Peptides 1988: Proceedings of the 20th European Peptide Symposium. University of Tübingen, Tübingen, FRG, September 4–9, 1988 9783110884708, 9783110109498

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Peptides 1988

Peptides 1988 Proceedings of the 20th European Peptide Symposium September 4-9,1988 University of Tübingen Tübingen, Federal Republic of Germany Editors Günther Jung • Ernst Bayer

W DE G Walter de Gruyter • Berlin • New York 1989

Editors Günther Jung, Professor, Dr. rer. nat. Ernst Bayer, Professor, Dr. rer nat. Institut für Organische Chemie Universität Tübingen Auf der Morgenstelle 18 D-7400 Tübingen Federal Republic of Germany

Library of Congress Cataloging-in-Publication

Data

European Peptide Symposium (20th : 1988 : University of Tübingen) Peptides 1988 : proceedings of the 20th European Peptide Symposium, University of Tübingen, Tübingen, F R G , September 4 - 9 1988 / editors, Günther Jung, Ernst Bayer. Bibliography: p. Includes index. ISBN 0-89925-594-9 : $20.00 (est.) 1. Peptides-Congresses. I. Jung, Günther. Bayer, Ernst. III. Title. QP552.P4E9 1988 574.19'2456-dc 19

Deutsche Bibliothek Cataloging-in-Publication

Data

Peptides . . . : proceedings of the . . . European Peptide Symposium. - Berlin ; New York : de Gruyter. Auf d. U m s c h l a g : . . . EPS N E : EPS 20.1988. University of Tübingen, Tübingen, F R G , September 4-9,1988 ISBN 3-11-010949-2

Copyright © 1989 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o 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 G m b H , Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Preface The history of the European Peptide Symposia reflects well the recent development of this field of chemistry and biochemistry. Thirty years ago, in 1958, Joseph Rudinger called a small meeting of European peptide chemists in Prague in order to promote the exchange and discussion of ideas and results.

At this time peptide chemistry

seemed to be a very specialized area of organic chemistry and it is remarkable that the increased importance of this research area was foreseen by the group gathered in Prague. We have to admire the courage of this group of chemists in initiating the symposium series, which then became the European Peptide Symposia, and in forming the European Peptide Committee.

In a very informal and, despite this fact,

very efficient manner the European Peptide Committee directed the European Peptide Symposia. It was and is an outstanding example of cooperation between scientists all over Europe. The initial idea of limiting the attendance only to those scientists who have been doing research in peptide chemistry over a long period restricted the attendance to less than 150 participants, always including colleagues from outside of Europe.

The development of peptide chemistry and its increased importance in various fields of biochemistry, biology and medicine has been well documented in the Proceedings of the European Peptide Symposia. The continuous contributions on polymer-supported peptide synthesis since the symposium in 1966, the papers on hormones and releasing hormones, the application of NMR in peptide chemistry starting in 1971, the implementation of HPLC in peptide chemistry reported on at the symposium in 1974, the first papers on peptides in immunochemistry in 1982, as well as the structure-activity relations and conformational studies at numerous symposia, and the great total syntheses, e.g. of insulin and secretin, are milestones in the development of peptide chemistry, to mention only a few.

VI

The number of scientists interested in peptides and attending the European Peptide Symposia has increased steadily. Next to preparative peptide chemistry, aspects of structure, activity and of biology and biochemistry have acquired equal importance. Therefore the European Peptide Committee suggested an increase in the number of participants at the 20th European Peptide Symposium in Tiibingen, as well as the inclusion of all topics of modern peptide chemistry and biochemistry. Even if participation was not completely open because only approximately 750 first circulars were mailed out, approximately 500 attendants were in Tübingen.

The programme committee had a rather difficult task screening the submitted papers. For the first time it was decided not to include any plenary or main lectures because the scientific quality of a large number of the submitted papers were excellent and did not justify to allowing more lecture time to only a few topics. Due to the limited time-schedule quite a number of excellent papers had to be assigned as poster contributions.

It was felt, however, that parallel sessions should not be held.

The

attendance of the lectures in Tübingen was excellent, until the very last lecture on Friday afternoon. We axe grateful to all the scientists who contributed to the scientific success of this meeting. The advice and support of the programme committee during the preparation of the symposium has to be gratefully acknowledged.

We also have to thank the companies and organizations who contributed funds to this symposium. Especially the Bachem Travel Funds provided by Bachem Switzerland, and the funds donated by the Deutscher Akademischer Austauschdienst were of great help in enabling participation of scientists who otherwise could not have attented the meeting. This is especially the case with younger peptide chemists.

It was through the generosity of

Ferring A.G. Pharmaceuticals, Sweden, that the

Joseph Rudinger Memorial Lecture could be financed. Based on the sponsorship of

VII

Bachem

Inc., U.S.A., the Leonidas Zervas Award weis established. Finally, we must

mention the excellent cooperation with the Walter de Gruyter Verlag for the relatively fast preparation of the Proceedings of the 20th European Peptide Symposium.

It was not easy for the local organization committee to deal with the unexpectedly large number of participants, and we certainly apologize for any inconvenience which might have arisen.

However, we hope, that, besides the scientific events, the par-

ticipants appreciated the charm of Tübingen. The organization was not done by a professional company but by the joint effort of our coworkers under the excellent guidance of Mrs.Karin Reichle.

We hope that this meeting has contributed to the advance of peptide chemistry, as is reflected in the Proceedings, and that the opening of the symposium to related disciplines and more participants will continue in forthcoming symposia.

Tübingen, October 1988

Ernst Bayer

Günther Jung

Committees EUROPEAN PEPTIDE COMMITTEE Austria: Belgium: Bulgaria: Czechoslovakia: Denmark: France: FRG: GDR: Greece: Hungary: Israel: Italy: Netherlands: Norway: Poland: Portugal: Spain: Sweden: Switzerland: United Kingdom: USSR:

E. Haslinger A. Loffet B. Aleksiev J . Hlavacek K. Brunfeldt P. Fromageot G. J u n g H . Niedrich D. Theodoropoulos K. Medzihradszky A. P a t c h o r n i k R . Rocchi G.I. Tesser J . Boler G. Kupryszewski M . J . S . A . A m a r a i Trigo E. Giralt U. R a g n a r s s o n C . H . Schneider R.C. Sheppard V . T . Ivanov

ORGANIZING COMMITTEE

PROGRAMME COMMITTEE

E. Bayer G. J u n g K. Reichte

E. Bayer D. B r a n d e n b u r g K.D. Jakubke G. Jung K. Medzihradszky G . T . Young

The 20th European Peptide Symposium was supported Dy Deutsche Forschungsgemeinschaft D e u t s c h e r Akademischer Austauschdienst University of T ü b i n g e n

and t h e following companies Bachem Switzerland

Grünenthal

Bachem Inc., USA

Hewlett-Packard

BASF

Hoechst

Bayer

Hoffmann-La Roche

Behringwerke

Merck

Bissendorf A G

Novabiochem

Boehringer Ingelheim

Orpegen

Boehringer M a n n h e i m

Ortho

Carlbiotech

Reichelt

Ciba-Geigy

Sandoz

Degussa

Schering

Diamalt

Thomae

Ferring

U C B (Bioproducts)

