r o t o x i c e f f e c t of the p a r e n t d r u g . Whether t h i s d i f f e r e n c e i s due t o the i n a b i l i t y o f the d i f l u o r o compound t o be c o n v e r t e d t o a n e u r o t o x i c m e t a b o l i t e as happens w i t h p-chloroamphetamine o r to some o t h e r f a c t o r s cannot be known a t present. The i n i t i a l l o w e r i n p-chloroamphetamine amine depends on the a c t i v e t r a n s p o r t o f t h o s e drugs i n t o s e r o t o n i n neurons, s i n c e t h e i r e f f e c t s are b l o c k e d by i n h i b i t i o n o f t h a t a c t i v e uptake (19). Continual r e u p t a k e o f p-chloroamphetamine i s n e c e s s a r y f o r the d e p l e t i o n o f s e r o t o n i n t o be m a i n t a i n e d , s i n c e t r e a t ment w i t h an uptake i n h i b i t o r a t e a r l y t i m e s a f t e r p-chloroamphetamine i n j e c t i o n can r e v e r s e the depletion of s e r o t o n i n (20). Thus one p o s s i b i l i t y i s t h a t the d i f l u o r o compound i s not so e f f i c i e n t l y r e - t a k e n up by the membrane pump on the s e r o t o n i n neuron. Consistent w i t h t h i s p o s s i b i l i t y . i s the r e c e n t f i n d i n g (D. T. Wong and F. P. Bymaster, u n p u b l i s h e d data) t h a t the d i f l u o r o compound has o n l y about o n e - t e n t h of the a f f i n i t y f o r the s e r o t o n i n uptake pump as does p-chloroamphetamine (determined by t h e i r r e l a t i v e a b i l i t y t o i n h i b i t h i g h a f f i n i t y s e r o t o n i n uptake i n t o synaptosomes i l l v i t r o ) . O t h e r Amines We have been i n t e r e s t e d i n a p p l y i n g the p K lower i n g t h r o u g h β , β - d i f l u o r o s u b s t i t u t i o n t o amine drugs whose l o c a l i z a t i o n i n a d i p o s e t i s s u e would be e x p e c t e d t o enhance t h e i r p h a r m a c o l o g i c a l a c t i o n s , i . e . whose t a r g e t t i s s u e would be f a t . One l i n e o f i n v e s t i g a t i o n has d e a l t w i t h agents that i n h i b i t l i p o l y s i s . N i c o t i n i c a c i d (I) lowers serum f r e e f a t t y a c i d l e v e l s and i s used i n the t r e a t ment o f h y p e r l i p o p r o t e i n e m i a (21). One of the s i d e e f f e c t s t h a t l i m i t s the u s e f u l n e s s o f n i c o t i n i c a c i d i s f l u s h i n g due t o v a s o d i l a t a t i o n (21) . We knew t h a t 3 - a m i n o m e t h y l - p y r i d i n e (II) had a n t i l i p o l y t i c a c t i v i t y l i k e n i c o t i n i c a c i d b o t h i n v i t r o and i n v i v o ; the a
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
A N D MOLLOY
Aliphatic
91
Fluorine
CF2CH2NH2
•CH2CH2NH2
Ν
III
IV
amine i s m e t a b o l i z e t i n i c a c i d i n v i v o , so i t s i n v i v o a c t i v i t y i s due almostly e n t i r e l y to n i c o t i n i c a c i d . T h i s amine t h e n o f f e r s no advantages o v e r n i c o t i n i c a c i d as an a n t i l i p o l y t i c drug. We thought t h a t s u b s t i t u t i o n o f f l u o r i n e s t o reduce t h e pKa o f an amine o f t h i s s o r t might a c h i e v e two o b j e c t i v e s : ( 1 ) r e t a r d the o x i d a t i v e deamination o f t h e amine and ( 2 ) cause t h e amine t o l o c a l i z e p r e f e r e n t i a l l y i n adipose t i s s u e . The u l t i m a t e purpose would be t o reduce s i d e e f f e c t s w h i l e r e t a i n i n g t h e a n t i l i p o l y t i c a c t i v i t y of n i c o t i n i c acid. Though t h e p r e c i s e mechanisms o f f l u s h i n g caused by n i c o t i n i c a c i d are n o t known, i t seemed l i k e l y t h a t an amine r a t h e r t h a n an a c i d — a n d i n p a r t i c u l a r an amine t h a t was con c e n t r a t e d m o s t l y i n f a t — m i g h t be f r e e o f t h i s s i d e effect. To approach t h i s o b j e c t i v e , i t was f i r s t n e c e s s a r y to lengthen the side chain o f 3-aminomethylpyridine t o provide a s i t e f o r f l u o r i n e s u b s t i t u t i o n . 3-Aminoe t h y l - p y r i d i n e ( I I I ) was s y n t h e s i z e d and found t o have a c t i v i t y as an a n t i l i p o l y t i c agent i n v i v o and i n v i t r o ; t h e a c t i v i t y i n v i t r o i n d i c a t e s t h a t t h e amine i t s e l f i s a c t i v e and does n o t r e q u i r e c o n v e r s i o n t o t h e acid. F i n a l l y t h e β , β - d i f l u o r o d e r i v a t i v e o f 3-aminoe t h y l - p y r i d i n e (IV) was s y n t h e s i z e d and s t u d i e d as an a n t i l i p o l y t i c drug. Some méthodologie d i f f i c u l t i e s were e n c o u n t e r e d i n measuring drug l e v e l s , b u t semiq u a n t i t a t i v e d a t a i n d i c a t e t h e drug was l o c a l i z e d t o some e x t e n t (though perhaps n o t as much as expected) i n adipose t i s s u e . However, a c t i v i t y as an a n t i l i p o l y t i c agent was n o t enhanced. A p p a r e n t l y t h e d i f l u o r o
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
92
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
s u b s t i t u t i o n d i d not a d e q u a t e l y r e t a r d the o x i d a t i v e d e a m i n a t i o n o f t h i s compound. Furthermore the s u b c e l l u l a r l o c a l i z a t i o n o f the drug may not have been p r o p e r f o r optimum a n t i l i p o l y t i c a c t i o n . N i c o t i n i c a c i d may a c t on the a d e n y l c y c l a s e c o n t a i n e d i n the c e l l membrane (22), whereas the d i f l u o r o s u b s t i t u t e d amine may be p r e 3 o m i n a n t l y l o c a l i z e d i n s i d e the a d i p o c y t e such t h a t the c o n c e n t r a t i o n a t the p r e c i s e s u b c e l l u l a r s i t e o f a c t i o n i s not h i g h e r t h a n t h a t o f n i c o t i n i c acid. Whatever the e x p l a n a t i o n , the a p p l i c a t i o n o f pK r e d u c t i o n t h r o u g h d i f l u o r o s u b s t i t u t i o n was not s u c c e s s f u l i n t h i s instance. We have a l s o s t u d i e d 3 , 3 - d i f l u o r o s u b s t i t u t e d N - c y c l o p r o p y 1 - p h e n e t h y l a m i n e s , compounds which are i r r e v e r s i b l e i n h i b i t o r s o f monoamine o x i d a s e . Monoamine o x i d a s e i n h i b i t o r s have been used c l i n i c a l l y t o t r e a t mental d e p r e s s i o pharmacologic a c t i o t h a t has p o t e n t i a l c l i n i c a l a p p l i c a t i o n i n v o l v e s a d i p o s e t i s s u e as a t a r g e t o r g a n . Stock and Westermann (2_3) have shown t h a t monoamine o x i d a s e i n h i b i t o r s e l e v a t e n o r e p i n e p h r i n e l e v e l s i n adipose t i s s u e . The n o r e p i n e p h r i n e i s presumably c o n t a i n e d i n a d r e n e r g i c nerve endings t h a t c o n t r o l the c y c l i c A M P - a c t i v a t e d l i p a s e i n the f a t c e l l s and c o n s e q u e n t l y the r a t e o f l i p o l y s i s . Exposure t o c o l d l e a d s t o i n c r e a s e d m o b i l i z a t i o n o f f a t depots and e l e v a t i o n o f plasma f r e e f a t t y a c i d l e v e l s . In r a t s whose a d i pose t i s s u e n o r e p i n e p h r i n e l e v e l s had been i n c r e a s e d by monoamine o x i d a s e i n h i b i t i o n , exposure t o c o l d produced a g r e a t e r i n c r e a s e i n plasma f r e e f a t t y a c i d s than o c c u r r e d i n c o n t r o l r a t s (23) . Of c o u r s e the use of monoamine o x i d a s e i n h i b i t o r s would have e f f e c t s i n the b r a i n and o t h e r organs i n n e r v a t e d by the a d r e n e r g i c system as w e l l , so one could not s e l e c t i v e l y a f f e c t f a t m o b i l i z a t i o n i n t h i s way. We wondered i f the r e d u c t i o n o f p K v a l u e s by 3/3d i f l u o r o s u b s t i t u t i o n might enhance the l o c a l i z a t i o n o f the monoamine o x i d a s e i n h i b i t o r i n f a t and thus make i t s e f f e c t s somewhat s e l e c t i v e , m a x i m i z i n g enhanced l i p i d m o b i l i z a t i o n w h i l e m i n i m i z i n g CNS e f f e c t s and e f f e c t s on the c a r d i o v a s c u l a r system. The p o s s i b l e use o f l i p o l y t i c agents i n t r e a t i n g o b e s i t y by m o b i l i z i n g f a t depots has l o n g been considered. The i d e a of u s i n g a drug t o enhance endogenous l i p o l y t i c s t i m u l i as opposed t o a drug t h a t d i r e c t l y caused l i p o l y s i s seemed e s p e c i a l l y i n t e r e s t i n g . Thus we have p r e p a r e d 3 , 3 - d i f l u o r o - N - c y c l o p r o p y l - p c h l o r o p h e n e t h y l a m i n e and s t u d i e d i t s p r o p e r t i e s as an i n h i b i t o r of monoamine o x i d a s e . N - C y c l o p r o p y l amines a
a
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
FULLER
A N D MOLLOY
Aliphatic
93
Fluorine
o f t h i s s o r t have l o n g been known t o i n h i b i t monoamine o x i d a s e i r r e v e r s i b l y (24). The 3 , β - d i f l u o r o compounds, l i k e t h e p a r e n t N - c y c l o p r o p y l a m i n e s , were found t o i n h i b i t t h e enzyme i n a manner t h a t was n o t r e v e r s i b l e by d i a l y s i s . The 3 / 3 - d i f l u o r o d e r i v a t i v e had t h e e x p e c t e d lower p K v a l u e (Table X ) . However when we i n j e c t e d t h e drugs i n t o r a t s and measured monoamine o x i d a s e i n h i b i t i o n i n v a r i o u s t i s s u e s , we d i d n o t observe any s e l e c t i v i t y i n t h e i n h i b i t i o n o f t h e enzyme i n f a t (Table XI). A g a i n méthodologie d i f f i c u l t i e s have kept us from o b t a i n i n g adequate d a t a on d i s t r i b u t i o n o f t h e d r u g s , but we can assume from d a t a on s i m i l a r drugs t h a t t h e d i f l u o r o compound would l o c a l i z e i n f a t . Why then was i t n o t a more e f f e c t i v e i n h i b i t o r o f monoamine o x i d a s e in this tissue? P o s s i b l y t h e monoamine o x i d a s e i n t h e fat tissue i s locate much g r e a t e r e x t e n t drug i s l o c a l i z e d c h i e f l y i n t h e a d i p o c y t e s . F o r whatever reason, s e l e c t i v e i n h i b i t i o n o f adipose t i s s u e monoamine o x i d a s e was n o t a c h i e v e d . The 3 / 3 - d i f l u o r o s u b s t i t u t e d monoamine o x i d a s e i n h i b i t o r s may n o n e t h e l e s s have i n t e r e s t i n g uses as pharmacological t o o l s . The f l u o r o s u b s t i t u e n t s might p e r m i t NMR s t u d i e s t h a t would r e v e a l something o f t h e n a t u r e o f t h e a c t i v e s i t e o f monoamine o x i d a s e t o which the i n h i b i t o r i s i r r e v e r s i b l y bound. a
TABLE Ionization
X
of N-Cy and its & &-Difluoro
alopropyl-4-ohlorophenethylamine Derivative
3
At Compound
Pa K
8.
pH
7.
4
y
RNH2
3
20
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
%
as
RNH
80
3+
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Control Drug
ControI Drug
ControI
Liver
Brain
Adrenals
ControI Drug
Fat
were injected activity in
ControI Drug
Heart
Drugs oxidase
Control Drug
Kidneys
Drug
Group
Tissue
Inhibition
i.p. tissue
Monoamine
at
H
61
60
± 1 ± 1 ± 1 ± 1
+
1
hour before was assayed
59
62
± 5 ± 1
± 1
51
± 2 ± 1
1
=
Inhibition
R
59
50 mg/kg homogenates
17 7
25 10
57 22
106 40
107 52
1,
in
Rat
the with
In_
F
62
60
46
55
± 5 ± 2 ± 1 ± 1 ± 2 ± 1 ± 1 ± 1
Monoamine substrate.
63
± 1 ± 1
+
69
Inhibition
R =
6
± 23
2.
rats were killed. ^-^C-tryptamine as
24 11
25 13
51 20
109 42
114 42
Vivo
Activity
Experiment
450 137
MAO
Tissues
Cl^~^-CR2CH2NH<^
Oxidase
XI
± 21 ± 15
Activity
Experiment
513 213
MAO
of
TABLE
5.
FULLER
AND
Aliphatic
MOLLOY
95
Fluorine
Summary S u b s t i t u t i o n o f one o r two f l u o r i n e atoms on t h e 8 carbon o f a r y l a l k y l a m i n e drugs markedly reduced the pK o f the amines. Monofluoro s u b s t i t u t i o n reduced the p K more than 1 pH u n i t , but the p K was s t i l l above p h y s i o l o g i c a l pH. In g e n e r a l , monofluoro s u b s t i t u t i o n l e d t o s m a l l changes o r no change i n p h a r m a c o l o g i c p r o p e r t i e s o f the d r u g s . Difluoro substitution further reduced the p K t o below p h y s i o l o g i c a l pH. Thus a t p h y s i o l o g i c a l pH the d i f l u o r o compounds a r e m o s t l y n e u t r a l whereas p a r e n t amines a r e n e a r l y c o m p l e t e l y cationic. Presumably as a r e s u l t o f t h i s e f f e c t on pK , d i f l u o r o s u b s t i t u t i o n markedly a l t e r e d the p r o p e r t i e s o f amphetamine and p h e n e t h y l a m i n e s . As a r e s u l t o f d i f l u o r o s u b s t i t u t i o n amphetamine became a b e t t e r s u b s t r a t whereas phenethylamin m i t o c h o n d r i a l monoamine o x i d a s e i r i v i t r o ; amphetamine was bound l e s s r e a d i l y t o albumin; amphetamine and phenethylamine became b e t t e r s u b s t r a t e s f o r l u n g N - m e t h y l t r a n s f e r a s e ; amphetamine, phenethylamine and p-chloroamphetamine were d i s t r i b u t e d d i f f e r e n t l y i n mouse and r a t t i s s u e s , l o c a l i z i n g l e a s t i n f a t w i t h o u t d i f l u o r o s u b s t i t u t i o n b u t most i n f a t w i t h t h a t s u b s t i t u t i o n ; amphetamine metabolism i n the r a t was s h i f t e d from p a r a - h y d r o x y l a t i o n t o o x i d a t i v e deaminat i o n ; the metabolism o f d i f l u o r o a m p h e t a m i n e but not amphetamine i n r a t s was a c c e l e r a t e d by phénobarbital p r e t r e a t m e n t t o i n d u c e microsomal enzymes i n l i v e r ; amphetamine h y p e r t h e r m i a i n r a t s and l o c o m o t o r s t i m u l a t i o n i n mice d e c r e a s e d p r o p o r t i o n a l t o drug l e v e l s i n b r a i n ; amphetamine l o s t the a b i l i t y t o d e p l e t e h e a r t and b r a i n n o r e p i n e p h r i n e even a t h i g h doses; p-chloroamphetamine s a b i l i t y t o lower b r a i n s e r o t o n i n l e v e l s i n i t i a l l y was n o t a l t e r e d e x c e p t t o the e x t e n t t h a t drug l e v e l s i n b r a i n were lower, but the neurot o x i c e f f e c t o f p-chloroamphetamine on s e r o t o n i n neurons was l o s t . These r e s u l t s i l l u s t r a t e the importance o f i o n i z a t i o n i n the i n t e r a c t i o n o f amine drugs w i t h b i o l o g i c a l macromolecules b o t h i n v i t r o and i r i v i v o . Fluorine s u b s t i t u t e d near the n i t r o g e n , because o f i t s s t r o n g e l e c t r o n e g a t i v i t y , can reduce the b a s i c i t y o f t h e amino group t o the e x t e n t t h a t i t s i o n i z a t i o n a t p h y s i o l o g i c a l pH i s s h a r p l y r e d u c e d . F l u o r i n e s u b s t i t u t i o n thus a l t e r s many o f the p h a r m a c o l o g i c p r o p e r t i e s o f such amine drugs. a
a
a
a
a
1
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Acknowledgments We thank D r s . W. S. M a r s h a l l , W. N. Shaw, and R. E . Toomey f o r t h e i r c o l l a b o r a t i o n i n t h e s t u d i e s on t h e p y r i d i n e compounds. We a l s o a r e g r a t e f u l f o r the t e c h n i c a l a s s i s t a n c e o f Kenneth L. Hauser i n t h e s y n t h e s i s o f f l u o r i n a t e d d e r i v a t i v e s and o f H a r o l d D. Snoddy, B e t t y W. Roush, and John C. Baker i n t h e biological studies.
Literature Cited 1. Pauling, L. "The Nature of the Chemical Bond" p. 93 & 260, 3rd ed., Cornell University Press, Ithaca, 1960. 2. Loncrini, D. F. and Filler, R., Advances in Fluorine Chemistr 3. Henne, A. L. an (1955) 77, 1901-1902. 4. Lewis, G. P., Brit. J. Pharmacol. (1954) 9, 488493. 5. Vree, Τ. Β., Muskens, A. Th. J. Μ., and van Rossum, J. Μ., J. Pharm. Pharmacol. (1969) 21, 774-775. 6. Fuller, R. W. and Roush, B. W., Res. Comm. Chem. Pathol. Pharmacol. (1975) 10, 735-738. 7. Fuller, R. W., Molloy, Β. B., and Parli, C. J. In "Psychopharmacology, Sexual Disorders, and Drug Abuse" pp. 615-624, Avicenum Press, Prague, 1973. 8. Fuller, R. W., Parli, C. J., and Molloy, Β. B. Biochem. Pharmacol. (1973) 22, 2059-2061. 9. Fuller, R. W., Molloy, Β. B., Roush, B. W. and Hauser, K. L., Biochem. Pharmacol. (1972) 21, 1299-1307. 10. Dring, L. G., Smith, R. L., and Williams, R. T., Biochem. J. (1970) 116, 425-435. 11. Gessa, G. L., Clay, G. Α., and Brodie, Β. B., Life Sci. (1969) 8, 135-141. 12. Fuller, R. W., Shaw, W. Ν., and Molloy, Β. B., Arch. Int. Pharmacodyn. (1972) 199, 266-271. 13. Moore, Κ. E., J. Pharmacol. Exptl. Therap., (1963) 142, 6-12. 14. Fuller, R. W., Snoddy, H. D., Molloy, Β. B., and Hauser, K. L., Psychopharmacologia (1973) 28, 205-212. 15. Sanders-Bush, Ε., Bushing, J. Α., and Sulser, F., Eur. J. Pharmacol., (1972) 20, 385-388. 16. Fuller, R. W. and Snoddy, H. D., Neuropharmacol., (1974) 13, 85-90.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
5.
17. 18. 19. 20. 21. 22. 23. 24.
FULLER
AND
MOLLOY
Aliphatic
97
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Harvey, J. A., McMaster, S. E., and Yunger, L. Μ., Science (1975) 187, 841-843. Fuller, R. W., Snoddy, H. D., and Molloy, Β. B. J. Pharmacol. Exptl. Therap. (1973) 184, 278-284. Fuller, R. W. and Molloy, Β. B., Adv. Biochem. Psychopharmacol. (1974) 10, 195-205. Fuller, R. W., Perry, K. W., and Molloy, Β. B., Eur. J. Pharmacol. (1975) 33, 119-124. Levy, R. I. and Frederickson, D. S., Postgrad. Med. (1970) 47, 130-136. Peterson, M. J., Hillman, C. C. and Ashmore, J . , Mol. Pharmacol. (1968) 4, 1-9. Stock, K. and Westermann, E. O., J. Lipid Res., (1963) 4, 297-304. Mills, J . , Kattau, R., Slater, I. H. and Fuller, R. W., J. Med Chem (1968) 11 95-97
Question and Answer Period Q. Have you prepared any ring-hydroxylated compounds?
A. No. We particularly wanted Ç> Ç>-difluoro-phydroxy amphetamine but have not been able to synthesize it. There may be a problem of stabil ity with compounds having both a hydroxy I and -CF2R group on the ring. Q. Is fluoride ion a metabolite of these difluoro compounds? y
A.
We do not know whether any of the fluorine is removed metabolically.
Q. About the possibility of changing the basicity of the nitrogen in the difluoro compounds—have you looked at the effect of the 3,3-difluoro substituent on transport across biological membranes? A.
Mostly in indirect ways. I think the large change in tissue distribution produced by the 3,3difluoro substitution i s a manifestation of altered transport across various biological membranes in the complex system of the whole animal. Dr. David Wong has looked at the ability of 3*3difluor ο-p-chlorο amphetamine to inhibit the trans port of monoamines across synaptosomal membranes in vitro; the difluoro compound is less effective as an inhibitor than is p-chloroamphetamine itself.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Q.
Do you know what t h e p r o d u c t s a r e from o x i d a t i v e d e a m i n a t i o n o f t h e β , β - d i f l u o r o compounds?
A.
Dr. John Parti has isolated the oxime> the and the alcohol as in vitro metabolites of difluoroamphetamine. He has found that the of difluorophenylacetone i s formed as a in vivo in rats and is excreted in urine in and conjugated form.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
ketone, βoxime metabolite free
6 Metabolic and Transport Studies with Deoxyfluoro-monosaccharides N.F.TAYLOR,A.ROMASCHIN,andD.SMITH Department of Chemistry, University of Windsor, Ontario N9B 3P4 Canada
The rational fluorocarbohydrates and related compounds as probes for the study of enzyme specifity, carbohydrate metabolism and transport in biological systems has been elaborated in a previous symposium (1). Such compounds have now been used to study carbohydrate metabolism in yeast cells (2), Ps. fluorescens (3) and E. coli (4); enzyme specificity of glycerol kinase (5), yeast hexotinase (6), phosphoglucomutase and UDPG pyrophosphorylase (7) and glycerol-3-phosphate dehydrogenase (8); carbohydrate transport in hamster intestine (9) and the human erythrocyte (10), (11). The wide range of synthetic fluorinated carbohydrates and related compounds now available has been extensively reviewed (12). As will be evident from our recent studies, however, many detailed biochemical studies will be limited until the introduction of C and/or into fluorocarbohydrates has been accomplished. l 4
The Transport of D-Glucose Across the Human Erythrocyte Membrane. The question of the exact nature of the carrier protein(s) and translocation mechanism for the transport of D-glucose across the erythrocyte membrane is still debated (13). However, the saturation kinetics obtained for D-glucose and glucose analogues are in accordance with a facilitated transport mechanism which allows binding of the sugar molecule to one or more sites of a receptor protein in the membrane. A comprehensive study of the specificity of this binding has been undertaken by a number of workers (14), (15), (16), (17), In particular the comparative inhibition and comparative transport studies of D-glucose with a number of monodeoxy-D-glucoses and monodeoxymonofluoro99 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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D - g l u c o s e s p r o v i d e k i n e t i c parameters which p e r m i t assignment o f hydrogen bonds between s p e c i f i c oxygen atoms i n D-glucose and r e c e p t o r s i t e s i n t h e c a r r i e r protein. Thus u s i n g the o p t i c a l method o f Sen and Widdas (1_5) and t h e s i m p l i f i e d r a t e e q u a t i o n (18) f o r the e x i t o f g l u c o s e from p r e - l o a d e d e r y t h r o c y t e s , we have shown (11_) t h a t r e p l a c i n g the oxygen f u n c t i o n a t C 3 o f D-glucose by f l u o r i n e t o g i v e 3 - d e o x y - 3 - f l u o r o D-glucose does n o t s i g n i f i c a n t l y change t h e h a l f - s a t u r a t i o n c o n s t a n t (K ) f o r t h e c a r r i e r p r o t e i n (Table 1). In c o n t r a s t 3-deoxy-D-glucose has l o s t t h i s a b i l i t y t o hydrogen bond a t C 3 and c o n s e q u e n t l y has a lower a f f i n i t y f o r the c a r r i e r p r o t e i n ( h i g h e r Κ v a l u e ) . In a d d i t i o n the K v a l u e f o r 5 - t h i o - D - g l u c o s e ( T a y l o r , N.F. & Gagneja, G.L. u n p u b l i s h e d r e s u l t ) s u g g e s t s t h a t t h e r i n g oxygen a t C 5 o f D-glucose i s a l s o i n v o l v e d w i t h hydrogen bonding (Tabl x
χ
x
T a b l e 1.
K and V v a l u e s o f D-glucose and d e r i v a t i v e s a t 37° x
m a x
V Sugar
Κ (mM) χ
D-Glucose 3-Deoxy-3-fluoroD-glucose 3-Deoxy-D-glucose 5-Thio-D-glucose
max (mmol. L i t r e
3.9 2.3
640 600
15.3 15.0
340 500
^ min
^)
K i n e t i c parameters were d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d (11). These r e s u l t s a r e i n agreement w i t h B a r n e t t e t a l . (10} who showed by i n h i b i t i o n s t u d i e s t h a t u n l i k e 3-deoxyD - g l u c o s e , 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e b i n d s t o the c a r r i e r p r o t e i n as w e l l as D - g l u c o s e . Furthermore, t h e importance o f the β - o r i e n t a t i o n o f -OH a t in D-glucose was s u g g e s t e d by the h i g h K i v a l u e s f o r 1-deoxy-D-glucose and α - D - g l u c o s y l f l u o r i d e and t h e low Ki value f o r 3-D-glucosylfluoride. These t r a n s p o r t and i n h i b i t i o n s t u d i e s p r o v i d e e v i d e n c e f o r t h e p r o p o s a l by Kahlenburg and Dolansky (19) t h a t t h e oxygen f u n c t i o n s l o c a t e d a t C i , C 3 and t h e r i n g oxygen a t C 5 o f the C l - c o n f o r m a t i o n o f 3-D-glucopyranose ( F i g u r e 1 ) , a r e c o n s i d e r e d t o be n e c e s s a r y f o r e f f e c t i v e hydrogen bonding o f D-glucose t o t h e t r a n s p o r t protein. T h i s model a g r e e s w i t h the d e t e c t i o n o f three d i f f e r e n t r e c e p t o r groups (- N H 2 , - SH and i m i d a z o l e ) on t h e p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t (20) and i s a l s o c o n s i s t e n t w i t h a r e c e n t l y proposed model
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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E T
A L .
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101
(21) f o r t h e mode o f a c t i o n o f c y t o c h a l a s i n Β (22) i n h i b i t i o n o f g l u c o s e t r a n s p o r t i n t h e human e r y t h r o cyte. Thus a D r i e d i n g m o l e c u l a r model ( F i g u r e 2) o f c y t o c h a l a s i n Β (I) r e v e a l s an almost i d e n t i c a l s p a t i a l d i s t r i b u t i o n o f t h e f o u r oxygen atoms l o c a t e d a t C I , C19, C18 and C4 t o t h o s e l o c a t e d a t C5, C l , C2 and C3 o f t h e C l - c o n f o r m a t i o n o f 3-D-glycopyranose ( F i g u r e 1 ) . At l e a s t t h r e e o f t h e s e s i t e s a t R, R l and R3 ( F i g u r e 2) a r e i m p l i c a t e d i n hydrogen bonding t o t h e c a r r i e r p r o t e i n f o r D-glucose and p a r t i a l l y e x p l a i n why c y t o c h a l a s i n Β i s a c o m p e t i t i v e i n h i b i t o r o f D-glucose t r a n s p o r t i n t h e human e r y t h r o c y t e w i t h K i , 1.2 χ 10"' M (21) . A f u r t h e r point o f i n t e r e s t r e s i d e s i n the f a c t t h a t when whole r e d b l o o d c e l l s a r e i n c u b a t e d w i t h 3d e o x y - 3 - f l u o r o - D - g l u c o s e f o r 24 h r s a t 37°C a s m a l l but s i g n i f i c a n t r e l e a s ( H a l t o n , D. & T a y l o r 200mM 3 - d e o x y - 3 - f l u o r o g l u c o s e C-F c l e a v a g e r e a c h e s a maximum (^ 1%) a f t e r 24 hours i n c u b a t i o n ( F i g u r e 3 ) . U n l i k e t h e c o n t r o l s such c e l l s c o m p l e t e l y l o s e t h e i r a b i l i t y to transport glucose. The s i g m o i d a l c u r v e i n d i c a t e s m u l t i p l e k i n e t i c s f o r t h e mechanism o f f l u o r i d e r e l e a s e and 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 e œ r e s u l t s would be t h e concommittant c o - v a l e n t attachment o f t h e g l u c o s e r e s i d u e t o one o f t h e r e c e p t o r s i t e s o f the c a r r i e r p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t . Such a mechanism i s shown ( F i g u r e 4) i n which 3-deoxy3-fluoroglucose (II) hydrogen bonds t o t h e r e c e p t o r p r o t e i n , e l i m i n a t e s HF t o produce t h e e p o x i d e ( I I I ) which i s a t t a c k e d by t h e n u c l e o p h i l i c p r o t e i n t o p r o duce ( I V ) . We have r e c e n t l y s y n t h e s i s e d 3H-C-(3)-3d e o x y - 3 - f l u o r o - D - g l u c o s e (Lopes, D. & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) i n o r d e r t o e s t a b l i s h whether g l u c o s y l a t i o n o f a membrane p r o t e i n has i n f a c t o c c u r r ed. Such a r e a c t i o n may p e r m i t i s o l a t i o n o f t h e c a r r i e r p r o t e i n f o r D-glucose. M i c r o b i a l Metabolism o f Deoxyfluoro-D-glucoses Our p r e v i o u s s t u d i e s (3) demonstrated t h a t 3-deaxyfluoro-D-glucose (II) i s m e t a b o l i s e d by whole r e s t i n g c e l l s o f P £ . f l u o r e s c e n s , w i t h r e t e n t i o n o f t h e C-F bond, t o produce 3 - d e o x y - 3 - f l u o r o - D - g l u c o n i c a c i d ( V ) . C e l l - f r e e e x t r a c t s o f t h i s organism o x i d i s e d 3FG f u r t h e r t o 3 - d e o x y - 3 - f l u o r o - 2 - k e t o - D - g l u c o n i c a c i d (VI). I t has a l s o been shown t h a t t h e same enzymes t h a t o x i d i s e D-glucose ( g l u c o s e o x i d a s e and g l u c o n a t e dehydrogenase) o x i d i s e (II) and t h a t (II) and (V) a r e c o m p e t i t i v e i n h i b i t o r s o f g l u c o n o k i n a s e (23) . In o r d e r t o study f u r t h e r t h e s p e c i f i c i t y o f t h e s e enzymes
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Figure 1. Stereospecific binding sites of β-D-glucopyranose in the Cl-conformation to a transport protein . . . Hydrogen bonds. R, Rl and R3 represent receptor sites on the transport protein.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
6.
T A Y L O R
E T
A L .
103
Deoxyfluoro-monosaccharides
Ί9Ν
Figure 2.
R1
Model of cytochalasin B
gen bonds. X = -CH ?h. R, RI, R2 and R3 represent receptor sites on the transport protein. t
R2
R3
C
Figure 3. Fluoride ion released after incubation of human erythrocytes at 37° with different concentrations of 3-deoxy-3fluoro-D-glucose: A 50 mM, · 100 mM, Ο ^50 mM, • 200 mM. Fluoride ion determinations were by the fluoride elec trode (26) and the preparation of the erythrocytes as previ ously described (11). The initial control fluoride ion concent ration was 1.0 X 10' M. 4
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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towards o t h e r d e o x y f l u o r o - D - g l u c o s e s we have examined the b i o c h e m i c a l e f f e c t s o f t h e i s o m e r i c 4-deoxy-4fluoro-D-glucose (VII) {24) on whole r e s t i n g c e l l s and c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s ( H i l l , L. & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) .
CH OH 2
COOH
COOH
-H H—I—OH
H-f-OH
CH OH 2
(ID
(V)
(VI)
(VII)
Warburg r e s p i r o m e t r y i n d i c a t e d t h a t u n l i k e (II) (VII) i s n o t o x i d i s e d by whole c e l l s o f P£. f l u o r e s c e n s b u t t h e r e i s an immediate r e l e a s e o f f l u o r i d e a n i o n f F i g u r e 5). U s i n g 2.5mM o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e t h e r a t e o f C-F c l e a v a g e i s l i n e a r o v e r t h e f i r s t f o u r hours and a f t e r 24 hours 94% o f t h e c o - v a l e n t f l u o r i n e i s r e l e a s e d as f l u o r i d e a n i o n (no F " was d e t e c t e d i n the absence o f c e l l s ) . The p o s s i b l e d e f l u o r i n a t e d products o f t h i s r e a c t i o n are D-glucose, D-galactose, 4-deoxy-D-glucose and 3,4-anhydro-D-galactose. Only the l a t t e r two compounds a r e l i k e l y s i n c e D - g a l a c t o s e and D - g l u c o s e , even i n t h e p r e s e n c e o f f l u o r i d e a n i o n , are o x i d i s e d by t h e organism. T . l . c . a n a l y s i s o f t h e c e l l s u p e r a t a n t s and i n t r a c e l l u l a r c o n t e n t s , however, has f a i l e d t o r e v e a l any new c a r b o h y d r a t e components. When c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s a r e c h a l l e n g e d w i t h 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) we f i n d t h a t no s i g n i f i c a n t de-flùorination o c c u r s . Thus i n the c o n c e n t r a t i o n range 2 - 2 0 ymoles t h e i n i t i a l r a t e and e x t e n t o f o x i d a t i o n o f (VII) by c e l l - f r e e e x t r a c t s is shown (Table 2 ) . The o x i d a t i o n o f (VII) i s comp l e t e w i t h i n 2 hours and 2 g atoms o f oxygen/mole o f s u b s t r a t e a r e consumed.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
6.
TAYLOR ET A L .
H" (III)
(ID
105
Deoxyfluoro-monosaccharides
(IV)
Figure 4. Possible mechanism of fluoride release from 3deoxy-3-fluoro-D-glucose. · protein X = Ν or S.
60
Hours Figure 5. Release of fluoride anion from 2.5 mM 4-deoxy-4-fluoro-Dglucose by whole cells of Ps. fluorescens. • Fluoride anion. A Oxygen uptake. Six Warburg flasks were used. Each flask contained 5 fimoles 4-deoxy-4-fluoro-D-glucose, 20 mg dry wt cells and 0.67M phosphate buffer to 2.0 ml in main well. Flask contents were incubated at 30°C with shaking. At time intervals the contents of each flask were centri fugea at 4 040 g for 20 min and fluoride ion determinations made with a fluoride electrode (26) on the supernatants. Warburg conditions for oxygen uptake: 30°C, reaction vol 2.0 ml. Fhsk contained 1.0 ml 0.67M phosphate buffer and 0.5 ml 5 ^moles of 4-deoxy-4-fluoro-D-glucose in main well and 0.2 ml 20% KO H aq. in center well. Reaction was initiated by tipping 0.5-ml cell suspension (24 mg dry wt) in 0.67M phosphate buffer from side arm. Endogenous respiration subtracted (1434 μΐ in 480 min).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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T a b l e 2.
