Chemistry of peptides and proteins: Vol. 3 Proceedings of the Fifth USSR-FRG Symposium on Chemistry of Peptides and Proteins, Odessa, USSR, May 16–20, 1985 9783110858846, 9783110106138

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Chemistry of Peptides and Proteins Volume 3

Chemistry of Peptides and Proteins Volume 3 Proceedings of the Fifth USSR-FRG Symposium on Chemistry of Peptides and Proteins Odessa, USSR, May 16-20,1985 Editors Wolfgang Voelter • Ernst Bayer Yuri A. Ovchinnikov • Vadim T Ivanov

W DE G Walter de Gruyter • Berlin • New York 1986

Editors Wolfgang Voelter, Professor, Dr. rer. nat. Head Department of Physical Biochemistry Institute for Physiological Chemistry Hoppe-Seyler-Straße 1 D-7400 Tübingen Ernst Bayer, Professor, Dr. rer. nat. Director Institute for Organic Chemistry Auf der Morgenstelle 18 D-7400 Tübingen Yuri A. Ovchinnikov, Professor, D. Sc. Director Shemyakin Institute of Bioorganic Chemistry USSR Academy of Sciences Moscow, USSR Vadim T Ivanov, Professor, D. Sc. Shemyakin Institute of Bioorganic Chemistry USSR Academy of Sciences Moscow, USSR

CIP-Kurztitelaufnahme der Deutschen Bibliothek Chemistry of peptides and proteins : proceedings of the . . . USSR FRG Symposium on Chemistry of Peptides and Proteins. - Berlin ; New York : de Gruyter In d. Vorlage a u c h : . . . USSR FRG Symposium on the Chemistry of Peptides and Proteins ISSN 0723-6271 NE: USSR FRG Symposium on the Chemistry of Peptides and Proteins Vol. 3 = 5. Odessa, USSR, May 16 - 20,1985. 1986 ISBN 3-11-010613-2 (Berlin) ISBN 0-89925-193-5 (New York)

Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Lüderitz & Bauer GmbH, Berlin. - Printed in Germany.

PREFACE

The fifth USSR-FRG Symposium on the Chemistry of Peptides and Proteins took place in Odessa in May 1985. This bilateral series of symposia began in 197 6 in Dushanbe and since then has not lost any actuality. On the contrary, Peptide Chemistry and Biochemistry have grown considerably and entered many new fields such as for example Immunology and Biotechnology. This growing interest in peptides requires a solid methodological basis in Peptide Chemistry. Therefore analytical methods and peptide syntheses have played a fundamental role in previous symposia and also at the Odessa meeting. However, increasingly, parts of the symposia have been devoted to biological problems, and, for the first time, immunological topics were discussed in Odessa.

Again, very fruitful discussions and exchange of knowledge long before publication contributed to the success of the Odessa symposium. The excellent local organisation of the symposium in Odessa has to be gratefully acknowledged as well as the support of the USSR Academy of Sciences and the Deutsche Forschungsgemeinschaft.

For the Editors

E. Bayer

CONTENTS

PEPTIDE SYNTHESIS AND ANALYTICAL PROBLEMS OF SYNTHETIC PEPTIDES

New Polymer Supports for Solid-Liquid-Phase Peptide Synthesis E.Bayer, W.Rapp

3

New Supports for Solid-Phase Peptide Synthesis R.P.Evstigneeva, E.I.Filippovich, E.N.Zvonkova

9

New Stratety for Peptide Synthesis Using Highly Lipophilic and Chromophoric Groups. Synthesis of Octapeptide Sequence [24 - 31]H-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-0H of Human ß-Endorphin H.Eckert, Y.Kiesel, C.Seidel, C.Kaulberg, H.Brinkmann

19

Use of the Sulfenyl-Hydrazide Method for the Synthesis of Unsymmetrical Cystine-Peptides S.Romani, L.Moroder, E.Wünsch

29

Application of Crown Ethers in Peptide Synthesis S.Andronati, A.A.Mazurov , T. I. Korotenko

37

Synthesis and Biological Activity of the Sauvagine Fragment 18-40 G.Wendlberger, P.Thamm, E.Wünsch

43

Synthesis and Properties of a New Somatostatin Structural Analog Yu.P.Shvachkin, A.P.Smirnova, N.M.Ermak, V.P.Fedotov, N.V.Sadovnikjva, G.N .Pluzhnikova

49

Immunologically Active Peptides of W.Voelter, H.Echner, H.Kalbacher

55

the Thymus

Synthesis and Biological Activity of Immunoglobulin G Peptide Fragments O.A.Kaurov, N.I.Kolodkin, G.P.Kazakov, B.D.Halaypin..