Fluka

Previous European Peptide Symposia 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Czechoslovakia, 1958; Prague. Collect. Czech. Chem. Commun. 24, Special Issue (1959) 1-160. FRG, 1959; Munich. Angew. Chem. 71 (1959) 741-743. Switzerland, 1960; Basel. Chimia 14 (1960) 366-380; 393-418. USSR, 1961; Moscow. Collect. Czech. Chem. Commun. 27 (1962) 2229-2262, also in Zhurnal Mendeleyevskovo Obshchestva 7 (1962). Great Britain, 1962; Oxford. Peptides (G.T. Young, ed.) Pergamon Press, Oxford (1963). Greece, 1963; Athens. Peptides (L. Zervas, ed.) Pergamon Press, Oxford (1966). Hungary, 1964, Budapest. Acta Chimica Academiae Scientarium Hungaricae (V. Bruckner and K. Medzihradszky, eds.) 44 (1965) 1-239. The Netherlands, 1966; Noordwijk. Peptides (H.C. Beyerman, A. van de Linde, W. Maassen van der Brink, eds.) North-Holland, Amsterdam (1967). Ftance, 1968; Orsay. Peptides 1968 (E. Bricas, ed.) North-Holland, Amsterdam (1968). Italy, 1969; Abano Terme. Peptides 1969 (E. Scoffone, ed.) North-Holland, Amsterdam (1971). Austria, 1971; Vienna. Peptides 1971 (H. Nesvadba, ed.) North-Holland, Amsterdam (1973). GDR, 1972; Reinhardsbrunn Castle. Peptides 1972 (H. Hanson and H.D. Jakubke, eds.) North-Holland, Amsterdam (1973). Israel, 1974; Kiryat Anavim. Peptides 1974 (Y. Wolman, ed.) Keter Press, Jerusalem (1975). Belgium, 1976; Wepion. Peptides 1976 (A. LofFet, ed.) Editions de l'Universite de Bruxelles. Poland, 1978; Gdansk. Peptides 1978 (I.Z. Siemion and G. Kupryszewski, eds.) Wroclaw University Press (1979). Denmark, 1980; Helsingor. Peptides 1980 (K. Brunfeldt, ed.) Scriptor, Copenhagen (1981). Czechoslovakia, 1982; Prague. Peptides 1982 (K. Blaha and P. Malon, eds.) Walter de Gruyter, Berlin-New York (1983). Sweden, 1984; Stockholm. Peptides 1984 (U. Ragnarsson, ed.) Almqvist & Wiksell Tryckeri, Uppsala (1984). Greece, 1986; Porto Carras, Chalkidiki. Peptides 1986 (D. Theodoropoulos, ed.) Walter de Gruyter, Berlin-New York (1987).

Contents

In Memoriam Yuri Ovchinnikov Karel Bläha Rolf Geiger

XIII XVII XXI

Awards Leonidas-Zervas Award Josef Rudinger Memorial Lecture

Contents - Scientific Contributions 1.

Methodology of Synthesis

1.1. 1.2. 1.3. 1.4. 1.5. 1.6.

Second Josef Rudinger Memorial Lecture Activation, Coupling, Racemization Protection and Deprotection Purification and Analysis Solid Phase Synthesis Rapid and Parallel Methods, Monitoring of Synthesis Enzymatic Synthesis

2.

Peptides with Unusual and Modified Residues

2.1. Peptidomimetics and Glycosylated Peptides 2.2. Peptide Antibiotics and other Peptidic Agents 2.3. Enzyme Inhibitors

3.

Physical Studies on Peptides

3.1. 3.2. 3.3. 3.4.

Molecular Dynamics, Cyclic Peptides Linear Peptides with Unusual Residues Hormones and Analogues Miscellaneous

XXV XXIX XXXIII

1 13 52 103 133 196 244

289 337 372

426 459 480 507

XII

4.

Hormones, Receptors and Structure-Activity Relationships

4.1. 4.2. 4.3. 4.4. 4.5.

Oxytocin and Vasopressin Bradykinin, Angiotensin, ANF, NPY and Bombesin LHRH, aMSH, GRF and HGH Opoid Peptides and Receptors Miscellaneous Biologically Active Peptides

5.

Immunochemistry

5.1. 5.2. 5.3. 5.4. 5.5.

Viruses Bacteria and Parasites Toxins Immunoregulatory Peptides Miscellaneous

Author Index Subject Index

534 559 592 610 649

676 710 721 733 751 769 779

Yuri Ovchinnikov

(1934 - 1988)

O n F e b r u a r y 17, 1988, a f t e r a t w o y e a r s s t r u g g l e w i t h a d v a n c i n g d i s e a s e a t t h e a g e o f 53 d i e d Y u r i O v c h i n n i k o v ,

the

v i c e p r e s i d e n t of U S S R A c a d e m y of S c i e n c e s a n d d e v o t e d m e m b e r of the international peptide community. He was b o r n on August 2, 1934 i n M o s c o w . H a v i n g c o m p l e t e d t h e p o s t g r a d u a t e

course

a t t h e c h e m i c a l d e p a r t m e n t of L o m o n o s o v M o s c o w S t a t e

Univer-

s i t y h e e n t e r e d in 1960 t h e l a b o r a t o r y o f M . S h e m y a k i n in t h e Institute of Chemistry of Natural Products

(presently

Shemyakin Institute of Bioorganic Chemistry) where he worked until the very last day.

XIV

The scientific heritage of Yu. Ovchinnikov

(over 300 publica-

tions) deals with chemistry of peptides and proteins. At the beginning it was the chemistry of depsipeptides: structures, total syntheses, mechanisms of action, the structure-functional relationship of enniatins, sporidesmolides, esperin, angolide, serratamolide, valinomycin. This was followed by developing mass-spectrometric approaches to the amino acid sequences of peptides. In 1964 Yu. Ovchinnikov spent a year in the laboratory of V. Prelog (Swiss Federal Institute of Technology, Zürich) where he acquired a taste of stereochemical studies of peptides. He was among the pioneers of dynamic conformational studies of peptides in solution by spectral means. The above mentioned depsipeptides as well as the membrane active antibiotics gramicidin S, and gramicidin A, and a large series of linear and cyclic model peptides were the objects of these studies. Topochemical approaches to structure-functional analysis of peptides were also formulated in the work of Yu. Ovchinnikov, e.g. the concept of the similarity of a peptide to its retro-enantio isomer. In more recent years the interests of Yu. Ovchinnikov shifted towards larger protein molecules, such as RNSA-polymerases, ion transporting ATPase, bacteriorhodopsin, sensory rhodopsin and other membrane receptors. Still, with these extremely complicated systems he continued to employ the precise organo- and physico-chemical methods and approaches which he mastered so well with peptides. The story of Ovchinnikov will be incomplete if limited only to scientific achievements. His was a unique personality. Generously gifted by Nature, he had a fantastic capacity for work, he easily made friends with both young and old, transferring his enthusiasm to people surrounding him? he was an outstanding organizer.