O x i d a t i o n o f 4-deoxy-4-fluoro-D-glucose by c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s . Rate o f Moles 0 / Amount o f oxidation mole o f Net 4-fluoroglucose y l oxygen substrate ymoles O 2 / ymoles oxidised hr/mg p r o t e i n consumed 2
2.0 5.0 10.0 20.0 5.0
(glucose)
0.07 0.18 0.26 0.34 0.38
1.12 0.99 1.00 0.96 1.03
50 110 225 440 115
Warburg c o n d i t i o n s : 30°C, gas phase, a i r . R e a c t i o n volume, 2.0 m l . Each f l a s k c o n t a i n e d 37 mg c e l l - f r e e e x t r a c t p r o t e i n , 1 ymole NAD, 0.67 M phosphate b u f f e r , pH, 7.0 made up t o 1.5 l i th mai compartment S i d e arm c o n t a i n e d 0. w e l l 0.2 ml 20% KOHag pape was i n i t i a t e d by t y p i n g c o n t e n t s from t h e s i d e arm. Endogenous r e s p i r a t i o n (317 y l oxygen i n 150 min) subtracted. A f t e r o x i d a t i o n o f (VII) was complete, s i l i c a g e l t . l . c . a n a l y s i s (EtOAC:ACOH:H 0 3:3:1) o f t h e c e l l - f r e e e x t r a c t r e v e a l e d t h e absence o f ( V I I ) ( R , 0.6) and t h e presence o f a new component (RF 0.45). By analogy w i t h t h e e s t a b l i s h e d m e t a b o l i c pathway o f g l u c o s e (27) and 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e {23) i n t h i s organism, t h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e two s t e p o x i d a t i o n o f (VII) by g l u c o s e o x i d a s e and g l u c o n a t e dehydrogenase t o 4 - d e o x y - 4 - f l u o r o - D - g l u c o n i c a c i d (VIII) and 4-deoxy-4-fluoro-2-keto-D-gluconic a c i d (IX) r e s p e c tively. 2
F
ÇOOH -OH
HCH-H (VII)
H-
H CH °H 2
(VIII)
COOH H H-
h o
H &H2OH
(IX)
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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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 o f (IX) i s c u r r e n t l y being i n v e s t i g a t e d . The e x t e n s i v e d e f l u o r i n a t i o n o f (VII) by whole c e l l s o f Ps. f l u o r e s c e n s and the r e t e n t i o n o f the C-F bond on t r e a t m e n t o f fVTI) w i t h c e l l f r e e e x t r a c t s suggest t h a t C-F c l e a v a g e o c c u r s a t the cell-wall/membrane l e v e l o f the organism. The mode o f uptake o f D-glucose by Ps. f l u o r e s c e n s i s not known. However, i n a c l o s e l y r e l a t e d s p e c i e s Ps. a e r u g i n o s a , i t has been shown (28) t h a t a l t h o u g h the phosphoenolp y r u v a t e p h o s p h o t r a n s f e r a s e system (29) i s not i n v o l v e d , the t r a n s p o r t o f D-glucose i s energy dependant and, t h e r e f o r e , l i k e l y t o have a c a r r i e r p r o t e i n system. A s i m i l a r g l u c o s e t r a n s p o r t system may be p r e s e n t i n Ps. f l u o r e s c e n s and t h e f a i l u r e o f t h e whole c e l l t o o x i d i s e 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) may be due t o a s t e r e o s p e c i f i c r e a c t i o n of (VII)with a c a r r i e r p r o t e i n system i n whic i s r e l e a s e d as f l u o r i d p e r m i t the sugar r e s i d u e (at C 4 ) t o become a t t a c h e d t o p r o t e i n i n the membrane and account f o r the f a c t t h a t we a r e unable t o d e t e c t any m e t a b o l i t e e i t h e r o u t s i d e o r i n s i d e the c e l l d u r i n g the i n c u b a t i o n p e r i o d . Some s u p p o r t f o r t h i s p o s s i b i l i t y i s p r o v i d e d by (a) the f a c t t h a t when whole c e l l s are p r e - i n c u b a t e d w i t h 2.5mM 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e f o r 12 hours and c h a l l e n g e d w i t h 2 - 8mM g l u c o s e s i g n i f i c a n t i n h i b i t i o n o f the r a t e o f r e s p i r a t i o n o c c u r s ( F i g u r e 6 ) . T h i s would be c o n s i s t e n t w i t h a b l o c k e d g l u c o s e t r a n s p o r t s i t e ( s ) a l t h o u g h as t h e g l u c o s e c o n c e n t r a t i o n i s i n c r e a s e d t o 20 ymoles some r e c o v e r y o f r e s p i r a t i o n i s apparent. The p o s s i b i l i t y t h a t a s m a l l u n d e t e c t a b l e amount o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e o r a n o n - o x i d i s able f l u o r i n a t e d metabolite i s i n h i b i t i n g glucose metabolism and/or t r a n s p o r t i s o f c o u r s e not e x c l u d e d by t h e s e r e s u l t s . (b) Ps. f l u o r e s c e n s i s unable t o grow on a m i n e r a l s a l t s medium w i t h 4 - d e o x y - 4 - f l u o r o g l u c o s e (VII) as a s o l e carbon source a l t h o u g h f l u o r i n e i o n i s r e l e a s e d i n t o the medium. S e v e r a l examples a r e known where the r e l e a s e o f f l u o r i d e from a C-F compound by a b a c t e r i u m a l l o w s the f r e e n o n - f l u o r i n a t e d fragment t o s e r v e as a source o f carbon and energy f o r growth. Thus a Pseudomonad has been i s o l a t e d which grows on f l u o r o a c e t a t e as a r e s u l t o f C-F c l e a v a g e (30). S i m i l a r l y , a Pseudomonad has been i s o l a t e d which w i l l grow on m o n o f l u o r o c i t r a t e a f t e r f l u o r i d e r e l e a s e (31). The i n a b i l i t y o f 4 - d e o x y - 4 - f l u o r o g l u c o s e ( V I I ) , t o a c t as a carbon source f o r Ps. f l u o r e s c e n s , d e s p i t e C-F c l e a v a g e , may be due t h e r e f o r e , t o the attachment o f the sugar r e s i d u e t o some c e l l u l a r component. It
i s expected
t h a t the s y n t h e s i s o f
^-^C-(l)-4-
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Figure 6. Oxidation of D-glucose by rest ing whole celh of Ps.fluorescensafter pre incubation with 2.5 mM 4-deoxy-4-fluoroD-glucose. Pre-incubation conditions: 30°C, reaction volume 2.0 ml, time 12 hr. Each Warburg flask contained 1.0 ml 0.67M phos phate buffer, 0.5 ml 5 μmoles of 4-deoxy-4fluoro-D-glucose or 5 ^moles D-glucose in main well and 0.5-ml cell suspension (28 mg dry wt) in 0.67M phosphate buffer in side arm. Pre-incubation was initiated by tipping contents from side arm. Oxidation of added D-glucose, Warburg conditions: 30°C, reaction volume 2.5 ml, gas phase, air. After preincubation period, 0.5 ml of μmole quantities of D-glucose were added from the side arm, 0.3 ml 20% KOHag added to the center well. Endogenous res piration subtracted (879 μΐ in 240 min). I Average oxidation of 5, 10, μτηοΙβ8 glucose after pre-incubation 5 μmole glucose. • 10 μmole, Δ 15 μmole, A 20 μmole glu cose added after pre-incubation of celh with 2.5 mM 4-deoxy-4-fluoro-D-glucose.
Temp.
^
Figure 7. (a) Temperature programmed gas chromatogram of locust haemolymph and (b) Locust fat body utilizing OV-17 (7.5%) phase at 170°C followed by a 4°C/min heating rate. Peaks: A = a-D-glucose, Β = β-D-glucose, C = trehalose.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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f l u o r o - D - g l u c o s e , based on a K i l i a n i e x t e n s i o n o f 3- d e o x y - 3 - f l u o r o - D - a r a b i n o s e {22) with Na CN, w i l l a l l o w us t o a s c e r t a i n the a c t u a l m e t a b o l i c f a t e o f 4- d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) and i t s d e f l u o r i n a t e d product. In o r d e r t o examine the b a c t e r i a l s p e c i f i c i t y o f t h i s C-F c l e a v a g e we have r e c e n t l y examined the b i o c h e m i c a l e f f e c t s o f (VII) on E. c o l i (ATCC 11775) and shown t h a t no s i g n i f i c a n t C-F c l e a v a g e by whole c e l l s or c e l l - f r e e e x t r a c t s occurs. A s m a l l but s i g n i f i c a n t uptake o f (VII) i s o b s e r v e d (0.06 mg/mg d r y weight o f b a c t e r i a ) . U s i n g a n o t h e r s t r a i n o f E. c o l i (ATCC 1494 8) we have a l s o demonstrated ( L o u i e , L i - Y u & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) t h a t (VII) p r e v e n t s u t i l i z a t i o n o f l a c t o s e i n t h i s o r g a n i s m by u n c o m p e t i t i v e i n h i b i t i o n o f the i n d u c t i o n o f β galactosidase. Our r e s u l t s are s i m i l a r t o t h o s e r e p o r t e d f o r the e f f e c t on E. c o l i ( 4 ) . 14
T o x i c i t y of 3-deoxy-4-fluoro-D-glucose i n Locusta înigratoria and S c h i s t o c e r c a g r e g a r i a A l t h o u g h 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e (II) d i s p l a y s s e v e r a l p h y s i o l o g i c a l and b i o c h e m i c a l e f f e c t s i n r a t s (33), i t i s not t o x i c . Coupled w i t h the f a c t t h a t r a t s e x c r e t e l a r g e q u a n t i t i e s o f unchanged (II) v i a u r i n e and f a e c e s and a l s o the r e l a t i v e l y l a r g e q u a n t i t i e s o f (II) (5g/Kg body weight) n e c e s s a r y t o evoke a b i o c h e m i c a l response i t was c o n s i d e r e d t o be o f some i n t e r e s t t o s t u d y an a n i m a l o r g a n i s m which r e q u i r e d a s m a l l e r dosage o f (II) and r e t a i n e d water t o a g r e a t e r extent. Two c l o s e l y r e l a t e d A f r i c a n l o c u s t s p e c i e s , S c h i s t o c e r c a g r e g a r i a and L o c u s t a m i g r a t o r i a were chosen f o r t h i s p u r p o s e . In b o t h 10-14 day a d u l t i n s e c t s (II) was t o x i c (LDCQ 5mg/g l o c u s t t i s s u e ) when i n j e c t e d i n t o the haemocoel. T h i s compound was a l s o found t o be t o x i c when o r a l l y i n g e s t e d . T o x i c i t y was e v i d e n c e d by p r o g r e s s i v e l o s s o f m o t o r a c t i v i t y w i t h d e a t h o c c u r r i n g between 30 hours t o 4 d a y s . These symptoms s u g g e s t e d t h a t a slow m e t a b o l i c p o i s o n i n g was occurring. U s i n g the gas c h r o m a t o g r a p h i c p r o c e d u r e o u t l i n e d by F o r d and Candy (34) f o r o b t a i n i n g a c a r b o h y d r a t e scan, l e v e l s of v a r i o u s steady s t a t e n e u t r a l carboh y d r a t e m e t a b o l i t e s were assayed from v a r i o u s l o c u s t t i s s u e s (Figure 7). I t was found t h a t l e v e l s o f (II) d i s a p p e a r e d r a p i d l y from hemolymph, f a t body and f l i g h t muscle w i t h i n two hours o f i n j e c t i o n . A n a l y s i s of e x c r e t a i n d i c a t e d t h a t a s i g n i f i c a n t p o r t i o n o f the i n j e c t e d dose was l o s t . Gas c h r o m a t o g r a p h i c r e s u l t s
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E BONDS
Figure 8. (a) Temperature programmed gas chromatogram of Locust haemolymph after 3deoxy-3-fiuoro-D-glucose injections. M = me tabolite, A = a-D-glucose, Β = β-D-glucose, C = trehalose, (b) Locust fat body after similar treatment.
T i me
Figure 9. (a) Gas chromatogram (isothermal conditions) of locust hemolymph after 3-deoxy-dfluoro-D-glucose and glucose administration using OV-17 stationary support. M = 3-deoxy3-fluoro-glucitol G = glucitol, A = a-D-glucose Β = β-D-glucose. (b) Using E301 stationary support. y
y
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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i n d i c a t e d t h a t a new m e t a b o l i t e appeared i n the n e u t r a l carbohydrate f r a c t i o n f o l l o w i n g a d m i n i s t r a t i o n of ( I I ) . T h i s c a r b o h y d r a t e was shown t o be the a l d i t o l o f ( I I ) , namely, 3 - d e o x y - 3 - f l u o r o - D - g l u c i t o l (X) by gas chroma t o g r a p h i c and TLC comparison o f the s y n t h e t i c a l l y made a l d i t o l (Lopes, D. and T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) . t o the one o b t a i n e d from l o c u s t t i s s u e e x t r a c t s ( F i g u r e s 8 and 9 ) . Furthermore, a f t e r i n s e c t s were p o i s o n e d w i t h a 10.8 mg dose o f (II) and 12 hours l a t e r hemolymph g l u c o s e l e v e l s a r t i f i c a l l y r a i s e d by a 10.8 mg dose o f g l u c o s e the presence o f 0.4 - 0.5 mg/100mg t i s s u e o f (X) and 0.3 - 0.6 mg/100mg t i s s u e D - g l u c i t o l was d e t e c t e d . T h i s i s the f i r s t example o f s o r b i t o l metabolism t o be d e t e c t e d i n e i t h e r o f t h e s e species. Under normal c o n d i t i o n s s o r b i t o l was not d e t e c t a b l e i n the n e u t r a l carbohydrate f r a c t i o n . This phenomenon may hav (a) the r a t e o f norma g r e a t e r than i t s r a t e o f f o r m a t i o n , (b) T h i s was not an important m e t a b o l i c pathway i n the l o c u s t and was o n l y d e t e c t a b l e due t o i n h i b i t i o n . S o r b i t o l m e t a b o l i s m has been r e p o r t e d i n the s i l k worm (35) and mosquito (36). An e x t e n s i v e r e v i e w o f p o l y o l s and t h e i r metabolism has been o f f e r e d by T o u s t e r and Shaw (37) . Our r e s u l t s suggested t h a t the pathway from g l u c o s e t o f r u c t o s e v i a s o r b i t o l was a c t i v e i n t h e s e l o c u s t s p e c i e s and t h a t (II) b l o c k e d the subsequent c o n v e r s i o n o f s o r b i t o l t o f r u c t o s e t h r o u g h the i n h i b i t o r y a c t i o n o f (X) on t h e enzyme s o r b i t o l dehydrogenase ( F i g u r e 10). A precursor product r e l a t i o n s h i p i s e v i d e n t between (II) and (X) i n b o t h hemolymph and f a t body ( F i g u r e 11). Initially i t was thought t h a t the enzyme s o r b i t o l dehydrogenase was found i n t h e hemolymph o f the l o c u s t . T h i s i s the case i n silkworm (35). No a c t i v i t y , however, was a s s a y a b l e i n l o c u s t hemolymph u s i n g 0.1M s o r b i t o l and lOmM n i c o t i n a m i d e adenine d i n u c e l t i d e (NAD ) as substrates. Subsequent study r e v e a l e d t h a t the enzyme a c t i v i t y was c o n f i n e d t o the f a t body. U s i n g about 10 f o l d p u r i f i e d enzyme p r e l i m i n a r y r e s u l t s have suggested t h a t t h i s enzyme i s not v e r y a c t i v e i n l o custs having a K f o r s o r b i t o l i n the o r d e r o f 0.05M. P r e l i m i n a r y r e s u l t s suggest t h a t (X) i s a c o m p e t i t i v e i n h i b i t o r o f the enzyme w i t h a Ki^O.lM. These r e s u l t s a l s o suggest t h a t the pathway o f s o r b i t o l m e t a b o l i s m from g l u c o s e t o f r u c t o s e i s not v e r y important t o the t o t a l m e t a b o l i s m o f the l o c u s t . Therefore, i t i s d i f f i c u l t t o r a t i o n a l i z e how the i n h i b i t i o n o f such a minor pathway can m a n i f e s t t o x i c a c t i o n . It i s q u i t e p o s s i b l e t h a t some h i t h e r t o u n d e t e c t e d +
M
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Dehydrogenase
Figure 10.
Sorbitol conversion pathway.
Time
in m i n u t e s
Figure 11. Levels of 3-deoxy-3-fluoro-D-glucose (II) and 3-deoxy-3-fluoro-D-glucitol (X) in locust hemolymph and fat body after a 3.6-mg injection of (II). (II) in hemolymph (X) in hemolymph (II) in fat body.
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metabolites are responsible for t o x i c i t y . C h e f u r k a (38), (39.) has suggested a means o f e v a l u a t i n g glucose metabolism i n i n s e c t s using 1 - C and 6 - C l a b e l l e d D-glucose by examining t h e r e l a t i v e r a t e s o f ^ C02 e v o l u t i o n . T h i s t e c h n i q u e may be a u s e f u l t o o l f o r m o n i t o r i n g any o t h e r changes i n t h e pathways o f g l u c o s e m e t a b o l i s m induced by (II) o r i t s metabolites. Our s t u d i e s on t h e mode o f t o x i c i t y w i l l be f u r t h e r e d when and/or H - l a b e l l e d f l u o r o g l u c o s e s become a v a i l a b l e b u t t h e s e p r e l i m i n a r y r e s u l t s i n d i c a t e t h a t some f l u o r i n a t e d c a r b o h y d r a t e s a r e n o t o n l y t o x i c t o i n s e c t s b u t may a l s o a c t as probes f o r t h e d e t e c t i o n o f unsuspected m e t a b o l i c pathways. 14
14
4
3
Acknowledgment T h i s work i s s u p p o r t e C o u n c i l o f Canada.
Abstract (a) The use of deoxyfluoro-D-glucose as probes for the study of glucose transport across the human erythrocyte membrane is discussed. These studies are also related to the mode of action of cytochalasin Β inhibition of D-glucose across this membrane, (b) Evidence is presented to show that 4-deoxy-4fluoro-D-glucose is not oxidised by whole resting cells of Ps. fluorescens (ATCC 12633) but an extensive release of fluoride anion occurs. With cell-free extracts of Ps. fluorescens however, 4-deoxy-4-fluoroD-glucose is oxidised to the extent of 2 g atoms of oxygen/mole of substrate. Possible reasons for this C-F cleavage are discussed. (c) 3-Deoxy-3-fluoro-Dglucose is toxic to Locusta migratoria (L.D.50 5mg/g and is metabolised by an NAD-linked sorbitol dehydro genase, which is located in the fat body of the insect, to 3-deoxy-3-fluoro-D-glucitol. The activity of the partially purified enzyme is low with Km, 0.05M for sorbitol. 3-Deoxy-3-fluoro-glucitol is a competitive inhibitor with K i , 0.1M.
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Literature Cited. 1. Ciba Fdn. Symposium: "Carbon-Fluorine Compounds: Chemistry, Biochemistry and Biological Activities." pp 1-417. Associated Scientific Publishers, New York, 1972. 2. Woodward, B., Taylor, N.F. & Brunt, R.V., Biochem. Pharmacol. (1971), 20 1071-1077. 3. Taylor, N.F., White, F.H. & Eisenthal, R.E., Biochem. Pharmacol. (1972), 21 347-353. 4. Miles, R.J. & S.J. J. Gen. Microbiol. (1973) 76 305-318. 5. Eisenthal, R.E., Harrison, R., Lloyd, W.J. & Taylor, N.F., Biochem. J. (1972), 130 199-205. 6. Bessel, E.M. & Thomas, P., Biochem. J. (1973) 131 843-850. 7. Wright, J.A., Taylor N.F. Brunt R.V & Brownse R.W., Chem. Commun 8. Silverman, J.B., Barbiatz, P.S., Mahajan, K.P., Buschek, J. & Foudy, T.P., Biochemistry (1975) 14 2252-2258. 9. Barnett, J.E.G., Ralph, A. & Monday, K.A., Biochem. J. (1970) 118 843-850. 10. Barnett, J.E.G., Holman, J.D. & Monday, K.A., Biochem. J. (1973)131211-221. 11. Riley, G.J. & Taylor, N.F., Biochem. J. (1973) 135 773-777. 12. Kent, P.W., Ciba Fdn. Symposium: "Carbon-Fluorine Compounds". pp 169-208. 13. Lieb, W.R. & Stein, W.D., Biochim. Biophys. Acta (1972), 265 187-207. 14. LeFevre, P.G., Pharmacol. Rev. (1961) 13 39-70. 15. Sen, A.K. & Widdas, W.F., J. Physiol (London) (1962) 160 392-403. 16. Lacko, L. & Burger, M. Biochem. J. (1962) 83 622-625. 17. Baker, G.F. & Widdas, W.F., J. Physiol. (London) (1972) 271 10p. 18. Miller, D.M. in "Red Cell Structure and Function". (Jamieson, G.A. & Greinwalt, Τ.A. Eds.) pp 240-292, J.B. Lippincott & Co. Philadelphia & Toronto, 1969. 19. Kahlenberg, A. & Dolansky, D., Canad. J. Biochem. (1972) 50 638-643. 20. Bloch, R.J. Biol. Chem. (1974) 249 1814-1822. 21. Taylor, N.F. & Gagneja, G.L., Proc. Canad. Fed. Biol. Soc. (1975) 18 p 4. 22. Aldridge, D.C., Armstrong, J.J. & Speake, R.N., J. Chem. Soc. (1967) 1667-1676. 23. Taylor, N.F., Hill, L. & Eisenthal, R.E., Canad. J. Biochem. (1975) 53 57-64.
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AL.
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24. Barford, A.D., Foster, A.B., Westwood, J.H., Hall, L., & Johnson, R.N., Carbohydr. Res. (1971), 19 49-61. 25. Umbreit, W.W., Burris, R.H. & Stauffer, J.F., "Manometric Techniques" pp 1-61. Burgess Publish ing Co., Minneapolis, Minn. 1964. 26. Woodward, B., Taylor, N.F., & Brunt, R.V., Analyt. Biochem. (1971) 36 303-309. 27. Wood, W.A. & Schwerdt, R.F., J. Biol. Chem. (1953) 201 501-511. 28. Midgley, M. & Dawes, E.A., Biochem. J. (1973) 132 141 29. Kundig, W., Ghosh, S. & Roseman, S., Proc. Nat. Acad. Sci. (1964) 52 1067-1074. 30. Goldman, P. J. Biol. Chem. (1965) 240 3434-3438. 31. Kirk, Κ., Goldman P. Biochem J (1970) 117 409-410. 32. Wright, J.A., an (1967) 3 333-339. 33. Riley, G.J., Ph.D. Thesis University of Bath U.K. (1973). 34. Ford, W.C.L., Candy, D.J., Biochem. J. (1972) 130, 1101. 35. Faulkner, P., Biochem. J. (1958) 68 375. 36. Handel, E. Van, Comp. Biochem. Physiol. (1969) 29 1023. 37. Touster, Ο., Shaw, D.R.D., Physiol. Rev., (1962) 42 181. 38. Chefurka, W., in "The Physiology of Insecta"., Volume 2, pages 641-656, Academic Press Inc., New York, 1965. 39. Chefurka, W., Ela, R., and Robinson, J.R., J. Insect Physiol. (1970) 16 2137-2156. 40. Hall, L.D., Johnson, R.N., Foster, A.B. and Westwood, J.H., Canad. J. Chem. (1971) 49 236-240. 41. Lin, S. and Spudich, J.A., J. Biol. Chem. (1974) 249 5778-5783.
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Q.
Is the conformation o f glucose, 3-deoxy-3-fluoro g l u c o s e and 3-deoxy-glucose t h e same?
A.
Although there is no direct experimental evidence, t h e s e t h r e e hexoses p r o b a b l y have t h e same p r e dominate C l - c o n f o r m a t i o n i n aqueous s o l u t i o n w i t h a l l t h e s u b s t i t u e n t s equitorial. There is some e v i d e n c e , however, t h a t t h e i n t r o d u c t i o n o f f l u o r i n e can d i c t a t e which c o n f o r m a t i o n t h e sugar r i n g w i l l adopt. Thus Hall et al (40) have shown t h a t i n the case o f t h e a c e t y l a t e d D - x y l o s y l f l u o r i d e d e r i v a t i v e s t h e ß-anomer adopts t h e unexpected c o n f o r m a t i o n i n which all t h e s u b s t i t u e n t s a r e axially disposed.
Q.
Does g l u c o s e b i n d w i t h c y t o c h a l a s i n B?
A.
No. The b i n d i n and our own kinetic results (21) in which c y t o c h a l a s i n Β is shown t o be a c o m p e t i t i v e inhibitor ( K i , 10- M) o f g l u c o s e t r a n s p o r t in t h e human e r y t h r o c y t e suggest t h a t c y t o c h a l a s i n Β b i n d s t o a c a r r i e r p r o t e i n and n o t t o g l u c o s e . 7
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
7 Organic Fluorocompounds in Human Plasma: Prevalence and Characterization W. S. GUY Department of Basic Dental Sciences, University of Florida, Box J424, Gainsville, Fla. 32610 D. R. TAVES Department of Pharmacology and Toxicology, University of Rochester, Rochester, N.Y. 14642 W. S. BREY, JR. Department of Chemistry, Universit Taves discovered tha tained two distinct forms of fluoride (1-4). Only one of these was exchangeable with radioactive fluoride. The other, non -exchangeable form was detectable as fluoride only when sample preparation included ashing. This paper is concerned with three aspects of this newly discovered, non-exchangeable form: 1) its prevalence in human plasma, 2) how its presence in human plasma affects the validity of certain earlier conclusions about the metabolic handling of the exchangeable form of fluoride, and 3) its chemical nature. Preliminary work in this laboratory suggested that the non -exchangeable form was widespread in human plasma but did not exist in the plasma of other animals. Ashing increased the amount of fluoride an average of 1.6 ± 0.25 SDμM(range 0.4-3.0) in samples of plasma from 35 blood donors in Rochester, N.Y. (5). No such fluoride was detectable (above 0.3 μM) in blood serum from eleven different species of animal including horse, cow, guinea pig, chicken, rabbit, sheep, pig, turkey, mule and two types of monkey (6). Standard methods for analysis of exchangeable fluoride in serum have in the past included ashing as a step in sample preparation (7). Taves showed that the amount of fluoride in serum that would mix with radioactive fluoride was only about one-tenth the amount generally thought to be present based on analyses using these older methods (4). When plasma samples from individuals living in cities having between 0.15 and 2.5 ppm fluoride in their water supply were analysed by these older methods, no differences were found between the averages for the different cities. This led to the conclusion that "homeostasis of body fluid fluoride content results with intake of fluoride up to and including that obtained through the use of water with a fluoride content of 2.5 ppm" (8). If the non-exchangeable form of fluoride predominated in these samples, differences in the exchangeable fluoride concentration would probably not have been apparent, and it would be unnecessary to postulate such rigorous 117 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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homeostatic c o n t r o l mechanisms f o r f l u o r i d e . In t h i s study plasma samples were c o l l e c t e d from a t o t a l of 106 i n d i v i d u a l s l i v i n g i n f i v e d i f f e r e n t c i t i e s with between 0.1 and 5.6 ppm f l u o r i d e i n t h e i r p u b l i c water supply. These were analyzed f o r both forms of f l u o r i d e . I n t h i s way the r e l a t i o n ship between exchangeable f l u o r i d e c o n c e n t r a t i o n i n the plasma and the consumption of f l u o r i d e through d r i n k i n g water was r e evaluated, and the prevalence of the non-exchangeable form was further studied. With respect to the chemical nature of the non-exchangeable form of f l u o r i d e s e v e r a l l i n e s of evidence suggested that i t was some s o r t of organic fluorocompound of intermediate p o l a r i t y , t i g h t l y bound to plasma albumin i n the blood. I t migrated with albumin during e l e c t r o p h o r e s i s of serum a t pH nine (3) and was not u l t r a f i l t e r a b l e from serum ( 2 ) . Attempts at d i r e c t e x t r a c t i o n from plasma with s o l v e n t petroleum ether and e t h y Treatment of albumin s o l u t i o n (prepared by e l e c t r o p h o r e s i s of plasma) with c h a r c o a l a t pH three d i d remove the bound f l u o r i n e f r a c t i o n . And f i n a l l y , when plasma p r o t e i n s were p r e c i p i t a t e d with methanol a t low pH the f l u o r i n e f r a c t i o n o r i g i n a l l y bound to albumin appeared i n the methanol-water supernatant i n a form which s t i l l r e q u i r e d ashing to r e l e a s e f l u o r i n e as i n o r g a n i c f l u o r i d e (5) . Based on these c o n s i d e r a t i o n s the non-exchangeable form of f l u o r i d e i n human plasma i s r e f e r r e d to as "organic f l u o r i n e " throughout the r e s t of t h i s paper. In order to f u r t h e r c h a r a c t e r i z e the organic f l u o r i n e f r a c t i o n , i t was p u r i f i e d from 20 l i t e r s of pooled human plasma and c h a r a c t e r i z e d by f l u o r i n e nmr. M a t e r i a l s and Methods A n a l y t i c a l Methods. Values f o r organic f l u o r i n e were c a l c u l a t e d by taking the d i f f e r e n c e between the amount of i n o r g a n i c f l u o r i d e i n ashed and unashed p o r t i o n s of the same m a t e r i a l . The f o l l o w i n g procedure was used to prepare ashed samples: 1) samples (sample s i z e f o r plasma was 3 ml) were placed i n platinum c r u c i b l e s and mixed with 0.6 mmoles of low f l u o r i d e MgCl and 0.1 mmoles of NaOH, 2) these were d r i e d on a h o t p l a t e and then ashed (platinum l i d s i n place) f o r 2-4 h r a t 600° C i n a muffle furnace which had been modified so that the chamber received a flow of a i r from outside the b u i l d i n g (room a i r increased the blank and made i t more v a r i a b l e ) , and 3) ashed samples were d i s s o l v e d i n 2 ml of 2.5 Ν H S0^ and t r a n s f e r r e d t o polystyrene d i f f u s i o n dishes using 2 r i n s e s with 1.5 ml of water. The f o l l o w i n g procedure was used f o r s e p a r a t i o n of f l u o r i d e from both ashed and unashed samples: 1) samples (sample s i z e f o r unashed plasma was 2 ml) were placed i n d i f f u s i o n dishes (Organ C u l t u r e Dishes, Falcon P l a s t i c s , Oxnard, C a l i f . , absorbent 2
2
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removed, r i n s e d with water), a c i d i f i e d with 2 ml of 2.5 Ν H SO^, and a g i t a t e d with a g e n t l e s w i r l i n g a c t i o n on a l a b o r a t o r y shaker f o r 30 min to remove C0 ; 2) f o r each sample the trapping s o l u t i o n (0.5 ml, 0.01 Ν NaOH + p h e n o l t h a l e i n - p - n i t r o p h e n o l i n d i c a t o r ) was placed i n a small polystyrene cup i n the c e n t e r - w e l l of the d i f f u s i o n d i s h , 1 drop of 10% T r i t o n - X 100 was added to the sample to decrease surface t e n s i o n and make the d i f f u s i o n r a t e more uniform between samples c o n t a i n i n g plasma and those not, the l i d with a small hole made near i t s l a t e r a l margin was sealed i n t o p l a c e with petroleum j e l l y , 0.02 ml of 4% hexamethyldisiloxane (Dow Corning, F l u i d 200, 0.65 c s , Midland, Mich.) i n ethanol was i n j e c t e d through the hole i n the l i d i n t o the sample, and the hole was sealed Immediately with petroleum j e l l y and a s t r i p of p a r a f f i n f i l m ; and 3) samples were d i f f u s e d with g e n t l e s w i r l i n g f o r a t l e a s t 6 hr, d i f f u s i o n was terminated by breaking the s e a l and trapping s o l u t i o n checked a t t h i s p o i n t t and d r i e d i n a vacuum oven (60° C, 26 in-Hg vacuum, i n the presence of a NaOH d e s i c c a n t ) . 2
2
F l u o r i d e was determined by potentiometry with the f l u o r i d e e l e c t r o d e . The system used c o n s i s t e d of a f l u o r i d e e l e c t r o d e o r i e n t e d i n an i n v e r t e d p o s i t i o n (model 9409A, Orion Research Inc. Cambridge, Mass.), a calomel reference e l e c t r o d e ( f i b e r t y p e ) , a p l a s t i c vapor s h i e l d which j u s t f i t t e d over the bodies of both e l e c t r o d e s forming an enclosed sample chamber i n which watersaturated t i s s u e paper was placed above the sample to prevent e v a p o r i z a t i o n of the sample, and a high impedence voltmeter (model 401, O r i o n ) . Samples were prepared and read i n the f o l l o w i n g way: 10 μΐ of 1 M HAc was drawn i n t o a polyethylene m i c r o p i p e t t e (Beckman Micro Sampling K i t , Spinco Div., Beckman I n s t . Co., Palo A l t o , C a l i f . ) and deposited i n t o the cup c o n t a i n i n g the r e s i d u e from the trapping s o l u t i o n a f t e r d r y i n g ; the f l e x i b l e t i p of the m i c r o p i p e t t e was used to wash down the w a l l s of the cup; and the s o l u t i o n was then t r a n s f e r r e d to the surface of the f l u o r i d e e l e c t r o d e and the reference e l e c t r o d e brought i n t o p o s i t i o n . Surfaces of the two e l e c t r o d e s were b l o t t e d dry between samples. Samples were read i n order of i n c r e a s i n g expected concentra t i o n and sets of samples were read between bracketing c a l i b r a t i o n standards. These standards were used i n two d i f f e r e n t ways during a run. F i r s t , they were flooded onto the e l e c t r o d e surfaces to e q u i l i b r a t e them to concentrations expected f o r samples and to make them uniform. This procedure permitted the a n a l y s t to take reasonably s t a b l e readings f o r samples w i t h i n one minute. Secondly, they were used i n 10 y l volumes f o r readings used i n preparing the standard curve. Values f o r i d e n t i c a l samples ( u s u a l l y t r i p l i c a t e s ) were averaged and the average blank was subtracted from sample means. These were then d i v i d e d by the average f r a c t i o n a l recovery of
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f l u o r i d e (usually 90 to 95%) i n standards treated the same way as the sample s e t . P l a s t i c w a r e (Falcon P l a s t i c s ) was used f o r a l l a n a l y t i c a l procedures to avoid contamination by f l u o r i d e from g l a s s . Liquid volume measurements were made with 1, 5 and 10 ml p o l y s t y r e n e p i p e t t e s and a polycarbonate volumetric f l a s k (100 ml). Reagents were p u r i f i e d to i n s u r e uniformly low blanks. Water was r e d i s t i l l e d and d e i o n i z e d . A c e t i c a c i d and ammonia were r e d i s t i l l e d . F l u o r i d e contamination i n MgCl2 ( a n a l y t i c a l grade) was reduced by preparing a 1 M s o l u t i o n c o n t a i n i n g HC1 to pH 1 and scrubbing with hexamethyldisiloxane vapor i n a column through which the s o l u t i o n was continuously r e c y c l e d . Following scrubbing the s o l u t i o n was b o i l e d to one t h i r d volume to remove any r e s i d u a l v o l a t i l e s i l i c o n e s and then made j u s t b a s i c with NHi+OH. F l u o r i d e contamination i n I^SO^ was reduced by repeated e x t r a c t i o n s of a 6.7 Ν then b o i l i n g to one t h i r Buffered c a l i b r a t i o n standards were made from the same NaOH and HAc stock s o l u t i o n s as f o r samples. The blanks f o r ashed samples ranged between 0.2 and 1.5 nmoles f l u o r i d e and were t y p i c a l l y about 0.5 nmoles. The blanks were smaller f o r unashed samples; these ranged between 0.05 and 0.2 nmoles f l u o r i d e and were t y p i c a l l y about 0.1 nmoles. Factors a f f e c t i n g recovery of f l u o r i d e during d i f f u s i o n were i n v e s t i g a t e d w i t h F~ t r a c e r . Recovery during d i f f u s i o n was 97% a f t e r 80 min from 5 ml containing 2 ml of plasma. Increasing the a c i d i t y of the sample up to 5 N, the volume of the sample up to 7.5 ml, the amount of c o l d F~ up to 1 pmole, the amount of f l u o r i d e complexors up to 1 ymole of ThiNC^)^ had no m a t e r i a l e f f e c t on the r a t e of f l u o r i d e d i f f u s i o n . The absence of both plasma and detergent i n the sample compartment markedly slowed the r a t e of d i f f u s i o n . Not shaking the sample a l s o slowed the r a t e of d i f f u s i o n . Increasing the a l k a l i n i t y of the trapping s o l u t i o n to 0.1 Ν increased the r a t e of d i f f u s i o n but the lower concentration, 0.01 N, was r e q u i r e d here to permit a lower i o n i c strength i n the sample reading s o l u t i o n . O v e r a l l recovery of added cold f l u o r i d e was measured. In samples containing n e i t h e r plasma nor detergent the recovery a f t e r 6 hr d i f f u s i o n averaged 93% and 95% f o r ashed and unashed samples, r e s p e c t i v e l y . In samples containing plasma the recovery was 95% a f t e r 3 hr d i f f u s i o n . The degree to which f l u o r i n e from organic fluorocompounds could be f i x e d as i n o r g a n i c f l u o r i d e by ashing v a r i e d from l e s s than 1% f o r v o l a t i l e compounds l i k e p - a m i n o b e n z o t r i f l u o r i d e , m-hydroxybenzotrifluoride, b e n z y l f l u o r i d e and benzotrifluoride to over 80% f o r l e s s v o l a t i l e compounds l i k e 5 - f l u o r o u r a c i l , f l u o r o a c e t a t e and p - f l u o r o p h e n y l a l a n i n e . Methods used here f o r s e p a r a t i o n of f l u o r i d e ( d i f f u s i o n at rm. temp.) (9) and i t s q u a n t i t a t i o n ( f l u o r i d e e l e c t r o d e ) (10) are considered to be q u i t e s p e c i f i c f o r f l u o r i d e . One p o t e n t i a l l y
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important i n t e r f e r e n c e , however, was c o d i f f u s a b l e organic a c i d s which might p a r t i a l l y n e u t r a l i z e the trapping s o l u t i o n and thus lower the pH of the b u f f e r e d reading s o l u t i o n . Indeed, i t was found that samples c o n t a i n i n g r e l a t i v e l y l a r g e concentrations of a c e t i c acid (e.g., f r a c t i o n s 2, 3 and 4 from step 4 i n the p u r i f i c a t i o n system) completely n e u t r a l i z e d the trap w i t h i n a few hours. The s i g n i f i c a n c e of t h i s problem i n the a n a l y s i s of f l u o r i d e i n blood plasma was i n v e s t i g a t e d i n two ways. F i r s t , four samples of human plasma were allowed to d i f f u s e f o r three weeks, and no change i n the c o l o r of the phenolphthalein i n d i c a tor i n the trapping s o l u t i o n was observed. Secondly, samples c o n t a i n i n g the same plasma were d i f f u s e d f o r d i f f e r e n t periods up to 158 hr and the apparent f l u o r i d e was determined. No changes were observed between samples which c o r r e l a t e d with d i f f u s i o n time. The s e n s i t i v i t y o the blank r a t h e r than th R e p r o d u c i b i l i t y v a r i e d with the amount of f l u o r i d e being measured. The c o e f f i c i e n t of v a r i a t i o n averaged 55% i n the low range (samples c o n t a i n i n g 0.25 to 0.75 nmoles F ) and 6.6% i n the high range (10-12 nmoles F " ) . Blood Plasma. Human plasma was obtained from blood banks i n f i v e c i t i e s . According to p u b l i c records these c i t i e s had not changed the f l u o r i d e c o n c e n t r a t i o n of t h e i r p u b l i c water supply f o r a t l e a s t s i x years p r i o r to o b t a i n i n g the samples. Samples were r e c e i v e d i n i n d i v i d u a l polyethylene bags which were p a r t of the Fenwall ACD blood c o l l e c t i o n system. In blood c o l l e c t i o n using t h i s system 450 ml of blood i s drawn i n t o a bag containing 67.5 ml of anticoagulant a c i d c i t r a t e dextrose (ACD) s o l u t i o n . When the c e l l s are removed the ACD s o l u t i o n remains i n the plasma. Because of t h i s d i l u t i o n of plasma a c o r r e c t i o n f a c t o r of 1.3 was a p p l i e d to values obtained here f o r the c o n c e n t r a t i o n of f l u o r i d e . The p o t e n t i a l e r r o r i n t h i s f a c t o r was ± 0 . 1 because of v a r i a t i o n between standard l i m i t s f o r hematocrit and minimum volume of the blood donation. Bovine blood was obtained a t slaughter and mixed immediately with ACD s o l u t i o n i n 1 l i t e r polyethylene b o t t l e s . E l e c t r o p h o r e s i s . A continuous flow e l e c t r o p h o r e t i c separator (model FF-3, Brinkman I n s t . , Inc., Westbury, N.Y.) was employed. Sample flow r a t e was 2.3 ml/hr, b u f f e r flow r a t e was 72 ml/hr, v o l t a g e was 0.67 kv, and current was 140 mamp. Separa t i o n took 19 hr. P l a t e s e p a r a t i o n was 1 mm and operating tempera ture was between 2 and 4° C. The b u f f e r was 0.12% (NHi ) C0 , made by bubbling C 0 from dry i c e i n t o r e d i s t i l l e d ΝΗίψΟΗ u n t i l the pH reached 9.0. +
2
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P u r i f i c a t i o n System. Steps i n the p u r i f i c a t i o n system are summarized i n t a b l e I. In the f i r s t step one l i t e r of plasma (pooled from 5-6 i n d i v i d u a l s ) was d i a l y s e d i n seamless c e l l u l o s e tubing (1 i n . diameter) against 20 l i t e r s of water at 4° C. The d i a l y s a t e was changed twice at 24 hr i n t e r v a l s . In the second step d i a l y s e d plasma was f r e e z e d r i e d . In the t h i r d step the d r i e d powder from e l e c t r o p h o r e s i s was extracted with methanol i n a soxhelet e x t r a c t i o n apparatus (model 6810 G, Ace Glass, Inc., Vineland, N.J.). C e l l u l o s e e x t r a c t i o n thimbles (model 6812 G, Ace Glass) were soaked overnight i n methanol. Operating c o n d i t i o n s were 25° to 30° C under a vacuum of 24 in-Hg. Coolant f o r the condenser was 80% ethanol; i n l e t temperature was -10° to -20° C and o u t l e t tempera ture was -10° to 0°. Two l i t e r s of methanol were r e f l u x e d through the apparatus f o r a period of 4 hr and approximately 400 ml were l o s t to evaporatio were placed i n the f l a s In the f o u r t h step the r e s i d u e from the methanol e x t r a c t was f r a c t i o n a t e d according to the method described by Siakotos and Rouser (11) f o r separating l i p i d and n o n - l i p i d components. The method i s based on l i q u i d - l i q u i d p a r t i t i o n i n a column containing a dextran g e l (Sephadex G-25, coarse, beaded, Pharmacia F i n e Chemicals, Inc., N.Y.). Four eluents are used: 1) 500 ml chloroform/methanol, 19/1, saturated with water, 2) 1000 ml of a mixture of 5 p a r t s of chloroform/methanol, 19/1, and 1 part of g l a c i a l a c e t i c a c i d , saturated with water, 3) 500 ml of a mixture of 5 p a r t s chloroform/methanol, 19/1, and 1 part g l a c i a l a c e t i c a c i d , saturated with water, and 4) 1000 ml of methanol/water, 1/1. T h e i r method was modified f o r use here by i n c r e a s i n g the column length to that a t t a i n e d by using a f u l l 100 grams of dextran beads. Sample s i z e corresponded to that from 2.5 l i t e r s of the o r i g i n a l plasma. In the f i f t h step the residues from eluents 2 and 3 from two runs of step four were combined, a p p l i e d to a s i l i c i c a c i d column, and e l u t e d by reverse flow with an exponential gradient of i n c r e a s i n g amounts of methanol i n chloroform. The column (model SR 25/45, 2.5 cm i . d . χ 45 cm, Pharmacia) was f i l l e d to a height of 30 cm with s i l i c i c a c i d ( U n i s i l , 100-200 mesh, Clarkson Chem. Co., Inc., W i l l i a m s p o r t , Pa., heat a c t i v a t e d at 110° C f o r 2 days) and was washed with a complete set of e l u t i o n s o l v e n t s before use. The gradient maker (model 5858, set 4, Ace Glass Co.) was f i l l e d with 1 l i t e r of methanol i n the upper chamber and 2 l i t e r s of chloroform i n the lower. The flow r a t e was adjusted by the height of the s o l v e n t r e s e r v o i r s to an average of 3 ml/min f o r the f i r s t l i t e r of eluent. The sample had to be t r a n s f e r r e d to the column by repeated washings with chloroform because of i t s low s o l u b i l i t y i n t h i s s o l v e n t . This u s u a l l y r e q u i r e d about 30 ml of chloroform t o t a l . Dead volume for the system as 90 ml. F r a c t i o n s of 15 ml volume were c o l l e c t e d i n c a r e f u l l y cleaned g l a s s tubes.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
7.