63

Synthesis of Peptide Fragments of Horse Heart Cytochrome c R.P.Evstigneeva

73

VIII Structure of Blastolysin. Synthesis and Properties of Related Glycopeptides M.V.Bezrukov, 0.S.Reshetova, B.V.Rosynov, Ya.D.Gavrilov, S.A.Kozmin, M.V.Astapova, T.I.Barkova, T.M.Andronova, V.T.Ivanov

85

Enzymatic-Chemical Exchange of Tyrosine (B16) in the B-Cha.in of Insulin H.-G.Gattner, M.Leithäuser

99

Semisynthetic Conversion of the Bovine Trypsin Inhibitor (Kunitz) into an Efficient Leukocyte-Elastase Inhibitor by Specific Valine for Lysine Substitution in the Reactive Site H.R.Wenzel, J.Beckman, A.Mehlich, E.Schnabel, H.Tschesche

105

Unified System for High Speed Analysis of Dns- and PTH Amino Acids I.V.Nazimov, N.B.Levina

119

Conformation of Gramicidin A in Solution and Micelles: Two Dimensional H NMR Study A.S.Arseniev, 1 An Improved M.Baumeister,

I.L.Barsukov, V.F. Bystrov 9 F NMR Racemization Test K.Seeholzer, I.Ugi, B.Westinger

127 159

STRUCTURAL FEATURES OF PEPTIDES AND PROTEINS

Sequence Homology Between Phospholipase and its Inhibitor in Snake Venom. The Primary Structure of the Inhibitor of Vipoxin from the Venom of the Bulgarian Viper (Vipera ammodytes ammodytes, serpentes) I.Mancheva, B.Aleksiev, T.Kleinschmidt, G.Braunitzer...

167

Primary Structure and a Model for Molecular Organization of Adrenocortical Cytochrome P-450 see V.L.Chashchin, S.A.Usanov, V.N.Lapko, T.B.Adamovich, V.M.Shkumatov, A.A.Akhrem

177

Studies on the Structure of Protein Hormones Yu.A.Pankov, V.Yu.Butnev, V.S.Karasev , V.I.Pozdnyakov.

191

IX

Current Problems of Space Structure Description for Oligopeptide Molecules in Solution G.V.Nikiforovich, B.Vesterman, I.Sekacis, J.Betins, L.Podins, B.S.Kataev, J.Balodis

207

Prediction and Synthesis of Some Immunologically Active Fragments of Interferon Molecules G.Chipens, R.Vegners, 0.Papsuevich, N.Ievina, G.Rosenthal, T.Petrova, I.Rituma, G.Afanasyeva, A.Krikis

221

Antigenic Determinants of Proteins and Peptides S .V.Komissarenko

235

Genetic Engineering as a Tool for the Identification of Functional Domains on Proteins: The Major Histocompatibility Complex Antigens B.Arnold, U.Horstmann, S.Kvist, G.J.Hämmerling

249

Protein Chemistry of Halorhodopsin Based on its Cysteine Residue E.S.Schegk, J.Tittor, F.Lottspeich, D.Oesterhelt

259

Structural Investigation of GTP-Binding Protein from Bovine Retinal Rod Outer Segments V.M.Lipkin, T.M.Shuvaeva, K.A.Ishchenko, A.N.Obukhov, I.N. Telezhinskaya, 0. G. Shamborant

273

Effect of Insulin Modification on its Conformation and Enzymatic Hydrolysis G.P.Vlasov, N.G.Illarionova, N.L.Izvarina, I.G.Denisov The Chemistry of Glycopeptide Antibiotics of the Vancomycin Group G. S .Katrukha, A.B.Silaev Structure Elucidation of the Antimycetic Glycolipodepsipeptide Herbicolin A W.A.König, M.Aydin, N.Lucht, G.Winkelmann, R.Lupp, G.Jung

281

289

307

MISCELLANEOUS AND BIOLOGICAL ACTIVITY OF PEPTIDES AND PROTEINS

Suggestion to the Mode of Phalloidin's Molecular Toxicity T.Wieland

323

X Raise of a Monoclonal Antibody Against et-Amanitin and Studies on its Molecular Interaction with Amatoxin Peptides H.Faulstich, K.Kirchner