XV A t the directory board of a large Institute at the age of 28, academician at 36, Vice President of the USSR Academy of Sciences at 40, he w a s a true leader in chemical and biological sciences. Along w i t h his family and teammates from the Shemyakin Institute many friends of Yuri around the world will grieve at his passing and will always remember him.

Vadim Ivanov

Karel Blaha, 1926 - 1987

Karel Blaha, the representative of Czechoslovakia in the European Peptide Committee and a member of the Progranme Committee, died suddenly on August 28, 1987. Even now, a year later, this seems hard to believe. Peptide chemistry has lost one of the most distinguished scientists, a man who has greatly contributed

to both the scientific research and

international

relations between peptide chemists. Karel Blaha was born on July 29, 1926 near Pilsen in West Bohemia. In 1949, after graduating in chemistry at the Technical University in Prague, he joined the laboratory of Heterocyclic Compounds led by one of the founders

of

modern

Czech

organic

chemistry,

Professor

Rudolf

Lukes.

Working in this prestigious laboratory for eleven years, Karel developed a deep knowledge of organic synthesis together with a fine feeling for organic chemistry systematics and spatial arrangement of molecules. In combination, these capabilities distinguished his whole scientific career. In 1960 Karel came to the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences, and started his long, highly motivated

and successful

research in the peptide chemistry field. He

worked first as a coworker and later as the successor of Professor Joseph Rudinger, the inspirer of European Peptide Symposia and spiritus agens of

XVIII

the international oonmunity of peptide chemists. In the Institute Karel gradually became one of the leading scientists and, in 1982, was appointed Head of the Department of Organic Chemistry. Karel's major contributions to both general organic chemistry and peptide chemistry are difficult to surrmarize. He left an imposing number of more than 200 scientific papers, over 20 review articles and six books. His first works deal with reactions of Grignard reagents, syntheses and configuration determinations of nitrogen-containing compounds, especially aminoalcohols, and alkaloid chemistry. Fran this period let us mention at least the monumental volume 'The Reactions of Organometallic Reagents', a part of the series 'Preparative Reactions in Organic Chemistry' (Prague 1961).

In

the

peptide

chemistry

field he was one of the first who

recognized the importance of interdisciplinary approaches to problems of structure and function of the ccmponents of living matter. He concentrated on conformational studies of basic constituent units of peptides in the first place on the amide group itself. Karel invented special - tailor made model lactams in which the amide group conformation is restricted by the rigid polycyclic skeleton. In his own words, it was necessary to find out the situations in which the effects under study were minimized or maximized. Spectroscopic investigation of these models resulted in his famous concept of the non-planar amide group acting as an

inherently

chiral chrcmophore. Further conformational studies included derivatives of noncoded amino acids, 2,5-piperazinediones, cyclohexapeptides, and basic sequential polypeptides simulating interactions within more complicated systems like DNA-histones or photosynthetic complexes. Although a wide range

of

chiroptical

physicochemical methods

methods

deserve

a

have

special

been used

for

these

comment. Karel Bláha

studies, supported

circular dichroism spectroscopy very much and gradually built a highly reputed laboratory in his group. For him, stereochemistry and symmetry considerations were truly lifetime hobbies. The tiny book on this subject 'Fundamentals of Stereochemistry and Conformational Analysis', which he wrote together with 0. Cervinka and J. Kovar, became widely popular and has subsequently been translated into English and Russian. Karel has also seriously

contributed

to

the

development

of

organic

chemistry

nomenclature, both on the domestic and the international scene as Chairman of the Czechoslovak Nomenclature Commission and a Vice-chairman of the

IXX I.U.P.A.C. Ncmenclature Commission. We cannot emit here his long standing endeavour to upkeep and raise the prestige of the world-wide circulating journal Collection of Czechoslovak Chemical Communications whose Editorin-Chief he was for 25 years. The same high merit distinguished his work for the Czechoslovak Chemical Society, where he acted as Vice-chairman. Since Oxford 1962 Karel regularly attended European Peptide Symposia and as a member of both the Programme Committee and the European Peptide Ccnmittee he devoted much of his time to the organization of these meetings. The 17th Symposium in Prague (1982) was organized under his chairmanship and he was also a coeditor of the Proceedings volume. Karel was a very good lecturer whose talks always brought a lot of new ideas and were a pleasure for the audience. He educated many younger colleagues who afterwards reached high posts in research and the pharmaco* chemical industry. For his merits in chemistry Karel Blaha received many i

awards. Let us just mention here the last one, the J.Heyrovsky Medal of the Czechoslovak Academy of Sciences. Besides science, Karel Blaha had a broad scope of other interests, especially painting, architecture, and history. Here he also expressed his sense of thinking in three dimensions. His knowledge of old Prague was outstanding. There was hardly a foreigner visiting the Prague group in the past 20 years who did not experience a walk in his company through the Old Town learning historical details about almost every building around. For those of us who were lucky to work with him, Karel Blaha was a man always radiating energy, a never ceasing source of inspiration, a sharp critique of any errors and mistakes made, a superb organizer, and a never failing friend. In summary, he was the best boss one could possibly imagine. His premature death leaves a big gap. (The Prague Peptide Chemistry Group)

ROLF GEIGER (1923 - 1988)

Rolf Geiger was born on July 22, 1923 in Bodmann at Lake Constance (West Germany). He had the cheerful and selfpossessed temper of a "homo bodensiensis". His maxim was "live and let live". He was head of the HOECHST peptide group for 19 years and led it in the field of peptide chemistry to an institution which found recognition world-wide. From 1931- 1942 Rolf Geiger visited schools in Güttingen and Überlingen. After the second world war he started to study chemistry at the University of Tubingen in 1949. Under the

XXII

supervison of Professor Friedrich Weygand he finished his doctoral thesis entitled "Synthesis and Cleavage of Ntrifluoro-acetylated Peptides" at the Technical University in Berlin in 1956. Thereafter he worked as a scientific assistant in the laboratories of Professor Friedrich Weygand, who remained for the rest of his life in close contact with him. In the laboratories of Pharma-Synthese at HOECHST AG in Frankfurt (Main) he started in May 1957 with peptide chemistry and became the leader of the peptide group in 1969. Besides his work at HOECHST AG, he lectured at the University of Frankfurt and was named honorary professor in 1979. Only a few weeks before his death he was honoured by the Deutschen Chemischen Gesellschaft with the Emil Fischer-Medal, which was dedicated after the second world war for the first time to a chemist working in industry. Besides methodical work on peptide synthesis he especially focussed his interest on ACTH, insulin and hypothalamic releasing hormones. 1964 he published the synthesis of ACTH-(1-23)-amide and 1971 a shorter 17 amino acid containing ACTH-peptide with prolonged ACTH-activity, which was later called alsactide. Alsactide was developed as a drug and became a sales-product in Italy. Unfortunately the suitability of alsactide as an interesting new tool for various therapies has not been recognized by other clinical departments. Another even shorter ACTH-peptide, an analogue of ACTH-(4-9), with the generic name ebiratide, was published 1986 and is currently under development. Ebiratide increases learning and memory and improves social behaviour. The elucidation of the structure of pro-insulin by D.F. Steiner (1968) was the signal for Rolf Geiger to shift his main interest to insulin. 1969 he published the synthesis of

XXIII porcine-proinsulin-(31-63)

and 1973 the C-peptide of human

proinsulin, which w a s used to develop a commercially

available

RIA for human insulin C-peptide. Intensive semisynthetic

work

on insulin, which included the coupling of insulin A- and Bchain by new developed reversible bridging compounds, the specific stepwise shortening of insulin A- and B-chains and the synthesis of new insulin substitution analogues, development of des-Phe(Bl)-insulins

led to the

("Insulin defalan").