Fluorocompounds
GUY E T A L .
in Human
123
Plasma
Table I PROCEDURE FOR PURIFICATION OF FLUOROCOMPOUNDS FROM BLOOD PLASMA Fraction Treated blood plasma
plasma p r o t e i n s & protein-bound substances i n water s o l u t i o n plasma p r o t e i n s & protein-bound substances
Treatment step 1: exhaustive d i a l y s i s against d i s t i l l e d water step 2:
lyophilization
Fraction Removed smaller, waters o l u b l e components
water
extraction—soxh e l e t , 25°C, 24 in-Hg vacuum
plasma l i p i d s
step 4: column chromatography— liquid-liquid p a r t i t i o n on Sephadex
polar l i p i d s
step 5: column chromatography— adsorption on s i l i c i c acid
l i p i d s of low p o l a r i t y and residual polar contaminants
unknown: severa1 yellow f r a c t i o n s
Figure 1. Relationship between the concentration of fluoride in human plasma and the concentration of fluoride in the drinking water
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E
Figure 2. Relationship between the concentration of organic fluorine in human pfosma and the concentration of fluoride in the drinking water
TUBE NO. Figure 3. Separation of fluoride and organic fluorine in human plasma by electrophoresis. A sample (about 45 ml) of human phsma was electrophoresed in pH 9 buffer and fractions between the sampling port (near tube 72) and the positive pole (near tube 1) were analyzed for the fluoride content of both ashed and unashed aliquots. Relative concentrations of proteins were estimated by absorbance at 280 nm.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
7.
GUY ET AL.
Fluorocompounds
in Human
Plasma
125
Tubing and f i t t i n g s to the columns were p o l y t e t r a f l u o r o e t h y lene (supplied l a r g e l y by Chromatronix, Inc., Berkeley, C a l i f . ) . A l l solvents were r e d i s t i l l e d . Methanol and chloroform were ACS c e r t i f i e d ( F i s h e r S c i e n t i f i c Co.) and a c e t i c a c i d was a n a l y t i c a l reagent grade, U.S.P. ( M a l l i n c k r o d t Chemical Works, St. L o u i s ) . Solvents were removed from samples i n a f l a s h evaporator. NMR. The nmr spectrum was obtained on a V a r i a n XL-100 spectrometer with N i c o l e t Technology F o u r i e r Transform accessory. The sample was d i s s o l v e d i n an approximately 1/1 mixture of CH OH and CDCI3 and s p e c t r a were run i n a 5 mm tube. External r e f e r e n c i n g to CFCI3 was used f o r the chemical s h i f t s , and these are expressed with p o s i t i v e numbers to lower f i e l d ( i . e . , higher frequency). E x t e r n a l l o c k was used. T y p i c a l c o n d i t i o n s were a pulse length of 15 microseconds c y c l e s of 2.5 sec, and a processing. 3
Results Values f o r i n o r g a n i c f l u o r i d e (F~) and organic f l u o r i n e (R-F) i n 106 plasma samples from humans l i v i n g i n f i v e c i t i e s are shown i n t a b l e I I . These data show that the average f l u o r i d e c o n c e n t r a t i o n i n plasma i s d i r e c t l y r e l a t e d to the f l u o r i d e c o n c e n t r a t i o n i n the water supply, and that the average organic f l u o r i n e concentration i n plasma i s not. No r e l a t i o n s h i p between f l u o r i d e i n plasma and organic f l u o r i n e i n plasma was apparent by i n s p e c t i o n of values f o r i n d i v i d u a l samples. The d i s t r i b u t i o n s of the values w i t h i n c i t i e s are shown i n f i g u r e s 1 and 2. In both cases the d i s t r i b u t i o n s appear to be l o g normally d i s t r i b u t e d with only 3 or 4 i n d i v i d u a l s s u r p r i s i n g l y deviant. In the cases of the two i n d i v i d u a l s with l i t t l e or no apparent organic f l u o r i n e ( f i g u r e 2, Andrews group, l e f t margin), the i n o r g a n i c f l u o r i d e l e v e l s were both i n excess of 7 μΜ, making the d i f f e r e n c e measure ment f o r organic f l u o r i n e d i f f i c u l t . The o v e r a l l mean value f o r organic f l u o r i n e was 1.35 ± 0.85 SD μΜ. Plasma was electrophoresed i n an attempt to reproduce the f i n d i n g s of Taves (3) using plasma from another i n d i v i d u a l . Results shown i n f i g u r e 3 c l o s e l y match those found e a r l i e r i n that a predominant form of organic f l u o r i n e appeared to migrate with albumin at pH 9, and i n that organic and i n o r g a n i c forms were c l e a r l y separated. The recovery, mass balance and p u r i f i c a t i o n f a c t o r s f o r steps i n the p u r i f i c a t i o n system l i s t e d i n t a b l e I are recorded i n t a b l e I I I . These data show that about o n e - t h i r d of the o r i g i n a l amount of organic f l u o r i n e i n plasma i s recovered i n the major peak from s i l i c i c a c i d chromatography. Another t h i r d i s accounted for i n other f r a c t i o n s and the r e s t i s not accounted f o r , presumably because of adsorption to surfaces of containers i n
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
126
BIOCHEMISTRY
INVOLVING CARBON-FLUORINE
BONDS
Table I I CONCENTRATION OF FLUORIDE (F~) AND ORGANIC FLUORINE (R-F) IN BLOOD PLASMA SAMPLES FROM FIVE CITIES HAVING DIFFERENT FLUORIDE CONCENTRATIONS IN THEIR WATER SUPPLY
3
a
[F"] i n Plasma , uM C i t y ([F~] i n Water, ppm)
Mean± SD(n)
Albany, N.Y. (<.D
0.38± 0.21 (30)
Rochester, N.Y.(1.0)
0.89± 0.75 (30)
Range
Diff.° P<.05
TR-Fl i n Plasma ***, yM Mean! SD(n)
Range
0.141.1
1.2± 0.6 (30)
0.32.6
4.2
1.2 (30)
6.8
1.3± 0.9 (12)
0.43.9
2.3± 0.6 (4)
1.52.8
1.1± 0.5 (30)
0.12.3
Diff. P<.05
n. s.
n.s. Corpus C h r i s t i , 1.0± Tex.(0.9) 0.35 (12)
0.601.7
Hillsboro, Tex.(2.1)
1.9± 0.9 (4)
0.602.6
Andrews, Tex. (5.6)
4.3± 1.8 (30)
1.48.7
C , d
n. s.
sig.
sig.
sig.
^Sach value used i n the computation was the average of a t l e a s t three r e p l i c a t e analyses and was corrected f o r d i l u t i o n by ACD s o l u t i o n by m u l t i p l y i n g i t by 1.3. taken to be the d i f f e r e n c e between the amount of inorganic f l u o r i d e measured i n ashed and unashed a l i q u o t s of the same sample ^by t - t e s t assuming equal v a r i a n c e i n each group The d i f f e r e n c e between Rochester and Andrews i s s t a t i s t i c a l l y significant.
b
c
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
7.
GUYE TAL.
Fluorocompounds
in Human
127
Plasma
Table I I I MASS BALANCE, RECOVERY AND PURIFICATION FACTOR FOR STEPS IN THE PURIFICATION SYSTEM
Fraction human plasma (ACD, 2.5 l i t e r batch)
Dry Wt. grams 200
Amt. R-F nmoles
a
b Recovery %
Purification
1725 ±273(6)
Methanol E x t r a c t i o n extract
10.
residue
—
105
6.1
±37(4)
±1.0
Sephadex Column Fraction I Fractions II + I I I
F r a c t i o n IV
— 1.29
—
125 ±18(4) 1195 ±129(6) 118
7.3 ±1.6 69.3 ±13.3
108 X
6.8
±29(4)
±1.2
S i l i c i c A c i d Column major peak other peaks combined
.03° —
630
d
240
d
36.5
2,440 X
13.9
fmean ± SD(n) percent of the amount of R-F i n the o r i g i n a l plasma sample, mean ± SD estimate based on weighing the contents of two tubes i n the center ^ of the major peak estimate based on area under peaks from graph
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
128
BIOCHEMISTRY
I
0
* I
1
U
1
<
1
" l
" » H »
I M
CARBON-FLUORINE
ι
Γ -
10 20 30 40 SO 60 70 80 90 100 TUBE
Figure 4.
INVOLVING
NO.
Distribution of organic fluorine from human and bovine plasma in fractions from silicic acid chromatography
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
7.
GUY E T AL.
Fluorocompounds
in Human
Pfosma
129
which samples were placed. The blank f o r the p u r i f i c a t i o n process was obtained by using bovine r a t h e r than human plasma. No organic f l u o r i n e was d e t e c t a b l e i n the o r i g i n a l bovine sample but as a f u r t h e r check the sample was d i a l y s e d to remove i n o r g a n i c f l u o r i d e to f a c i l i t a t e making the measurement f o r organic f l u o r i n e by d i f f e r e n c e . Some organic f l u o r i n e was apparent i n d i a l y s e d bovine plasma: 0.13 ± 0.11 SD μΜ (n=6), a s t a t i s t i c a l l y s i g n i f i c a n t though small d i f f e r e n c e . This t r a c e amount of organic f l u o r i n e c l e a r l y was not found i n the same s i l i c i c f r a c t i o n s as the dominant peak from human plasma as shown i n f i g u r e 4. Human plasma had been stored i n polyethylene bags w i t h ACD s o l u t i o n . A n a l y s i s of ACD s o l u t i o n from unused blood bags and a n a l y s i s of blood plasma before and a f t e r p l a c i n g i t i n the bags showed that not more than 5% of the organic f l u o r i n e i n human plasma could have come fro t h i The d i s t r i b u t i o n o s i l i c i c a c i d chromatography figur batches corresponding to 5 l i t e r s of the o r i g i n a l plasma each. There i s c l e a r l y one dominant peak l y i n g i n approximately the same e l u t i o n p o s i t i o n f o r each batch (the exact p o s i t i o n v a r i e d with column use and the degree of h y d r a t i o n of the s i l i c i c a c i d adsorbant). There were always some s m a l l e r secondary peaks, but they v a r i e d i n s i z e and p o s i t i o n r e l a t i v e to the major peak. The sample used f o r c h a r a c t e r i z a t i o n by nmr was obtained by combining the f r a c t i o n s c o n t a i n i n g the major peaks i n each of the four batches. Much of the m a t e r i a l from batches one and two had been used f o r other purposes p r i o r to t h i s combination. The combined sample was rechromatographed on s i l i c i c a c i d and a s i n g l e sharp peak obtained. The f i n a l sample was taken from the c e n t r a l p o r t i o n of that peak and contained 3.3 μπιοΐββ of organic fluorine. Four sample runs were made on the nmr spectrometer w i t h 15,000 to 17,000 scans each and with a sweep width of 15,151 Hz i n a l l but one run, where i t was 7,576. The r e s u l t s of a l l runs were c o n s i s t e n t w i t h the spectrum shown i n f i g u r e 5 and the chemical s h i f t s shown i n t a b l e IV. A blank run on the solvent mixture showed no i n s t r u m e n t a l a r t i f a c t s which might have contributed to the spectrum. Chemical s h i f t s determined f o r p e r f l u o r o - o c t a n o i c a c i d are a l s o included i n t a b l e IV. Compari son of the s h i f t s i n the unknown with that of p e r f l u o r o - o c t a n o i c a c i d show that there i s a constant d i f f e r e n c e i n s h i f t s of about 2 ppm except f o r the -CF - peak next to the f u n c t i o n a l group (peak E) where the s h i f t i s about 6 ppm. Only the l a t t e r i s enough to be considered a s i g n i f i c a n t d e v i a t i o n s i n c e e x t e r n a l r e f e r e n c i n g was used f o r each. The d i f f e r e n c e i n s h i f t f o r peak Ε i s c o n s i s t e n t with the presence of amide or e s t e r d e r i v a t i v e s , or p o s s i b l y w i t h the presence of a s u l f o n i c a c i d d e r i v a t i v e as the f u n c t i o n a l group. One e x p l a n a t i o n f o r the a d d i t i o n a l peaks i n the spectrum i s the presence of branched isomers, peaks A and Β 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
130
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Table IV RESULTS OF NMR SPECTROSCOPIC ANALYSIS
Chemical S h i f t
Peak Designation
Perfluorooctanoic Acid
Sample
A
-70.7
Β
-71.9
C
-80.0
D
-81.
Ε
-114.3
, ppm
Suggested Assignments -CF groups a t branch p o i n t s 3
terminal -CF i n branched isomers 3
-120.2
-CF - next to X
b
2
F
-120.3
-123.1
G
-121.5
-124.2
H
-122.3
I
-126.0
-CF - i n -CF -CF -CF 2
2
2
2
- C F - next to branch p o i n t s 2
-CF - next to terminal -CFo
-127.6
2
E x t e r n a l r e f e r e n c i n g t o CFC1 was used f o r the chemical s h i f t s , and these a r e expressed with p o s i t i v e numbers , to lower f i e l d ( i . e . , higher frequency), where X i s l i k e l y to be -CO-Y 3
wXjlLt ι ι I ι ι
ι
ι I ι ι ι ι I ι ι ι ι I
AB Figure 5.
CD
ι
ι
ι I ι ι ι ι I ι i ι ι I
i
ι ι
Ε
ι
I i ι ι ι I ι
1
FGH I
NMR spectrum of organic fluorocompound(s) isolated from human pfosma
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
7.
GUY
ET
AL.
Fluorocompounds
in Human
131
Plasma
representing - C F 3 groups at branch p o i n t s , peak C the -CF groups two carbons removed from the branch p o i n t s , and peak H representing -CF - next to the branch p o i n t s . The sample was reanalyzed f o r organic f l u o r i n e f o l l o w i n g c h a r a c t e r i z a t i o n by nmr to check f o r contamination; no a d d i t i o n a l f l u o r i n e was apparent. The degree to which f l u o r i n e from p e r f l u o r o - o c t a n o i c a c i d i s f i x e d as i n o r g a n i c f l u o r i d e during ashing was found to be 21 ± 3 SD % (n=3). 3
2
Discussion These f i n d i n g s suggest that there i s widespread contamination of human t i s s u e s with t r a c e amounts of organic fluorocompounds derived from commercial products. A l l a v a i l a b l e i n f o r m a t i o n on t h i s subject i s i n accordance with t h i s i n t e r p r e t a t i o n . A s e r i e s of compounds havin here f o r the predominan i s widely used commercially poten proper ties. For example, they are used as water and o i l r e p e l l e n t s i n the treatment of f a b r i c s and l e a t h e r . Other uses i n c l u d e the production of waxed paper and the formulation of f l o o r waxes (12). The f i n d i n g s presented here that the c o n c e n t r a t i o n of organic f l u o r i n e was not r e l a t e d to the c o n c e n t r a t i o n of inorgani c f l u o r i d e e i t h e r i n blood or i n the p u b l i c water supply, and the e a r l i e r f i n d i n g that there was l i t t l e or no organic f l u o r i n e i n the blood of animals other than human (6) are a l l i n keeping with environmental sources such as these. The prevalence of organic f l u o r i n e i n human plasma i s probably q u i t e high s i n c e 104 of the 106 plasma samples tested here and a l l 35 i n an e a r l i e r study (5) had measurable q u a n t i t i e s . The prevalence of the p a r t i c u l a r compounds i s o l a t e d and charact e r i z e d here, i . e . , p e r f l u o r o f a t t y a c i d ( C - C ) d e r i v a t i v e s , i s not known s i n c e the s t a r t i n g m a t e r i a l f o r each batch shown i n f i g u r e 4 was pooled from between 25 and 30 i n d i v i d u a l s and s i n c e only about one t h i r d of the o r i g i n a l organic f l u o r i n e content was accounted f o r i n the f r a c t i o n s c o n t a i n i n g these compounds (see t a b l e I I I ) . Peaks other than the one c h a r a c t e r i z e d by nmr appear i n the chromatograms shown i n f i g u r e 4 suggesting that human plasma contains other forms of organic fluorocompounds. They are probably not v o l a t i l e compounds l i k e freons s i n c e i t i s d o u b t f u l that these would be detected by the a n a l y t i c a l methods used i n t h i s study. They correspond i n s o l u b i l i t y to very p o l a r l i p i d s s i n c e they appear i n f r a c t i o n s two and three i n the f o u r t h p u r i f i c a t i o n step. According to the authors of the method used i n that step the f i r s t eluent contains most f a t s , the second and t h i r d eluents c o n t a i n very p o l a r f a t s l i k e g a n g l i o s i d e s and c e r t a i n b i l e a c i d s i n a d d i t i o n to compounds l i k e urea, phenylal a n i n e and t y r o s i n e . The l a s t f r a c t i o n contains water s o l u b l e n o n - l i p i d compounds (11). Components of these other peaks are 6
8
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
132
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INVOLVING
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l e s s p o l a r than the compounds i n the predominant peaks i n accordance with the methanol-in-chloroform gradient used to e l u t e them i n the f i f t h p u r i f i c a t i o n step. Other forms not seen i n s i l i c i c a c i d f r a c t i o n s may a l s o e x i s t s i n c e only about h a l f the o r i g i n a l organic f l u o r i n e was recovered i n these f r a c t i o n s . The a c t u a l amounts of the p e r f l u o r i n a t e d f a t t y a c i d d e r i v a t i v e s i n human plasma i s not known both because i n d i v i d u a l plasma samples were not assayed f o r these p a r t i c u l a r compounds and because the degree to which organic f l u o r i n e from these compounds i s converted to i n o r g a n i c f l u o r i d e during ashing i e not known. Metal s a l t s of p e r f l u o r i n a t e d f a t t y a c i d s have been reported to decompose at 175 to 250° C forming 00 , v o l a t i l e p e r f l u o r i n a t e d o l e f i n s one carbon s h o r t e r , and one atom of f l u o r i d e per molecule (13). About 3 f l u o r i n e atoms per molecule of p e r f l u o r o - o c t a n o i c a c i d were f i x e d as i n o r g a n i c f l u o r i d e by ashing methods used here f l u o r i d e a f t e r ashing f r a c t i o n 4 probably represent somewhere between o n e - t h i r d and one times the molar amount. L i t t l e has been published about the metabolic handling and t o x i c o l o g y of p e r f l u o r i n a t e d f a t t y a c i d d e r i v a t i v e s . Computer a s s i s t e d l i t e r a t u r e searches using Medline, T o x l i n e and Chemcon developed no i n f o r m a t i o n on these s u b j e c t s . This was s u r p r i s i n g with respect to the widespread commercial use of such compounds. I t would appear from information presented here that r a p i d e x c r e t i o n of such compounds i n t o u r i n e i s u n l i k e l y s i n c e they are bound to albumin i n the blood. On t h i s t o p i c i t can a l s o be s t a t e d that other chemicals are u s u a l l y not t o x i c i n blood con c e n t r a t i o n s s i m i l a r to those found here f o r organic f l u o r i n e . The c o n c e n t r a t i o n of organic f l u o r i n e i n human plasma may be changing with time. In 1960 Singer and Armstrong reported that the plasma of 70 i n d i v i d u a l s r e s i d i n g i n communities with 1 ppm or l e s s f l u o r i d e i n t h e i r p u b l i c water supply had an average c o n c e n t r a t i o n of f l u o r i d e of 8.8 μΜ (8). They prepared t h e i r samples by ashing them and then d i s t i l l i n g f l u o r i d e from the ash a c i d i f i e d with p e r c h l o r i c a c i d (7). Thus, i t seems l i k e l y that t h e i r values f o r " f l u o r i d e " would have included organic f l u o r i n e had i t been present. Assuming that i n o r g a n i c f l u o r i d e concentrations at that time were s i m i l a r to those found i n t h i s study (see t a b l e I I ) , the organic f l u o r i n e component would exceed 7 μΜ. In 1969 the same i n v e s t i g a t o r s using the same method reported an average f l u o r i d e c o n c e n t r a t i o n of 4.5 μΜ f o r 6 plasma samples each pooled from at l e a s t 3 i n d i v i d u a l s supposedly l i v i n g i n f l u o r i d a t e d communities (14). This corresponds to an organic f l u o r i n e component of only about 4 μΜ. Organic f l u o r i n e c o n c e n t r a t i o n presented here averages only 1.35 μΜ. Therefore, there may have been a decrease i n the c o n c e n t r a t i o n of organic f l u o r i n e i n human plasma s i n c e the l a t e 1950 s An a l t e r n a t e explanation might be that d i f f e r e n c e s i n 2
f
%
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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the a n a l y t i c a l methods or d i f f e r e n c e s i n the sample populations caused these values to vary. Organic f l u o r i n e i s the predominant form of f l u o r i n e i n human blood except where the c o n c e n t r a t i o n of f l u o r i d e i n d r i n k i n g water i s high ( i n which case f l u o r i d e predominates, see t a b l e I I ) . T h i s together with the f i n d i n g reported here that there i s no apparent r e l a t i o n s h i p between the concentrations of organic f l u o r i n e and i n o r g a n i c f l u o r i d e i n plasma helps e x p l a i n why i n e a r l i e r s t u d i e s (8) no r e l a t i o n s h i p was found between plasma f l u o r i d e determined i n ashed samples and the f l u o r i d e content of the p u b l i c water supply. The data i n t a b l e I I show that when methods s p e c i f i c f o r i n o r g a n i c f l u o r i d e are a p p l i e d , a c l e a r r e l a t i o n s h i p between f l u o r i d e i n plasma and f l u o r i d e i n the p u b l i c water supply (between 0.1 and 5.6 ppm) can be demonstrated. Thus, there i s no need to p o s t u l a t e the existence of such r i g o r o u s homeostati f l u o r i d e as suggested e a r l i e concentrations f o r i n d i v i d u a l s l i v i n g i n the same c i t y as reported here r e f l e c t the balance e s t a b l i s h e d between f l u o r i d e i n blood and that i n bone mineral over periods of years. These f i n d i n g s do not c o n t r a d i c t a passive homeostatic c o n t r o l mechanism i n which bone m i n e r a l damps swings i n blood f l u o r i d e c o n c e n t r a t i o n over r e l a t i v e l y shorter periods of time. The values presented here f o r the average i n o r g a n i c f l u o r i d e c o n c e n t r a t i o n of plasma from i n d i v i d u a l s l i v i n g i n a community having about 1 ppm f l u o r i d e i n the water supply are c o n s i s t e n t with recent f i n d i n g s of others using s i m i l a r methods (14, 15).