331

Synthesis and Reactivity of Peptides - Substrates and Inhibitors of Thrombin V.K.Kibirev, A.A.Hershkovich, S.A.Poyarkova, V.P. Romanova, S.B.Serebryany

339

Potent B Lymphocyte Mitogens as Covalently Bound Carriers for the Presentation of Antigens and Enhancement of Immune Response G.Jung, G.Becker, J. Metzger, K.-H.Wiesmüller, W.G. Bessler, L.Biesert, H.-J.Bühring, C.Muller

351

Electron Microscopy Studies of Fast Sodium Channel Structure V.V.Demin, E.V.Grishin, V.A.Kovalenko, S. N.Spadar

363

Further Experimental Evidence for the Flip-Flop Gating Mechanism of Alamethicin Pore Formation G.Jung, K.-P.Voges, G.Becker, W.H.Sawyer, V.Rizzo, G.Schwarz, G.Menestrina, G.Boheim

371

PEPTIDE SYNTHESIS AND ANALYTICAL PROBLEMS OF SYNTHETIC PEPTIDES

NEW POLYMER

SUPPORTS

E. Bayer, W.

FOR SOLID-LIQUID-PHASE

PEPTIDE

SYNTHESIS

Rapp

I n s t i t u t e f o r O r g a n i c C h e m i s t r y , U n i v e r s i t y of Auf d e r M o r g e n s t e l l e 18, 7400 T ü b i n g e n / F R G

Tübingen,

The increasing

in

requires different time.

importance

reliable

peptide

synthesis

synthesis

and often a large number

p e p t i d e s h a v e to b e p r e p a r e d

In p r i n c i p l e

polyoxyethylene

the Liquid Phase

in h o m o g e n e o u s

m e n t s of a u n i f o r m Liquid

of p e p t i d e

Peptide

Synthesis

solution fulfills

is d i f f i c u l t

synthesizers.

is m a i n l y u t i l i z e d b e c a u s e ever, b e s i d e s

in a r e a s o n a b l e

m e n t . D u e to t h e h i g h c o m p r e s s i b i l i t y that the mass

transfer

sequences

of

sult

Therefore

in a considerable

transfer,

require

liquids

in m i c r o p o r o u s

and assumes

improvement

a Gaussian

and consequently

of t h e r a t e

in a d e c r e a s e

like coupling,

the

improvere-

it is

beads

is

known

de-

type

size should limiting

of t h e t i m e

(2)

How-

of t h e p o l y m e r b e a d s

a r e d u c t i o n of p a r t i c l e

f o r all s y n t h e s i s s t e p s ,

used

(3,4)

( 6 0 - 2 0 0 p.) a r e u s e d . H o w e v e r

p e n d a n t u p o n the b e a d d i a m e t e r , distribution.

However,the

it c a n b e e a s i e r s t a n d a r d i z e d .

t h e g e n e r a l p r o b l e m of f a i l u r e

large diameters

on

require-

the Solid Phase Method

polystyrene beads show other disadvantages which latively

(1)

a n d to b e

to s t a n d a r d i z e

Therefore

of

short

these

reaction with high reaction rates.

Phase method

in a u t o m a t i z e d

biochemistry

re-

mass

required

washing procedures

and

cleavage . One approach Peptide

to u n i f y

of S o l i d a n d L i q u i d

S y n t h e s i s w o u l d b e to i m m o b i l z e

a polymer matrix, pected

the a d v a n t a g e s

polyoxyethylene

e.g. cross-linked polystyrene.

that starting

from

a certain

l e n g t h of

v a t i o n of s u c h p o l y s t y r e n e — p o l y o x y e t h y l e n e mers will be d o m i n a t e d by the POE, P O E ch'ain w i l l b e c o m p a r a b l e

(POE)

It c o u l d b e

the P O E c h a i n

(PS—POE)

and m o b i l i t y

to s o l u b l e

Phase

craft

of t h e

POE.