These

insulins with improved characteristics were only for a short period on the market. They were substituted by

semisynthetic

human insulin which was the result of a collaboration

with

Rainer Obermeier at HOECHST AG, the so called last student of Professor Friedrich

Weygand.

Another field of activity

started when the structure of the

hypothalamic releasing hormones were elucidated. The peptide group synthesized TRH, LH-RH and somatostatin. LH-RH and TRH were developed as diagnostics

("Relefact LH-RH,

TRH"). LH-RH is also used to treat

Relefact

cryptorchidism

("Kryptocur"). Buserelin, a highly active LH-RH agonist, which was synthesized

in collaboration with Wolfgang König

(also a

student of Professor Friedrich Weygand), was developed as a drug, which suppresses gonadotropins and gonadal sex hormones (testosteron and estrogen) by down regulation of LH-RHreceptors. It is used for the treatment of prostate ("Suprefact")

and endometriosis

cancer

("Suprecur").

Besides these three main projects and multiple small projects, Rolf Geiger developed also considerable activities in the gastrointestinal

field, on thymus hormones and on the

renin/angiotensin

system. In collaboration with Hans Wissmann,

he found a very active competitive angiotensin antagonist the gastrin-heptapeptide

desglugastrin. Secretin, which was

synthesized by Wolfgang König and Georg Jäger with the

support

of the whole peptide group, is now on the market as a diagnostic tool

and

("Sekretolin"). Furthermore Rolf

Geiger's

XXIV

coworkers succeeded in synthesizing the highly active enkephalin agonist ociltide and the angiotensin converting enzyme antagonist ramipril (Volker Teetz), in the development of novel suicide inhibitors of prolylhydroxylase (Stephan Henke) and in the synthesis of ANF- and somatoliberin analogues with promising properties (Jochen Knolle, Gerhard Breipohl). The methodical work of the peptide group under Rolf Geiger includes the development of protecting groups for amino-, imino-, hydroxy-, thio- (Georg Jäger, Wolfgang König) and amido-functions (Wolfgang König, Gerhard Breipohl) as well as the elaboration of new methods for peptide coupling (Wolfgang König, Hans Wissmann). The solid phase method, which was already studied by Hans Wissmann about twenty years ago, was recently reactivated with new techniques by Gerhard Breipohl and Jochen Knolle. The most important contribution in the methodical field was the introduction of 1-hydroxybenzotriazole as an additive to the dicyclohexylcarbodiimide mediated coupling reaction. It was published with Wolfgang König (Chem. Ber. 103 (1970) 788-798) and became a Citation Classic in 1982. The long list of outstanding results, which is documented in about 130 publications, might give an impression of the stimulating atmosphere created by Rolf Geiger, who was not only an excellent peptide chemist with a high insight into physiology, but also an expert in modern art and far east art. Besides his expertise, Rolf Geiger's modesty and kindness have won him many friends all over the world.

Wolfgang König HOECHST AG, Pharma Synthese 6230 Frankfurt am Main

THE LEONIDAS ZERVAS AWARD Leonidas Zervas was among the pioneering personalities of the European Peptide Symposia for many years. A summary of his outstanding contributions to natural product chemistry is found in the Proceedings of the Helsingor Symposium 1980 written by the late Iphigenia Photaki. Here only the most wellknown of his many important inventions in peptide chemistry is mentioned, the introduction of the benzyloxycarbonyl protecting group, which led to modern strategies and tactics in peptide chemistry. The first useful selectively removable urethane protecting group, which was abbreviated as the Z group, in a happy connection to the name of its inventor Zervas constituted a breakthrough in 1932. Bergmann, Zervas and coworkers applied the Z group to the synthesis of glutathione, and Du Vigneaud used it for the first synthesis of a hormone, oxcytocin, in 1953. The Leonidas-Zervas-Award

is sponsored by Bachem Inc. U.S.A.,

which is represented by Bissendorf-Biochemicals in central Europe. Our proposal to name this award in honour of Leonidas Zervas found agreement among the European Peptide Committee and the dominating opinion was to give this valuable award to a dedicated younger peptide chemist, who shows distinguished promise. On occasion of the 20th European Peptide Symposium an outstanding young scientist was proposed and elected for the First Leonidas

Zervas

award:

Dr. Alex Eberle from Zürich, Switzerland Alex Eberle finished his study of chemistry, biochemistry and molecular biology at the ETH Zürich and received his doctoral degree in 1976. The supervisor of his doctorate was Robert Schwyzer, and Alex Eberle received a prize for his thesis in peptide chemistry and biology. In 1980 - 81 he gained ex-

XXVI

perience as a scientific fellow in Cambridge in the laboratories of Walker, Sanger, Milstein and Sheppard.

From 1982 - 86 he built up at the Kantons Spital in Basel a laboratory for basic research in endocrinology with particular emphasis on peptide and protein chemistry. At present he is Dozent at the Medical Faculty of Basel and leader of a research group. One of the major topics of Alex Eberle's research is - and has always been - the field of the melanotropins

and related

peptides where he made numerous contributions not only to their synthesis but also to their biological testing and determination in biological fluids. He developed several new bioassays and binding assays as well as very sensitive and

XXVII

elegant radioimmuno-assays, whose principle he then applied t o t h e m e a s u r e m e n t of o t h e r h o r m o n e s s u c h as to a n u l t r a s e n sitive s a n d w i c h - t y p e of immunoassay for h u m a n g r o w t h hormone, now u s e d in t h e clinic. H e c a r r i e d o u t a v e r y e x t e n s i v e structure-activity

study

MSH, in w h i c h o n t h e one h a n d h e o b t a i n e d v a l u a b l e

informa-

t i o n a b o u t hormone-receptor

and on the

recognition

mechanisms

into

o t h e r h a n d h e l o c a t e d t h e p o s s i b l e sites in t h e M S H m o l e c u l e for introducing l a b e l e d groups. H e p r e p a r e d M S H p e p t i d e s c o n t a i n i n g v a r i o u s m a r k e r g r o u p s and a f f i n i t y or p h o t o a f f i n i t y labels. U s i n g a new t y p e of t r i t i a t i o n set-up a n d p r o p a r g y l - g l y c i n e s in t h e p e p t i d e sequence, he o b t a i n e d v e r y highly

tritiated

photolabels

linear

and cyclic

peptides. Applying MSH

to intact c e l l u l a r systems, h e d i s c o v e r e d

long-

lasting r e c e p t o r s t i m u l a t i o n a f t e r f o r m a t i o n of t h e c o v a l e n t h o r m o n e - r e c e p t o r complex. T h i s led to t h e c o n c e p t of sible