Literature Cited 1. Taves, D.R., Nature (1966), 211, 192. 2. Taves, D.R., Nature (1968), 217, 1050. 3. Taves, D.R., Nature (1968), 200, 582. 4. Taves, D.R., Talanta (1968), 15, 1015. 5. Guy, W.S., "Fluorocompounds of Human Plasma: Analysis, Prevalence, Purification and Characterization", doctoral thesis, University of Rochester, Rochester, N.Y., 1972. 6. Taves, D.R., J. Dental Res. (1971), 50, 783. 7. Singer, L. and Armstrong, W.D., Anal. Chem. (1959), 31, 105. 8. Singer, L. and Armstrong, W.D., J. Appl. Physiol. (1960), 15, 508. 9. Taves, D.R., Talanta (1968), 15, 969. 10. Frant, M.S. and Ross, Jr., J.W., Science (1966), 154, 1553. 11. Siakotos, A.N. and Rouser, G., J. Amer. Oil Chem. Soc. (1965) 42, 913. 12. Bryce, H.G., "Industrial and Utilitarian Aspects of Fluorine Chemistry" in "Fluorine Chemistry", Vol. V, J.H. Simon, ed., Academic Press, N.Y., 1964. 13. Hals, L.J., Reid, T.S. and Smith, G.H., J. Amer. Chem. Soc. (1951), 73, 4054. 14. Singer, L. and Armstrong, W.D., Arch. Oral Bio.(1969), 14, 1343. 15. Singer, L. and Armstrong, W.D., Biochem. Med. (1973), 8, 415.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Q. I wonder if you t r i e d to c o r r e l a t e w i t h i n i n d i v i d u a l s the l e v e l of organic f l u o r i n e with age. A. I t would c e r t a i n l y be i n t e r e s t i n g to have t h i s information but unfortunately we cannot supply i t at t h i s time. An expeditious approach might be to analyze cord blood from i n f a n t s of mothers who had not received f l u o r i n e - c o n t a i n i n g anesthetics at childbirth. I t would a l s o be of i n t e r e s t to know whether i n d i v i d u a l s l i v i n g i n i s o l a t e d regions have organic f l u o r i n e i n t h e i r blood plasma. Q. Did you say the sample analyzed by nmr contained methyl alcohol? A. Yes, I d i d . Q. Methyl a l c o h o l w i l l r e a c t very r a p i d l y with f l u o r i n a t e d acids. The nmr spectrum may, t h e r e f o r e , represent that of methyl ester d e r i v a t i v e s . A. Methanol was a l s o use p u r i f i c a t i o n system. Th presence of methyl ester d e r i v a t i v e s of p e r f l u o r i n a t e d f a t t y acids (C6-C8) and t h e i r branched isomers.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8 Intravenous Infusion of Cis-Trans Perfluorodecalin Emulsions in the Rhesus Monkey LELAND C. CLARK, JR., EUGENE P. WESSELER, SAMUEL KAPLAN, and CAROLYN EMORY Children's Hospital Research Foundation, Elland and Bethesda Aves., Cincinnati, Ohio 45229 ROBERT MOORE Sun Ventures, Inc., Marcus-Hook, Pa. 19061 DONALD DENSON Stanford Research Institute, Menl Introduction Interest in the possibility of making fluorocarbon-based artificial blood began following the discovery by Clark (1) that animals survived the breathing of oxygen-saturated FC75 (3M Company). Since that time over 150 papers have been published, a FASEB Symposium (2) and several industrial symposia have been held on the subject. Reviews by Sloviter (3) and Geyer (4) have been published. A large number of perfluorochemicals (PFC) have been screened for this purpose. Most PFC carry oxygen in biologically significant quantities. But most are ruled out for practical use because, after performing their oxygen-carrying function, they remain in the body, largely in the liver, after being taken up mainly by the mononuclear phagocytes as are all PFC. So far it seems that only those containing fluorine and carbon or fluorine, carbon and bromine in their structure leave the body. Although we have not completed our testing of the PFC on hand and expect to test new ones in the future, nonetheless, of the PFC tested so far, perfluorodecalin (PFD) emerges as the best because it leaves the liver in a reasonable time and has a vapor pressure compatible with intravenous use. As an emulsion, its acute intravenous toxicity in the mouse is very low. It is available in commercial quantities and can be partially purified by distillation. For these, and other reasons, it was selected for testing as artificial blood in a non-human primate, the rhesus monkey. We previously reported (5) on the infusion of perfluorodecalin in one awake rhesus monkey. This paper describes the first results of further tests in 19 monkeys and 10 dogs and outlines the beginning of a protocol for its possible evaluation as a blood substitute in man. Of the 19 monkeys, 10 were infused with 10% PFD, 4 with 20% PFD emulsions, and 4 with only a "hypotensive test dose". The data included here is only a part of that on hand; it has been selected to illustrate the salient problems and findings. It is to be interpreted in terms of our previous publications (6-18) on this subject. 135 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Data on oxygen s o l u b i l i t y i n PFC presented at t h i s meeting i s being expanded and w i l l be the subject of a separate report (19). Methods and m a t e r i a l s Nineteen monkeys were s e l e c t e d from the I n s t i t u t e of Developmental Research's r e s i d e n t p o p u l a t i o n . Many of these monkeys had been p r e v i o u s l y used f o r t e s t i n g drugs i n t e r a t o l o g i c a l research. Three were males and had not been given drugs. The monkeys were housed i n s t a i n l e s s s t e e l cages i n a i r c o n d i t i o n e d rooms and were fed a d i e t of Purina monkey chow (Code 5038) supplemented with bananas, oranges, and raw peanuts. They were given p e r i o d i c s k i n t e s t s f o r t u b e r c u l o s i s and were given 11 mg/kg i s o n i a z i d e d a i l y impregnated as a s o l u t i o n on a sugar cube. The monkeys, a l l of which presumably have lun mites chest d arrival and weighed weekly. P u b l i used f o r monkeys obtaine lowed i n the l a b o r a t o r y . The dogs were beagles obtained from a commercial source. The f i r s t monkey tested and reported (_5) , was a young male. Two of the monkeys i n the present s e r i e s were a l s o males. The v e t e r i n a r i a n s were unable to a s s i g n an approximate age to any monkey but, judging by t h e i r teeth and t h e i r behavior, many were very old. The p e r f l u o r o d e c a l i n used i n these experiments was purchased from ISC Chemicals, L t d . , Avonmouth, B r i s t o l , England and p u r i f i e d by s p i n n i n g band d i s t i l l a t i o n using a Perkin-Elmer NFA-200 Autoannular s t i l l . The f r a c t i o n b o i l i n g between 91°C and 93°C at 180 mm pressure was used f o r these s t u d i e s . The P l u r o n i c (PF68) s o l u t i o n was made by d i s s o l v i n g 200 gm i n s t e r i l e water (Abbott), brought up to 1 l i t e r , and f i l t e r i n g succ e s s i v e l y through 5.0, 0.8, and 0.22 uM M i l l i p o r e f i l t e r s at 4°C. This stock was d i l u t e d j u s t before use, the i o n i c composition was adjusted so the emulsion contained h a l f the s a l t s required f o r Ringer's and the pH was adjusted with Tham (28). Emulsions were made i n a G a u l i n Model 15M l a b homogenizer. Parts which were exposed to the emulsion were autoclaved. The shear pressure was preset to 6000 l b s / i n ^ and the gauge was r e moved. Homogenization was continued u n t i l the o p t i c a l d e n s i t y plateaued. The emulsion was placed i n s t e r i l e Pyrex v i a l s or one l i t e r b o t t l e s , frozen and preserved at -70°C. S t e r i l i t y t e s t s , performed by adding 2 ml i n t o 20 ml of a supplemented peptone broth prepared by Becton-Dickinson and Co., were conducted on random samples of the emulsion. A l l t e s t s showed the emulsion to have no b a c t e r i a l growth. Chemical methods o f s t e r i l i z a t i o n i n c l u d i n g the use of prop i o l a c t o n e were avoided, except with one monkey, Melvin 05), who r e c e i v e d emulsion s t e r i l i z e d with t h i s compound.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Two batches of 10% by volume PFD i n 5% PF68 and two batches of 20% by volume PFD i n 10% PF68 were prepared. A l l of the blood pressure t e s t doses were from the same batch (1 l i t e r ) of 10% PFDE and were taken from small Pyrex v i a l s which were thawed j u s t before use. A l l of the monkeys i n f u s e d with 10% PFDE r e c e i v e d emulsion (4 l i t e r s ) from the second batch where i n d i v i d u a l bott l e s were thawed j u s t before use. Two of the monkeys, C2 and C3, r e c e i v e d 20% PFDE from a t h i r d batch (1.5 l i t e r ) . Two, 74 and 113, r e c e i v e d 20% PFDE from a f o u r t h batch (4 l i t e r ) . The f i r s t three batches were prepared from pooled PFD (ISC a n a l y t i c a l r e f erence S173/A) while the f o u r t h batch was prepared from PFD (ISC a n a l y t i c a l references S172/Aand S148/B). A l l the 10% PFDE and the f i r s t two 20% PFDE emulsions were s t o r e d i n i c e water u n t i l used but the 20% PFDE f o r 74 was warmed before a d m i n i s t r a t i o n and that used f o r 113 was bubbled with oxygen at room temperature f o r three hours before i n f u s i o n Blood samples wer through i n d w e l l i n g catheter syringes having the dead space f i l l e d with heparin (1000 USP u n i t s / m l ) . The samples from awake r e s t r a i n e d monkeys were obtained by venipuncture. Precautions were taken to be sure that the blood samples were not d i l u t e d with the i s o t o n i c s o l u t i o n s used to r i n s e the sampling l i n e s . Samples f o r blood gases, pH, and packed c e l l volume were analyzed immediately. The remaining blood was c e n t r i f u g e d and the plasma analyzed by Autotechnicon SMA 12 procedures (21)• Venous blood samples were taken at random from L. C l a r k , to determine the v a r i a b i l i t y of the same blood f a c t o r s being measured i n the monkey. Most of the samples from L. C l a r k were taken during the f a s t i n g s t a t e , a l l those reported here were taken while L. C l a r k was i n a s t a t e of w e l l - b e i n g , and a l l were processed l i k e monkey blood. Samples were taken p e r i o d i c a l l y from the monkey p r e v i o u s l y published (5). A l l samples not analyzed immediately were c h i l l e d i n an ice-water bath. Approximately 4800 determinations were performed as part of the research reported here. Test f o r hypotensive
effect
The monkeys were f a s t e d overnight and a n e s t h e t i z e d with i n travenous sodium p e n t o b a r b i t a l i n the morning. They breathed hum i d i f i e d oxygen. The a r t e r i a l pressure was measured by means of a p e d i a t r i c c u f f and a Model 802 Doppler (Parks E l e c t r o n i c s Labo r a t o r y , Oregon) using the r a d i a l a r t e r y . 0.05 ml/kg of emulsion was i n j e c t e d i n t r a v e n o u s l y and r i n s e d i n with 5 ml of Ringer's s o l u t i o n . A second t e s t dose was given w i t h i n f i v e minutes a f t e r the f i r s t i n the monkey but a f t e r the blood pressure returned to normal, 10 or 20 minutes, i n the dog. A s i m i l a r procedure was used f o r each v i a l of emulsion using the beagle w i t h i n one day of the t e s t i n the monkey. The blood pressure i n the dog was measured with a mercury manometer and d i r e c t cannulation of the femoral
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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artery. Twelve monkeys were subjected to biopsy before PFDE i n f u s i o n and nine a f t e r . The o v e r n i g h t - f a s t e d monkeys were anesthetized with sodium p e n t o b a r b i t a l and a 2 or 3 gram piece of l i v e r was exc i s e d under d i r e c t v i s i o n , f i x e d , and examined by l i g h t and e l e c tron microscopy using methods p r e v i o u s l y published (18). Parts of these post i n f u s i o n samples were analyzed f o r PFD by sodium b i phenyl combustion and by GLC. C o n t r o l blood samples were taken p r i o r to making the biopsy i n c i s i o n . Post biopsy blood samples were taken one hour a f t e r the i n c i s i o n was c l o s e d and again on the f o l l o w i n g day from the r e s t r a i n e d animal. For the i n f u s i o n , the animal i s anesthetized with pentobarbit a l and kept asleep with pentothal. An endotracheal tube i s i n serted and the animal's head covered with a transparent bag being flushed with humidified oxygen. The femoral v e i n s and the r a d i a l a r t e r y are cannulated with s t e r i l e p l a s t i c tubes and the r i g h t heart i s c a t h e t e r i z e d wit On the day of i n f u s i o and ECG of the monkey have been recorded f o r s e v e r a l minutes and look s t a b l e , the f i r s t samples of blood are taken f o r blood gas analyses. A 2 ml sample of blood i s drawn into a h e p a r i n i z e d 2 ml syringe from the r i g h t heart and the r a d i a l a r t e r y . I f the p02 and pC0 readings obtained on these samples are w i t h i n normal l i m i t s , a l a r g e (12.5 ml/kg) sample of blood i s removed. This blood is c e n t r i f u g e d and analyzed as described elsewhere. At t h i s p o i n t 21 ml/kg of 57o human albumin i s infused with a Sage Instrument syringe pump at 6 ml/min. Another 12.5 ml/kg of blood i s removed c a r e f u l l y so that the blood pressure does not f a l l below 80 mm Hg. The hematocrit of t h i s blood i s determined and i f i t i s not one h a l f the o r i g i n a l hematocrit, another 4 ml/kg i s infused and another hematocrit run. To date, t h i s procedure has always reduced the hematocrit approximately 507o. In f i v e of the f i r s t s i x monkeys infused 50 ml/kg of Ringer's s o l u t i o n was given i n place of the albumin. The PFDE i s infused i n four batches and a r t e r i a l and mixed venous blood gases and pH are measured a f t e r each of the four batches are i n . A f t e r the i n f u s i o n , the a r t e r i a l cannula and a l l but one of the venous cannulae are removed and the i n c i s i o n s c l o s e d . The animal i s kept i n the operating room u n t i l i t begins to waken. Then i t i s t r a n s f e r r e d to a recovery room and c l o s e l y watched f o r the next 12 hours. F l u i d s may be given intravenously i f r e q u i r e d . U s u a l l y the monkeys are awake and d r i n k i n g l i q u i d s or even e a t i n g by l a t e afternoon. The autopsies are conducted under the auspices of the Medical School's V e t e r i n a r y Department. The organs are inspected, photographed, weighed and h a l f of each organ i s placed i n n e u t r a l 10% phosphate-buffered f o r m a l i n s o l u t i o n and h a l f i s placed i n 957o ethanol a f t e r portions of each organ are removed f o r f i x i n g , s t a i n ing and examining under l i g h t microscopy. 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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Results and d i s c u s s i o n I n t e r l a b o r a t o r y v a r i a t i o n i n the a n a l y s i s of three p e r f l u o r i nated l i q u i d s i s given i n Table 1. I t i s apparent that the d i f f i c u l t y l i e s i n g e t t i n g q u a n t i t a t i v e recovery of f l u o r i n e and not of carbon. Some a n a l y t i c a l l a b o r a t o r i e s obtained such inaccurate r e s u l t s that they abandoned attempts to analyze the samples. Another l a b o r a t o r y i s continuing i t s e f f o r t s to develop a method. No d i f f i c u l t y was encountered, i n preparing the PFD emuls i o n s . Those preserved at -70°C remained f o r months with no d i s c e r n i b l e change. The seven day LD50 i n mice of the i n f u s e d 10% PFDE was 140 ml/kg; the LD50 of the 20% PFDE was 69 ml/kg. Measurement of p a r t i c l e s i z e d i s t r i b u t i o n i n three emulsions was performed at Sun Ventures by a technique (26) i n v o l v i n g e l e c tron microscopy and the r e s u l t s are shown i n F i g u r e 1. Tables 2 and 3 represent the changes i n blood chemistry found upon biopsy of the l i v e r l i t t l e meaning because glycolysis. The increased l a c t i c dehydrogenase a c t i v i t y may have come from the damaged l i v e r . There i s no doubt that c r e a t i n e kinase increased as a r e s u l t of the procedure but i t i s known to increase a f t e r any kind of surgery (25). C o n t r o l values f o r blood samples from a human primate are shown i n Table 7 and those from awake monkeys i n Table 5. One reason the monkey was s e l e c t e d f o r these experiments i s that i t very o f t e n behaves l i k e the human i n i t s response to drugs. The dog sometimes shows b i z a r r e responses to emulsions and s o l u t i o n s c o n t a i n i n g s u r f a c t a n t s . In Table 4 i t can be seen that no monkey t e s t e d , but a l l dogs, s u f f e r e d a drop i n blood pressure. It sometimes takes h a l f an hour f o r the dog to recover but once i t has recovered a second dose has very l i t t l e or no e f f e c t . In Tables 10 and 11 i t can be seen that n e i t h e r the anesthes i a nor the s u r g i c a l manipulations i n v o l v e d i n g i v i n g a t e s t dose had a s i g n i f i c a n t e f f e c t upon the blood components analyzed except f o r c r e a t i n e kinase where a small but s i g n i f i c a n t increase occurred. The abnormally high values f o r twenty-four hours on monkey 80 are due to the f a c t that she was moribund and died two hours l a t e r a f t e r s e v e r a l attempts at r e s u s c i t a t i o n . The d e t a i l s concerning the replacement of withdrawn blood by Ringer's s o l u t i o n and/or 5% human albumin are given i n Table 16. The amounts of PFDE i n f u s e d are shown. For our purpose here we have r e f e r r e d to Dextran 40 and albumin as osmotic or o n c o t i c l i q quids. Albumin, however, has a longer h a l f l i f e i n the body and of course the PF68 i n PFDE has o n c o t i c a c t i v i t y . 5% human a l b u min was given p r e v i o u s l y to one monkey with no d i s c e r n i b l e e f f e c t . That we had considerable d i f f i c u l t y i n maintaining blood b a l ance a f t e r phlebotomy and PFDE i n f u s i o n i s apparent from the t a b l e . Most of the d i f f i c u l t i e s happened during the evening of the day of the i n f u s i o n and were thought to be due to the f a c t that most of the PF68 and water administered had been excreted l e a v i n g the
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Particle size distribution as determined by electron microscopy
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animals hypovolemic and although awake they were not a l e r t and were weak. When Dextran 40 was given on two occasions (C2, C3) four or f i v e hours post i n f u s i o n the animals responded to the point where they were d i f f i c u l t to r e s t r a i n . They a l s o drank Gatorade and ate. One which r e c e i v e d Dextran twenty-four hours l a t e r , f o l l o w i n g an a l l night slow b l e e d i n g from a p o o r l y t i e d i n cannula, r e v i v e d only b r i e f l y . Most of the 10% PFD monkeys that r e c e i v e d albumin during the phlebotomy r e q u i r e d l e s s f l u i d l a t e r . Two (74, 113) that r e c e i v e d 20% PFDE d i e d , p o s s i b l y because they were overloaded, but more l i k e l y because the PFDE they were given had been a t room temperature too long and coalescence of p a r t i c l e s had begun. Our experience i n maintaining blood and f l u i d balance i n thousands of dogs and human p a t i e n t s has not served us w e l l with the rhesus. I t seems to us that the rhesus monkey, p a r t i c u l a r l y the o l d monkey, i s a very d e l i c a t e and f r a g i l e animal. The main f i n d i n g i Tabl 6 i tha th mixed tension increased i n a l none of those r e c e i v i n g p0 beyond 100 mm, the approximate p o i n t where a l l the hemoglobin i s saturated. In three of the monkeys r e c e i v i n g 20% PFDE the mixed venous p 0 went above 100 mm. In monkey C3 the a r t e r i a l p 0 was on the low s i d e and t h i s could not be increased by chest and/or heart massage. At autopsy t h i s monkey was found to have severe emphysema. T h i s low a r t e r i a l p 0 may account, at l e a s t i n p a r t , f o r the low venous p0 . At autopsy C3 was found to have lungs h e a v i l y i n f e s t e d with mites. The heart r a t e decreased and the r e s p i r a t i o n increased as a r e s u l t of PFDE i n f u s i o n as shown i n Table 8. The Τ t e s t s shown i n t h i s t a b l e i n d i c a t e there i s no d i f f e r e n c e i n these responses to the 10% and 20% PFDE. There was no change i n a r t e r i a l pressure as a r e s u l t of PFDE i n f u s i o n as shown i n Table 9. From these and other s t u d i e s we have concluded that there i s an increase i n pulmonary a r t e r i a l pressure followed by a r e t u r n to normal i n about an hour. Because of u n c e r t a i n t y about the l o c a t i o n (RV or PA) of the catheter t i p (x-ray equipment was not a v a i l a b l e to us during these s t u d i e s ) the data i n Table 8 cannot be analyzed s t a t i s t i c a l l y . However i t can be seen that i n a l l of the cases where the pressure was low before i n f u s i o n , probably i n d i c a t i n g that pulmonary a r t e r i a l pressure was being monitored, there was a d i s t i n c t i n c r e a s e . Tables 12 and 13 give the r e s u l t s of a n a l y z i n g the blood of monkeys r e c e i v i n g 10% PFDE and Table 13 gives the average values and standard e r r o r s f o r t h i s data. Some of the apparent decreases i n c o n c e n t r a t i o n of blood components are due to the f a c t that about h a l f the blood was removed and d i l u t e d by Ringer's, albumin, and PFDE. The extent to which d i l u t i o n per se a f f e c t e d the r e s u l t s can be best judged by the extent to which the hematocrit de creased. While the t a b l e shows a decrease i n a l l components ex cept t o t a l b i l i r u b i n , i f these are " c o r r e c t e d " f o r d i l u t i o n by 2
2
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2
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Figure 2. Light microscopic view of the liver of a mon key before infusion. H, hepatocyte; N, nucleus; S, sinus oid; Toluidine blue 0. χ 850.
Figure 3. Liver specimen taken at sacrifice two weeks after infusion with a 20% emulsion shows hepatocytes essentially unchanged morphologically. Cytoplasm of is completely filled with particles of PP5, and bulges into the lumen of sinusoids. H, hepatocyte; N, nucleus; M, mononuclear phagocyte. Toluidine blue 0. X 850.
Figure 4. Eleven months after infusion, the liver shows normal morphology and no structures remained which could be identified unequivocally as fluorocarbon. Other features were within normal limits. H, hepatocyte; N, nucleus; S, sinusoid. Toluidine blue 0. X 850.
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d i v i d i n g by 0.32, the hematocrit f a c t o r , the r e s u l t s i n d i c a t e an increase i n everything except c h o l e s t e r o l . The percentage change i n the components before and a f t e r c o r r e c t i o n are as f o l l o w s : TP 41,128; AB 71,222; CA 71,222; IP 90,281; GL 74,231; BN 88,275; VA 44,138; CT 80,250; TB 140,438; AP 51,159; LD 75,234; GO 45,141; CK 142,444; CH 18,56; PV 32,100. The blood chemistry changes found before and a f t e r i n f u s i o n of 20% PFDE are shown i n Table 14. Twenty-eight samples were taken from the monkey i n f u s e d with 10% PFDE about a year ago and the mean values are shown i n Table 15. The mean a l k a l i n e phosphatase i s higher than that shown i n Table 3 and Table 5 but t h i s would be expected because Melvin i s growing r a p i d l y . A l l the other data are normal according to the information we have accumulated here. Over a three month p e r i o d the average weight gain of the eight s u r v i v i n g monkeys i n f u s e d with PFDE t significantl d i f f e r e n t from s i x monkey Morphology. Three of nineteen monkeys are p i c t u r e d here which represent the p r e i n f u s i o n , post i n f u s i o n and long term r e covery of the l i v e r of a l l monkeys examined to date. The normal, or p r e i n f u s i o n morphology of the l i v e r d i f f e r e d s l i g h t l y from that of other commonly used l a b o r a t o r y mammals ( f i g . 2). V a r i a t i o n s included a marked e l e v a t i o n i n hemosiderin depos i t s i n Kupffer c e l l s , g e n e r a l l y a s s o c i a t e d with age, numerous large autophagic vacuoles (10 μ) and the presence of long tubular s t r u c t u r e s i n the mitochondria. These p e c u l i a r i t i e s have a l s o been observed by others (27). Within twenty four hours a f t e r i n f u s i o n of emulsions of PP5, p a r t i c l e s begin to appear w i t h i n the phagocytic c e l l s of the s i n u s o i d s of the l i v e r , other organs, and the c i r c u l a t i o n . Just a f t e r i n f u s i o n , while p a r t i c l e s are i n the bloodstream, the poly-' morphonuclear c e l l s show a few cytoplasmic p a r t i c l e s . Most a f fected m o r p h o l o g i c a l l y by the i n f u s i o n of emulsions are the c e l l s of the mononuclear phagocytic system. These c e l l s engulf i n d i v i dual p a r t i c l e s or clumps of p a r t i c l e s u n t i l they reach very l a r g e proportions ( f i g . 3) as they r e s t i n the l i v e r s i n u s o i d s , s p l e n i c pulp or s i m i l a r area. A f t e r a few days they may aggregate i n t o small nodules of e p i t h e l i a l - t y p e c e l l s s i m i l a r to those seen i n other f o r e i g n body responses ( f i g . 3). The hepatocytes are not changed d r a m a t i c a l l y by the i n f u s i o n of emulsions ( f i g . 4) but a small number of p a r t i c l e s do enter the cytoplasm. These d i m i n i s h , however, and u l t i m a t e l y disappear by unknown means. Residual e f f e c t s of the occupation of the l i v e r (and of course other comparable c e l l systems i n the body) by PFC i n f a c t have not been observed m o r p h o l o g i c a l l y , s i n c e mito chondria, smooth and rough endoplasmic r e t i c u l u m , lysosomes,microbodies, n u c l e i , e t c . , appear i n d i s t i n g u i s h a b l e from c o n t r o l s ( f i g s . 5,6). A l l the specimens shown were obtained at biopsy.
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Figure 5. Electron microscopic view of liver from the same monkey as figure 3. A very small percent of the cytoplasm is occupied by particles of fluorocarbon (small arrow). Organelles appear unchanged. H, hepatocyte cytoplasm; N, nucleus; M, mononuclear phagocyte with cytoplasm full of fluorocarbon particles (large arrows). Uranyl acetate, lead citrate. X 8,500.
Figure 6. Liver from the same monkey as figure 4 shows organelles which appear normal. Fluorocarbon particles, no longer identified in the cytoplasm of hepatocytes or mononuclear phagocytes, have apparently left the liver unchanged. M, mitochondria; N, nucleus; H, hepatocyte cytoplasm. Uranyl acetate, lead citrate. X 4,000.
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Post mortem f i n d i n g s . Of ten monkeys which r e c e i v e d 10% PFDE; eight are a l i v e . One, M e l v i n , has s u r v i v e d over a year. One died the day a f t e r i n f u s i o n from slow b l e e d i n g around a venous cannula. One (117) died eleven weeks a f t e r the i n f u s i o n from anesthesia preparatory to surgery f o r l i v e r biopsy. Of the four monkeys which r e c e i v e d 20% PFDE two died w i t h i n twenty four hours f o l l o w ing the i n f u s i o n and were thought to have died because the emuls i o n had begun to d e t e r i o r a t e . P o s s i b l y t h e i r c i r c u l a t i o n s were overloaded. Two s u r v i v e d i n apparent good h e a l t h but were s a c r i f i c e d at one and two weeks post i n f u s i o n because t h e i r l e g s became ischemic and p o s s i b l y gangrenous, f o l l o w i n g l i g a t i o n of t h e i r femoral a r t e r i e s as a part of the process of r e c o r d i n g t h e i r a r t e r i a l pressure. Femoral a r t e r i e s can be l i g a t e d i n cats and dogs with no v i s i b l e e f f e c t but primates are s u s c e p t i b l e to n e c r o s i s i f the femoral a r t e r i a l c i r c u l a t i o n i s compromised. One monkey's femoral a r t e r y was s u c c e s s f u l l y r e p a i r e d ; t h i s animal (117) l i v e d eleven weeks with norma femoral a r t e r y , d i r e c t pressure tery. One monkey s u f f e r e d p a r t i a l l o s s of the f i n g e r s of the l e f t hand f o l l o w i n g l i g a t i o n of the r a d i a l . One i n j u r e d monkey, 6, was s a c r i f i c e d s h o r t l y a f t e r i t was purchased, and before i t was used as a c o n t r o l f o r organ weights. Monkey 80 b l e d to death overnight because a femoral a r t e r y was punctured during an attempt at catheterization. The organ weights obtained at autopsy are shown i n Table 17. Because of the d e a r t h of normal data i n the rhesus very l i t t l e can be s a i d about the e f f e c t s of i n f u s i o n . The l i v e r of C2 and the spleen of C3 are q u i t e p o s s i b l y enlarged. A l l the monkeys were found to have lung mites, and some were considered h e a v i l y i n f e s t e d . Many of the lungs were s t i f f and d i d not c o l l a p s e . More d e t a i l e d r e p o r t s of the f i n d i n g s with l i g h t microscopy w i l l be the s u b j e c t of a separate r e p o r t . Sixteen p i e c e s from v a r i o u s p a r t s of the l i v e r of one monkey were analyzed and were found to range from 0.61% to 2.05% with a mean of 1.45%. Information obtained by sodium biphenyl combustion of the l i v e r and GLC are shown i n Table 18. There was of course c i r c u l a t i n g PFDE i n monkeys 62, 74, and 113. Depending on the dose, i t r e q u i r e s s e v e r a l days f o r i t to completely disappear from the blood. The PFD disappears from the blood both by evaporation through the lungs and s k i n and by being engulfed by the scavenger c e l l s of the body. I n t e r e s t i n g l y , a t r a c e could be detected i n Melvin's l i v e r almost a year a f t e r the i n f u s i o n . There i s c o n s i d e r a b l e v a r i a b i l i t y between monkeys i n the way i n which they sequester the PFD i n t h e i r l i v e r s and spleen, and the r a t e at which i t leaves. The h a l f l i f e of PFD i n the l i v e r i s probably about two weeks. In the mouse, most PFD i s gone i n two weeks.
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General d i s c u s s i o n Although most of our r e s u l t s have been discussed above we think i t may be d e s i r a b l e to add some general comments about our concepts and our f i n d i n g s . Our work on a r t i f i c i a l blood i s prompted by our experience with thousands of open heart surgery p a t i e n t s and a f a m i l a r i t y with the problems surrounding plasma, plasma s u b s t i t u t e s , pare n t e r a l s o l u t i o n s and the use of donor blood. Various substances have been used over the past s e v e r a l decades as plasma s u b s t i t u t e s . C e r t a i n of these substances, such as Dextran and plasma albumin, have earned a d e f i n i t e place i n c l i n i c a l medicine f o r maintaining c i r c u l a t o r y volume. G e n e r a l l y , such s o l u t i o n s d i s s o l v e only 2 or 3 ml of oxygen per 100 ml, while whole blood d i s s o l v e s about 20 ml per 100 ml. In order to meet the oxygen demands of the body the c a r d i a c output must be increase words, the way i n which c a r r y i n g c a p a c i t y of blood i s to work harder and pump more blood. Because fluorocarbons c a r r y l a r g e q u a n t i t i e s of oxygen they can be used to increase the oxygen c a p a c i t y of the c i r c u l a t i n g blood and t h e r e f o r e decrease the work of the heart. Of course, a weak, f a i l i n g , or i n j u r e d heart may not be able to increase i t s output to meet oxygen needs and shock and death may f o l l o w . Therefore, a primary f u n c t i o n of a r t i f i c i a l blood i s to decrease the work of the heart. Oxygen c a r r y i n g l i q u i d s w i l l be most u s e f u l i n c o n d i tions where the c a r d i a c output i s low or the blood volume i s below normal. The only other way to increase the oxygen c a p a c i t y of blood i s to add stroma-free hemoglobin but t h i s has not proven to be p r a c t i c a b l e yet. Hemoglobin i s e a s i l y s t o r e d and i t may someday be p o s s i b l e to use hemoglobin from other animals, such as the cow and the p i g . One of i t s f a u l t s i s i t s high rate of e x c r e t i o n . Synthetic oxygen chelates seem to be f a r i n the f u t u r e . PFC a r t i f i c i a l blood, l i k e stroma-free hemoglobin, has no blood types. Our research on l i q u i d breathing of p e r f l u o r o c h e m i c a l l i q u i d s formed the e a r l y b a s i s of the work reported here. It indicated, f o r example, that c e r t a i n h i g h l y f l u o r i n a t e d l i q u i d s were probably biologically inert. I t has l e d to the use by Dr. David M. Long, as we hear today, of brominated p e r f l u o r i n a t e d l i q u i d as X-ray contrast agents i n the d i a g n o s i s of lung disease. Perfluorocarbon l i q u i d s may a l s o be u s e f u l some day i n the treatment of lung d i s eases, such as by washing out o b s t r u c t i v e m a t e r i a l s . I t would be h i g h l y d e s i r a b l e to have a s u i t a b l e water s o l u b l e s y n t h e t i c oxygen solvent to increase the oxygen c a p a c i t y of plasma or plasma s u b s t i t u t e s . PFC i n general are good oxygen and carbon d i o x i d e s o l v e n t s but they are completely i n s o l u b l e i n water. Therefore they can only be used as emulsions. This introduces at l e a s t three problems. (a) B i o l o g i c a l l y s u i t a b l e e m u l s i f i e r s must be found and used. (b) C e r t a i n PFC cannot be e m u l s i f i e d and
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c e r t a i n others form only unstable emulsions. (c) Because emulsions c o n s i s t of suspended p a r t i c l e s they are g r a d u a l l y removed from the c i r c u l a t i o n and deposited i n part i n the l i v e r and spleen. We have e l e c t e d to concentrate our e f f o r t s on a study of the use of p e r f l u o r o d e c a l i n p r i m a r i l y because i t g r a d u a l l y leaves the body (16), but a l s o because i t has an acceptable vapor pressure, and i s a good oxygen and carbon dioxide s o l v e n t . I t r e a d i l y forms an emulsion having a low o p t i c a l d e n s i t y but must, at present, be preserved at -70°C. For comparison, we and others have found that FC47 forms a f i n e p a r t i c l e emulsion, as judged by o p t i c a l d e n s i t y , which i s f a r more s t a b l e than that produced by PFD but once entrapped i n the l i v e r i t remains f o r years. PFD i n a d d i t i o n , unl i k e , f o r example, p e r f l u o r o m e t h y l d e c a l i n with ten isomers, has only two. A fluorocarbon having but a s i n g l e molecular c o n f i g u r a t i o n such as adamantane woul mers such as PFD. PFD a f t e l a t i o n as described i n t h i s paper, was analyzed at Stanford Research I n s t i t u t e by GLC and f i e l d i o n i z a t i o n mass spectrometry and reported to be 96.4% pure with p o s s i b l y eight d i s t i n c t compounds present having molecular weights ranging from 68 to 484. A few m i l l i t e r s of the two isomers of PFD, having the publ i s h e d p r o p e r t i e s , were l a b o r i o u s l y prepared by gas chromatography. It may be that the very property of being i n e r t , which makes some of these fluorocarbons p o t e n t i a l l y u s e f u l i n medicine, makes them d i f f i c u l t to p u r i f y , c h a r a c t e r i z e , and i d e n t i f y . Of course, high p u r i t y and u n i f o r m i t y are r e q u i r e d f o r medical use. The l i s t of u s e f u l e m u l s i f i e r s i s very short indeed because they must be non-toxic, non-hemolytic and cause no undesirable p h y s i o l o g i c a l r e a c t i o n s . Only two such substances e x i s t at the present time. S p e c i a l egg p h o s p h o l i p i d (Vitrum, Sweden) has been used e x t e n s i v e l y o u t s i d e of the United States as an e m u l s i f i e r f o r intravenous f a t emulsions f o r c l i n i c a l use. This e m u l s i f i e r can only be used to formulate emulsions under very c a r e f u l l y c o n t r o l l ed c o n d i t i o n s , as to p r e p a r a t i o n , temperature, exposure to a i r and other f a c t o r s . I t i s capable of e m u l s i f y i n g PFD. The other emuls i f i e r , P l u r o n i c F68 (Wyandotte, USA), at the present time i s not used c l i n i c a l l y i n the USA as a component of intravenous emulsions of any kind. I t has found extensive use as an e m u l s i f i e r for PFC emulsions i n research with animals. I t was s e l e c t e d f o r the PFDE used here i n the rhesus because i t forms emulsions with PFD and i s r e l a t i v e l y non-toxic and s t a b l e . Most of previous work with PFDE was done using the mouse, the cat, and the dog. We decided to perform the t e s t s reported here i n the rhesus (macaca raulatta) monkey to determine i f the primate responded d i f f e r e n t l y . We found that the hypotensive e f f e c t of small doses of emuls i o n d i d not occur i n the primate. Measurements of the LD50 of plasma from the dog at the depth of the blood pressure drop
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INVOLVING C A R B O N - F L U O R I N E
BONDS
i n d i c a t e d that no t o x i c substance was present and we tend t h e r e fore to agree with W r e t l i n d (23, 24) that t h i s i s some k i n d of a cardiovascular reflex. Emulsions prepared from PFD and PF68 are d i f f i c u l t to charact e r i z e as to p a r t i c l e s i z e and c o n c e n t r a t i o n of PF68 i n the aqueous phase a f t e r e m u l s i f i c a t i o n . There i s no one method f o r measu r i n g p a r t i c l e s i z e i n the ranges i n v o l v e d here which i s g e n e r a l l y accepted. O p t i c a l d e n s i t y measurement continues to be the best means to monitor the making of emulsions from any given PFC. I t probably cannot be used to compare p a r t i c l e s i z e when d i f f e r e n t PFC s t r u c t u r e s are i n v o l v e d . Space w i l l not permit d i s c u s s i o n of the complex f a c t o r s i n volved i n maintaining f l u i d balance by means of osmotic, o n c o t i c , and h y d r o s t a t i c f o r c e s . S u f f i c e to say that we attempt to maint a i n o n c o t i c a c t i v i t y by the s h o r t - a c t i n g P l u r o n i c and Dextran and by the long a c t i n g plasma albumin We found that Dextran given s e v e r a l hours a f t e r the f i c i a l e f f e c t ; i t has brough s t a t e to one of near normal. The d i f f e r e n c e i n the f a t e of the two monkeys given a warmed 20% emulsion and a cooled 20% emulsion would seem to suggest that warming an emulsion i n v i t r o i s apparently completely d i f f e r e n t than warming i t i n v i v o . Not only does the emulsion appear to be much more s t a b l e i n v i v o than i n v i t r o , even though f a r above room temperature, but judging from the b l u i s h haze i n the plasma of some of these monkeys, the p a r t i c l e s i z e may even have decreased. This may be due not only to the e m u l s i f y i n g p r o p e r t i e s of blood, but to the mechanical mixing e f f e c t s i n the c a r d i o v a s c u l a r system and the unique c h a r a c t e r i s t i c s of the l i n i n g of the blood vessels. I t should be borne i n mind that most of the chemical analyses of blood reported here are done on plasma d i a l y z e d through a c e l lophane membrane and should t h e r e f o r e not be a f f e c t e d by the presence of PFD. T o t a l p r o t e i n , albumin, t o t a l b i l i r u b i n , and l a c t i c dehydrogenase were analyzed without passing through a d i a l y s i s membrane and the measurement could have been a f f e c t e d . Summary Of nineteen monkeys s e l e c t e d f o r t h i s study, fourteen were used f o r the i n f u s i o n of p u r i f i e d p e r f l u o r o d e c a l i n i n the form of 10 and 20% by volume emulsions. Four received only t e s t doses and one was s a c r i f i c e d before use as a c o n t r o l . P l u r o n i c F68 was used as the e m u l s i f y i n g agent. Previous to the i n f u s i o n of PFDE, blood was removed to decrease the hematocrit by h a l f and t h i s blood was replaced by e i t h e r Ringer's s o l u t i o n or 5% human albumin. Nine out of ten monkeys infused with 10% PFDE survived. Two of four monkeys infused with 20% PFDE survived. Mixed venous p 0 i n c r e a s ed i n a l l monkeys and exceeded 100 t o r r i n three monkeys, which r e c e i v e d 20% of PFDE. The s u r v i v i n g monkeys appear to be i n good 2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8.
CLARK
ETAL.
Cis-Trans
Perfluorodecalin
Emulsions
149
h e a l t h . Three monkeys were s a c r i f i c e d , one as a c o n t r o l , two be cause o f compromised c i r c u l a t i o n t o the l e g . One succumbed during anesthesia preparatory to biopsy and one from a s u r g i c a l accident during cannulation. Aside from a drop i n c h o l e s t e r o l there were only questionable changes i n about 20 blood components analyzed. Small (0.05 ml/kg) doses o f PFDE given t o dogs i n v a r i a b l y caused a pronounced drop i n blood pressure but none occurred i n the mon keys. The morphologic changes i n the l i v e r of the monkeys were r e v e r s i b l e with no s i g n of damage. The major problems encountered were d i f f i c u l t y i n o b t a i n i n g pure PFD, making s t a b l e emulsions, o b t a i n i n g a n a l y s i s of compounds and t i s s u e s f o r PFD, working with the f r a g i l e c a r d i o v a s c u l a r and r e s p i r a t o r y systems of the rhesus and judging optimum f l u i d balance post phlebotomy and post i n f u sion. Abbreviations NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK CH PV PF PFC PFD PFDE C GLC MK PTD M SE Τ RI BL LD50 ME PP5
Number Samples T o t a l P r o t e i n (gm%) Albumin (gm%) Calcium (mg%) Inorganic Phosphrous (mg%) Glucose (mg%) Blood Urea Nitrogen (mg%) U r i c A c i d (rag%) C r e a t i n i n e (mg%) T o t a l B i l i r u b i n (mg%) A l k a l i n e Phosphatase (mU/ml) (EC 3.1.3.1) L a c t i c Dehydrogenase (mU/ml) (EC 1.1.1.27) Glutamic-oxaloacetic Transaminase (mU/ml) (EC 2.6.1.1) Creatine Kinase (mU/ml) (EC 2.7.3.2) C h o l e s t e r o l (mg%) Packed C e l l Volume (%), hematocrit Packed PFD Volume (%), f l u o r o c r i t Perfluorochemical D i s t i l l e d Perfluorodecalin P e r f l u o r o d e c a l i n Emulsion Control Gas-Liquid Chromatography Monkey Post Test Dose Mean
Ringer's s o l u t i o n Blood Mean l e t h a l dose, i . v . i n j e c t i o n The monkey ( r e f . 5), named Melvin In the s e c t i o n on morphology t h i s i s used to designate d i s t i l l e d perfluorodecalin.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
150
BIOCHEMISTRY
INVOLVING C A R B O N - F L U O R I N E
BONDS
Acknowled gemen t s The authors wish to thank Dr. George M i l l e r f o r guidance i n s u r g i c a l techniques used f o r t i s s u e biopsy. We consulted C h r i s t Taraborski concerning chemical problems. The a s s i s t a n c e of the f o l l o w i n g persons i s g r a t e f u l l y acknowledged: Dr. Marian L. M i l l e r , Dr. Steele F. M a t t i n g l y , Dr. Jag L a i , Frank Knapke, Pat Turner, L i l a m Stanley, Margaret Kelm, Steven Jones, David DeForest, Barbara Cincush, Stanley Gaines, Eleanor C l a r k , Eleanor Brinkmoeller, Sandra Hoffman, and Dotty O ' R e i l l y of Sun Ventures. The P l u r o n i c F68 was a g i f t from Dr. I r v i n g Schmolka of Wyandotte. This research i s supported i n part by grants HL17586, HL17353, GM21475, HD05221, from the N a t i o n a l I n s t i t u t e s of Health, grant 74 619 from the American Heart A s s o c i a t i o n and a grant from the Southwestern Ohio Chapter of the American Heart A s s o c i a t i o n . Shortly a f t e r mailin Technicon I n d u s t r i a l Systems, Tarrytown, New York 10591, v i a Richard Carr, 1000 Crest C i r c l e , C i n c i n n a t i , Ohio 45208, a d d i t i o n a l data f o r normal values f o r c l i n i c a l blood chemistry i n the rhesus. One report (29) gives the normal values f o r blood from f o r t y - s i x female and t h i r t y - t h r e e male rhesus. Another report (30) gives the mean and range f o r eleven blood component analyses f o r f i f t y rhesus.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
F
2
2
3
Comparison of a n a l y t i c a l
results.