Chemistry of Peptides and Proteins, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany.

to ex-

sol-

copoly-

crafted

4 Relatively

small

methylated

polystyrene

latively polymer with

short

yields

the p r o p e r t i e s

cyclic oxide

of

functionalized

After preparation lene

oxide

increasing

chain

of

the p o t a s s i u m to

such

render

not of

decrease, terminal

PS-POE

and a

the consider-

OH-groups of

beads was

was crafted

rethe

very

POE

polymerization

glycol

polystyrene

is p o l y m e r i z e d

length

polystyrene

1, t e t r a e t h y l e n e

cross-linked

chloro-

to s y n t h e s i z e

dalton were

(5,9). Therefore direct

scheme

to

(5-8). However

attempts

of 5 0 0 - 1 0 . 0 0 0

synthesis dramatically

to s u i t a b l y

methylated

synthesis

the POE reacts w i t h b o t h

ethers

Following

immobilized

u p to 5 0 0 d a l t o n d o n o t y e t

of P O E . O u r

size

because with

of e t h e r

part

have been

by ether

POE chains

POE molecular

successfull able

POE chains

developped

to

beads

(J_) b y e t h e r

s a l t of

the p o l y m e r

forming ethylene chloro-

synthesis . {2)

ethy-

(3).

(2)

n

*

©- CD

4

a) S >-

CD

1

ai S Q a) s n (1) H Pu u u a

4

O SB 3 jmj

3 m 4J o

3 m

~7

3 m

3 m •M o ~7

" 7

3 m •p o

3 m

3 m

«3

o

7

"7

7

7 " o 3 m

7 ' o 3 m •p

X O

\

\

\

" V

ai S

(1)

s

M dl h

a) h

X

(O >-

7

7 1 — 7 1 — 7 1 — 7 1 — 7 u u U u u u u U X o o O O o o o o O m m m m m m m a o u u u EH E-i Eh H

"7

«

S CD

4 u u u u o o o o m m m u u EH H

m

~A

u o m

U DC O m

N

8

~7T o x 3 m 4->

i/u s o n m ai u fa H

"7"

o3

O 3 m 4J

05 •P

X o

X 2 CO C

u o m

Ci

O

- 3 àO ~7l 3 O m m m m •u u 4J u Eh < c

o O m

o a. o u

33

o >-:

" 7 " u o m

V o co

m

Ol

ai u s o m m a; u tu EH

a/u S O n m ai u fa EH

—a eu S ^ ai fa

u o m u eh

~ 7 ai s S M ai fa

ai S IH ai fa

Sa)M

ai fao 04

X in

Ift ~7~

4 u u u X o o o o o m m m m m u u EH EH

27 Secondly for the dipeptide sequence [25 rating both isomers, TCBOC-Asn(FerMe)-OH FerMe, formerly obtained, is simplier on dipeptide derivative than separating the

26] -Asn-Ala-, where sepaand TCBOC-Asp(OH)-NHthe stage of the neutral free acids.

Except coupling of FerMe-Gly-OMe, all coupling reactions are carried out with MEI. Yields vary thereby considerably, the average yield amounts to 66 %. The TCBOC rest as an intermediate protection for a-amino groups has proved best, cleavable quantitatively on all stages without exception. The average yield amounts to 96 %. Purification of intermediate products succeeds excellently by simple and quick column chromatography on low-priced silicagels, by reason of the intermediates' colour coming from the ferrocene chromophores, and the application of very unpolar solvents on the basis of hexane, due to intermediates' high lipophilicity. Capacities of about 4 % of the weight of silicagel and a performance on separating products differing by 0.1 Rp-value results in nearly quantitative isolation of the pure product. Final

acidolysis of all BOC, tBu, and FerMe groups from the

protected octapeptide derivative with TFA and 3-thionaphthol as a scavenger in dichloromethane is achieved in 2 - 4 h, and yields 76 % of the product H-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-OH.

Acknowledgement

This work was supported by Deutsche Forschungsgemeinschaft.

References 1. Wünsch, E.. 1974. In: Houben-Weyl: Methoden der organischen Chemie, vol. 15. Thieme, Stuttgart. 2. Hubbuch, A.. 1979. Kontakte (Merck). 1 979 , 1 4. 3. Büllesbach, E.E.. 1 980. Kontakte (Merck). 1 980 , 23. 4. Weygand, F., W. Steglich, J. Bjarnason, R. Akhtar, N. Chytil. 1968. Chem. Ber. 101, 3623.

5. M a r q u a r d i n g , D . , P. H o f f m a n n , H. H e i t z e r , I. U g i . C h e m . Soc. 92, 1969.

1970. J. A m .

6. E c k e r t , H., M. L i s t l , I. Ugi. 1978. A n g e w . C h e m . 90, A n g e w . C h e m . Int. Ed. E n g l . V7, 361. 7. F e r M e w i l l be s u g g e s t e d as the a b b r e v i a t i o n for methyl.