agonism,

irrever-

w h i c h also found its a p p l i c a t i o n in many o t h e r

h o r m o n a l systems. Recently, the u s e of a v e r y p o t e n t M S H p h o t o l a b e l led to t h e i d e n t i f i c a t i o n of t h e M S H receptor, p r o b a b l y t h e first amongst the r e c e p t o r s of POMC p e p t i d e s . A l t h o u g h A l e x Eberle h a s d e v e l o p e d m a n y a c t i v i t i e s

ranging

from p e p t i d e s y n t h e s i s and b i o l o g y to t i s s u e culture,

physio-

logical studies, m o n o c l o n a l antibody p r o d u c t i o n a n d DNA s e quencing, h i s m a j o r interest are peptides, in p a r t i c u l a r t h e i r a p p l i c a t i o n to t h e d i a g n o s i s a n d p o s s i b l y t h e r a p y of diseases. Finally it s h o u l d b e m e n t i o n e d t h a t A l e x E b e r l e p r e p a r e d a v o l u m i n o u s o u t s t a n d i n g m o n o g r a p h , The Melanotropins

(Karger,

Basel 1988), w h i c h is a very v a l u a b l e source for all t h o s e w o r k i n g in t h e field of h o r m o n e s and t h e i r

receptors.

Günther Jung

THE JOSEF RUDINGER MEMORIAL LECTURE:

AN INTRODUCTORY NOTE

The "Joseph Rudinger Memorial Lecture " was installed through the generosity of Ferring A.B., Malmoe, Sweden, commemorating the contributions of Prof. Joseph Rudinger to the advancement of peptide chemistry. The Memorial Lecture should be awarded to an outstanding peptide chemist. It was awarded for the first time at the 19th European Peptide Symposium in Porto Carras, Greece, and Prof. Young has written a detailed history of the Joseph Rudinger Memorial Lecture in the Proceedings of the 19th European Peptide Symposium.

XXX The second "Joseph Rudinger Memorial Lecture" was w i t h overwhelming majority by the European Peptide

awarded

Committee

o n t h e o c c a s i o n of t h e 2 0 t h E u r o p e a n P e p t i d e S y m p o s i u m t o P r o f . Dr. E r i c h W ü n s c h , D i r e c t o r o f t h e D e p a r t m e n t of

Peptide

Chemistry, M a x - Planck-Institut für Biochemie, Munich

in

r e c o g n i t i o n of h i s o u t s t a n d i n g c o n t r i b u t i o n s in t h e f i e l d of synthesis of biologically active

peptides.

P r o f . E r i c h W ü n s c h w a s b o r n o n M a r c h 17, 1923

in

R e i c h e n b e r g . H e s t a r t e d h i s s t u d i e s of c h e m i s t r y 1 9 4 1 - 1 9 4 2 t h e C h a r l e s U n i v e r s i t y in P r a g u e a n d r e s u m e d h i s s t u d i e s

at

1951

a t t h e U n i v e r s i t y of R e g e n s b u r g . A f t e r r e c e i v i n g t h e g r a d e of a D i p l o m c h e m i k e r 1951 h e w o r k e d for h i s d o c t o r a l t h e s i s a t t h e M a x - P l a n c k - I n s t i t u t für E i w e i ß - u n d L e d e r f o r s c h u n g r e c e i v e d 1956 h i s Dr. rer. n a t . . B e g i n n i n g f r o m 1960 W ü n s c h w a s t h e l e a d e r of t h e D e p a r t m e n t o f P e p t i d e

and

Prof.

Chemistry

a t t h e M a x - P l a n c k - I n s t i t u t für E i w e i ß - u n d L e d e r f o r s c h u n g

in

M u n i c h . H e r e c e i v e d 1963 t h e v e n i a l e g e n d i f o r O r g a n i c C h e m i s t r y a t t h e T e c h n i c a l U n i v e r s i t y of M u n i c h a n d 1973

the

t i t l e of a p r o f e s s o r . I n t h e s a m e y e a r h e w a s a p p o i n t e d as director of the Department of Peptide Chemistry at the M a x P l a n c k - I n s t i t u t für B i o c h e m i e in M a r t i n s r i e d a n d m e m b e r of the

Max-Planck-Gesellschaft. Prof. Erich Wünsch received many honors and awards,

among

t h e m t h e d o c t o r h o n o r i s c a u s a in m e d i c i n e of t h e U n i v e r s i t y of Erlangen and the State Award of the Bavarian Academy S c i e n c e s . H e h a s a u t h o r e d m o r e t h a n 410 p u b l i c a t i o n s . o u t s t a n d i n g c o n t r i b u t i o n s to e x p e r i m e n t a l p e p t i d e

of

Another

chemistry

is t h e e x c e l l e n t r e v i e w o n p e p t i d e s y n t h e s i s in t h e s e r i e s of Houben-Weyl-Müller, Methoden der Organischen Chemie, (Vo. 15/1 a n d II).

1974

XXXI

His contributions have lead to highlights in synthetic peptide chemistry, like the total syntheses of glucacon, secretin, somatostatin by the solution method. He was always interested on structure-activity relation and has made significant contributions in strategies for peptide synthesis including the device of protecting groups and coupling methods. An excellent review of his work will be found in his Joseph Rudinger Lecture.

Ernst Bayer

Scientific Contributions 1. M E T H O D O L O G Y O F S Y N T H E S I S SECOND J O S E F RUDINGER MEMORIAL LECTURE: KAISER-WILHELM-/MAX-PLANCK-GESELLSCHAFT. RESEARCH IN P E P T I D E CHEMISTRY FROM 1921 T O 1991? E. Wünsch

1

1.1. Activation, Coupling, R a c e m i z a t i o n

13

FACILITATION OF OXAZOLINONE FORMATION BY BULKY AMINO ACID SIDE CHAINS A.M.Freitas and H.L.S.Maia

13

APPLICATION OF NEW CARBODIIMIDES TO P E P T I D E SYNTHESIS J.Izdebski and A.Orlowska

16

DIACYLAMINES AS ACYLATING AGENTS IN P E P T I D E CHEMISTRY W. Gruszecki, M.Gruszecka, and H.Bradaczek

19

A NEW WAY T O NON-RACEMIZING SEGMENT CONDENSATION R.Jacquier, V.Pévere, and J.Verducci

22

SYNTHESIS OF DNA-BINDING PROTEIN II (HBs) BY THE USE OF P R O T E C T E D - P E P T I D E S-ALKYL THIOESTERS S.Aimoto, C.Maegawa, S.Yoshimura, and H.Hojo

25

STUDIES ON RACEMIZATION USING FMOC AMINO ACID CHLORIDES IN P E P T I D E SYNTHESIS M.Beyermann, D.Granitza, M.Bienert, M.Haussner, and L.A. Carpino

28

DEVELOPMENT OF EFFICIENT FMOC SYNTHETIC METHODS AND THEIR COMPARISON WITH BOC STRATEGIES D.Chaturvedi, J.Ormberg, and H.Wolfe