74.22 74.09
Lab D
Table 1
68.60 68.78
66.35 77.02 76.72
Lab C
25.93 25.78
65.76 73.59 73.79
78.24 78.32
Lab Β
73.53 73.94
84.05 83.90
21.81 21.59
70.14 70.35
21.31 21.30
21.90 22.01 72.93 73.39
21.14 21.32
72.65 72.73 71.73 72.32 71.85
26.10 26.35
74.19
Lab A
73.1b
21.43
70.28
21.40
67.70
25.99
74.01
Calculated
CFo
4
CF CHF-(OCF2CF> F
%C
n=6
%F
n
%C
£o-CF-CF ]-
CF,
E4
%F
• Fa
F
Fomblin
%c
h
h
PP5
%F
Structure
Trade Name
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
4.6 3.9 3.9 3.9 3.5 3.3 3.6 4.0 3.8 3.7 3.7 3.8 3.9 5.7 5.2 4.5 4.9 4.5 4.2 3.9 3.7 4.1
4.8
AB gm%
10.0 9.0 9.2 10.1 9.8 10.1 10.0 9.3 9.2 9.7 8.8 9.4 10.0 10.5 10.1 9.5 9.5 9.0 9.6 8.9 8.9 9.6
9.3
CA mg%
4.1 2.6 2.4 5.9 3.0 3.5 4.6 5.4 5.0 5.7 2.4 4.0 4.2 4.6 4.3 4.6 4.7 4.5 6.0 3.8 3.4 2.9
4.7
IP mg%
93 116 97 149 105 108 122 81 78 185 119 80 109 77 72 225 75 36 122 72 70 111
198
GL mg%
16 11 14 16 20 21 12 14 15 15 15 15 21 16 16 27 18 18 19 22 24 16
14
BN mg%
0.15 0.29 0.31 0.29 0.20 0.15 0.19 0.30 0.19 0.47 0.28 0.19 0.25 0.19 0.09 0.28 0.12 0.10 0.19 0.17 0.12 0.21
0.07
UA mg%
0.9 1.0 1.0 1.3 0.9 0.9 0.9 0.8 0.8 1.1 1.1 0.8 1.1 1.1 0.8 1.5 1.1 0.9 1.1 0.8 0.7 0.8
0.8
CT mg%
0.2 0.1 0.2 0.3 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.3 0.6 0.2 0.3 0.1 0.2 0.2 0.3
0.2
TB rag%
227 152 167 167 580 610 > 350 110 107 135 128 156 170 194 195 232 138 140 186 223 234 236
187
AP mU/ml
330 226 480 336 175 439 294 660 493 381 317 569 373 583 820 > 600 195 252 224 192 109 207
—
242
LD mU/ml 40 48 18 42 36 20 38 48 48 47 51 50 66 57 86 113 > 300 38 44 40 53 60 83
—
CK mU/ml
71 >778 183 <27 192 250 40 830 380 58 500 220 67 640 245 > 778 > 778 > 788 39 210 435 52 144 86
GO mU/ml 40 38 38 43 43 38 41 42 37 40 40 37 39 42 39 46 43 31 39 35 37 40 42 38
%
PV
Blood chemistry before and a f t e r l i v e r biopsy and before t e s t dose o r i n f u s i o n with PFDE i n the monkey.
8.0 7.7 7.6 8.2 8.2 8.3 8.4 8.1 7.4 7.6 7.2 7.4 7.8 8.8 7.5 7.5 7.2 6.8 7.6 7.3 7.2 7.6
7.3
TP gm%
continued on Table 3
Table
113
74
58
33
117
71
2
C 1H 48H C 1H 24H C 1H 24H C 1H 24H C 1H 24H C 1H 24H C 1H 48H C 1H 24H
62
86A
SP
NO
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
C 1H 24H
S.Ε.
0.15 0.16 0.14
9.4 9.3 9.8
9.9 10.0 9.0 9.0 9.3 9.0 8.6 9.5 9.3 9.1 10.7
CA mg%
0.36 0.29 0.93
10 7 13
103 77 143
87 176 111 97 122 92 62 110 104 66 119
4.4 12.0 1.3 2.9 2.1 4.0 5.0 3.3 2.9 3.2 3.3 3.6 3.8 4.9
GL mg%
IP mg%
0. 9 1.1 1.6
16 17 18
19 24 16 17 15 14 15 15 18 19 17
BN mg%
0.06 0.05 0.07
0.01 0.03 0.04
45 52 20
171 186 156
0.2 0.2 0.2
0.02 0.02 0.04
122 177 65 68 83 97 108 99 75 80 105
0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.4 0.1
1.0 1.3 1.0 0.9 1.1 1.0 0.8 1.1 1.5 1.2 1.2 1.0 0.9 1.1
AP mU/ml
TB mg%
CT mg%
0.20 0.17 0.27
0.1 0.5 0.3 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.1
UA mg%
50 64 20
298 463 326
185 380 191 388 321 410 509 282 204 570 364
LD mU/ml
5.5 7.4 7.4
41 59 60
38 78 25 51 96 38 55 38 33 76 51
6.5 84 75
51 365 305
<27 835 <25 210 300 27 280 220 < 25 280 210
GO CK mU/ml mU/ml
0, 1, 1,
42 41 39
45 43 42 45 39 42 38 40 44 46 44
%
PV
*bone marrow b i o p s i e d .
IH = sample one hour a f t e r i n c i s i o n c l o s
Blood chemistry before and a f t e r l i v e r biopsy and before t e s t dose or i n f u s i o n with PFDE i n the monkey.
0.19 0.19 0.15
4.4 4.2 4.2
4.5 4.0 4.4 4.3 4.3 4.5 4.4 4.6 4.9 4.7 5.1
AB gm%
C sample taken from the a n e s t h e t i z e d monkey before biopsy. ed. 24 and 48H = 24 and 48 hours a f t e r i n c i s i o n c l o s e d .
β
Table
3
7.7 7.4 7.8
C 1H 24H
Mean
97A
98
0.17 0.14 0.14
7.4 7.6 6.9 6.9 7.2 7.9 7.5 8.0 8.1 7.8 8.4
C 24H C 1H 24H C 1H 24H C 1H 24H
80*
95
TP gm%
SP
NO
BIOCHEMISTRY
INVOLVING
c
m rH rH '— 1 1
Ο CM rH
Ο CM rH —^ CO 1 1
rH
1
ο CM vO rH 00 • \ CO 1 rH 1 rH rH
ο
CO CM rH
CO ON '—^
Ο Ο rH
I 1
rH
m CO rH \ m vO rH
CO Ο CM rH rH *>^» — CO Ο CM CM CM rH rH rH rH Ο rH
•H
Ο
G ο •Η CO rH
CO
Β Φ <4-t Ο 00
00 co
co
ο m
m vO
m co rH
m CO rH "—^
m
Ο vO rH
1 1
1 1
m vO
m rH **»^ CM co CM CM
Ο
co co ~»— ••-^ 1 ο Ο 1 m
m CM \ vO CO
CO rH
CO CO *•*•—
CM CM
m
m
φ Β CO CO
rH rH rH
φ
*ιC
φ
Q Β
4->
00 CM rH
vO
•
m ^— vO
•
vO
Ό Φ φ 4-1 4-1 •Η CO CO Φ 4-1
ο α α ο
Φ μ φ
CO
•
CO 00
ο • Qο ο
CM \ •—^ ON co
•
of
1 1
CM CM
ν. rH Β
CARBON-FLUORINE
1 1
rH
rH
j
CO rH
1 1
NJ
CM
CM
co
CO
m CM
CO
rH
rH
rH
rH
rH
rH
οrH
rH
Φ Τ")
CO
CM
w
S5 < CO
<
ON Ο
00 1 1
CM Ο
Μ CM
ω
CO 1 1
1
< co CsJ
Ο
& PQ
ω CO
1 1
PL, CO Ν
Ο W >* N3
> >
vO CO rH vO rH CO 00
ON CO
c
c •Η Ο
Σ*
4->
14-1 Ο
ο
rH Ο m •—_ —
ON
co ο rH
rH CO rH
vO CM rH
rH m rH
ο
rH Ο m m — "--^ vO
m 00
οCM rH
CM m —. ON rH rH
Ο m *-«^. ON CM rH rH rH
1 1
1 1
co
ο 4-1
α
Ο rH
rH m
CM CM Ο m m •—·— — ^ ο 00 σ\ CM CM rH rH rH rH rH
vO ON rH VO m —„ — co
<* rH
00 rH rH
ο CO rH
m 00 \ . ο rH rH
rH rH VO m —^ vO ON rH CO rH rH
Φ Μ-Ι 14-4 Φ
CM m
m CM rH
Φ j
1 1
U U-4 Ο
c οCO
Ο m \ rH Ο rH
rH Ο ON — •—^ **—. ^ » rH
m 00 \ m
οrH
CM m
CM ON vO —•—
vO rH rH
in CO rH
CM rH
•
Β
β •Η Ο •Η rH
1 1
1 1
co
rH
vO
00 m
CO
CO 00
ο
Ο
? *
•
ο
υ
m
ο
Ο vO \
c
CM m •—
ο
4-1 CO CO CO Τ 3 CO CO Φ φ •Η U Ό Β •— CO CO rH •Η (0 rH φ •Η 4-1 4-» U Φ CO 4-1 C U CO
α
m m
C CO
α
Φ U
rH «n
Ο
•
00
ο
CM rH
φ c u CO • Η CO CO rH φ M Β Φ CO CO Φ 0 φ u cO CO
•
ο
00 m —^
α φ
m
CM rH
φ
co co
•
ON
•
CO
4-1
Η
C Φ > • •Η • Η CO U OOrH CO rH Φ Π3 a C U >> CO Φ Φ CJ C >> Φ Φ rH s Xi CO CO φ Β 4-> CO rH Φ φ r>% cd CO 4-1 Ό Η U
•
00
ο
9* ο
00 m rH CM rH
m co* •—^ rH
•
ο
^· α ο CM vO
< vD 00
rH
rH rH
CO CO
00 m
CO rH rH r
Ο 00
m ON
00 ON
< ON
1 1
I
1
C • CO Φ • S CO
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
7.9 7.7 8.3 8.2 7.5 7.5 8.1 7.9 7.5 7.3 7.7 7.6 7.5 7.9 7.8 8.0 8.0 8.3 8.0 8.2 7.6 7.9
4.4 7.9 4.2 9.2 4.9 9.2 4.6 9.1 4.3 8.7 4.1 7.8 4.5 8.1 4.5 8.6 4.1 8.6 3.8 8.4 4.2 9.0 4.1 9.1 4.3 9.4 4.3 10.0 4.1 9.1 4.3 9.6 4.4 10.2 4.5 9.8 4.3 9.6 4.5 9.8 3.9 7.9 4.1 9.6
3.1 84 3.3 77 4.0 90 4.4 96 3.6 111 3.3 99 3.9 82 4.0 88 3.6 69 3.3 74 3.8 62 3.4 85 2.5 58 3.2 67 2.8 74 3.2 94 3.6 89 4.1 82 2.0 77 2.8 85 4.3 104 4.4 90
Table
0.16 0.11 0.19 0.38 0.29 0.29 0.20 0.28 0.12 0.20 0.15 0.20 0.29 0.19 0.30 0.21 0.29 0.17 0.20 0.15 0.31 0.20
0.9 0.9 0.9 0.9 1.0 0.9 0.9 1.0 0.9 0.9 0.9 1.0 0.8 1.0 0.9 1.1 0.9 1.0 0.7 0.9 0.6 0.8
0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.1 0.2 0.1 0.2 0.1
112 106 157 135 192 156 169 150 84 77 130 128 110 188 119 133 78 80 119 124 73 73
0.22 0.9 0.2 122 16 0.45 0.02 0.02 0.01 7.9
18 18 18 20 17 18 15 16 15 14 15 15 18 14 17 14 19 14 14 12 15 16
39 38 42 58 53 54 47 50 44 49 52 60 42 47 39 30 81 37 30 25 27 28
21 38 104 450 91 86 87 188 50 30 83 124 42 70 18 40 188 40 15 32 16 34 308 44 84 19 2.8 21
395 370 148 297 280 312 404 476 351 383 310 324 347 250 306 166 427 254 252 155 323 240 6.9 5.6 4.3 4.9 4.8 6.3 6.5 5.4 4.6 4.8 5.3 4.5 5.0 3.6 5.4 3.8 5.5 3.7 5.4 4.0 5.1 4.3
103 104 108 106 104 105 107 105 104 103 107 106 108 110 109 115 110 108 107 110 111 113 146 5.0 107 1.1 0.19 0.70
138 138 151 145 148 141 143 139 141 138 144 142 148 150 148 150 151 150 150 152 150 152 157 145 133 162 133 129 143 134 167 148 210 207 175 156 170 145 170 144 190 159 460 159
42 42 43 42 42 40 45 44 41 42 45 42 43 44 42 43 41 42 43 43 39 42 16 173 42 0.84 14.8 Ο.ι
14 18 25 14 13 16 13 15 18 20 13 18 20 20 14 12 10 13 18 19 9 13
5 A n a l y s i s of blood and plasma from awake monkeys a f t e r biopsy and blood pressure t e s t dose but before PFDE i n f u s i o n
Mean 7.8 4.3 9.0 3.5 84 S.Ε. 0.06 0.05 0.15 0.14 2.9
117
86A
97A
33
58
71
95
62
74
113
98
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
349
15.7 10.5
6
Mean
S.E.
Table
22.5
430
369 478 524 452 489 318 494 306 448 440 354 494
A
pOo
1.6
39
43 40 39 46 33 34 31 45 33 36 38 42 48
9.3
68
68 53 62 50 43 57 45 57 38 64 69 122 150
19
95
56 58 81 61 59 52 63 71 52 135 87 233 233
VENOUS A D B 33 41 39 33 26 39 30 34 34 30 36 35
34 34
39 38 39 35 26 38 28 34 32 36 39 23
2
39
39 47 47 30 33 50 40 43 42 46 45 26 23
40
36 47 48 44 35 45 40 37 42 41 42 42 22
2. 1 2.5 1.9
39
42 44 44 45 37 45 34 35 36 46 43 28 22
VENOUS D A B
pC0
2. 1 1.7 1.,3
34
36 36 41 35 30 43 25 29 28 42 40 23
AORTIC D A B
0.22
0.28 0.28
0.19
0.24
7.37
" A o r t i c " r e f e r s to samples removed from the cannula i n s e r t e d i n t o the a o r t a v i a the femoral a r t e r y . "Venous" r e f e r s to mixed venous samples removed from a catheter e i t h e r i n the r i g h t v e n t r i c l e or the pulmonary a r t e r y . Blood gas tensions are i n mm. o f mercury. Β = before, D = during i n f u s i o n , A = a f t e r . The average uncorrected barometric pressure i n C i n c i n n a t i i s 744ramHg.
0.25
7.36 7.42 7.44
7.42
7.39
7.44 7.43 7.36 7.40 7.43 7.40
7.41 7.50
7.42
•
7.28 7.26 7.30 7.43 7.38 7.37 7.37 7.46 7.27 7.23 7.31 7.42 7.37 7.42 7.31 7.46
A
7.30 7.33 7.37 7.48 7.43 7.45 7.35 7.36
VENOUS D
7.34 7.32 7.41 7.54 7.43 7.44 7.41 7.54
B
7.34 7.32 7.34 7.48 7.46 7.49 7.38 7.54
A
PH
7.34 7.36 7.40 7.52 7.44 7.52 7.39 7.43
B
AORTIC D
Blood gases and pH before, during, and a f t e r i n f u s i o n o f PFDE i n the monkey.
430
378 400 360 311 378 369 416 227 341 332 295 379
Cl C4 C5 62 86A 71 117 33 58 C2 C3 74 113
455 415 484 436 416 420 461 407 430 414 352 466
AORTIC D B
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
0.30
S.E.
Table 7
3.5 5.0
0.28
4.97
4.8 5.0 4.8 5.1 5.3 5.2 5.0 5.0 5.1 5.5 4.4 4.9 4.6
AB gm%
8.5 10.5 2.5 4.5
0.35
0.22
50 120
18.3
108
3.4
9.80
GL mg%
81 94 111 133 111 100 77 97 130 119 125 118
IP mg% 3.6 2.8 3.6 3.6 3.4 3.5 3.3 3.3 4.1 3.6 3.1 3.3 2.8
9.6 9.5 9.6 9.9 10.0 10.1 9.8 9.3 10.1 9.6 9.5 9.9 9.6
CA mg%
10 20
2.0 7.0
0.7 1.5
0.2 1.0
0.07
0.06
0.73
2.5
0.4
1.0
7.9
0.4 0.4 0.4 0.4 0.3 0.4 0.5 0.4 0.5 0.5 0.5 0.6 0.5
TB mg%
12.2
1.0 1.0 1.1 1.1 1.0 1.0 1.1 0.9 1.0 1.0
CT mg%
7.5 7.8 6.8 8.7 8.5 9.2 7.7 8.7 7.7 7.1 6.9 8.1 8.1
UA mg%
14 14 18 11 11 9 10 15 11 10 12 12 11
BN mg%
25 125
06.9
60.5
71 71 57 52 55 61 58 64 56 72 53 57 60
100 225
3.6
206
205 215 221 222 220 201 217 166 210 219 225 260 190
15 110
33.0 01.9
7 40
65.5
82 104 40 36
CK mU/ml
79.8
64 88 74 67 70 69 86 68 68 82 92 138 72
AP LP GO mU/ml mU/ml mU/ml
Random blood samples from L.C. used as l a b o r a t o r y c o n t r o l f o r the monkeys.
1 6.0 ) 8.0
7.60
Mean
Normal Range
7.4 7.9 8.1 7.6 8.1 7.8 7.8 7.1 7.7 7.6 7.3 8.0 7.6
TP gm%
11-08-73 12-07-73 01-09-74 05-08-74 06-27-74 07-24-74 09-03-74 10-10-74 11-09-74 12-09-74 03-19-75 07-10-75 08-14-75
DATE
150 300
16.8
235
220 250 221
235 213 252 225 248 247 244 206 254
CH mg%
158
BIOCHEMISTRY
INVOLVING
HEART RATE (BEATS/MINUTE)
CARBON-FLUORINE
BONDS
RESPIRATION RATE (BREATHS/MINUTE)
MK NO
BEFORE INFUSION
DURING INFUSION
AFTER INFUSION
CI CA C5 62 86A 71 117 33 58
207 216 213 193 187 209 238 212 213
200 203 200 148 189 190 205 189 186
210 212 198 140 170 171 179 186 179
42 37 32 36 25 25 30 41 31
66 42 47 36 31 58 40 43 36
87 52 42 51 36 84 48 42 42
Mean S.E.
210 5.10
190 6.0
C2 C3 74 113
214 174 170 206
201 156 174 176
180 152 162 164
34 70 23 47
39 77 32 65
51 72 34 70
Mean S.E.
191 12.8
177 10.7
165 6.7
44 11.7
53 12.3
57 10.3
186 1.25
177 1.52
36 -1.45
47 -1.01
55 -0.27
Mean 204 T-Test 1.85
Table 8
BEFORE INFUSION
DURING INFUSION
Pulse and r e s p i r a t i o n before during and a f t e r of 10% PFDE (Cl-58) and 20% PFDE (C2-113)
AFTER INFUSION
infusion
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8.
CLARK
E T
AL.
Cis-Trans
Perfluorodecalin
MK NO
ARTERIAL PRESSURE mm H g BEFORE DURING AFTER INFUSION INFUSION INFUSION
CI C4 C5 62 86A 71 117 33 58
134 111 115 88 111 112 100 88 102
126 122 120 79 111 105 108 95 102
Mean S.E.
107 5.07
108 5.1
C2 C3 74 113
101 89 88 107
159
Emulsions
RV OR PA PRESSURE mm H g BEFORE DURING AFTER INFUSION INFUSION INFUSION
107 121 121 75 110 101 100 102 96
10 8 2 3 8 11 7 12 6
14 16 9 15 17 19 19 19 11
16 12 18 20 12 19 20 18 9
111 93 71 124
120 97 82 124
8 16 8 5
14 18 18 19
19 18 21 19
96 5.36
100 13.28
106 11.44
Mean 104 T-Test 1.33
105 0.75
104 -0.22
Mean S.E.
Table 9
Blood pressures i n the rhesus monkey before during and a f t e r i n f u s i o n of 10% PFDE (Cl-58) and 20% PFDE (C2-13)
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Table 10
95
80
113
74
58
33
117
71
4.0 3.5 2.9 4.1 4.3 4.2 4.5 3.2 2.6 3.4 3.3 3.0 3.9
9.8 8.0 7.5 8.5 8.4 8:4 9.1 8.9 14.9 7.6 8.4 8.4 9.6
9.0 8.7 8.8 10.1
3.7 3.6 3.6 4.4
2.9 3.6 2.2 5.5 3.9 4.2 5.6 4.0 6.6 10.8 3.4 3.0 2.3
5.3 3.8 1.4 2.6
3.5 2.9 3.4 4.7
9.2 9.1 9.8 8.9
3.8 3.8 3.9 4.0
1.8
IP
2.5 3.8 2.6
8.7
CA
9.1 9.1 8.7
4.7 3.7 3.4
4.0
AB
14 16 16 16 19 19 14 27 24 70 16 17 16
0.28 0.12 0.08 0.22 0.12 0.09 0.30 0.20 0.23 0.47 0.25 0.13 0.14 1.3 0.6 0.5 1.0 0.7 0.6 1.2 0.8 0.7 2.4 0.9 0.8 1.2
1.0 1.0 0.8 1.2
0.21 0.21 0.15 0.28
14 16 15 17
132 132 68 103 96 74 71 128 81 71 125 168 102 218 92 78 97
1.1 1.0 1.1 0.7
0.18 0.20 0.20 0.10
22 21 12 15
0.4 0.2 0.2 0.2 0.2 0.2 0.1 0.3 0.2 0.4 0.1 0.2 0.1
0.2 0.2 0.2 0.2
0.1 0.2 0.2 0.1
0.1 0.1 0.1
163 207 193 197 116 128 145 122 104 137 76 79 88
156 86 108 102
153 154 152 75
236 158 164
168
0.1
0.8 1.0 0.9 0.8
AP
TB
CT
126 89 105 75
0.35
UA
0.15 0.32 0.21
19
BN
15 14 14
77 94 91
67
GL
444 162 249 256
292 420 394 225
333 162 212
364
302 125 138 352 184 143 174 367 362 3950 207 286 256
LP 98
47 26 26 36 38 38 34 63 67 480 40 44 53
82 30 34 40
38 52 117 23
105 32 34
GO
Blood chemistry before and a f t e r t e s t dose of PFPE continued on Table 11
8.1 7.0 6.7 QNS 7.1 7.0 7.8 7.1 QNS 7.2 6.6 6.7 7.8 QNS 7.1 6.7 5.8 7.0 7.1 6.9 8.2 6.3 5.0 6.8 7.1 6.6 7.0
7.1
C 1u ±n 24H C 1H 24H C 1H 24H C 1H 24H C 1H 24H 1H 24H C 1H 24H C 1H 24H C 1H 24H C 1H 24H
62
86A
TP
SP
NO 65
196 39 112 82 110 132 54 104 130 56 102 130 854 854 847 48 720 112
95 46 250 90 110 420 550 170
CK
165 107 104 102 142 138 141 84 72 111 116 133 128
126 149 159 146
133 178 175 172 189 196 191 163
138
CH 41.0 44.0 43.5 40.0 43.0 38.5 39.0 38.5 40.0 42.0 39.0 37.0 39.5 41.0 40.0 34.0 39.0 39.5 44.0 40.0 38.0 42.0 43.0 34.0 28.0 32.0 41.0 49.5 40.0
PV
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 0.02 0.02 0.04
0.20 0.14 0.23
0.18 0.06 0.09 0.21 0.12
UA
28 4.5 .15
28 8.1 .29
Ν M SE
IP
28 5.0 .37
CA
28 10.2 .27
28 112 4.6
GL 28 19.3 1.21
BN 27 .29 .02
UA
14 15 15
0.02 0.01 0.04
0.04 0.05 0.14
28 0.2 .02
28 1.17 0.37
28 28 390 387 25.8 52
AP
Na 28 26 58 160 11 1.60
Gp
26 35 29
224 241 309
187 204 275 189 156
LD
26 5.1 0.33
Κ
7.1 5.2 11.5
40 38 60
23 28 30 24 18
GO
a f t e r i n f u s i o n of PFDE i n M e l v i n .
TB
LD
120 121 148
0.2 0.2 0.2
0.8 0.8 1.2
86 98 101 72 64
0.1 0.2 0.2 0.2 0.2
0.8 0.7 1.0 0.8 0.9
AP
TB
CT
CT
Blood chemistry f o r one year's sampling
AB
TP
Table 15
10 5 13
0.31 0.55 0.84
1. 2 1. 1 0. 6
18 18 15
97 78 117
3.0 1.9 3.2 1.3 2.4
3.3 3.0 4.5
BN 17 17 13 18 17
GL 82 76 89 76 56
IP
Blood chemistry before and a f t e r t e s t dose of PFDE
NO
Table 11
.13 .77 .24
0.10 0.19 1.13
S.E.
C 0.09 1H 0.24 24H 0.17
8.7 9.2 9.2
3.8 3.5 4.1
6.9 Mean C 1H 6.5 24H 7.5
97A
8.4 8.4 9.0 9.3 8.9
4.0 3.8 4.3 3.9 4.0
7.0 6.7 7.7 7.1 7.1
C 1H 24H C 1H
CA
98
AB
TP
SP
NO
26 112 0.91
Ç1
76 98 79
140 277 219
30 60 44 72 38
CK
26 15 1.7
CO?
28 37 1.6
HCT
0.65 1.66 0.9Ç
39.5 40.8 39.1 140 141 139 9.7 13.0 8.6
39.0 44.0 37.0 41.5 42.0
PV
131 141 119 143 151
CH
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
SP
TP
CI
1DPP 7.8 IPP 7.2 PI 4.9 C4 8DPP 8.7 1DPP 9.2 IPP 6.0 PI 5.8 C5 8DPP 7.9 1DPP 7.8 IPP 6.8 PI 2.2 62 1DPP 7.4 IPP 6.5 PI 1.7 2HPI 2.1 86A 1DPP 7.8 IPP 6.5 MP 6.0 PI 3.1 1DPI 6.2 71 8DPP 7.2 IPP 6.0 MP 6.0 PI 3.1 3DPI 6.1 117 1DPP 7.4 IPP 6.0 MP 5.9 PI 3.3 2DPI 6.3 Table 12
NO
CA
IP
GL
BN
3.8 9.1 5.7 140 11 4.0 8.7 7.4 12 83 2.6 7.3 7.3 55 10 4.1 9.7 5.2 134 12 92 4.7 11.3 4.5 21 3.1 8.9 4.4 63 20 3.0 8.2 6.2 68 18 4.2 9.9 4.8 112 18 4.3 10.3 5.5 76 18 3.9 9.2 4.6 99 19 1.2 6.6 4.2 81 14 4.4 10.0 1.8 72 19 3.6 8.4 2.6 85 16 0.9 5.9 2.9 64 14 1.1 6.0 2.5 123 15 4.5 9.4 3.4 82 15 3.9 8.4 4.0 97 11 4.4 8.5 3.6 95 11 2.2 6.8 3.5 65 10 3.8 9.2 5.8 125 18 4.3 9.5 3.0 93 17 3.6 8.5 3.2 102 18 4.6 8.7 3.0 92 16 2.4 7.2 2.8 69 14 3.4 8.7 4.8 97 24 4.0 10.0 5.0 102 18 3.2 8.2 3.8 93 18 4.1 8.5 3.7 79 18 2.2 7.0 3.4 70 15 3.4 9.1 3.8 122 16 Blood chemistry before and
AB
CT
0.50 0.8 0.19 0.6 0.21 0.7 0.18 0.8 0.36 0.9 0.10 0.6 0.10 0.6 0.28 0.8 0.33 0.9 0.19 0.6 0.09 0.5 0.20 1.0 0.20 0.8 0.12 0.7 0.18 0.9 0.28 1.0 0.09 0.7 0.02 0.3 0.02 0.7 0.34 1.1 0.29 1.0 0.12 0.6 0.10 0.7 0.08 0.7 0.30 0.9 0.01 0.8 0.04 0.5 0.06 0.6 0.02 0.6 0.21 0.8 a f t e i: 10%
UA
AP
LD
GO
CK
0.2 "500 340 30 194 0.2 "500 340 30 148 0.2 348 292 24 142 0.1 202 406 33 0.2 182 377 52 66 0.1 158 194 28 88 0.2 166 519 51 190 0.2 208 258 112 31 0.2 200 252 31 114 0.3 207 299 36 210 82 0.1 155 17 80 0.2 145 42 313 50 0.2 162 286 102 43 92 0.1 47 17 38 0.1 69 148 26 80 162 0.1 135 38 68 0.2 129 183 52 27 0.3 69 133 17 38 102 0.3 43 10 38 0.1 88 1520 325 857 0.1 104 190 26 43 0.2 114 32 62 233 17 26 0.3 58 147 0.3 44 117 90 13 0.2 230 " 1160 183 1502 0.1 79 170 16 8 0.2 83 200 23 54 0.2 52 12 125 36 0.2 68 94 8 40 0.2 214 265^ > 600 PFDE. Continued on Tab
TB
149 92 12 200 183 181 44 156 122 24 48 220 152 98 36 46 220 190 124 55 190 182 145 82 34 123
131
—
CH 42 39 28 44 38 38 20 44 43 36 14 44 38 11 13 46 39 25 13 13 45 40 24 9 15 40 32 20 8 17
PV 0 0 12 0 0 0 12 0 0 0 12 0 0 13 13 0 0 0 12 6 0 0 0 13 4 0 0 0 13 4
PF
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 0.04 0.02 0.04 0.02 0.05
0.9 0. 9 1. 0 2. 0 1. 8
0 0 0 0.3 0.9 10 11 9 5 37 19 38 46 21 4993 7 8 9 5 56 29 26 27 44 207 36 44 14 32 41
0.02 0.02 0.07 0.03 0.07
0.03 0.05 0.12 0.04 0.09
1. 0 1. 1 1. 3 0. 8 2. 0
(DPP = days pre-phlebotomy, IPP = immediately pre-phlebotomy, MP = mid-phlebotomy, PI = immediately p o s t - i n f u s i o n , HPI = hours p o s t - i n f u s i o n , DPI = day (post-infusion). *This value i s considered erroneous and has been e l i m i n a t e d from the mean and standard e r r o r .
8. 3 4. 4 5. 8 7. 1 10.4
Blood chemistry before and a f t e r i n f u s i o n of 10% PFDE i n 9 monkeys.
0.39 0.57 0.55 0.11 0.49
Table 13
0.28 0.12 0.16 0.22 0.31
0.20 0.13 0.22 0.75 0.82
0.22 0.17 0.11 0.42 0.10
0 0 0 12.1 3.8 44 37 23 14 13 188 144 94 34 107 74 147 95 105 6601 41 38 24 22 296
260 268 167 194 1473
194 188 75 98 192
0.15 0.20 0.28 0.21 0.23
0.89 0.69 0.68 0.71 0.98
0.25 0.14 0.10 0.11 0.28
16 16 15 14 19
S.E. DPP IPP MP PI DPI
94 87 83 70 108
4.2 3.9 3.1 3.8 4.7
9.7 8.6 8.7 6.9 9.2
220 153 87 23 70
90 374 240 204 3640
105 94 57 42 412
256 408 258 207 1740
258 199 122 62 237
0.1 0.3 0.5 0.4 0.4
0.9 0.9 0.9 0.8 1.1
0.23 0.19 0.21 0.15 0.40
14 17 15 13 19
4.1 3.5 4.1 2.9 4.1
53 82 82 58 89
4.0 1.5 1.2 0.9 4.4
7.7 2.4 3.1 8.9 3.9 9.2 2.2 7.2 3.3 10.0
6.7 5.7 5.5 2.9 5.9
7.8 6.3 5.8 3.2 6.1
2DPP IPP MP PI 2DPI
0 0 0 11 1 JL 0 0 0 10 4 44 36 22 12 11 Α.Λ. 48 35 23 9 8
166 130 78 34
8 232 134 144
28 28 18 15
137 273 174 213
116 137 73 54
0.1 0.1 0.1 0.2
0.9 0.9 0.9 0.9
PF
0.11 0.10 0.11 0.15
16 16 15 14
PV
CH
CK
GO
LD
AP
TB
CT
UA
BN
Mean DPP IPP MP PI DPI
58
83 76 66 42
3.2 4.0 3.9 3.5
9.7 8.2 8.4 7.1
4.5 3.1 3.5 2.2
8.0 5.9 5.7 3.3
7DPP IPP MP PI Ο TV Ό jDïr 1 Τ
33
GL
IP
CA
AB
TP
SP
NO
164
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In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Τ 3 MM G II · Η I PM 4-I PM
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In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 69 55 53 52 53 52 53 52 53 56 53 53 53
4 54 55 53 32 29 24 35 30 49 53 35 32
18 3 6 7 4 3 4 3 3 1 8 8 4
4 5 3 12 2 3 1 1 1 5 9 27 0
0 0 0 19D 3A 0 3A 0 0 0 16D 16A 0
0 0 0 11D 0 0 9A 0 0 23D 0 8A 0
0 0 0 11 0 0 16 0 0 23 0 24 0 34 35 35 35 36 31 32 21 31 41 37 35 31
OVER 4 HR. POST INFUSION IN OUT RI OS TO BL
4 0 5 0 3 0 31 0 5 0 3 0 4 7 1 0 1 0 5 0 25 0 43 16 0 0
4 HR. POST INFUSION IN OUT BL RI OS TO 17 64 61 68 8 9 14 5 9 60 65 51 10
0 0 0 30D 31A 25A 33A 33A 25A 23D 16D 54A 25A
60 50 50 50 50 50 50 50 50 50 50 50 50
77 43 114 79 111 76 148 113 89 53 84 53 97 65 88 67 84 53 133 92 131 94 155 120 85 54
TOTALS IN AND OUT IN RI OS PFDE TO BAL
Monkey weight i s i n kilograms. Blood out and f l u i d s i n are expressed as ml/kg. OS = osmotic or o n c o t i c , D = Dextran 40, A = albumin, BL = blood, RI = Ringer's s o l u t i o n , TO = t o t a l , B a l = balance or t o t a l i n minus t o t a l out.
i n b e f o r e , during and a f t e r i n f u s i o n of PFDE.
9 5 3 2 3 2 3 2 3 6 3 3 3
60 50 50 50 50 50 50 50 50 50 50 50 50
TO
6 4 3 2 2 2 2 2 2 4 3 2 1
TO
INFUSION IN OUT PFDE RI BL
Blood out and f l u i d s
4 54 55 53 3 4 3 2 5 49 53 5 7
0 0 0 0 29 25 21 33 25 0 0 30 25
10 28 26 26 30 26 26 16 26 26 26 25 26
3.2 3.2 4.4 6.6 6.1 8.0 6.8 7.4 7.1 3.0 3.8 6.2 5.6
Table 16
C5 62 86A 71 117 33 58 C2 C3 74 113
OA
Cl
NO
IN AB
PHLEBOTOMY OUT RI WT BL
166
BIOCHEMISTRY
EMULSION RECEIVED 7.PFDE 10 10 20 20 20 20
NO. 62 117 C2 C3 74 113 M S.E.
0 0 0
6 80 C7 M S.E. FAS
-0
Table 17
NO. 62 74 113 C3 C2 CI C4 C5 86A 71 117 58 ME Table 18
BODY WEIGHT kg 7.2 6.4 3.0 4.2 6.4 5.7 5.5 0.7
LIVER
INVOLVING
SPLEEN
CARBON-FLUORINE
LUNG
KIDNEYS
26..3 22..9 47,.5 37..7 32..3 29..0 32..6 4,.0
1.1 1.9 2.3 6.3 1.2 1.5 2.4 0.9
11.8 7.2 12.3 13.6 14.9 9.9 11.6 1.2
3.7 4.4 5.7 5.1 5.0 4.6 4.8 0.3
7.2 4.0 3.9
17..0 36..6 26 8
0.7 0.9 1.0
5.1 8.8 13.3
3.4
3.6
19..2
-
19.0
BONDS
-
5.8
-
Organ weights obtained at autopsy. FAS = normal organ weights f o r the rhesus monkey reported by FASEB (20). A l l organ weights expressed as gms/kg of body weight. DAYS POST INFUSION 1 1 1 8 14 42 47 56 64 70 77 84 332
PFD INFUSED gm 64 120 109 74 58 37 31 43 59 78 66 69 25
PFD FOUND gm % 12 7.7 20 24 14 13 17 23 22 13 2.1 5.6 4.2 1.3 2.1 4.8 0.3 0.5 3.4 4.4 0.96 1.0 0.2 0.3 0.1 0.4
GLC 8,000 12,000 1,400 7,000 10,000 1,500 1,700 1,800 4 1,000 800 40 1
PFD content of l i v e r obtained a t biopsy or autopsy as determined by sodium biphenyl combustion or vapor phase GLC. Data on Monkeys C l , C4, C5, 86A, 71, 58 and ME were obtained from samples obtained a t biopsy; the l i v e r was assumed to be 37« of the body weight. R e l a t i v e peak area was obtained from a pen w r i t i n g i n t e g r a t o r on the s t r i p chart.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8.