388.

ferrocenyl-

8. E c k e r t , H., C. S e i d e l , D. M a r q u a r d i n g . P u b l i c a t i o n in p r e p a ration . 9. E c k e r t , H., G. F a b r y , Y. K i e s e l , G. R a u d a s e h l , C. S e i d e l . 1983. A n g e w . C h e m . 9j>, 894. A n g e w . C h e m . Int. Ed. E n g l . T2, 881. A n g e w . C h e m . S u p p l . 1983, 1291. 10. E c k e r t , H., A . S c h i e r . 1979. A n g e w . C h e m . 9J_, 841. A n g e w . Int. Ed. E n g l . J_8< 794. 11. E c k e r t , H., I. Ugi. 1976. A n g e w . C h e m . 88, 717. A n g e w . Int. Ed. E n g l . V5, 681. 12. E c k e r t , H . , I. U g i .

1976. J. O r g a n o m e t . C h e m .

13. E c k e r t , H . , I. Ugi.

1 979. L i e b i g s A n n . C h e m .

14. S c h w a n d t , P., W . R i c h t e r , J.S. M o r l e y . 21 1 .

Chem.

Chem.

118, C 5 9 . 1979,

278.

1981. N e u r o p e p t i d e s J_,

USE OF THE SULFENYL-HYDRAZIDE METHOD FOR THE SYNTHESIS OF UNSYMMETRICAL CYSTINE-PEPTIDES

S. Romani, L. Moroder and E. Wünsch Max-Planck-Institut für Biochemie, Abteilung Peptidchemie, 8033 Martinsried, FRG

Introduction Among the various methods known to produce symmetric and unsymmetric disulfides from thiol compounds, the dialkyl azodicarboxylate procedure

(1,2) seemed the most adequate for an adaptation

to peptide chemistry particularly in view of a selective cysteine pairing to unsymmetrical cystine-peptides. Our model studies clearly indicated that stable and analytically well defined sulfenyl-hydrazides are readily formed upon reacting cysteinecompounds with azodicarboxylic acid derivatives without any side reaction at the level of other amino acid residues, e.g. methionine, tyrosine, histidine and particularly tryptophan

(3-5).

These sulfenyl-hydrazide derivatives react smoothly with a second cysteine component to generate the desired unsymmetrical cystinepeptides or cystine derivatives in high yields. The usefulness of this new procedure in peptide chemistry is demonstrated on selected examples.

Results Synthesis of S-tert-butylthio-cysteine In the last years the S-tert-butylthio-cysteine

(6) has found

widespread application in the synthesis of cysteine- and cystinepeptides because of its high stability under the usual conditions of peptide synthesis and its clean and selective removal via reduction with phosphines

(7). For the preparation of this useful

cysteine-derivative we have elaborated a new method (8) to avoid the use of larger excesses of tert-butylmercaptan as needed in

Chemistry of Peptides and Proteins, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany.

30 the previous procedure

(6,9). The new method proceeds via the

sulfenyl-hydrazide intermediates as shown in fig. 1.

o

CM

o

OJ

W

f4

•

VOVO

m

et"

0 vo VO VfCM • • O 1 O 1 1-3 O *

fc

a a> H Ü

O F4 1 1 O.® H i ! C5P4

r-

CM

*

\—

a>

1

>>

tí

H H O C3 >> 1 H H 1 1 CS !>> O >> P< 1 H 1 » O H i-l Cs) Cl tS3 P FQ C5 C5 "=t-

Ln

VO

V '

tu •H 1 p< H C5

41

References 1. Roeske, R.W., P.D.Gesellchen. 1976. Tetrahedron Lett., U 38, 3369. 2. Chorev, M., Y.S. Klausner. 1976. Chem. Commun., N 15, 596. 3. Klausner, Y.S., M . Chorev. 1977. J. Chem. Soc., Perkin I, N 6, 627. 4. Sasaki, S., M . Shinoya, K. Kogak. 1983. Heterocycles, 20, 124. 5. Rips, R., E. Morier. Patent Gr. Br. 1592552. Int. cl. C 07 C 103/52 6. Sievertsson, H., J.-K. Chang, K. Folkers, C.Y. Bowers. 1972. 8. J. Med. Chem., 7. Voelter, W., H. Kaibacher, E. Pietrzik. 1976. Z. Uaturforsch., 31b, 1015. 8. Vilkas, E., M . Vilkas, J. Sainton. 1980. Int. J. Peptide and Protein Res., 29. 9. Reig, P., J.M. Garcia Anton, G. Valencia, J.J. Garcia Dominique. 1981. Fette, Seifen, Anstrichmittel, 82, 367.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE SAUVAGINE FRAGMENT 18-40