31

THE USE OF PHOSPHINYL CHLORIDES FOR CARBOXYL ACTIVATION AND N a-AMINO PROTECTION IN P E P T I D E SYNTHESIS C.Poulos and R.Ramage 34 NEW COUPLING REAGENTS IN P E P T I D E CHEMISTRY R.Knorr, A.Trzeciak, W.Bannwarth, and D. Gillessen

37

XXXIV RP-HPLC DETECTION OF EPIMERIZATION DURING THE SYNTHESIS AND HYDROLYSIS OF AMINOSUCCINYL PEPTIDES Zs.Va.dasz, J.Seprödi, J.Erchegyi, T.Teplan, and I.Schön

40

KINETICS OF THE REACTIONS OF 4-ISOBUTYL-5(4H)-OXAZOLONE IN THE PRESENCE OF TRIETHYLAMINE M.Slebioda, A.M.Kolodziejczyk, N.L.Benoiton, and F.M.F.Chen 43 SOME APPARENT ANOMALIES IN THE TEMPERATURE DEPENDENCE OF RACEMIZATION OF ACTIVATED N-ACYLAMINO ACIDS N.L.Benoiton, Y.Lee, F.M.F.Chen 46 EPIMERIZATION OF AN UNACTIVATED C0 2 H-TERMINAL ASPARTYL RESIDUE IN THE PROTECTED FORM OF RGH 0205 I.Schön, L.Kisfaludy, O.Nyéki, B.Herényi and S.Görög

49

1.2. Protection and Deprotection

52

3-NITRO-2-PYRIDINESULFENYL (Npys): A VERSATILE PROTECTING GROUP IN PEPTIDE SYNTHESIS O.Rosen, S.Rubinraui and M.Fridkin

52

DEPROTECTION PROCEDURES IN PEPTIDE SYNTHESIS. A "REDUCTIVE ACIDOLYTIC" DEPROTECTION METHOD AND FLUORIDE ION DEPROTECTION METHOD Y.Kiso, M.Yoshida, T. Kimura, M.Shimokura, and T.Mimoto

55

SYNTHETIC STUDIES ON CYSTINE-CONTAINING PEPTIDES N.Fujii, A.Otaka, A.Okamachi, T.Watanabe, H.Arai, H.TamamuTa, S.Funakoshi, and H. Yajima

58

CLEAVAGE OF THE DISULFIDE BONDS IN PEPTIDES BY CATALYTIC HYDROGENATION USING POLYMER BOUND METALLIC CATALYSTS E.Bayer, Z.Chen, W.Schumann, K.Reichle, and B.Hemmasi

61

SYNTHESIS OF ß-\ AND /3-2-ADAMANTYLASPARTATES AND THEIR APPLICATION TO THE PEPTIDE SYNTHESIS IN SOLID PHASE AND CONVENTIONAL SOLUTION METHODS Y.Okada and S.Iguchi

64

XXXV AN IMPROVED METHOD FOR THE PREPARATION OF LARGE AMOUNTS OF w-CYCLOHEXYLESTERS OF ASPARTIC AND GLUTAMIC ACID B.Penke and G.K.Tóth

67

THE Mpc-GROUP: A NEW BASE-LABILE AMINO PROTECTIVE GROUP W.J.G.Schielen, E.C.A.C.van de Ree, and G.I.Tesser 70 SYNTHESIS OF A SOMATOSTATIN ANALOGUE WITH THE ACID LABILE t-BUMEOC AMINO PROTECTING GROUP E. Jungfleisch, H.Kaibacher, W.Voelter, and C.Tzougraki

73

SYNTHESIS, PROPERTIES AND APPLICATIONS OF N,N-bis-BOC-AMINO ACIDS K.Gunnarsson and U.Ragnarsson

76

ELECTROCHEMICAL OXIDATION OF PROTECTED AMINO ACIDS AND DIPEPTIDES E.Steckhan, K.-D.Ginzel, C.Herborn, A.Papadopoulus, B.Lewall, and P.Brungs 79 DIRECT USE OF THE 2-(4-NITROPHENYLSULPHONYL) ETHYL ESTER GROUP IN PEPTIDE SYNTHESIS M.J.A.Amarai

Trigo and M.J.R.Gomes

82

PYRIDOXYLESTERS - A NOVEL PROTECTION L.Skylyarov, A.Nickolayev, and N.Kopina

85

USE OF 2,2,2-TRICHLOROETHYL ESTER, A SELECTIVELY REMOVABLE C-PROTECTING GROUP, IN LARGE SCALE P E P T I D E SYNTHESIS 88 R.Forino, R.de Castiglione, and M. Galantino N-TRITYLATED DERIVATIVES OF cis-4-HYDROXYL-L-PROLINE AND THEIR APPLICATION IN PEPTIDE SYNTHESIS D.Papaioannou,

G.Stavropoulos, and K.Karagiannis

91

SOLUTION - SYNTHESIS OF ENDOTHELIN H.Immer, I.Eberle, W.Fischer, and E.Moser

94

SOLUTION SYNTHESIS OF THYMOSIN ßA A.Kapumiotu, P.Link, and W.Voelter

97

XXXVI

SYNTHESIS OF Glu 65 -C5a ANAPHYLATOXIN BY THE SOLUTION PROCEDURE AND CONFIRMATION OF THE REPORTED STRUCTURE N.Chino, S.Kubo, T.Kimura, and S.Sakakibara

100

1.3. Purification and Analysis

103

LARGE SCALE REVERSED PHASE PURIFICATION OF PEPTIDES AND SMALL PROTEINS A.Lifferth, G.Becker, and C.Birr

103

POROUS COPOLYMERS FROM ACRYLATES AND VINYLAROMATES WITH DIFFERENT CARBON CHAIN LENGTHS AS CHROMATOGRAPHIC SUPPORTS P.Slonina, K.-D.Kaufmann, K.Häupke, and G.Schwachula

106

EPIMERIZATION AND ENANTIOMER RESOLUTION OF TRIPEPTIDES BY GC ON L-CHIRASIL-VAL B.Koppenhoefer, L.Bingcheng, V.Muschalek, U.Trettin, H. Willisch, and E.Bayer 109 SLOW CIS-TRANS ISOMERIZATION OF SOME PROLINE CONTAINING PEPTIDES INDUCES PEAK SPLITTING DURING REVERSED PHASE HPLC J.C.Gesquiere, E.Diesis, and A.Tartar

112

BYPRODUCTS OF Trp-PEPTIDES SYNTHESIZED ON A p-BENZYLOXYBENZYLALKOHOL POLYSTYRENE RESIN B.Riniker and B.Kamber

115

UNEXPECTED SIDE REACTION CAUSED BY RESIDUAL METHANESULFONIC ACID P.B.W.Ten Kortenaar, W.P.A.Janssen. B.M.M Hendrix, and J.W.van Nispen

118

ANALYSIS OF SYNTHETIC PEPTIDES BY PLASMA DESORPTION MASS SPECTROMETRY G.Lindenberg, Ä.Engström, A.G.Craig, and H.Bennick 121