CLARK
ET
AL.
Cis-Trans Perfluorodecalin Emulsions
167
Literature cited 1. Clark, Jr., Leland C. and Gollan, Frank. Science (1966) 152, 1755-1756. 2. Clark, Jr., Leland C., Editor. Federation Proceedings (1970) 29, 1696-1820. 3. Sloviter, Henry A. Medical Clinics of North America (1970) 54, 787-795. 4. Geyer, Robert P. The New England Journal of Medicine (1973) 289, 1077-1082. 5. Clark, Jr., Leland C.; Kaplan, Samuel; Emory, Carolyn and Wesseler, Eugene P. "Progress in Clinical and Biological Research, Vol. I. Erythrocyte Structure and Function", edited by George J. Brewer, 589-600, Alan R. Liss, Inc., New York (1975). 6. Gollan, Frank and Clark, Jr., Leland C. The Alabama Journal of Medical Sciences (1967) 4 336-337 7. Gollan, Frank and (1966) 9, 191. 8. Gollan, Frank and Clark, Jr., Leland C. Transaction of the Association of American Physicians (1967) 80, 102-110. 9. Clark, Jr., Leland C.; Kaplan, Samuel; Becattini, Fernando and Benzing III, George. Federation Proceedings (1970) 29, 1764-1770. 10. Spitzer, Hugh L.; Sachs, George and Clark, Jr., Leland C. Federation Proceedings (1970) 29, 1746-1750. 11. Clark, Jr., Leland C.; Kaplan, Samuel and Becattini, Fernando. Presented at the American Association for Thoracic Surgery 50th Annual Meeting, Washington, D.C. (Abstract No. 34), April 8, 1970. 12. Clark, Jr., Leland C.; Kaplan, Samuel and Becattini, Fernando. Pediatric Research (1970) 4, 464 (Abstract 113). 13. Clark, Jr., Leland C.; Kaplan, Samuel and Becattini, Fernando. The Journal of Thoracic and Cardiovascular Surgery (1970) 60, 757-773. 14. Clark, Jr., Leland C.; Bacattini, Fernando and Kaplan, Samuel. Triangle (1972) 11, 115-122. 15. Clark, Jr., Leland C.; Becattini, Fernando and Kaplan, Samuel. The Alabama Journal of Medical Sciences (1972) 9, 16-29. 16. Clark, Jr., Leland C.; Becattini, Fernando; Kaplan, Samuel; Obrock, Virginia; Cohen, David and Becker, Charles. Science (1973) 181, 680-682. 17. Clark, Jr., Leland C.; Wesseler, Eugene P.; Kaplan, Samuel; Miller, Marian L.; Becker, Charles; Emory, Carolyn; Stanley, Lilam; Becattini, Fernando and Obrock, Virginia. Federation Proceedings (1975) 34, 1468-1477. 18. Miller, Marian L.; Clark, Jr., Leland C.; Wesseler, Eugene P.; Stanley, Lilam; Emory, Carolyn and Kaplan, Samuel. The Alabama Journal of Medical Sciences (1975) 12, 84-113. 19. Wesseler, Eugene P.; Iltis, Ron and Clark, Jr., Leland C. The solubility of oxygen in highly perfluorinated liquids. In preparation.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
168
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
20. Altman, P h i l i p L. and Dittmer, Dorothy S., E d i t o r s . " B i o l o g i c a l Handbooks: Growth i n c l u d i n g r e p r o d u c t i o n and morphol o g i c a l development". Federation of American S o c i e t i e s f o r Experimental Biology, Washington, D.C. (1962). 21. Technicon SMA12 procedures were used f o r most of these a n a l yses. The exact methods used are on f i l e here. 22. Calderwood, H. W.; Modell, J . H.; Rogow, L.; Tham, M. K. and Hood, C. I. Anesthesiology (1973) 39, 488-495. 23. W r e t l i n d , A r v i d . J o u r n a l o f N u t r i t i o n , Metabolic Diseases and D i e t e t i c s (1972) 14, 1-57. 24. W r e t l i n d , A r v i d . The pharmacological basis f o r the use of f a t emulsions i n intravenous n u t r i t i o n . Department of N u t r i t i o n and Food Hygiene. The N a t i o n a l of I n s t i t u t e of P u b l i c Health Stockholm 60, Sweden. 25. Galen, R. S. and Gambino, S. R. C l i n i c a l Chemistry (1975) 21, 272. 26. Pooley, F. D.; Kanellopoulos (1972) 3, 486-496. 27. S t e i n , R. J . ; R i c h t e r , W. R.; Rdzok, E. J.; Moize, S. M. and B r y n j o l f s s o n , G. "Use of nonhuman primates i n drug e v a l u a t i o n " , e d i t e d by Harold Vogtborg, 187-199, U n i v e r s i t y of Texas Press, A u s t i n (1968). 28. Here we have given a summary of the procedure used f o r making emulsion. A p r e c i s e d e t a i l e d account f o r making a v a r i e t y of PFC emulsions i s on f i l e and can be provided on request. 29. Busey, W i l l i a m and W i l l n e r , Howard. Normal biochemical p a r a meters o f rhesus monkeys and beagle dogs. Presented at the Technicon Symposium, "Automation i n A n a l y t i c a l C h e m i s t r y , " New York, N . Y . , October 4, 1967. Published by Technicon C o r p o r a t i o n . 30. Technicon I n d u s t r i a l System brochure e n t i t l e d , "The Technicon SMA 6/60 micro multichannel biochemical a n a l y z e r , " August 23, 1974. Technicon number 4229-9-4-2.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
8.
CLARK
ET
AL.
Cis-Trans
Perfluorodecalin
169
Emulsions
D i s c u s s i o n of the paper of Dr. Leland C. Clark, J r . Q.
How long does i t take f o r the pulmonary a r t e r i a l pressure to r e t u r n to normal?
A.
About an hour.
Q.
So that i t was back to normal before the m a t e r i a l was from the blood?
A.
Yes, i t takes over a day before i t i s cleared from the blood.
Q.
Do you have any studies on the s t r u c t u r e you showed?
A.
We have made p r e l i m i n a r y studies on a small batch which somewhat impure bu and a l s o leaves th
Q.
In the a b s t r a c t you mentioned brominated and i o d i n a t e d perfluorocarbons as X-ray c o n t r a s t agents. Would you l i k e to comment on that?
A.
Dr. Long w i l l soon t e l l us the s t o r y of the X-ray c o n t r a s t agents. We have found that the i o d i n a t e d compounds are very l i t t l e , i f any, more X-ray opaque than the brominated compounds, at 55KV. The i o d i n a t e d compounds are unstable i n the presence of l i g h t and even though the l i b e r a t e d iodine could be removed by m e t a l l i c s i l v e r there would be other decomposit i o n products there. We have j u s t about given up on i o d i n e .
Q.
How
A.
I f they decompose i n the presence of l i g h t you could almost count on t h e i r breakdown i n the body. We've had some animals survive small doses. Some of t h i s information has been publ i s h e d . (Clark, Leland C , J r . , Eugene P. Wesseler, Samuel Kaplan, Marian L. M i l l e r , Charles Becker, Carolyn Emory, Lilam Stanley, Fernando B e c a t t i n i and V i r g i n i a Obrock. Emulsions of p e r f l u o r i n a t e d solvents f o r i n t r a v a s c u l a r gas transport. F e d e r a t i o n Proceedings 34, 1468-1477 (1975); C l a r k , Leland C., J r . , Eugene P. Wesseler, Marian L. M i l l e r and Samuel Kaplan. Ring versus s t r a i g h t chain p e r f l u o r o c a r b o n emulsions as a r t i f i c i a l blood. J o u r n a l of M i c r o v a s c u l a r Research 8, 320-340 (1974)).
Q.
What are your l i m i t a t i o n s on v o l a t i l i t y of the p e r f l u o r o carbons?
cleared
perfluoromethyladamatane
was
do the iodinated compounds hold up i n the animal?
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
170 A.
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
When used as a r t i f i c i a l blood the vapor pressure must be below 50 torr at 38°C. Higher vapor oressures, perhaps much higher vapor pressures can be w e l l t o l e r a t e d f o r l i q u i d breathing i f 1007 oxygen i s used as the gas phase. o
Q.
Do you think these compounds w i l l ever be u s e f u l i n man?
A.
I f I didn't I wouldn't be working so hard. major e f f o r t of ours f o r over eight years.
Q.
How about
A.
When p e r f l u o r o d e c a l i n i s p u r i f i e d by c a r e f u l d i s t i l l a t i o n i n a spinning band column the t o x i c i t y decreases to the point where the LD50 of a 10% by volume emulsion i s over 200 ml/kg. 200 ml/kg i s about three times the blood volume of the mouse The lower b o i l i n g f r a c t i o n r i a l as r e c e i v e d .
This has been a
toxicity?
Assessment of the t o x i c i t y of PFC emulsions i s complicated by the f a c t that i t i s s t i l l d i f f i c u l t to c h a r a c t e r i z e the f i n a l product with a high degree of c e r t a i n t y . Hence one i s often unsure whether d i f f e r e n c e s are due to the p h y s i c a l p r o p e r t i e s of the emulsions themselves or to the v a r i o u s impurities i n d i f f e r e n t PFC. Generation of f l u o r i d e ion by s o n i c a t i o n , a former complicat i o n of emulsion t o x i c i t y t e s t i n g , has been p r a c t i c a l l y eliminated by the discovery by Geyer that by s a t u r a t i n g the l i q uids being sonicated with carbon dioxide, f l u o r i d e i o n i s not generated. Nitrogen, helium, or hydrogen do not work.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9 Radiopaque Applications of Brominated Fluorocarbon Compounds in Experimental Animals and Human Subjects D. M. LONG Department of Radiology, School of Medicine, University of California, San Diego, Calif. M. S. LIU, G. D. DOBBEN, and P. S. SZANTO Hektoen Institute for Medical Research, Cook County Hospital, Chicago,Ill.60612 A. S. ARAMBULO College of Pharmacy, Universit Interest in the biological application of perfluorocarbon compounds was begun with the imaginative fluid ventilation studies of Clark and Gollan in 1965. In the first decade since the publication of those experiments, there has been a slowly growing expansion of background information on the effects of perfluorocarbons on the biological systems. The industrial use of perfluorocarbon compounds has helped by making more molecules available in pure states at reasonable costs. Imaginative biomedical workers have asked where these new compounds could help solve existing medical problems. Our aggressive chemical industry extended itself in seeking new markets in medicine for the products of their laboratories and plants. Fluid ventilation as a tool for delivering oxygen to damaged lungs was our initial goal in experiments with perfluorocarbon compounds. This application and the dreams of Jacques Cousteau of a fluid breathing diver in the depths of the ocean seem frustrated for the time being by the unsolved problems of the work of fluid ventilation and by the mechanical injury to the lungs resulting from long periods of fluid ventilation. We became interested in the possibility of developing a radiopaque fluorocarbon molecule that would be less irritating and safer than currently available radiopaque media which can be quite irritating to the lungs. The inert synthetic fluids, either silicone oils or perfluorocarbons, that were available in 1966 did not possess radiopaque properties. Brominated fluorocarbon liquid was found to be radiodense. Iodinated fluorocarbon compounds were found to be chemically unstable and, therefore, unsuitable for biomedical application. Brominated organic compounds were used in the past and were found to be inferior to iodinated compounds when studied with the usual high kilovoltage used in x-rays of humans. It was argued that brominated compounds, although satisfactory for small animal studies, would be unsatisfactory in the larger human subjects. As can be seen in Figure 1, brominated perfluorocarbon is most satisfactory for use in humans. Lower kilovoltage or energies are used to get maximum absorption from 1
23 ,
4
171 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
172
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
Figure 1. X-ray of the abdomen in a healthy subject ten min oral administartion of 500 neat radiopaque fluorocarbon. Note the rapid visualization of the jejunal portion of the small intestine.
Figure 2. Bronchogram after administration of 6:1 emulsion of radiopaque fluorocarbon in a patient with hemoptysis. A calcified nodule or broncholith can be seen in continuity with an eighth generation bronchus in the lower right hand corner.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
9.
LONG ET A L .
Brominated
Fluorocarbon
173
Compounds
t h e b r o m i n e , b u t t h i s a l t e r a t i o n i n t e c h n i q u e h a s p r e s e n t e d no problems w i t h c u r r e n t l y a v a i l a b l e x - r a y equipment. Emulsions of r a d i o p a q u e f l u o r o c a r b o n (RFC) were p r e p a r e d t o o b t a i n a m a t e r i a l w i t h a h i g h e r v i s c o s i t y t o produce a t h i c k e r c o a t i n g o f the t r a cheobronchial tree (Figure 2). The e m u l s i o n s w e r e p r e p a r e d i n high concentrations of fluorocarbon i n physiologic s a l t solution w i t h P l u r o n i c F-68 as t h e e m u l s i f y i n g a g e n t and were a l s o n o n irritating. A l t h o u g h we h a v e e x a m i n e d a number o f b r o m i n a t e d f l u o r o c a r b o n m o l e c u l e s , most o f o u r s t u d i e s h a v e b e e n p e r f o r m e d w i t h p e r f l u o r octylbromide. T h i s compound i s b i o l o g i c a l l y i n e r t and p o s s e s s e s a v e r y low t o x i c i t y . The LD50 ( T a b l e I) o f C g F B i s g r e a t e r than 64 m l . / k g . when a d m i n i s t e r e d i n t o t h e g a s t r o i n t e s t i n a l t r a c t . We h a v e u s e d h i g h e r d o s a g e s o f as much as 128 m l . / k g . w i t h o u t a d v e r s e e f f e c t s , but these experiments are f a c e t i o u s s i n c e the g a s t r o i n t e s t i n a l t r a c t was l o a d e d and o v e r f l o w i n g a t one o r b o t h e n d s b e f o r e a l l t h e d o s e was a d m i n i s t e r e d 8 17 R injected int a n i m a l s h a v e b e e n c o m p l e t e l y submerged i n C Q F 7 B and b r e a t h e d t h i s f l u i d f o r s h o r t p e r i o d s o f time w i t h s u r v i v a l . The L D ^ Q o f t h e 10:1 e m u l s i o n o f C g F ^ B j ^ i n t h e l u n g s was g r e a t e r t h a n 4 m l . / kg. R e p e t i t i v e dosage programs have been p e r f o r m e d i n a n i m a l exp e r i m e n t s w i t h no a d v e r s e e f f e c t s . The e f f i c a c i o u s d o s e s o f t h e RFC i n human d i a g n o s t i c x - r a y s t u d i e s a r e g i v e n i n T a b l e I . There i s o b v i o u s l y a w i d e m a r g i n o f t h e r a p e u t i c s a f e t y when RFC i s u s e d i n these areas of a p p l i c a t i o n . 4
1 7
C
F
B
w
n
e
R
n
1
r
Gastroenterography Our i n i t i a l t o x i c o l o g i c a l and d i a g n o s t i c s t u d i e s w e r e d i r e c t e d t o w a r d e x a m i n a t i o n o f t h e e f f e c t s o f RFC i n t h e GI t r a c t . It s h o u l d be remembered t h a t p e r f l u o r o c a r b o n compounds a r e new i n b i o m e d i c a l f i e l d s , and none o f t h i s f a m i l y o f compounds h a d e v e r b e e n a d m i n i s t e r e d p u r p o s e f u l l y i n l a r g e d o s e s t o humans. The l a b o r a t o r y s t u d i e s i n d i c a t e d RFC w o u l d be among t h e s a f e s t o f d r u g s or d i a g n o s t i c agents. When g i v e n t o e x p e r i m e n t a l a n i m a l s and h u man s u b j e c t s b y t h e GI t r a c t , t h e r e h a v e b e e n no a d v e r s e e f f e c t s . S p e c i f i c a l l y , t h e r e was no c h a n g e i n t h e b l o o d c o u n t s , s e r u m e n zymes, and u r i n a l y s i s . The a n i m a l s showed no c h a n g e i n g r o w t h p a t t e r n s e v e n when g i v e n r e p e t i t i v e h i g h d o s e s o v e r s h o r t p e r i o d s of time o r over prolonged i n t e r v a l s . RFC h a s b e e n g i v e n r e p e t i t i v e l y i n newborn a n i m a l s u n t i l t h e y a c h i e v e y o u n g a d u l t h o o d and to adult animals. The RFC i s o d o r l e s s and t a s t e l e s s , and h a s a low v i s c o s i t y s o t h a t i t i s easy t o d r i n k . Because o f the e x c e l l e n t w e t t a b i l i t y , t h e mouth and o r o p h a r y n x a r e c o a t e d i m m e d i a t e l y a f t e r t a k i n g t h e material. Some s u b j e c t s h a v e c o m p l a i n e d o f an u n p l e a s a n t o i l y f e e l i n g i n t h e mouth, b u t m o s t h a v e h a d no s u c h r e s p o n s e . RFC t r a v e r s e s t h e GI t r a c t more r a p i d l y t h a n f o o d o r o t h e r c o n t r a s t agents. When RFC i s m i x e d w i t h f o o d and f e d t o d o g s , t h e RFC
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
174
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE BONDS
Table I
LD50 Gastroenterology
(Animals)
64 m l . / k g . Dogs Rats
Efficacious
Dose
1-6 m l . / k g .
Alveolography
> 35 m l . / k g . H a m s t e r s ^> 12 m l . / k g . C a t s
1-2 m l . / k g .
Bronchography
>
0.3-0.6 m l . / k g .
4 m l . / k g . Dogs
Figure 3. Gastrointestinal series in an "asymptomatic" volunteer forty minutes after oral administration of 225 ml of neat RFC. This subject had a small channel ulcer with pylorospasm and delayed gastric emptying. Note that RFC is still present in the stomach while the left colon is beginning to show filling with RFC.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG
ET
AL.
Brominated
Fluorocarbon
Compounds
175
l e a v e s t h e f o o d and t r a v e r s e s t h e GI t r a c t a h e a d o f t h e f o o d . T h i s b e h a v i o r o f RFC h a s b e e n a t t r i b u t e d t o t h e " c r e e p i n g " p r o p e r t y o f s u b s t a n c e s w i t h low s u r f a c e t e n s i o n . I n i t i a l l y , we t h o u g h t t h a t r a p i d g a s t r i c e m p t y i n g m i g h t be a d i s a d v a n t a g e i n c e r t a i n d i s e a s e s t a t e s ; h o w e v e r , we f o u n d t h i s p r o p e r t y t o be an a d v a n t a g e i n c l i n i c a l p r a c t i c e . I n F i g u r e 3, we s e e t h e x - r a y o f a s u b j e c t w i t h a s m a l l , c h a n n e l u l c e r c r a t e r . G a s t r i c e m p t y i n g o f RFC was d e l a y e d b e y o n d f o r t y m i n u t e s due t o p y l o r o s p a s m o f a v e r y modest degree. T h i s s u b j e c t was a h e a l t h y v o l u n t e e r m e d i c a l s t u d e n t who s t a t e d a f t e r t h e s t u d y t h a t he r e g u l a r l y had m i l d e p i g a s t r i c d i s c o m f o r t e s p e c i a l l y b e f o r e s l e e p and d u r i n g t i m e s o f s t r e s s . R e p e a t u p p e r GI s e r i e s w i t h b a r i u m s u l f a t e d i d n o t r e v e a l any u l c e r c r a t e r o r d e l a y e d g a s t r i c emptying. One d i s a d v a n t a g e o f RFC i n t h e GI t r a c t o f humans i s t h a t t h e r e i s i n a d e q u a t e r a d i o p a c i f i c a t i o n o f t h e e s o p h a g u s and f u n d u s o f t h e stomach. T h i s inadequac animals. I n d i c a t i o n s f o r Use o f RFC i n t h e GI T r a c t . O b v i o u s l y , RFC c a n n o t compete i n c o s t w i t h b a r i u m s u l f a t e f o r GI s t u d i e s . There a r e s p e c i f i c c i r c u m s t a n c e s i n w h i c h RFC c a n and s h o u l d be u s e d , and t h e s e i n d i c a t i o n s r e p r e s e n t an e s t i m a t e d one p e r c e n t o f t h e t o t a l m a r k e t o f GI x - r a y s t u d i e s . I n any s u b j e c t w h e r e p u l m o n a r y a s p i r a t i o n o f c o n t r a s t m e d i a i s l i k e l y , RFC w o u l d be u s e f u l due to i t s lack of t o x i c i t y t o the lungs. These c i r c u m s t a n c e s i n c l u d e i n f a n t s , e l d e r l y p a t i e n t s , and weak c a c h e c t i c p a t i e n t s a s w e l l as a l l s u b j e c t s w i t h symptoms o f t r a c h e o e s o p h a g e a l f i s t u l a . We h a v e a l s o f o u n d RFC u s e f u l i n p a t i e n t s w i t h i n t e s t i n a l f i s t u l a s , perforations, or i n t e s t i n a l obstruction, or patients with suspected postoperative i l e u s or obstruction. The x - r a y p i c t u r e s o f t h e s m a l l i n t e s t i n e s h a v e b e e n j u d g e d s u p e r i o r t o t h o s e obt a i n e d w i t h b a r i u m s u l f a t e and c a n be o b t a i n e d i n l e s s t h a n an hour. P e r i t o n e a l Contamination
w i t h Radiopaque
Agents
Leakage o f c o n t r a s t m a t e r i a l such as b a r i u m s u l f a t e i n t o t h e p e r i t o n e a l c a v i t y c a r r i e s p o t e n t i a l l y s e v e r e c o m p l i c a t i o n s due t o t h e a c u t e and c h r o n i c i n f l a m m a t o r y r e a c t i o n i n c i t e d b y b a r i u m sulfate. W a t e r - s o l u b l e o r g a n i c i o d i d e compounds may be u s e d when p e r f o r a t i o n i s s u s p e c t e d , b u t t h e s e compounds a l s o p r o d u c e a c u t e inflammation o f the peritoneum. Water-soluble organic iodide compounds p r o d u c e d i a r r h e a and do n o t g i v e s a t i s f a c t o r y r a d i o p a c i f i c a t i o n o f t h e GI t r a c t . RFC i s n o n - i o n i c and d o e s n o t r e s u l t in diarrhea. RFC h a s b e e n i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y o f e x p e r i m e n t a l a n i m a l s i n d o s a g e s o f 16 m l . / k g . , many t i m e s t h e l e t h a l dosage o f barium s u l f a t e o r o r a l Hypaque. T h e r e was no a c u t e o r c h r o n i c i n f l a m m a t i o n e v e n i n a n i m a l s o b s e r v e d f o r more than four years. No e v i d e n c e o f c a r c i n o g e n i c i t y was o b s e r v e d on R
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
176
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BONDS
histological studies. The C g F ^ B l e a v e s t h e p e r i t o n e a l c a v i t y v e r y s l o w l y by v a p o r i z a t i o n and by p h a g o c y t o s i s . Some o f t h e 8 17 R f d i n s u b c u t a n e o u s lymph n o d e s o f t h e a n t e r i o r a b dominal w a l l . P h a g o c y t o s i s o f RFC o c c u r s b y m o n o c y t e s i n t h e p e r itoneal cavity. A c c u m u l a t i o n s o f v a c u o l e - l a d e n p h a g o c y t e s c a n be s e e n i n t h o s e a r e a s w i t h r a d i o d e n s i t y on x - r a y s ( F i g u r e 4). Large c y s t - l i k e a c c u m u l a t i o n s o f RFC a r e s e e n w i t h i n t h e p e r i t o n e a l c a vity. T h i s r e a c t i o n i s comparable t o t h e f o r e i g n body r e a c t i o n seen w i t h T e f l o n o r S i l a s t i c * , t h e most i n e r t m a t e r i a l s u s e d i n medical implants. Such f o r e i g n body r e a c t i o n s a r e f o u n d w i t h any i n e r t m a t e r i a l with long residence time. 7
c
f
b
w
a
s
o
u
n
R
Fluorocarbons
R
1
o f High Vapor
Pressure
The r a d i o p a q u e f l u o r o c a r b o n C ^ F ^ B R was a l s o s t u d i e d i n t h e peritoneal cavity. T h i s compound h a s a b o i l i n g p o i n t o f 9 8 ° a n d a v a p o r p r e s s u r e o f 90 T o r r vity, C Fi3B vaporize tended w i t h f l u o r o c a r b o n gas. C^F-^Bj^ l e a v e s t h e b o d y more r a p i d l y than C g F ^ B j ^ and i s c o m p l e t e l y e l i m i n a t e d from t h e p e r i t o n e a l c a v i t y i n weeks e v e n w i t h l a r g e d o s e s o f 16 m l . / k g . This p r o p e r t y o f r a p i d v a p o r i z a t i o n and i n t r a c a v i t a r y gas f o r m a t i o n d o e s n o t p r o d u c e a n y a d v e r s e e f f e c t s i n t h e GI t r a c t o r l u n g s ; h o w e v e r , when i n j e c t e d i n t o t h e s u b a r a c h n o i d s p a c e , g a s f o r m a t i o n r e s u l t s i n n e u r o l o g i c a l i n j u r y i n a s i g n i f i c a n t number o f a n i m a l s . F o r t h i s r e a s o n , we s e l e c t e d C s F i B f o r o u r i n i t i a l i n v e s t i g a t i o n s o f RFC s i n c e e c o n o m i c c o n s i d e r a t i o n s d i d n o t p e r m i t s i m u l t a n e o u s a n d p a r a l l e l s t u d i e s w i t h more t h a n o n e RFC compound. T h e C-6 17 R compound was a l s o n o n - t o x i c a n d may p r o v e t o b e u s e f u l f o r GI a n d p u l m o n a r y s t u d i e s . C £ F i B v a p o r i z e s w i t h i n t h e GI t r a c t thus p r o v i d i n g u s e f u l d i a g n o s t i c p r o p e r t i e s o f gas c o n t r a s t . In some m a n u f a c t u r i n g p r o c e s s e s , C F B i s the principle contaminant o r i m p u r i t y . Up u n t i l now, we h a v e h e l d s t a n d a r d s o f b e t t e r t h a n 99.9 p e r c e n t p u r i t y f o r C F B . T h i s h i g h p u r i t y s t a n d a r d may n o t b e n e c e s s a r y , a n d i f n o t , c o s t s o f raw m a t e r i a l w o u l d b e r e duced s i g n i f i c a n t l y . 6
R
7
F
R
B
7
6
Q
Submersion
1 7
R
1 7
R
R
Experiments
S u b m e r s i o n o f h a m s t e r s i n RFC h a s b e e n p e r f o r m e d f o r p e r i o d s up t o t e n m i n u t e s . When t h e h a m s t e r s w e r e removed f r o m t h e l i q u i d , x - r a y s showed t h e l u n g s t o b e f i l l e d w i t h RFC a n d RFC was a l s o p r e s e n t i n t h e GI t r a c t . L a t e r x - r a y s showed g r a d u a l c l e a r ing o f t h e lungs. S i n c e RFC c l e a r s p r i m a r i l y b y v a p o r i z a t i o n , t h e a r e a s o f t h e l u n g s t h a t a r e b e t t e r v e n t i l a t e d show more r a p i d clearing. T h u s , t h e a l v e o l o g r a m s w i t h RFC p r o v i d e a p h y s i o l o g i c picture of different ventilatory patterns i n different parts of the lungs. in
Microscopic examination RFC r e v e a l e d t h e p r e s e n c e
o f t h e l u n g s one d a y a f t e r submersion o f h y p e r i n f l a t i o n o f t h e a l v e o l i due
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG E T A L .
Brominated
Fluorocarbon
177
Compounds
Figure 4.
Photomicrograph of
phagocytic cells one month after intraperitoneal injection of 16 ml of neat RFC into the peritoneal cavity of a rat. Note the large vacuoles in the mono cytic cells at the top of the Hematoxylin and eosin stain. 17 5χ.
Figure
5.
Lung
macrophage
of radiopaque fluorocarbon in cupying the alveoli in a rabbit two days after a bronchogram with 10:1 emulsion of RFC. Hematoxylin and eosin stain. 290X.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
178
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INVOLVING
CARBON-FLUORINE
BONDS
to the m e c h a n i c a l e f f e c t s o f the dense f l u o r o c a r b o n ( d e n s i t y 1.93 gm./ml.). M a c r o p h a g e s w i t h v a c u o l e s o f RFC were s e e n i n t h e a l v e o l a r spaces ( F i g u r e 5). No e v i d e n c e o f p n e u m o n i t i s due t o RFC was o b s e r v e d when compared w i t h c o n t r o l a n i m a l s . A t one month, t h e r e were few m a c r o p h a g e s w i t h v a c u o l e s o f RFC. The m a c r o p h a g e s w i t h v a c u o l e s o f f l u o r o c a r b o n showed no e v i d e n c e o f i n t r a c e l l u l a r damage, and t h e a l v e o l a r a r c h i t e c t u r e h a d r e t u r n e d t o n o r m a l . Alveolographic
Studies
with
RFC
X - r a y v i s u a l i z a t i o n o f t h e a l v e o l a r compartment o f t h e l u n g s c a n n o t be o b t a i n e d w i t h c u r r e n t l y a v a i l a b l e m a t e r i a l . Theoretic a l l y , i t s h o u l d be h i g h l y d e s i r a b l e t o o b t a i n a l v e o l a r s t u d i e s in l i v i n g subjects without r e s o r t i n g to lung b i o p s i e s . Such i n f o r m a t i o n s h o u l d be u s e f u l i n d i f f e r e n t i a t i n g v a r i o u s t y p e s o f lung diseases. In other areas of medicine precision i n diagnosis has been e s s e n t i a l i n p r e s c r i b i n understanding e t i o l o g i disease. A t f i r s t , we w e r e d i s a p p o i n t e d t o o b s e r v e t h a t RFC f i l l e d t h e a l v e o l a r compartment and d i d n o t p r o v i d e r a d i o p a c i f i c a t i o n o f t h e t r a c h e a and b r o n c h i . I t t h e n became n e c e s s a r y t o d e velop unique emulsions f o r tracheobronchography. In t i m e , the a l v e o l o g r a p h i c s t u d i e s may p r o v e more u s e f u l . A l v e o l o g r a p h y c a n be a c c o m p l i s h e d w i t h d o s e s t h a t a r e a f r a c t i o n o f t h e LD50 d o s e . We h a v e b e e n u n s u c c e s s f u l i n o u r a t t e m p t s t o n e b u l i z e n e a t RFC i n t o t h e l u n g s . A catheter i s inserted into t h e t r a c h e a w i t h t o p i c a l a n e s t h e s i a o f t h e l a r y n x and t r a c h e a . The n e a t RFC d o e s n o t i n d u c e c o u g h i n g o r o t h e r phenomena o f i r r i tation. The t o p i c a l a n e s t h e s i a d o e s i n d u c e b r o n c h o s p a s m and hyp o x e m i a , b u t t o p i c a l a n e s t h e s i a has b e e n n e e d e d f o r c a t h e t e r p l a c e m e n t i n s u b j e c t s s t u d i e d t h u s f a r . The s u b j e c t s h a v e comp l a i n e d o f p h a r y n g i t i s and l a r y n g i t i s f r o m t h e t o p i c a l a n e s t h e s i a and t h e c a t h e t e r . We h a v e a l s o o b s e r v e d m i l d e l e v a t i o n s i n o r a l t e m p e r a t u r e s , w h i t e b l o o d c e l l c o u n t , and o c c a s i o n a l m i l d e l e v a t i o n s i n t h e s e r u m enzymes SGOT o r LDH. These e f f e c t s have d i s a p p e a r e d i n t w e n t y - f o u r t o f o r t y - e i g h t h o u r s , and i t i s d i f f i c u l t t o d e t e r m i n e what p a r t o f t h e s e a d v e r s e e f f e c t s a r e due t o t h e c a t h e t e r p l a c e m e n t , t h e t o p i c a l a n e s t h e s i a and t h e f l u o r o c a r b o n . I n p a t i e n t s w i t h b u l l o u s emphysema, t h e a l v e o l o g r a m s h a v e a c c u r a t e l y o u t l i n e d the areas o f normal lung. The emphysematous b u l l a e h a v e n o t f i l l e d w i t h RFC ( F i g u r e 6 ) . Areas of c e n t r i l o b u l a r emphysema h a v e a l s o showed a v o i d on x - r a y s ( F i g u r e 7 ) . A r e a s o f n o r m a l a l v e o l a r s t r u c t u r e were c l e a r l y d e m o n s t r a t e d i n p a t i e n t s and h e a l t h y v o l u n t e e r s . Those areas o f l u n g compressed by emphysematous b u l l a e f i l l e d s l o w l y on a l v e o l o g r a p h y . Similarl y , t h e a r e a s o f p o o r l y v e n t i l a t e d o r c o m p r e s s e d l u n g c l e a r e d RFC more s l o w l y t h a n d i d a r e a s o f w e l l v e n t i l a t e d l u n g . Radiographic e v i d e n c e f o r t h e RFC h a d c l e a r e d by f o r t y - e i g h t h o u r s .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG
E T A L .
Brominated
Fluorocarbon
Compounds
Figure 6.
179
Alveologram with neat RFC
hemithorax. Note that the fluorocarbon does not fill the emphysematous bullae.
Figure 7. Close up view of the lower portions of the left hemithorax in the same patient as seen in Figure 6. The compressed areas of the lung can be seen to contain radiodense material which is RFC. The punched out areas in the alveologram represent areas of centrilobuhr emphysema verified by histological examination of the tissue.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
180
BIOCHEMISTRY
Emulsions
of
INVOLVING
CARBON-FLUORINE
BONDS
RFC
D i l u t e e m u l s i o n s o f RFC (2:1 v o l u m e t o v o l u m e ) a r e e a s i l y p r e p a r e d by m i x i n g RFC i n a 6% s o l u t i o n o f P l u r o n i c F-68 i n p h y siologic salt solutions. C o n c e n t r a t e d e m u l s i o n s (up t o 15:1) can be p r e p a r e d b y g r a d u a l a d d i t i o n o f n e a t RFC. The c o n c e n t r a t e d emulsions are u s e f u l i n bronchography. We u s e e m u l s i o n s w i t h c o n c e n t r a t i o n s v a r y i n g f r o m 6:1 t o 10:1. Emulsions w i t h concent r a t i o n s l e s s t h a n 6:1 c a u s e c o u g h i n g i n e x p e r i m e n t a l a n i m a l s s i m i l a r t o t h a t produced by p h y s i o l o g i c s a l t s o l u t i o n . Emulsions o f c o n c e n t r a t i o n s 6:1 o r g r e a t e r b e h a v e more l i k e n e a t RFC and d o not produce coughing. The e m u l s i o n s h a v e a w i d e r a n g e o f p a r t i c l e s i z e i n c l u d i n g some p a r t i c l e s a s l a r g e a s 1 mm. T h e r e i s some c h a n g e i n p a r t i c l e s i z e d i s t r i b u t i o n as w e l l as v i s c o s i t y w i t h t i m e , b u t t h e s h e l f l i f e o f the emulsions i s v e r y s a t i s f a c t o r y f o r our purpo ses. Only s l i g h t creamin r e - e m u l s i f i e d by s h a k i n s i o n s i s n o n - N e w t o n i a n and t h i x o t r o p i c . 5
Bronchography w i t h
RFC
Emulsions
B r o n c h o g r a p h y was p e r f o r m e d w i t h t o p i c a l a n e s t h e s i a o f t h e p h a r y n x and t r a c h e a and i n s e r t i o n o f a t r a c h e a l c a t h e t e r . The t r a c h e a l c a t h e t e r was p o s i t i o n e d i n t o t h e b r o n c h u s o f c h o i c e , and 10 t o 20 m l . o f 6:1 o r 10:1 e m u l s i o n was i n j e c t e d i n t o t h e m a i n bronchus w i t h the p a t i e n t apneic i n e x p i r a t i o n . The p a t i e n t was then asked t o t a k e a deep b r e a t h . Additional injections into l o b a r b r o n c h i were p e r f o r m e d as i n d i c a t e d . A p p r o p r i a t e x - r a y s were t a k e n t o o b t a i n c o m p l e t e i n f o r m a t i o n on t h e s t r u c t u r e o f t h e t r a cheobronchial tree. B i l a t e r a l bronchograms are u s u a l l y d e s i r e d and h a v e b e e n p e r f o r m e d s i m u l t a n e o u s l y w i t h o u t i n c i d e n t . The x - r a y s o f t h e c h e s t w e r e o b t a i n e d w i t h k i l o v o l t a g e o f 70 t o 75, somewhat l e s s t h a n t h a t o b t a i n e d w i t h c o n v e n t i o n a l x - r a y s o f t h e chest. S a t i s f a c t o r y bronchograms have been o b t a i n e d i n a l l h e a l t h y v o l u n t e e r s and p a t i e n t s s t u d i e d . E v e n p a t i e n t s w i t h s e v e r e and f a r a d v a n c e d p u l m o n a r y d i s e a s e h a v e t o l e r a t e d t h e RFC e m u l s i o n s . Three p a t i e n t s had e x p e r i e n c e d s e v e r e r e s p i r a t o r y d i s t r e s s p r e v i o u s l y when t h e y r e c e i v e d b r o n c h o g r a m s w i t h c u r r e n t l y a v a i l a b l e o r g a n i c i o d i d e b r o n c h o g r a p h i c media. These t h r e e p a t i e n t s t o l e r a t e d t h e RFC b r o n c h o g r a m s w e l l . D e c r e a s e s i n a r t e r i a l p02 were o b s e r v e d i n s u b j e c t s f o l l o w i n g t o p i c a l a n e s t h e s i a and h y p o x e m i a and b r o n c h o s p a s m h a v e b e e n r e p o r t e d by o t h e r s f o l l o w i n g t o p i c a l a n e s t h e s i a o f t h e t r a c h e o bronchial t r e e . ' A f t e r t h e RFC was i n j e c t e d , t h e a r t e r i a l p 0 e i t h e r d e c r e a s e d o r i n c r e a s e d o r s t a y e d t h e same. A r t e r i a l hypo x e m i a was c o r r e c t e d by t h e u s e o f s u p p l e m e n t a l n a s a l o x y g e n breathing. 6
7
2
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG ET
AL.