G. Wendlberger, P. Thamm

*

and E. Wünsch

Max-Planck-Institut für Biochemie, Abteilung Peptidchemie, 8033 Martinsried, FRG V. Erspamer Istituto di Farmacologia Medica, Università di Roma, Roma, Italy

Introduction The skin of frogs and toads has been found to represent an inexhaustible source of physiologically important compounds such as biogenic amines and peptide factors (1-2). A straight correlation, both, concerning structure homology and pharmacological activities, was revealed for the skin peptides and their counterparts in mammalian brain and gut,leading to the concept of a skin-gutbrain peptide triangle

(2). In this context a new tetracontapeptide

has recently been isolated by Montecucchi et al. (3-4); it was named sauvagine according to its source,the Phyllomedusa sauvagei (fig. 1). This peptide factor displays characteristic pharmacological actions as intense mucosal vasodilatation, hypotension, stimulation of ACTH and B-endorphin release and potent inhibition of prolactin, somatotropin and tyrotropin release. The observed

Pyr-Gly-Pro -Pro-1 le-Ser-lie-Asp-Leu-Ser-Leu-Glu- Leu-Leu-Arg -Lys-Met-lle-Glu-Ile 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

- Glu-Lys-Gin-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-lle-NH2 21

22

23

24

25

26

27

2 8 29

30

31

32

33

34

35

36

37

38

Fig. 1. Primary structure of sauvagine . 1 * " ' • ' Present address: Diamalt AG, Munich

Chemistry of Peptides and Proteins, Vol. 3 © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany.

39

40

44

biological activities parallel those of the mammalian counterpart, the corticotropin-releasing factor (CRF) isolated subsequently by Vale et al. (5) from ovine hypothalamus. An alignement of the primary structures of sauvagine and CRF indicates a high degree of homology with almost half of the residues indentical and 12 additional amino acids resulting from single point mutations. The presence of two adjoining basic residues Arg-Lys in positions 15-16 in the sauvagine sequence as possible site for ulterior processing of these tetracontapeptide to shorter peptide hormone(s) prompted us to synthetize for structure-function studies in a first approach the sequence portion 18-40 corresponding to the C-terminal fragment resulting from the cyanogen bromide cleavage.

Results The synthesis of the tricosapeptide amide was performed following our general strategy (6) successfully applied in previous syntheses of peptide hormones (e.g. Ref. 7,8): i) a side-chain protection with acid-labile groups derived from tert-butanol and 1-adamantanol in combination with Na-benzyloxycarbonyl or Na-2-nitrophenylsulfenyl derivatives in the intermediate chain-elongation steps. The arginine guanido function is protected by protonation with hydrogen halides to enhance the solubility of both fragments and intermediate seqments via an interchange of hydrophobic and hydrophilic regions. The symmetry of the side-chain protection is additionally disrupted by using various protecting groups (Boc/Adoc) to further enhance the solubility. ii) assembly of shorter fragments in sequence order via dicyclohexylcarbodiimide in the presence of 1,2-dinucleophiles. iii) deprotection under relatively mild conditions, i.e. trifluoroacetic acid treatment. Consequently four suitably protected fragments corresponding to sequences 32-40, 27-31, 22-26 and 18-21 (fig. 2) were prepared by the stepwise active ester procedure using 4-nitrophenyl and N-hydroxysuccinimide esters in the acylation steps, and catalytic

45 Fragment I : H - A s n - A s n - A r g l H B r H e u - L e u - L e u - A s p l O B u t l - T M B u ' H I e - N H j

II:

Fig. 2.

N p s - L y s ( B o c ) - G I n - G I n - A l a - Al a - O H

[27-3l]

III : Nps-Lys(Adoc)-G[n-Glu(OBul)-Lys{Boc)-Glu(OBu')-OH

[22-26]

IV:

[l8-2l]

Boc-lle-Glu(OBu')-lle-Glu(OBu«)-OH

Protected fragments for the synthesis of sauvagine 18-40.

Nps-[27-3Î]-OH • H - [ 3 2 - i O ] - N H 2 DCC/HONSu Nps-[27-i0]-NH2 | HBr/2-Methy I indole Nps-[22-26j-0H

. H-[27-

0 0 pq pq

V

o pq

N pq

•-i pq Q

tSJ.

PH

cd o

«

O O pq

r-\ o.

ä ä i ± 4 ¿ S i

m fi

w