SYNTHESIS AND APPLICATION OF IMMOBILIZED PEPTIDE FRAGMENTS FOR CHROMATOGRAPHIC INVESTIGATIONS H.Eckstein

124

XXXVII

SYNTHESIS AND HPLC ANALYSIS OF LYSINE ISOPEPTIDES G.Szokân, G.Kelemen,

E.Tyihâk,

and B.Szende

127

DETECTION OF C-TERMINAL AMIDATED AMINO ACIDS IN PEPTIDES BY COMBINED PROTEOLYSIS/EI-MASS SPECTROMETRY A.Otto,

P.Franke,

R.Kraft,

and G.Etzold

1.4. Solid P h a s e Synthesis

130

133

RACEMIZATION-FREE COUPLING OF FMOC ACIDS TO ALKOXYBENZYL ALCOHOL TYPE RESIN M.Mergler,

J.Gosteli,

R.Nyfeler,

R. Tanner, and P.Grogg

133

DEVELOPMENT AND APPLICATION OF NEW ANCHOR GROUPS FOR FMOC-BASED SOLID-PHASE SYNTHESIS OF AMIDES AND AMINOALKYLAMIDES G.Breipohl,

J.Knolle,

R.Geiger,

and W.Stüber

136

APPLICATION OF A HIGHLY ACID-SENSITIVE TRIALKOXY-DIPHENYLMETHYL LINKAGE FOR THE SOLID-PHASE SYNTHESIS OF PROTECTED PEPTIDE FRAGMENTS AND OF NONPROTECTED PEPTIDES AMIDES 139

H.Rink and P.Sieber

PREPARATION AND APPLICATION OF A NEW RESIN FOR SYNTHESIS OF PEPTIDE AMIDES VIA FMOC-STRATEGY B.Penke

142

and L.Nyerges

FACILE RELEASE OF PROTECTED PEPTIDE SEGMENTS FROM Pam RESIN SUPPORT WITH TETRABUTYLAMMONIUM FLUORIDE TRIHYDRATE M.Ueki,

K.Kai,

H.Horino,

and H.Oyamada

145

FMOC-AMINO ACID-TDO ESTERS AS REAGENTS FOR PEPTIDE COUPLING AND ANCHORING IN SOLID PHASE SYNTHESIS R.Kirstgen

and W. Steglich

148

FMOC-AMINO ACID OXOBENZOTRIAZINYL ESTERS IN SOLID PHASE SYNTHESIS: USE IN AUTOMATED SYNTHESIS AND AS AN INVESTIGATIONAL TOOL O.Nguyen and R.C.Sheppard

151

XXXVIII

SOLID PHASE SYNTHESIS OF PEPTIDES AND GLYCOPEPTIDES ON RESINS WITH ALLYLIC ANCHORING GROUPS H.Kunz, B.Dombo, and W.Kosch

154

EVALUATION OF THE NEW ALLYLIC ANCHOR GROUP HYCRAM IN THE MERRIFIELD SOLID-PHASE PEPTIDE SYNTHESIS G.Becker, H.Nguyen-Trong, C.Birr, B.Dombo, and H.Kunz

157

COUPLING OF PEPTIDE SEGMENTS IN CONVERGENT SOLID PHASE PEPTIDE SYNTHESIS A.Grandas, F.Albericio, E.Pedroso, E.Giralt, J.M.Sabatier, and J.van Rietschoten

160

N,N-DIETHYLHYDROXYLAMINE AS A CLEAVAGE REAGENT FOR PEPTIDES AND FULLY PROTECTED PEPTIDE SEGMENTS FROM PHENOLIC SOLID (GEL) PHASE SUPPORTS P.A.Baker, R.Epton, and T.Johnson

163

PEPTIDE SYNTHESIS BY FRAGMENT ASSEMBLY ON A POLYMER SUPPORT K.Nokihara, H.Hellstern, and G.Höfle

166

BUILDING BLOCKS FOR THE COVALENT SEMISYNTHESIS OF APOCYTOCHROME c. SOLID-PHASE SYNTHESIS AND CHARACTERIZATION OF THE N-TERMINAL (1-66) SEQUENCE C.Di Bello, C.Vita, L.Gozzini, and A.Hong

169

METHODOLOGY AND STRATEGY IN PEPTIDE SYNTHESIS: AN APPROACH TO THE SYNTHESIS OF UBIQUITIN J. Green, O.H.Ogunjobi,

and R.Ramage

172

LARGE SCALE SYNTHESIS OF 7-ENDORPHIN W.A.A. J.Bijl, M.C.A.van Tilborg, and J.W.van Nispen

175

SOLID PHASE SYNTHESIS OF RHESUS MONKEY RELAXIN P.J.Kelly, P.F.Lambert, G.W.Tregear, J.D.Wade, and P.D.Johnston

178

COMPARISON OF FOUR APPROACHES TO THE SOLID-PHASE SYNTHESIS OF THE MAGAININS, SOME OF ITS SEGMENTS AND ANALOGUES H.Echner and W. Voelter

181

XXXIX CONVENTIONAL AND SOLID-PHASE SYNTHESIS OF Leu-ANALOGS OF RAT MINIGASTRIN I. AND THEIR SEGMENTS L.Baldspiri, Cs.Somlai, P.E.Menykdrt, K.Kovdcs, G.Rcmdk, J.Lonovics, and V. Varro

184

FMOC-MEDIATED SOLID PHASE ASSEMBLY OF HIV TAT PROTEIN R.M.Cook, D.Hudson, D.Tsou, D.B.Teplow, H.Wong, A.Q.Zou, and E. Wickstrom

187

SYNTHESIS OF A PROPOSED SEQUENCE FOR THE ASPARTIC PROTEASE OF THE HUMAN IMMUNODEFICIENCY VIRUS D.F.Veber, R.F.Nutt, S.F.Brady, E.M.Nutt, T.M.Ciccarone, V.M.Garsky, L.Waxman, C.D.Bennett, J.A.Rodkey, I.Sigal, P.Darke

190

TEMPLATE-ASSEMBLED SYNTHETIC PROTEINS (TASPS) CONTAINING TWO FOLDING DOMAINS M.Mutter, R.Gassmann, R.Hersperger, L.Kvrz, and G.Tuchscherer 193

1.5 R a p i d a n d Parallel M e t h o d s , M o n i t o r i n g of S y n t h e s i s

196

PEPTIDE SYNTHESIS ON POLYSTYRENE-GRAFTED POLYETHYLENE SHEETS R.H.Berg, K.Almdal, W.B.Pedersen, A.Holm, J.P. Tarn, and R.B.Merrifield

196

POLYSTYRENE-POLYOXYETHYLENE GRAFTCOPOLYMERS FOR HIGH SPEED PEPTIDE SYNTHESIS W.Rapp, L.Zhang, R.Häbich, and E.Bayer 199 CONTINUOUS FLOW ULTRA-HIGH LOAD POLYMER SUPPORTED PEPTIDE SYNTHESIS WITH SOFT GEL PACKINGS A.F.Coffey, R.Epton, and T.Johnson

202

SIMULTANEOUS PEPTIDE SYNTHESIS USING CELLULOSE PAPER AS SUPPORT MATERIAL J.Eichler, M.Beyermann, M.Bienert, and M.Lebl