Brominated
Fluorocarbon
Compounds
181
Biological Disposition The b i o l o g i c a l d i s p o s i t i o n o f RFC h a s b e e n e x a m i n e d i n e x p e r i m e n t a l a n i m a l s r e c e i v i n g RFC b y t h e s e v e r a l r o u t e s s t u d i e d . The a n i m a l s were s a c r i f i c e d a t d i f f e r e n t i n t e r v a l s a f t e r r e c e i v i n g RFC and t h e t i s s u e s were e x t r a c t e d i n h e x a n e . The e x t r a c t s were a n a l y z e d f o r f l u o r o c a r b o n u s i n g g a s l i q u i d c h r o m a t o g r a p h y . The b i o l o g i c d i s p o s i t i o n o f RFC g i v e n b y t h e g a s t r o i n t e s t i n a l r o u t e i s shown i n T a b l e I I . O n l y t h o s e t i s s u e s w i t h t h e h i g h e s t f l u o r o c a r b o n c o n c e n t r a t i o n s were l i s t e d a l t h o u g h o t h e r t i s s u e s were a l s o a n a l y z e d . T r a c e amounts o f RFC c o u l d b e s e e n i n x - r a y s o f t h e a n i m a l s t w e n t y - f o u r h o u r s a f t e r a d m i n i s t r a t i o n , b u t chemi c a l a n a l y s i s revealed s i g n i f i c a n t q u a n t i t i e s i n the g a s t r o i n t e s tinal tract. T r i v i a l amounts o f RFC were a b s o r b e d f r o m t h e GI t r a c t as e v i d e n c e d b y t h e s m a l l q u a n t i t i e s o f RFC p r e s e n t i n o t h e r t i s s u e s . A p r o g r e s s i v e d e c l i n e i n t i s s u e RFC c o n c e n t r a t i o n o c c u r r e d , and a t t h r e e r e s i d u a l RFC. T a b l e s I I I and I V p r e s e n t t h e b i o l o g i c d i s p o s i t i o n d a t a on RFC a d m i n i s t e r e d i n t o t h e l u n g s f o r a l v e o l o g r a p h y and f o r b r o n c h o g r a p h y , r e s p e c t i v e l y . Twenty-one d a y s a f t e r a d m i n i s t r a t i o n o f a l a r g e d o s e o f 4 m l . A g . o f n e a t RFC, t h e r e w e r e o n l y t r a c e amounts f o u n d i n t h e t i s s u e s . When RFC e m u l s i o n was g i v e n i n a d o s a g e o f 2 m l . / k g . , e l i m i n a t i o n was v i r t u a l l y c o m p l e t e b e f o r e t w e l v e weeks a f t e r a d m i n i s t r a t i o n . Lymphography R a d i o p a c i f i c a t i o n o f l y m p h a t i c s t r u c t u r e s was o b t a i n e d b y i n f u s i o n o f RFC i n t o l y m p h a t i c c h a n n e l s o r i n t o lymph n o d e s . N e a t RFC was p r e f e r r e d f o r l y m p h o g r a p h y . I n f u s i o n of emulsions r e s u l t e d i n r a d i o p a c i f i c a t i o n o f the lymphatics t o the f i r s t lymph node. The e m u l s i o n was r a p i d l y p h a g o c y t o s e d b y t h e c e l l s i n t h e lymph n o d e , t h e lymph node became e n g o r g e d and t e n s e , and t h e f l o w o u t o f t h e lymph node was b l o c k e d s o t h a t d i s t a l lymphat i c s and lymph n o d e s c o u l d n o t b e v i s u a l i z e d . When n e a t RFC was i n f u s e d , t h e u p t a k e by t h e lymph n o d e s was l e s s v o r a c i o u s . The lymph n o d e s became r a d i o p a q u e b u t n o t t e n s e , and t h e d i s t a l lymph n o d e s and c h a n n e l s were v i s u a l i z e d ( F i g u r e 8 ) . When e x c e s s i v e q u a n t i t i e s were i n j e c t e d , t h e n e a t RFC s p i l l e d o v e r i n t o t h e p u l monary v a s c u l a t u r e . The L D Q d o s e o f RFC i n c a t s was b e t w e e n 1 . 0 and 1 . 2 5 m l . / k g « The e f f i c a c i o u s d o s e was 0 . 2 m l . / k g . , w h i c h was t h a t q u a n t i t y r e q u i r e d t o f i l l t h e l y m p h a t i c s b e f o r e s p i l l over i n t o t h e venous system. The r e s i d e n c e t i m e o f RFC i n lymph n o d e s was g r e a t e r than t h r e e years i n dogs. T h i s l o n g r e s i d e n c e t i m e may be an a d v a n t a g e , p a r t i c u l a r l y when one i s s t u d y i n g t h e i n v o l v e m e n t o f lymph n o d e s b y m a l i g n a n t t u m o r s . I o d i z e d o i l , the agent c u r r e n t l y used f o r lymphography a l s o has a l o n g r e s i d e n c e t i m e , b u t t h i s a g e n t i n c i t e d i n f l a m m a t i o n and f i b r o s i s i n lymph n o d e s . 5
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
182
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
Table II Biologic
Disposition
after
Gastroenterography 3 Days
1 Day Large
Intestines
7 X ΙΟ"
5
1 X ΙΟ"
2
1.4 X Ι Ο "
3
Liver
1.6 X Ι Ο "
4
6 X ΙΟ"
Lungs
4 X ΙΟ"
5
6 X
ίο"
5
Lymph Nodes
5
2
ίο"
5
Stomach & S m a l l
Intestines
X ΙΟ"
5
5
X
X
ίο"
6
5
Fat T i s s u e l e v e l s i n m l . 8 1 7 ] p e r gram o f t i s s u e a t d i i n t e r v a l s a f t e r g a s t r o e n t e r o g r a p h y w i t h 16 m l . / k g . o f C
time C
F
B
8 17 R*
F
B
R
E x p e r i m e n t a l a n i m a l was t h e a d u l t r a t .
Figure 8. Lymphangiography in a dog with Etniodal injected into the right leg and neat radiopaque fluorocarbon injected into the left leg. The iodinated oil, Ethiodal , is more radiodense than RFC but the difference in radiodensity does not detract from diagnostic accuracy. R
R
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
9.
LONG
E T AL.
Brominated
Fluorocarbon
183
Compounds
Table I I I Biologie
Disposition
3 Days Lungs
8 x 1CT
after
Alveolography
1 Week 4
3 Weeks
8 χ ΙΟ"
5
5 χ ΙΟ"
5
9 χ ΙΟ"*
6
3 χ ΙΟ"
5
Lymph Nodes Fat
7 χ ΙΟ"
5
7 χ ΙΟ"
5
4 χ 1(Γ
4
4 χ 10-5
T i s s u e l e v e l s i n m l . C Q F I 7 B p e r gram o f t i s s u e a t d i f f e r e n t time i n t e r v a l s a f t e r a l v e o l o g r a p h y w i t h 4 ml./kg. Experimental a n i m a l was t h e a d u l t r a t r
Table IV Biologic
1 Day 4
Lungs
6 χ 10"
Lymph Nodes
2.4 χ 1 0 "
Fat
7 χ 10"
5
Disposition
a f t e r Bronchography
3 Days
1 Week
2 χ 10~
5
4
3.8 χ 1 0 " 9 χ 10-5
4
4 Weeks
12 Weeks
1 χ 10~
4
1 χ 10" < 1 χ 10"
3 χ 10"
5
4 χ 10~
χ
x
IQ-5
5
4
X
IQ-5
6
6
<2 χ 10* 6
T i s s u e l e v e l s i n m l . C Q F ^ B R p e r gram o f t i s s u e a t d i f f e r e n t t i m e i n t e r v a l s a f t e r b r o n c h o g r a p h y w i t h 2 m l . / k g . o f a 10:1 e m u l sion. E x p e r i m e n t a l a n i m a l was t h e d o g .
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
Figure 9. Ventriculomyelogram in a rabbit with neat RFC. The ventriculomyelogram was well tolerated and resulted in excellent visualization of central nervous system structures.
Figure 10. Hepatography and splenography in a rat given 15 ml/kg of a 67% emulsion of radiopaque fluorocarbon intravenously. The particle size of the emulsion was 1-1.5 microns. The x-ray on the left was made prior to injection, and the x-ray on the right was made four days after injection of the emulsion. The structures of the thin spleen are more accurately outlined than those of the more dense liver.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
BONDS
9.
LONG
ET AL.
Brominated
Fluorocarbon
185
Compounds
Ventriculomyelography Radiopaque p e r f l u o r o c a r b o n has been used t o enhance r a d i o l o g i c a l v i s u a l i z a t i o n o f c e n t r a l nervous system s t r u c t u r e s i n e x p e r i mental a n i m a l s . These s t u d i e s have been performed w i t h C Q F ^ B R , C7F15BR and C ^ F ^ B R . N e a t RFC h a s b e e n u s e d s i n c e e m u l s i o n s w e r e too v i s c o u s f o r t h i s purpose. The RFC compounds p r o v e d t o be e f f i c a c i o u s f o r o u t l i n i n g appropriate s t r u c t u r e s of the c e n t r a l nerv o u s s y s t e m ( F i g u r e 9 ) . The r e s i d e n c e t i m e o f C Q F B i n the suba r a c h n o i d s p a c e was g r e a t e r t h a n t h r e e y e a r s . T h e r e was no a c u t e i n f l a m m a t o r y r e a c t i o n e l i c i t e d b y CgF-^BR a s e v i d e n c e d by t h e l a c k o f s i g n i f i c a n t c e l l u l a r and c h e m i c a l c h a n g e s i n t h e c e r e b r o s p i n a l fluid. No d e m o n s t r a b l e n e u r o l o g i c a l i n j u r y was p r o d u c e d i n e x p e r imental animals, e i t h e r i n the acute or c h r o n i c phases. A mild a r a c h n o i d i t i s was s e e n i n c h r o n i c e x p e r i m e n t s and was m a n i f e s t e d by t h e a c c u m u l a t i o n o f p h a g o c y t i c m o n o c y t e s i n t h e a r e a s o f f l u o rocarbon a c c u m u l a t i o n . w i l l be o f c l i n i c a l s i g n i f i c a n c s u p e r i o r t o PantopaqueÇ the c u r r e n t l y a v a i l a b l e c o n t r a s t agent f o r myelography. 8 , 9
1
7
r
P e r f l u o r o h e x y l b r o m i d e was s t u d i e d b e c a u s e o f t h e r a p i d v a p o r i z a t i o n o f t h i s compound a t b o d y t e m p e r a t u r e . The v a p o r p r e s s u r e o f C ^ F Q ^ B R was a p p r o x i m a t e l y 9 0 T o r r compared t o 1 4 T o r r f o r C8F17BR and 5 5 T o r r f o r C 7 F B . Previous s t u d i e s demonstrated t h a t C5F13BR v a p o r i z e d r a p i d l y and d i s a p p e a r e d r a d i o l o g i c a l l y w i t h i n weeks when i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y o r s u b c u t a neous s p a c e . When i n j e c t e d i n t o t h e s u b a r a c h n o i d s p a c e , C ^ F ^ B R f o r m e d v a p o r p o c k e t s , and t h e g a s p h a s e was n o t r e a b s o r b e d r a p i d l y e n o u g h , s o t h a t n e u r o l o g i c a l i n j u r y and e v e n m o r t a l i t y o c c u r r e d i n animals. The r a t e o f d i s a p p e a r a n c e o f C 7 F 5 B f r o m t h e b o d y and the s u b a r a c h n o i d space i s under i n v e s t i g a t i o n i n our l a b o r a t o r y . 1 5
R
9
1
Hepatography
and
R
Splenography
I n t r a v e n o u s i n f u s i o n s o f e m u l s i o n s o f RFC r e s u l t i n r a d i o p a c i f i c a t i o n o f t h e s p l e e n o r t h e s p l e e n and l i v e r . When we t e s t e d t h e b r o n c h o g r a p h i c e m u l s i o n s f o r i n t r a v e n o u s t o x i c i t y , we o b s e r v e d r a d i o p a c i f i c a t i o n of the spleen. These emulsions have a l a r g e particle size. The r a d i o p a c i f i c a t i o n was a p p a r e n t i n t h i r t y m i n u t e s and i n c r e a s e d b y f o u r h o u r s . The r a d i o p a c i f i c a t i o n d i m i n i s h ed g r a d u a l l y and d i s a p p e a r e d b y a b o u t f o u r weeks. When s m a l l p a r t i c l e s i z e e m u l s i o n s o f 1 t o 1.5 m i c r o n were i n j e c t e d i n t r a v e n o u s l y , r a d i o p a c i f i c a t i o n o f t h e s p l e e n and l i v e r o c c u r r e d ( F i g u r e 10). E m u l s i o n s o f s m a l l e r p a r t i c l e s i z e have been t e s t e d w i t h equivocable r e s u l t s . T h e s e s t u d i e s o f h e p a t o g r a p h y and s p l e n o g r a phy a r e i n an e a r l y s t a g e o f d e v e l o p m e n t s o t h a t a c o n s i d e r a b l e amount o f r e s e a r c h i s r e q u i r e d t o d e f i n e t h e optimum p a r t i c l e s i z e and d o s a g e . The RFC i s p h a g o c y t o s e d b y t h e r e t i c u l o e n d o t h e l i a l c e l l s o f t h e l i v e r and s p l e e n ( F i g u r e 1 1 ) . As s e e n i n F i g u r e 1 1 , the r a d i o d e n s i t y achieved i s s u f f i c i e n t f o r d i a g n o s t i c purposes.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
186
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Figure 11. Photomicrograph of the spleen of a rabbit one week after receiving intravenous injection of large particle size emuhion of RFC. The spleen contained foci of mononuclear cells with foamy cytoplasm due to the presence intracellular fluorocarbon.
Other Areas
of Application
B o t h n e a t RFC and e m u l s i o n s o f RFC h a v e b e e n t e s t e d f o r a r t h r o g r a p h y a n d h a v e b e e n f o u n d t o be i n a d e q u a t e . RFC h a s a l s o b e e n e v a l u a t e d i n r e t r o g r a d e u r o g r a p h y , c h o l e c y s t o g r a p h y , and pancreatography i n experimental animals. The r e s u l t s o b t a i n e d were comparable t o t h o s e o b t a i n e d w i t h c u r r e n t l y a v a i l a b l e c o n t r a s t agents. D e t a i l e d e f f i c a c y and t o x i c i t y s t u d i e s h a v e n o t been performed t o date i n t h e s e areas o f l i m i t e d frequency o f use. RFC may b e a f e a s i b l e a l t e r n a t i v e f o r u s e i n p a t i e n t s w i t h h y p e r s e n s i t i v i t y t o organic iodide contrast agents. I t may a l s o p r o v e t o be s a f e r i n v i s u a l i z a t i o n o f i n f l a m e d o r f r a g i l e d u c t a l s y s t e m s where e x t r a v a s a t i o n o f c o n t r a s t m e d i a s u c h a s o r g a n i c i o d i d e compounds w o u l d e x a c e r b a t e t h e i n f l a m m a t o r y p r o c e s s i n an organ such as t h e p a n c r e a s . Summary M o n o b r o m i n a t e d p e r f l u o r o a k y l compounds h a v e b e e n t e s t e d a s x-ray c o n t r a s t agents. T h e compound p e r f l u o r o c t y l b r o m i d e i s t h e agent o f c h o i c e a t t h i s time because o f i t s b i o l o g i c a l i n e r t n e s s , low s u r f a c e t e n s i o n , e a s e o f e m u l s i f i c a t i o n and f a v o r a b l e r a t e o f e l i m i n a t i o n from t h e body. Gastroenterography, bronchography, and a l v e o l o g r a p h y h a v e b e e n p e r f o r m e d w i t h r a d i o p a q u e f l u o r o c a r bon i n e x p e r i m e n t a l a n i m a l s and humans. O t h e r a r e a s o f a p p l i c a t i o n are being investigated i n experimental animals.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG
E T A L .
Brominated
Fluorocarbon
Compounds
187
Acknowledgements: The a u t h o r s w i s h t o e x p r e s s t h e i r g r a t i t u d e t o M i s s P a t L e e , M r s , F r a n c e s M u l t e r , and M i s s Margo N i e l s o n f o r e x c e l l e n c e i n l a b o r a t o r y e x p e r i m e n t s , a n d t o D o c t o r s Hugh G. B r y c e , Raymond J . S e f f l , J . Dana McGowen, a n d D o n a l d L a Z e r t e o f t h e 3M Company.
Supported by g r a n t s from t h e C h e m i c a l D i v i s i o n , M i n n e s o t a and M a n u f a c t u r i n g Company, S t . P a u l , M i n n e s o t a Grant
Mining
a n d t h e U.S.P.H.S.
GM20998.
Literature Cited 1. Clark, L.C., Jr., an 56: "Survival of mammals breathing organic liquids equilibra ted with oxygen at atmospheric pressure." 2. Clark, L.C., Jr., Kaplan, S., Becattini, F. J. Thoracic Car diovascular Surg. (1970) 60, p. 757-773: "The physiology of synthetic blood." 3. Geyer, R.P. Fed. Proc. (1975) 34, p. 1499-1505: "'Bloodless' rats through the use of artificial blood substitutes." 4. Long, D.M., Liu, M., Szanto, P.S., Alrenga, D.P., Patel, M.M., Rios, M.V., and Nyhus, L.M. Radiology (1972) 105, p. 323-332: "Efficacy and toxicity studies with radiopaque perfluorocar bon." 5. Arambulo, A.S., Liu, Μ., Rosen, A.L., Dobben, G., and Long, D.M. Drug Devel. Commun. (1975) 1, p. 73-87: "Perfluoroctyl bromide emulsions as radiopaque media." 6. Salisbury, B.G., Metzgir, L.F., Altrose, M.D., Stanley, N.N., and Cherniak, N.S. Am. Rev. Respir. Dis. (1974) 109, p. 691: "Effect of fiberoptic bronchoscopy on respiratory performance in patients with chronic obstructive pulmonary disease." 7. Miller, W.C. and Awe, R. Am. Rev. Respir. Dis. (1975) 111, p. 739-741: "Effect of nebulized lidocaine on reactive air ways . " 8. Dobben, G.D., Long, D.M., Szanto, P.S., Mategrano, V.C., and Liu, M. Neuroradiology (1973) (6, p. 17-19: "Experimental studies with radiopaque fluorocarbon in the subarachnoid space." 9. Brahme, F., Sovak, Μ., Powel, Η., and Long, D.M. Acta Radiol. Scan. In Press: "Perfluorocarbon bromides as contrast agents in radiography of the central nervous system."
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
188
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Discussion Q. Dr. A.
How c a n t h e b r o m o p e r f l u o r o c a r b o n s compete w i t h b a r i u m s u l f a t e i n s t u d i e s o f t h e GI t r a c t ? Long P e r f l u o r o c t y l b r o m i d e and t h e o t h e r s c a n n o t compete w i t h BaSO/4 on a c o s t b a s i s . However, f o r s e l e c t e d c a s e s , b a r i u m c a n be d a n g e r o u s . F o r a b o u t 1% o f t h e GI s t u d i e s d o n e , w a t e r s o l u b l e c o n t r a s t agents are used at the present time. These c o n t r a s t a g e n t s a r e n o t w e l l t o l e r a t e d , c a u s i n g d i a r r h e a and i r r i t a t i o n , and d o n ' t g i v e v e r y g o o d x - r a y c o n t r a s t . The fluorocarbon i s vastly superior. The f l u o r o c a r b o n i s s u p e r i o r t o barium i n the s m a l l i n t e s t i n e s a l s o . There are other ways w h e r e t h e b a r i u m i s n ' t as a d e q u a t e as t h e fluorocarbon. F o r r e a s o n s t h a t a r e n o t c l e a r , we g e t g o o d c o a t i n g o f t h e e s o p h a g u s and o t h e part f th stomach i dog with fluoro carbons , but not i a b l e t o g e t good c o a t i n the stomach, so t h a t ' s a l i m i t a t i o n . R a d i o l o g i s t s and s u r geons a r e v e r y e n t h u s i a s t i c about t h e s e f l u o r o c a r b o n s i n the GI t r a c t , a l t h o u g h t h e r e w i l l be an i n c r e a s e d c o s t .
Q. A.
Would y o u comment on t h e C7 compound? We h a v e t r i e d t h e C compound and i t i s v e r y i n t e r e s t i n g . I t i s n o t e l i m i n a t e d r a p i d l y enough f r o m t h e b o d y t o be u s e f u l i n myelography. F o r a l v e o l o g r a p h y , y o u c a n u s e t h e Cg, C7 o r C3 compounds. C5 may be b e t t e r t h a n t h e o t h e r s . For c l i n i c a l s t u d i e s , we h a v e u s e d t h e Ce compound.
Q. A.
What a b o u t i r r i t a t i o n ? T h e r e i s l i t t l e o r no i r r i t a t i o n
Q.
You m e n t i o n e d i n t h e c a s e o f m y e l o g r a p h y how t h e f l u o r o c a r bons are r e t a i n e d f o r a l o n g p e r i o d o f t i m e . Have y o u t r i e d t o r e l a t e t h a t t o Pantopaque r e t e n t i o n ?
A.
Pantopaque i s r e t a i n e d permanently. You d o n ' t remove a l l t h a t agent. The f l u o r o c a r b o n i s more a c c e p t a b l e t h a n P a n t o p a q u e s i n c e i t does not cause a r a c h n o i d i t i s l i k e Pantopaque does. There are water s o l u b l e c o n t r a s t agents t h a t are being used now i n m y e l o g r a p h y , and we a r e n o t s u r e how t h e s e a r e g o i n g t o work o u t . I n some o f t h e e a r l i e r s t u d i e s , t h e y d i d n o t app e a r t o be t o x i c when f i r s t i n j e c t e d . E v e n t h o u g h t h e y comp l e t e l y d i s a p p e a r e d , an a r a c h n o i d i t i s o r c h r o n i c i n f l a m m a t o r y p r o c e s s was i n c i t e d b y t h e i n i t i a l i n j e c t i o n o f t h e w a t e r s o l u b l e c o n t r a s t agents. The n o n - i o n i c w a t e r s o l u b l e c o n t r a s t a g e n t s a r e now b e i n g e v a l u a t e d c l i n i c a l l y t o s e e w h e t h e r t h e y w i l l be more a c c e p t a b l e , and t h e r e s u l t s h a v e b e e n e n c o u r a g ing. The f l u o r o c a r b o n i s v a s t l y s u p e r i o r t o P a n t o p a q u e , t h e m a t e r i a l c u r r e n t l y used i n almost a l l myelography.
7
from t h i s
material.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
9.
LONG
E T
AL.
Brominated
Fluorocarbon
Compounds
189
Q.
I am s u r p r i s e d t h a t y o u do n o t f e e l more f a v o r a b l e a b o u t t h e u s e o f f l u o r o c a ibons i n t h e s p e c i f i c a p p l i c a t i o n o f m y e l o g r a phy.
A.
I was n o t c o m p a r i n g f l u o r o c a r b o n s w i t h P a n t o p a q u e , b u t w i t h the n o n - i o n i c water s o l u b l e c o n t r a s t agent under study. I t h i n k t h i s c o n t r a s t agent i s p r e f e r r e d , b u t i t i s n o t approv e d b y t h e F.D.A. I t i s b e i n g used i n o t h e r c o u n t r i e s , and i t i s u n d e r c l i n i c a l i n v e s t i g a t i o n i n t h e U.S. I t looks l i k e t h e n o n - i o n i c c o n t r a s t a g e n t i s g o i n g t o be a v e r y g o o d a g e n t b u t f l u o r o c a r b o n s w i l l b e a b l e t o compete w i t h i t u n d e r some c i r c u m s t a n c e s , such as i n p a t i e n t s w i t h h y p e r s e n s i t i v i t y t o i o d i n a t e d compounds.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
10 Preparation and Physiological Evaluation of Some New Fluorinated Volatile Anesthetics DONALD D. DENSON, EDWARD T. UYENO, ROBERT L. SIMON, JR., and HOWARD M. PETERS Stanford Research Institute, 333 Ravenswood Ave., Menlo Park, Calif. 94025
Although several adequate fluorinated anesthetics are in clinical use today, all have disadvantages and possible hazards. We are synthesizing and evaluating fluorinated ethers for use as volatile anesthetics. Since these compounds are "inert" gases, they exert their biologic effects without undergoing any chemical transformation during administration, residence in the body, and elimination from the body. It is hoped they will provide the advantages of currently available fluorinated anesthetics but preclude their disadvantages (1). In discussing volatile anesthetics, it is important to understand the difference between analgesia, narcosis, and anesthesia. Analgesia (Stage I) is the loss of pain or numbing of sensory nerves without loss of consciousness. Narcosis (Stage II) is a reversible state of analgesia accompanied by stupor or unconsciousness. Surgical anesthesia (Stage III) is the reversible loss of all modalities of sensation and loss of consciousness. The planes of surgical anesthesia in humans (2) are: Plane 1: Swallowing reflex lost; respiration regular; muscle relaxation minimal Plane 2: Muscle relaxation increased Plane 3: Muscle relaxation further increased and suitable for intraabdominal surgery Plane 4: Skeletal muscle relaxation complete; possible cyanosis; blood and pulse pressure falls; pulse rate increases The level below Plane 4 of surgical anesthesia is respiratory arrest (Stage IV). While planes 1-3 of surgical anesthesia are the most important, some consideration must be given to the 190 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
10.
DENSON
E T
Fluorinated
AL.
dangerous a s p e c t s o f Stage l e v e l s must be c a r e f u l l y without
adverse
Volatile
I I I , Plane
avoided
191
Anesthetics
4, and S t a g e
i f the patient
IV.
These
i s to survive
affects.
Thus the major o b j e c t i v e s
of successful
a n e s t h e s i a (_2) a r e
to: (1)
Alleviate
(2)
Block mentation
(3)
Relax
(4)
Preclude adverse
anxiety
tages
that
than
to alleviate
resulting
from
or afferent
the mental
the fear
effects
fluorinated
ethers
o r motor
nerves.
of surgery or anesthesia.
of the f l u o r i n a t e d
fluorinated
nerves.
a n g u i s h and
of pain.
t h e m u s c l e s by b l o c k i n g e f f e r e n t
Examination suggests
p a i n by b l o c k i n g s e n s o r y
a n e s t h e t i c s i n use today
(as a c l a s s )
hydrocarbons
While
offer
more
advan-
these ethers are
more u n p r e d i c t a b l e i three
important
advantages
b e t w e e n a n a l g e s i a and a n e s t h e s i a , (2) t h e y tive
muscle r e l a x a t i o n ,
tion
of myocardial
and (3) t h e y
tissue
Halogenated
as
potential While
compounds have been u s e d
to
as a n e s t h e t i c agents
about
comprehensive
During
study
t h e 1940s,
of fluorocarbons
anesthetics(3).
any number o f f l u o r i n a t e d
anesthetic
effec-
sensitiza-
i n Use
1847 w i t h t h e d i s c o v e r y o f c h l o r o f o r m .
Robbins r e p o r t e d the f i r s t
less
to epinephrine.
H a z a r d s o f F l u o r i n a t e d A n e s t h e t i c s Now
since
p r o v i d e more
demonstrate
properties,
eight
wide s c a l e
compounds.
compounds have
clinical
potential
use has been
T h e f o u r compounds u s e d
limited
most o f t e n
are :
All
these
ities
that
Halothane
CF CHBrCl
Methoxyflurane
CH 0CF CHC1
Fluroxene
CF CH OCH=CH
Enflurane
CHF 0CF CHC1F
3
3
2
3
2
2
2
compounds a r e a s s o c i a t e d w i t h a c u t e must be c a r e f u l l y
Halothane acid (5).
considered
i s oxidatively NADPH/0
2
2
and c h r o n i c
metabolized
to t r i f l u o r o a c e t i c
2
CF CHBrCl 3
pathways
toxic-
(1, 4 ) .
Hepatocellular
damage
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
192
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
This transformation if
any t o x i c
aberrant
metabolic
implicated
h e p a t o c e l l u l a r damage
i n producing
production
o f few,
capa-
( 1 ) . Halothane i s
an u n p r e d i c t a b l e
a n d i n some c a s e s
rine
harmless with
I t i s q u i t e p o s s i b l e however t h a t
pathways produce r e a c t i v e i n t e r m e d i a t e s
ble of i n f l i c t i n g
(1),
i s probably
intermediates.
postanesthetic
s e n s i t i z e s myocardial
tissue
to
hepatitis epineph-
(6) . Methoxyflurane produces f r e e f l u o r i d e
i o n on metabolism ( 5 ) .
NADPH CH 0CF CHC1 3
2
CH OCF CH OH + C I " 3
2
2
2
\ Urinary
H0CH CC1 H
CH 0 + H0CF CC1 H 2
In f a c t lized, These
2
2
2
s i n c e 60-80% o f a l l absorbed m e t h o x y f l u r a n e relatively
high
serum f l u o r i d e
levels
t o the high output
expected.
renal failure
This nephrotoxicity i s further complicated p h é n o b a r b i t a l may
sensitize
Fluroxene
i s metabolized
humans t o t r i f l u o r a c e t i c CF CH 0CH 3
= CH
2
H i g h serum f l u o r i d e s
·*
2
cell
syndrome ( 4 ) .
by t h e o b s e r v a t i o n
a t h r e e f o l d o r greater extent of
methoxyflurane metabolism t o f l u o r i d e
in
i s metabo-
c a n be
l e v e l s a r e o f t e n h i g h enough t o cause r e n a l t u b u l a r
damage, g i v i n g r i s e
that
+ 2F'
2
ion (4).
i n animals
t o t r i f l u o r o e t h a n o l and
acid (5). CF C0 H + Urinary 3
2
Metabolites
a n d h e p t o c e l l u l a r damage a r e a s s o c i a t e d
its
u s e i n humans.
In a d d i t i o n fluroxene
and
i t i s primarily for this
reason
that
i s extremely i t has been
with
flammable, removed
from c l i n i c a l u s e . Enflurane ion
i s tranformed
CF H0CF CHC1F 2
Although
these
patients
they
normal
-»
2
fluoride reach
Unknown M e t a b o l i t e s
ion levels
a r e r o u t i n e l y low, i n some close t o the
level (2).
exposures t o t r a c e s o f these
be a h a z a r d .
+ F"
80 μ m / l , w h i c h i s d a n g e r o u s l y
fluoride tolerance
Chronic can
t o unknown m e t a b o l i t i e s a n d f l u o r i d e
i n humans ( 5 ) .
According
fluorinated
t o a r e c e n t ASA s t u d y
anesthetics
(7), operating
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
DENSON
10.
room p e r s o n n e l malformation liver
Fluorinated
ET AL.
Volatile
193
Anesthetics
h a v e shown a n i n c r e a s e i n s p o n t a n e o u s a b o r t i o n ,
of children,
cancer
d i s e a s e , and k i d n e y
i n female
anesthesiologists,
disease.
Synthesis of Fluorinated Ethers While
o n l y a few f l u o r i n a t e d
application, of
cyclic
(8) p a t e n t e d
methyl-l,3-dioxolane in
1967.
clinical
A n example o f a c l a s s
diether anesthetics i s 4,5-dihalo-2,2-(bis)trifluoro-
methyl-1,3-dioxolanes,
Gilbert
ethers are i n actual
o t h e r s have shown p r o m i s e .
Terrell
1.
the use o f the parent
2,2-(bis)trifluoro-
1 (X = Y = H) a s a n i n h a l a t i o n a n e s t h e t i c
a n d Moore (9) t h e n
patented
t h e u s e o f 4,5-
dihalosubstituted-2,2-(bis)trifluoromethy1-1,3-dioxolanes
1 in
1973.
~ Gilbert
(8).
While
Terrell
found
h i s m a t e r i a l t o be more p o t e n t
a r e more p o t e n t
than
has l i m i t e d
Our
that the halosubstituted materials
the parent
compound
( 9 ) ; 1,3-dioxolane
anesthetic properties (10).
experience
i n the conversion o f carbonates
formals
l e d us t o i n v e s t i g a t e
equally
as potent,
anesthetics.
halothane
no d e f i n i t i v e A n e s t h e t i c I n d e x d a t a a r e p r o v i d e d by
e t aJL., i t a p p e a r s
itself
than
the p o s s i b i l i t i e s
b u t more s t a b l e ,
Our approach i s based
cyclic
to difluoro-
of preparing
diether inhalation
on t h e f o l l o w i n g r e a c t i o n :
Ο Cat.
Δ Because o f t h e ready
availability
ease o f p r e p a r i n g a host
R-0-CF -0-R 2
of the starting
o f halogenated
analogs,
material, the t h e apparent
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
194
BIOCHEMISTRY INVOLVING
success as
C A R B O N - F L U O R I N E BONDS
of the 4 , 5 - d i h a l o - 2 , 2 - ( b i s ) t r i f l u o r o m e t h y l - 1 , 3 - d i o x o l a n e s
a n e s t h e t i c s ( 9 ) , we
carbonate,
initiated
our i n v e s t i g a t i o n
using
ethylene
2.
2
We
extended
include
the conversion
the p r e p a r a t i o n of
of carbonates
to difluoroformals to
4,5-dihalo-2,2-difluoro-1,3-dioxolanes:
where X and Y a r e h a l o g e n s
Ethylene
carbonate,
2, c a n be c h l o r i n a t e d t o g i v e
3, and 4 , 5 - d i c h l o r o e t h y l e n e
carbonate,
2
3
Monochloroethylene vinylidene
carbonate,
carbonate,
4-chloro,
4.
4
3, c a n be d e h y d r o h a l o g e n a t e d
5, a s shown b e l o w ( 1 1 ) .
CI Ο
+
>=
(C H ) N 2
5
3
*Ο
*0'
3
5
0
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
to
10.
Fluorinated
DENSON E T A L .
Vinylidene
carbonate,
halogenated
Volatile
5, c a n be c o n v e r t e d t o a number o f o t h e r
derivatives
a s shown i n Scheme 1.
8
Compounds
^7,
7
and £
are readily
polyhydrogenfluoride/pyridine (12).
o b t a i n e d by t h e u s e o f
reagent,
Straightforward hydrobromination
affords
compounds 10 and 11.
c a n be p r e p a r e d ethylene The
195
Anesthetics
A similar
a s d e s c r i b e d by O l a h e t a l . o r b r o m i n a t i o n (11) series of derivatives
from t h e d e h y d r o h a l o g e n a t i o n
of 4,5-dichloro-
c a r b o n a t e , 4^. fluorination
much e x c i t i n g
reactions
and u s e f u l
dichloroethylene
of these carbonates
chemistry.
carbonate,
have
The f l u o r i n a t i o n
4, f o r example,
led to
o f 4,5-
gives three products:
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
196
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
14
The
product
lyst/SF
4
results
mixture
ratio. i n the
ratio
An
i s extremely
increase of >
formation of
0.5
as the major p r o d u c t .
1,3-dioxolane,
^4,
suggests
that
t r i f l u o r o - 1 , 3 - d i o x o l a n e , ^3, f o l l o w e d by
r e a c t i o n a s we carbonate,
will
see
4
ratio
from
ΊΛ
i s an
i n the
4-chloro-2,2,5-
a dehydrochlorination addition
important
fluorination
reaction.
finding
in
The
this
of 4-chloroethylene
3.
B e c a u s e we
observed
the monochlorinated totally
formation of
results
halogen
4,5-dichloroethylene carbonate,
The
the c a t a -
The c h l o r i n a t e d - u n s a t u r a t e d
the
a hydrogen f l u o r i d e
unsaturated-1,3-dioxolane,
on
4-chloro-2,2,5-trifluoro-l,3-
d i o x o l a n e , J^3,
reaction
dependent
i n the c a t a l y s t / S F
we
began o u r
d e r i v a t i v e 3 u s i n g an H F / S F
unexpected
result
2,2,5-trifluoro-1,3-dioxolane,
3
exchange i n the 4,
was 13,
4
fluorination
of
investigation
of
ratio
of
0.45.
the formation of 4-chloroas the o n l y i s o l a t e d
product.
13
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
10.
DENSON
For t h i s be
Fluorinated
ET AL.
compound t o form,
occurring.
halogen
There
Volatile
197
Anesthetics
a hydrogen s u b s t i t u t i o n
i s no r e p o r t e d e v i d e n c e
exchange o r a d e h y d r o c h l o r i n a t i o n h y d r o g e n
tion
reaction.
tion
a n d o c c u r s w h e t h e r HF o r T i F
precursor results
This result
carbonate
with either
i n no r e a c t i o n .
with SF
i s independent
of the s t a r t i n g
carbonate,
3.
Heating the
c a r b o n a t e , 3^,
f o r 24 h r r e s u l t s
Hydrogen
addi-
concentra-
4
a t 150°C f o r 24 h r
4-chloroethylene
i n t h e absence o f c a t a l y s t
4
4
must
a direct
fluoride
of cat/SF
i s the catalyst.
4
HF o r T i F
Heating
reaction
f o reither
i n recovery
s u b s t i t u t i o n by S F
4
has
b e e n r e p o r t e d by A p p l e q u i s t a n d S e a r l e ( 1 3 ) . Experiments either
conducted
a t lower
temperatures
resulted i n
no r e a c t i o n o r i n t h e f o r m a t i o n o f 2 , 2 , 5 - t r i f l u o r o - 1 , 3 -
d i o x o l a n e , ^15, a s t h e m a j o r
product.
Cl-,
= 0 + SF
A
Cat./125°C
-0' 15
Hydroquinone
A
similar
ethylene prepare
was o b t a i n e d
2,
t o determine
to biological
i n attempts
The o b j e c t i v e o f t h i s
2,2-difluoro-1,3-dioxolane
dioxolane moiety
result
carbonate,
r a t i o was a g a i n m a i n t a i n e d
of the difluoroformal
In these
b e l o w 0.5.
experiments,
t h e HF/SF
4
T h e f o r m a t i o n o f 2,2,4,5-
t e t r a f l u o r o - 1 , 3 - d i o x o l a n e , J^6, was i n d e p e n d e n t t u r e and c a t a l y s t
was t o
f o r c o m p a r i s o n w i t h 1,3-
the contribution
activity.
to fluorinate experiment
o f both
tempera-
concentration.
16
The
hydrogen
substitution
of 4 - c h l o r o e t h y l e n e
reaction
carbonate,
observed
f o r the fluorination
3, i s t h e p r e d o m i n a n t
this
case.
pare
2,2-difluoro-1,3-dioxolane.
reaction i n
We have been u n s u c c e s s f u l i n a l l o u r a t t e m p t s
investigation
t o determine
This reaction
the f e a s i b i l i t y
t o pre-
i s under
of preparing
further this
compound.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
198
BIOCHEMISTRY INVOLVING
Structure-Activity The
•
Relationships
major s t r u c t u r e - a c t i v i t y
anesthetics
relationships
potency
o f h y d r o c a r b o n s and
i n the
order I >
•
Unsaturation
•
F l u o r i n e a d d i t i o n decreases point
and
Increased an
•
increases
volatile
halogen
potency
ethers
Br >
Cl >
increase
F.
potency.
f l a m m a b i l i t y and
of adjacent •
for
a r e as f o l l o w s :
Halogenation in
C A R B O N - F L U O R I N E BONDS
potency, increases
boiling stability
atoms.
i n a homologous s e r i e s
increase i n molecular
weight,
and
oil/gas
One
o r more h y d r o g e n atoms a r e
follows
boiling
point,
coefficient. necessary
for
CNS
depression. Since
molecules
oxygen are introduce 16.
not one
c o n t a i n i n g only carbon, u s u a l l y very
potent
chlorine into
These experiments are
fluorine,
hydrogen,
a n e s t h e t i c s , we
and
attempted
to
2,2,4,5-tetrafluoro-1,3-dioxolane, summarized
i n the
following
equations
s
16
Photochemical at
chlorination
room t e m p e r a t u r e
results
of
16
tetrafluoro-1,3-dioxolane, maximum y i e l d ther in
of t h i s
chlorination
the
with
one
significant
Photochemical 16,
with
chlorination excess
of
4-chloro-2,2,4,5products.
to date
results
The
i s 53%.
i n an
i n c r e a s e i n the
4-chloro-2,2,4,5-tetrafluoro-l,3-dioxolane,
oxolane,
of
five
obtained
r e a c t i o n mixture no
e q u i v a l e n t of c h l o r i n e
formation
along with
p r o d u c t , ^17,
of the
by-products,
with
i n the
Fur-
increase desired
17.
2,2,4,5-tetrafluoro-1,3-di-
chlorine results
i n the
4,4,5,5,-tetrachloro-2,2-difluoro-1,3-dioxolane,
formation JL8, a s
the
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
of major
Fluorinated
DENSON E T A L .
10.
product be
(>95%).
reported
This
Volatile
reaction
i n a future
199
Anesthetics
i s under
publication.
further
s t u d y and w i l l
We do n o t b e l i e v e
4,4,5,5-tetrachloro-2,2-difluoro-1,3-dioxolane, potent
anesthetic
cule.
Generally
since
t h e r e a r e no h y d r o g e n
one o r more h y d r o g e n s
that
18, w i l l
be a
atoms i n t h e m o l e -
are required
f o r CNS
depression. The that atom. the
structure-activity
relationships
a more p o t e n t member o f t h i s We have a t t e m p t e d
because
weak bond
i n metabolic environments.
determine
how w e l l
structure
reactivity
our c l a s s
reaction
suggest
bromine
potency without
i t i s a thermodynamically However i n a n e f f o r t t o
of d i e t h e r s adheres
r e l a t i o n s h i p s g i v e n above,
f l u o r i n a t e 4,5-dibromoethylene this
contain a
t o maximize a n e s t h e t i c
i n c o r p o r a t i o n o f bromine,
from
p r e s e n t e d above
c l a s s would
carbonate
11
t o the reported we a t t e m p t e d t o
The major
product
i
19.
11
As
19
i n the case o f the f l u o r i n a t i o n
of 4,5-dichloroethylene
carbonate,
3, we have o b s e r v e d h a l o g e n e x c h a n g e ,
conversion
i s independent
been a b l e t o i s o l a t e
of cat/SF
4
but here
concentration.
t h e u n s a t u r a t e d bromine
this
We h a v e n o t
c o n t a i n i n g 2,2-
d i f l u o r o - 1 , 3 - d i o x o l a n e , 20.
20
Experiments with T i F exchange i n t h e absence in
t h e absence
experiments ethylene 6
f
4
4
of catalyst
involving
a t 1 0 0 ° and 150°C
of SF .
Similarly,
resulted
i n no e x c h a n g e .
the f l u o r i n a t i o n
c a r b o n a t e , 11,
resulted
i n no
e x p e r i m e n t s w i t h SF, Additional
o f b o t h 4,5-dibromo-
and 4 - b r o m o - 5 - f l u o r o e t h y l e n e
carbonate,
are i n progress.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
200
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
Physiological The search
potential
a
by
Burgison
flushed with
synthesized
onto the stance
to the
the
of
the
j a r , and Every
after
15
0.5
the
interval
The
the
j a r was
by Ough and
Stone
j a r was
sub-
calculated (15).
compound t h a t w o u l d The
of
syringe
amount o f a t e s t
not
closed quickly
facilitated
by
gentle
F i v e m i c e were q u i c k l y d r o p p e d
but
noted.
no
later
t o become a n e s t h e t i z e d Since
the
t h a n 5 min
occurrence
into
between t h e s e
two
time
(loss
of
i s considered
i n d u c t i o n t i m e f o r known p o t e n t
m i c e were k e p t on
a pan.
the
of
induction to
represent
anesthetic
l i m i t s was
i n the
The
being
removed
every
15
record The
agents,
u s e d as
tail
a
crite-
latent
In the
toxicity
first
performed to determine
each of
the
another dose-range
e a c h compound were e x p r e s s e d
as
The
by
an
point.
hour
each
5 0
and
three
second
stage,
lower
con-
selected
anesthetic
margin of
(AC
of the
in
was
a n i m a l s were
In the
anesthetic
median l e t h a l
range,
t o chose t h r e e
p o t e n c y and
w h i c h 50%
hours
conducted
Fifteen
medium e f f e c t i v e
an
If
after
administered
concentration
median a n e s t h e t i c c o n c e n t r a t i o n concentration
A
end
i n m i c e was
a n i m a l s were t e s t e d a t
anesthetic
by
minutes.
compounds.
conducted
t o determine the
anesthetized.
was
concentrations.
was
concentration
The
the
was
tested
a dose-range experiment
lethal
three
removed
f o r twenty-four
were s e l e c t e d .
Fifteen
estimated
five
as
test
substances
the
study
of the
centrations.
The
pinch
stage,
concentrations
concentration.
every
s u b j e c t s were k e p t
e v a l u a t i o n of the
stages.
at
a n a l g e s i a was
considered
j a r , the
The
then
(to righting)
showed a n a l g e s i c r e s p o n s e a h a l f
from the
any
was
minutes,
recovery
Postanesthetic
the
minutes.
appropriate
j a r f o r 10
time of
base of each a n i m a l ' s t a i l
still
aminal
be
from a
seconds
an
to
ejected the
glass jar
a m e a s u r e d amount
bottl
pressing
tested
s c r e w cap,
and
" a i r " i n the
s u b s t a n c e was
container.
s q u e a k on
two
Initially the
of each t e s t
f o r each animal.
pinching
to
minute,
re-
those
induction.
placed
noted
jar.
f o r each animal
min
optimal
r i o n of
of
r e f l e x ) was
the
and
the
saturate
similar
A wide-mouth,
methods r e p o r t e d
the
time r e q u i r e d righting
to
" a i r " were e v a l u a t e d .
evaporation
rotation
in this
procedures
i n a s y r i n g e was
concentrations
saturate
the
compound
bottom of
according
(14).
agents synthesized
o x y g e n f o r one
t h a t would
Several
and
anesthetic
were t e s t e d i n m i c e by
described was
Evaluation
safety
of
index. )
i s defined
animals are
concentration
(LC
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
as
an
expected 5 0
)
is
10.
DENSON
an
E T
Fluorinated
AL.
Volatile
201
Anesthetics
e s t i m a t e d c o n c e n t r a t i o n by w h i c h 5 0 % o f t h e a n i m a l s a r e
expected to
AC
5 0
to die.
.
safety.
The lower
available
the A C
5 0
,
the lower
of
LC
safer.
5 0
of
the c o n c e n t r a t i o n o f drug
However, a d r u g
i s not n e c e s s a r i l y
methoxyflurane
(AI) i s the r a t i o
the a n e s t h e t i c index t h e g r e a t e r margin
f o r metabolism.
centration
for
The a n e s t h e t i c i n d e x
The l a r g e r
acting
at lower
con-
As a l r e a d y d i s c u s s e d ,
i s nephrotoxic, yet i t s A C
i s about
5 0
half
that
halothane. Before
reviewing are
p r e s e n t i n g our p h y s i o l o g i c a l
some d a t a a v a i l a b l e
summarized
i n Table
1 (2, 8 ) .
nephrotoxicity
data are a v a i l a b l e
1,3-dioxolane,
i t i s interesting
larger
than halothane
fluorine,
hydrogen,
reported
by T e r r e l l
noteworthy
that
(8)
data,
i t i s worthwhile
f o r other anesthetics. While
These
data
no h e p a t o c e l l u l a r o r
for 2,2-(bis)trifluoromethylt o note
that
T h i s molecule
i t s AI i s s l i g h t l y
contains only
carbon
an (9)
halogenate
t h e r e was
very
little
analogs,
increase i n activity.
Table 1 ANESTHETIC POTENCY FOR FLUORINATED
ANESTHETICS
B.P. Anesthetic
(°C)
AC
50
Halothane
0.3
Fluroxene
43
1.2-8
Enflurane
57
2-4
100
0.5
2,2
(Bis)trifluoro-
AI
MAC
5 0
2.74
0.78
105
Methoxyflurane
LC
5 0
0.78
3.50
-
-
0.23 6.0 2.2
-
4.70
2.38
methyl-1,3-dioxolane
We
began o u r p h y s i o l o g i c a l
standard. is
slightly
Table ized
1).
gained rapid
we o b t a i n e d a n A I o f 4.75,
h i g h e r t h a n t h e A I o f 3.50
previously
by p t o s i s
was c h a r a c t e r i z e d
the r i g h t i n g
reflex,
accompanied
by " f l a t
r e c o v e r y t i m e s were o b s e r v e d ,
elimination.
(Rapid e l i m i n a t i o n
lipophilicity.)
At l e t h a l
e a r s were o b s e r v e d
tails."
they maintained
by
i n addition
i s character-
lacrimation.
Once t h e a n i m a l r e it.
indicating
In a d d i t i o n , rapid
halothane
i s often associated with
c o n c e n t r a t i o n s , dark
T a b l e 2 summarizes our f i n d i n g s
t o p t o s i s and
as a which
reported (see
At a n e s t h e t i c concentrations, halothane
during induction
Recovery
e v a l u a t i o n s using halothane
In these experiments
eyes
lower
and b l a n c h e d
lacrimation.
f o r halothane.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
202
BIOCHEMISTRY
INVOLVING
CARBON-FLUORINE
BONDS
Table 2 EFFECTS OF HALOTHANE CF -CHBrCl, ON MICE (AI = 4.75) 3
Cone,
No of
Vol
Animals
%
Time, min 0.78
Deaths 0
5
0.83
0
1.00 1.06
15 15
-
7 12
1.03
0
4.00
15
8
15
7.72
3
5.00
15
13
15
8.13
9
15
8.83
12
15
14
anesthetic concentration 12, minimum h i n d
l e g movement and p t o s i s were
no l a c r i m a t i o n o r c o n v u l s i v e b e h a v i o r .
terized
by t h e a n i m a l s
had
a bouncy g a i t .
for
halothane,
running
Recovery
in circles.
times
elimination
suggests
M i c e were w o b b l y and
Ptosis
than
t h a t , e v e n a t low
a higher
4 , 5 - d i c h l o r o - 2 , 2 - d i f l u o r o - 1 , 3 - d i o x o l a n e , 12.
t r a t i o n s were c h a r a c t e r i z e d by i r r e g u l a r bradycardia.
charac
f o r 1£ were much l o n g e r
i s not n e a r l y as r a p i d
Slower e l i m i n a t i o n
observed,
R e c o v e r y was
a s shown i n T a b l e 3, s u g g e s t i n g
concentrations, halothane. for
Mean Recovery
15
dioxolane, but
Induced
0.94
5.50
At
Induced
before 30 sec before 5 min
as i t i s f o r
lipophilicity L e t h a l concen
respiration
a n d l a c r i m a t i o n were o b s e r v e d
and
apparent
i n a few
animals.
Table 3
EFFECTS OF
:c> |
\ F„ ON MICE
o
/
12
(ΑΙ = 7.88) Cone,
No of
Vol
Animals
%
Induced
Induced
before 30 sec before 5 min
Mean Recovery time, min
0
Deaths
0.25
15
-
0.30
15
2
0.63
0
0.35
15
-
13
10.60
0
2.00
15
-
15
60.08
0
2.50
15
-
15
60.47
6
3.00
15
1
15
120
0
14
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
10.
DENSON
ET
Anesthetic dioxolane, siveness, animals
Fluorinated
AL.
Volatile
concentrations of 4-chloro-2,2,5-trifluoro-1,3-
13, were c h a r a c t e r i z e d by h y p o a c t i v i t y , s l i g h t ptosis,
and o c c a s i o n a l l a c r i m a t i o n .
often ran i n c i r c l e s
Recovery times,
given
and some l o s t
i n Table
t h e AI d e t e r m i n e d
13^, was a p p r o x i m a t e l y
halothane,
recovery
mos)
times
those
reflexes.
reported
(see Table 3 ) .
t h e same a s t h e A I d e t e r m i n e d
were somewhat
the animals'
longer
f o r 13.
e y e s became e x t r e m e l y
and deep j e r k y r e s p i r a t o r y
breathing
righting
half 12
convul-
recovery the
f o r4-chloro-2,2,5-trifluoro-1,3-
dioxolane,
concentrations,
During
their
4, were a b o u t
for 4,5-dichloro-2,2-difluoro-1,3-dioxolane, Although
203
Anesthetics
response
was
At
dark
for
lethal
(exophthal-
f o l l o w e d by
shallow
and b r a d y c a r d i a .
Table
4
0<
E F F E C T S OF
F
13
(AI Cone,
No. o f
Vol
Animals
%
0.88
15
0.94
15
1.00
=
4.66)
Induced before
Induced
30 s e c
before
Mean R e c o v e r y
5 min
time,
min
Deaths
5
1.95
0
-
13
3.07
0
15
-
14
4.13
0
3.00
15
-
15
15.48
0
4.00
20
-
20
17.78
10
5.00
15
5
15
19.72
12
The
preliminary evaluation of
dioxolane, ptosis
During
shivered
recovery,
the animals
f o r a few m i n u t e s .
those
dioxolane, During
2,2,4,5-tetrafluoro-1,3-
i s summarized i n T a b l e
5.
and t a c h y p n e a were a s s o c i a t e d w i t h
tions.
than
1£,
observed
Recovery
for either
Rapid
hind
anesthetic
ran i n c i r c l e s , times
l e g movement concentraand some
were f a r more
1J2, o r 4 - c h l o r o - 2 , 2 , 5 - t r i f l u o r o - 1 , 3 - d i o x o l a n e ,
induction, lethal
1,3-dioxolane
rapid
4,5-dichloro-2,2-difluoro-1,31£.
concentrations of 2,2,4,5-tetrafluoro-
were c h a r a c t e r i z e d by t h e same o b s e r v a t i o n s
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
made f o r
204
BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
the
anesthetic concentrations.
During
l e g s were e x t e n d e d and d r a g g e d , front
recovery,
and a n i m a l s
however,
crawled
with
hind their
legs. Table
5
Ο
F^°v E F F E C T S OF
F
ON
2
MICE
0
16 Cone.,
No. o f
Vol
Animals
%
4.0
10
5.0
5
6.0
5
Mean R e c o v e r y
Induced before
Time,
5 min
Deaths
min
2
0.92
0
5
2.37
0
5.58
10.0 12.0
Statistical The
Analysis
data
obtained
from the a d m i n i s t r a t i o n o f t h e lower
c e n t r a t i o n s of halothane centage of animals
were a n a l y z e d
The p e r c e n t a g e s
c e n t r a t i o n on l o g a r i t h m i c p r o b a b i l i t y
three
were p l o t t e d p a p e r a s shown
of
induced
at each o f the three
13^, were p l o t t e d .
From t h e g r a p h ,
t o t h e method o f L i t c h f i e l d
i n Table
1,3-dioxolane, the
6.
Since A C
5 0
the A C
5 0
o f e a c h compound and W i l c o x o n
was (16)
of 4,5-dichloro-2,2-difluoro-
12, i s s i g n i f i c a n t l y
former i s considered
inducing
1.
of 4-chloro-2,2,5-trifluoro-1,3-dioxolane,
computed a c c o r d i n g shown
con
i n Figure
4,5-dichloro-2,2-difluoro-1,3-
12, and t h e p e r c e n t a g e
lower c o n c e n t r a t i o n s
as
against
t h e p e r c e n t a g e o f mice a n e s t h e t i z e d a t each o f t h e
lower c o n c e n t r a t i o n s
dioxolane,
con
the per
t h a t were a n e s t h e t i z e d a t e a c h o f t h e t h r e e
lower c o n c e n t r a t i o n s .
Similarly,
by c a l c u l a t i n g
lower than
t o be more p o t e n t
than
that
of
halothane,
the l a t t e r i n
anesthesia.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
10.
DENSON E T A L .
Fluorinated
0.2
0.4
VOLUME
PERCENT
2
4
VOLUME
PERCENT
0.6
Volatile
Figure
1
CONCENTRATION
6
10
CONCENTRATION
20
205
Anesthetics
1. Effects of inhalation anesthetics on mice
Figure 2. Effects of high doses of inhahtion anesthetics on mice
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
206
BIOCHEMISTRY
Table
INVOLVING
CARBON-FLUORINE
BONDS
6
ANESTHETIC POTENCY AND
MARGIN OF
STRUCTURE VS.
SAFETY
ACTIVITY
:t> ^0
X
Compound
Y
AC
Halothane
0.99
4.7
4.75
CI
Cl
0.33
2.6
7.88
13
CI
F
0.88
4.1
4.66
16
F
_
_
The
that
results
d i e d at
compound.
of the
a d m i n i s t r a t i o n of
the
LC
Further
pound
computing
three
high
the
high
concentrations
percentage of
concentration
p e r c e n t a g e s were p l o t t e d a g a i n s t 2.
From t h e
three
a n a l y s i s showed t h a t
indicating
i s considerably
derived
t h e AI
of
the
higher
levels
of
each
concentrations
as
margin of
than that
listed
than that
s a f e t y of
of
in
Table
4,5-dichloro-2,2-
i s s u b s t a n t i a l l y higher that
of
animals
concentration-response
o f e a c h compound was
5 0
difluoro-1,3-dioxolane halothane,
by
each of the
The
shown i n F i g u r e
curves, 6.
5 0
12
compounds were a n a l y z e d
as
AI
LC
5 0
the
of
new
com-
halothane.
Summary From t h e trends
o x o l a n e s do present. rine
summary o f o u r
data
in structure-activity not
As
contain
could
i s replaced
be
by
6,
we
can
see
some
A l t h o u g h our d i -
b r o m i n e , good a n e s t h e t i c a c t i v i t y
predicted,
fluorine.
chlorine i s replaced
cients will
be
determined
and
activity
increases
new
c l a s s of
potent
The
structure activity
the
The
d e c r e a s e as
Anesthetic
i n Table
relationships.
activity
lipophilicity
by
fluorine
reported as
the
in a
is
d e c r e a s e s as appears
(partition
chlo-
to coeffi-
later publication).
boiling
point
of
the
series
increases. A oped.
volatile
pounds a p p e a r t o f o l l o w t h o s e compounds i n t h i s
c l a s s are
anesthetics
has
r e l a t i o n s h i p s of t h i s reported
being
i n the
prepared
and
more d e t a i l e d p h y s i o l o g i c a l e x a m i n a t i o n s w i l l
been
devel-
c l a s s of
literature. evaluated, be
and
conducted.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
com-
Other
10.
DENSON
Fluorinated
E T A L .
Volatile
207
Anesthetics
Acknowledgement s The search
authors
gratefully
by I n s t i t u t e s
Division,
acknowledge t h e support
of Health,
under Grant
General
Medical
of this re
Sciences
Number 5-R01-GM-20082-02.
Discussion Q:
How l o n g d i d y o u o b s e r v e
the animals
f o r evidence
of latent
toxicity? A:
In a l l cases
Q:
A r e t h e compounds s t a b l e t o base?
A:
Y e s ! You c a n c o n v e r t carbonate are
Q:
f o r at least
using
t h e d i f l u o r o f o r m a l group
concentrated
also stable t o mild
sulfuric
acid.
back t o t h e
T h e s e compounds
acid.
I s t h e r e any s p l i t t i n
A:
No, a t l e a s t
Q:
The p o s s i b i l i t y on
A:
24 h o u r s .
we hav exists
f o r other
isomers.
C a n y o u comment
that?
We c a n n o t
predict
or d e a l i n g with isomers
w h e t h e r we a r e s t u d y i n g one a c t i v e
unknown m i x t u r e s .
a r e present,
We c a n s a y t h a t
we a r e n o t o b s e r v i n g
an a c u t e
isomer
i f other lethal
effect.
Literature Cited 1. Cascorbi, H. F., "Anesthesia Toxicity," in 1974 Annual Refresher Course Lectures, American Society of Anesthesi ologists Annual Meeting, Washington, D. C., October 12-16, 1974, Lecture number 227 and references cited therein. 2. Larsen, Ε. R., "Fluorine Compounds in Anesthesiology," in Fluorine Chemistry Review, P. Tarrant, Vol. 3, pp. 1-44 (1969) and references cited therein. 3. Robbins, J. H., J. Pharmacol. Exptl. Therap. (1946), 86 197. 4. Brown, B. R., Jr., "Enzymes and Anesthesia," in 1974 Annual Refresher Course Lectures, American Society of Anesthesiolo gists Annual Meeting, Washington, D.C., October 12-16, 1974, Lecture Number 225 and references cited therein. 5. Van Dyke, R. A. and Chenoweth, Μ. Β., Anesthesiology (1965), 26, 348. 6. Tucker, W. K., Rackstein, A. D., and Munson, E. S. Brit. J. Anaesth., (1974) 46, 392. 7. Cohen, E., et al., Anesthesiology (1974), 41, 321. 8. Gilbert, Ε. E., (to Allied Chemical Co.), U.S. Pat. 3,314,850 (1967).
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E
BONDS
9. Terrell, R. C., and Moore, G. L., (to Airco, Inc.), U.S. Pat. 3,749,791 (1973). 10. Virtue, R. W., Proc. Soc. Exptl. Biol. Med., (1950), 73, 259. 11. (a) Newman, M. S., and Addor, R. W., Amer. Chem. Soc. (1953) 75 1263. (b) Newman, M. S., and Addor, R. W., J. Amer. Chem. Soc., (1955), 77, 3789. 12. Olah, G., Nojima, Μ., and Kerekes, I., Synthesis, (1973) 779. 13. Applequist, D. E., and Searle, R., J. Org. Chem. (1964), 29, 987. 14. Burgison, R. Μ., "Animal Techniques for Evaluating Anesthetic Drug," in Animal and Clinical Pharmacologic Techniques in Drug Evaluation, J. H. Nodine and P. E. Siegler, eds., Yearbook Medical Publishers Inc, Chicago, Ill. (1964), pp. 369-372. 15. Ough, C. S., and Stone, , , (1961), , 16. Litchfield, J. T., Jr., and Wilcoxon, F. Α., J. Pharm. Exptl. Ther. (1949), 96, 99.
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
INDEX A AC 200 ACD 121 N-Acetylserotonin 39 iV-Acetyltransferase 39 adrenergenic induction of 41 cyclic A M P induction of 43 pineal 3 induction of 4 N-Acetyltryptamine 3 Acid(s) amino, analogs 37 electrophoresis of fluorocitric 10 erythrofluorocitric 8 fluoroacetic 8 fluorocarboxylic 1-19 fluoro-dicarboxylic 2 fluorourocanic, effects on urocanase 31 inhibitory properties of F-carboxylic 3 monofluorocitric 7 isomers of 9 nicotinic 90,91 perfluoro-octanoic 129 substrate properties of F-carboxylic 3 Aconitase, cytoplasmic 12 Actinomycin 28 Activities of fluorohistidines, antiviral 29 Activity, ornithine decarboxylase 46 Adenosine 3',5'-monophosphate 39 Adipose tissue 91 Adrenalin 41 Adrenergenic agents 41 Adrenergenic induction of N-acetyltransferase 41 AI 201 5-AICAR 32,33 Alditol Ill Aliphatic fluorine 77 Alphamethyldopa 37 Alveolographic studies with R F C 178 Amethopterin 57 Amine drugs 77 Amino acid analogs 37 p-Aminobenzoylglutamate 60 3-Aminomethyl-pyridine 90,91 A M P , cyclic 43 Amphetamine 78 in rat brain 81 A M P induction of N-acetyltransferase, cyclic 43 8 0
Analgesia Analogs, amino acid Anesthesia, surgical Anesthetic(s) concentration, median cyclic diether fluorinated volatile hazards of fluorinated
190 37 190 200 193 190-206 191
3,4-Anhydro-D-galactose 104 Antilipolytic drug 91 Antiviral activities of fluorohistidines 29 A T P synthesis 17
Β Benz[a]anthracene induction 43 Biochemistry of ring-fluorinated imidazoles 23-33 Biological disposition of R F C 181 Bonds, phosphate 38 Brain, amphetamine in rat 81 Brain, /?,/?-difluoroamphetamine in rat 81 Brominated fluorocarbons 171-186 Bronchography with R F C emulsions 180
C F-Carboxylic acids inhibitory properties of 3 substrate properties of 3 Catalysis of M D H 2 Catecholamines 41 CF dUMP 66 Chlorination, photochemical 198 p-Chloroamphetamine 88 Chloroform 191 Cholecystography 186 Chymotrypsin 38 Cis-trans perfluorodecalin emul sions 135-167 Citrate-isocitrate exchange 6 Complexes, Michaelis-Menten 2 Cyanosis 190 Cyclic A M P induction of N-acetyl transferase 43 Cyclic diether anesthetics 193 Cycloheximide 28 N-Cyclopropyl amines 92 Cytochalasin Β 101 Cytoplasmic aconitase 12 3
211 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
212
BIOCHEMISTRY INVOLVING CARBON—FLUORINE BONDS
D Deaminase, liver microsomal 79 Deamination, oxidative 82 Defluorination of fluorocitrate 14 4- Deoxy-D-glucose 104 Deoxyfluoro-D-glucoses 101 Deoxyfluoro-monosaccharides 99-113 Dephosphorylation 38 Dibutyryl adenosine 3',5'-monophosphate 43 Dibutyryl cyclic AMP 43 Dibutyryl adenosine 3',5'-monophosphate 43 Dibutyryl cyclic AMP 43 Diether anesthetics, cyclic 193 Difluoro-oxalacetate 2 0,/?-Difluoro substitution 79-80 β,/^Difluoroamphetanune 83,86 in rat brain 81 Drug, antilipolytic 9 Drugs, amine 7 dUMP 57
Fluoro-dicarboxylic acids 2 Fluorohistidines, antiviral activities of 29 Fluorohistidines, effects on histidine ammonia-lyase 30 2-Fluorohistidine 26 4-Fluorohistidine 30 2-Fluoro-L-histidine 37 Fluoroimidazole(s) 26 ribosides 33 4-Fluoroimidazoles, synthesis of 24 Fluorourocanic acids, effects on urocanase 31 Fluroxene 191,192
G D-.Galactose Gastroenerography GI tract, RFC in the
104 173 175
Gluconeogenesi Glucose-6-phosphatase Gregaria, Schistocerca
38 109
£ E. coli, effect offluorocompounds on 29 Efflux, inhibition of isocitrate 16 Efflux, rates of isocitrate 15 Electrophoresis offluorocitricacid 10 Emulsions bronchography with RFC 180 cis-trans perfluorodecalin 135-167 of RFC 180 Enflurane 191,192 Enzymatic probes 1-19 Enzyme induction 37, 43 Enzymes, histidine in 37 Erythrocyte membrane, human 99 Erythrofluorocitrate 11 Ervthrofluorocitric acid 8 Etners, synthesis of fluorinated 193 Ethylamine 77 Ethylene carbonate 194 Exchange, citrate-isocitrate 6 F FdUMP 59 5- FICAR 32,33 Fluorinated volatile anesthetics 190-206 Fluorine, aliphatic 77 Fluoroacetic acid 8 Fluoro compounds, effect on E. coli 29 Fluorocarbons, brominated 171-186 Fluorocarbons of high vapor pressure 176 Fluorocarboxylic acids 1-19 Fluorocitrate 7 defluorination of 14 Fluorocitric acid, electrophoresis of 10 5-Fluoro-2'-deoxyuridylate 59
H Halothane 191 effects on mice 202 Hazards offluorinatedanesthetics .... 191 Hepatocytes 143 Hepatography 185 Histidine 28 ammonia-lyase, effects of fluoro histidines on 30 in enzymes 37 incorporation into protein 50 Human erythrocyte membrane 99 Human plasma 117-133 HydTOxylindole-O-methyltransferase.
Hyperlipoproteinemia Hyperthermia Hypotensive effect
50
90 83 137
I Imidazole(s) 26 biochemistry ofring-fluorinated..23-33 pharmacology of ring-fluorinated 23-33 Incorporation of ( H) 2-fluoro-Lhistidine into protein 52 Incorporation of histidine into protein 50 Induction of N-acetyltransf erase, adrenergic .. 41 of Ν-acetyltransferase, cyclic AMP 43 benz [ a ] anthracene 43 enzyme 37,43 of pineal N-acetyltransferase 41 steroid 43 Inhibition of isocitrate efflux 16 Inhibition of monoamine oxidase 94 3
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
213
INDEX
Inhibitory properties of F-carboxylic acids 3 Intraabdominal surgery 190 Isocitrate efflux, inhibition of 16 Isocitrate efflux, rates of 15 Isomers of monofluorocitric acid 9 Isoproterenol 41,55
L Lactobacillus casei LC Lethal concentration, median Lipolysis Lipophilicity Liver microsomal deaminase Locusta migratoria Lymphography 5 0
57,72 200 200 90 201 79 109 181
M M D H , catalysis of 2 Median anesthetic concentration 200 Median lethal concentration 200 Melatonin 39 Membrane, human erythrocyte 99 Metabolic probes 1-19 Metabolism, sorbital Ill Methoxyflurane 191,192,201 N-Methyltransferase 79 Mice, halothane effects on 202 Michaelis-Menten complexes 2 Microsomal deaminase, liver 79 Migratoria, Locusta 109 Mitochondrial monoamine oxidase .... 80 Monoamine oxidase, inhibition of 94 Monochloroethylene carbonate 194 Monodeoxy-D-glucoses 99 Monodeoxymonofluoro-D-glucoses 99-100 Monofluorocitric acid 7 isomers of 9 Monofluoro-malate 5 Mosquito 111 Multienzymatic processes 1 Muscle relaxation 190
Ν Narcosis Nicotinic acid Norepinephrine
190 90, 91 83,92 Ο
Organic fluoro compounds Ornithine decarboxylase activity Oxidase, inhibition of monoamine Oxidative deamination
117-133 46 94 82
Ρ Pancreatography Para-chlorophenylalanine Para-fluorophenylalanine Perfluorochemicals Perfluorodecaline emulsions, cis-trans Perfluorohexylbromide Perfluoro-octanoic acid Peritoneal contamination PFC PFD Phagocytosis of R F C Pharmacology of ring-fluorinated imidazoles Phenethylamine C-Phenethylamine Phosphate bonds Phospholipid 14
186 37 37 135 135,147 135-167 185 129 175 135 135 176 23-33 84 86 38 147
Pineal N-acetyltransferase 39 induction of . 41 Pineal gland 39 Polyols HI Potency for fluorinated anesthetics 201 Pressure, fluorocarbons of high vapor 176 Probes, enzymatic 1-19 Probes, metabolic 1-19 Processes, multienzymatic 1 Protein, incorporation of ( H ) 2 fluoro-L-histidine into 52 Protein, incorporation of histidine into 50 Ps. aeruginosa 107 Ps. fluorescens 107 Pseudomonad 107 Ptosis 203 Pyrimidines 67 3
R Radiopaque fluorocarbon 173 Rat brain, amphetamine in 81 Rat brain, /?,^difluoroamphetamine in 81 Rates of isocitrate efflux 15 Relaxation, muscle 190 RFC 173 alveolographic studies with 178 biological disposition of 181 emulsions 180 bronchography with 180 in the GI tract 175 phagocytosis of 176 submersion in 176 Ribavirin 32 Ring-fluorinated imidazoles biochemistry of 23^33 pharmacology of 23-33 RNAse 38
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
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BIOCHEMISTRY
S Schistocerca gregaria 109 Serine 38 Silk worm Ill Sorbitol dehydrogenase Ill Sorbitol metabolism Ill Splenography 185 Steroid induction 43 Submersion in R F C 176 Substrate properties of F-carboxylic acids 3 Surgery, intraabdominal 190 Swallowing reflex 190 Synthesis ATP 17 of fluorinated ethers 193 of 4-fluoroimidazoles 24 Synthetase, thymidylate 57-74
INVOLVING C A R B O N - F L U O R I N E
BONDS
Thymidylate synthetase 57-74 Tissue, adipose 91 TMP 57 Toxicity of 3-deoxy-4-fluoro-D-glucose 109 Triethyl fluorocitrate, N M R of 10 β,β,β-Trifluoroethylamine 77 5-Trifluoromethyl-2'-deoxyuridylate .. 66 Tryptamine 39
U Urocanase, effects of fluorourocanic acids on Urography
31 186
V
Τ Tachypnea Threonine
203 38
Ventriculomyelography Vinylidene carbonate
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
185 195