205

MULTIPLE COLUMN PEPTIDE SYNTHESIS A.Holm and M.Meldal

208

XL METHODOLOGICAL INVESTIGATIONS BY SIMULTANEOUS SOLID PHASE PEPTIDE SYNTHESIS D.Hudson

211

THE RAPID PREPARATION OF LARGE NUMBERS OF DISCRETE PEPTIDES FOR BIOLOGICAL; IMMUNOLOGICAL; AND METHOLOGICAL STUDIES: IMPROVED END-CAPPING REAGENTS R.A.Houghten and N.Lynam

214

THE EFFECTS OF INDUCED CONFORMATIONAL CHANGES ON THE ANTIGENICITY AND IMMUNOGENICITY OF SYNTHETIC BRANCHED PEPTIDE POLYMERS R.A.Houghten, J.R.Appel, and C.Pinilla

217

A RAPID APPROACH TO SYNTHETIC PEPTIDES AND EPITOPEDIRECTED MONOCLONAL ANTIBODIES Th.Böldicke, F.Maywald, E.Wingender, J.Collins, and R.Frank

220

ORGANOSILICON REAGENTS FOR RAPID AND PARALLEL PEPTIDE SYNTHESIS J.P.Tam, D.-X.Wang, A.Unden, and T.Bartfai

223

ANALYSIS OF ANTIPEPTIDE SERA BY PEPSCAN METHODS W.M.M.Schaaper, W.C.Puijk, H.Lankhof, A.Thomas, R.H.Meloen, J.M.Peters, and G.I.Tesser

226

ONLINE MONITORING OF PEPTIDE SYNTHESIS WITH N-PROTECTED AMINO ACID-TDO-ESTERS K.Friedrich and W.Steglich 229 COLOR MONITORED SOLID PHASE PEPTIDE SYNTHESIS V.Krchndk, J.Vagner, J.Eichler, and M.Lebl

232

REAL AUTOMATION BY ON-LINE NON-DESTRUCTIVE PHOTOMETRIC MONITORING IN SOLID PHASE PEPTIDE SYNTHESIS M.Horrn, C.Novak, and C.Birr 235 USE OF BOP REAGENT FOR RAPID AMINO ACID ACTIVATION AND COUPLING IN CONTINUOUS FLOW FMOC-POLYAMIDE PEPTIDE SYNTHESIS W.K.Rule, J.-H.Shen, G.W.Tregear, and J.D. Wade

238

XLI IN SITU ACTIVATION OF FMOC-AMINO ACIDS B Y BOP IN SOLID PHASE PEPTIDE SYNTHESIS H.Gausepohl, M.Kraft, and R.Frank

241

1.6. Enzymatic Synthesis

244

CHYMOTRYPSIN CATALYSED PEPTIDE BOND SYNTHESIS G.G.Whittaker, E.A.Hamilton, K.J.Bryant, L.T.McVittie, and, P.A.Schober

244

ENZYMATIC SAFETY-CATCH COUPLING:AN APPROACH TO BROADEN THE SYNTHESIS POTENTIAL OF a-CHYMOTRYPSIN AND TO PREVENT PRODUCT HYDROLYSIS IN KINETICALLY CONTROLLED PEPTIDE SYNTHESIS V.Schellenberger, U.Schellenberger, A.Kucharski, and H.-D.Jakubke

247

PEPTIDE SYNTHESIS CATALYZED B Y PAPAIN IN ORGANIC SOLVENTS CONTAINING MINIMUM WATER Yu.V.Mitin, V.Schellenberger, and H.-D.Jakubke

250

PEPTIDE SYNTHESIS CATALYZED B Y a-CHYMOTRYPSIN IN ULTRA LOW WATER SYSTEMS U.Slomczynska and T.Leplawy,Jr.

253

APPLICATION OF 2-GUANIDINOETHANOL AS A SOLUBILIZING PROTECTING GROUP IN ENZYMATIC PEPTIDE SYNTHESIS L.Andtrsson

256

DES-HEXAPEPTIDE (B25-30) INSULIN-B24-/3-PHENYLETHYL AMIDE ENZYMATIC SEMISYNTHESIS AND PROPERTIES E.Krause, K.D.Kaufmann, and H.Niedrich

259

C-TERMINAL CARBOXYL FUNCTION (Thr-B30) MEDIATED CROSS-LINKING AND IMMOBILIZATION OF INSULIN H.-G.Gattner and V.K.Naithani

262

ENZYMATICALLY CATALYZED FRAGMENT CONDENSATION IN THE SYNTHESIS OF CHOLECYSTOKININ OCTAPEPTIDE ANALOGUES V.Öefovsky, J.Pirkovd, P.Majer, J.Slaninova, and J.Hlavdcek

265

XLII ENZYMATIC SYNTHESIS OF Z-KYOTORPHIN AMIDE P.Clapes,

G.Valencia,

F.Rcig, J. M. Garcia-Anton,

and. J. Mata-Alvarez

268

A SIMPLE,CPD-Y CATALYSED SYNTHESIS OF L,L-AND D.L-DIPEPTIDES P.Thobek,

G.Houen,

S.Aasmul-Olsen,

271

and F.Widmer

ATTACHMENT OF LINKER GROUPS TO CARBOXYL TERMINI USING ENZYME-ASSISTED REVERSE PROTEOLYSIS K.Rose,

R.M.L.Jones,

G.Sundaram,

274

and R.E.Offord

THE USE OF PENICILLIN ACYLASE FOR SELECTIVE N-TERMINAL DEPROTECTION IN PEPTIDE SYNTHESIS 277

H. Waldmann

PROTEASE MEDIATED SYNTHESIS OF THYMOPENTIN S.Aasmul-Olsen,

F.Widmer,

280

and A.J.Andersen

PROTEIN ENGINEERING OF CYTOCHROME C: SUBSTITUTIONS OF Tyr 67 Thr 78 , AND Ala83 OF THE HORSE PROTEIN BY SEMISYNTHESIS C.J.A. Wallace, A.E.I.Proudfoot,

P.Mascagni,

and S.B.H.

Kent

283

PREPARATIVE SYNTHESIS OF POLYPEPTIDES IN THE CELL-FREE TRANSLATION SYSTEM OF CONTINUOUS ACTION Yu.B.Alakhov,

V.I.Baranov,

S.Yu.Ovodov,

and L.A.Ryabova

286

2. P E P T I D E S W I T H U N U S U A L A N D MODIFIED RESIDUES 289

2.1. Peptidomimetics and Glycosylated Peptides CYCLIC Trh ANALOGUES J.H.Jones

289

and P.B.Wyatt

PEPTIDE AZOLES: A NOVEL APPROACH IN THE DESIGN OF PEPTIDE MIMETICS AND ITS APPLICATION TO TACHYKININ ANTAGONISTS T.Gordon, P.Hansen, D. Keif er, and S.Ward

F.McKay,

B.Morgan,

J.Singh,

E.Baizman, 292

XLIII TETRAZOLE P E P T I D E ANALOGS J.Zabrocki, G.D.Smith, J.B.Dunbar,Jr.,K.W.Marshall, and. G.R.Marshall

M.Toth, 295

CHEMISTRY A N D E N A N T I O M E R I C R E S O L U T I O N O F a - H Y D R O X Y M E T H Y L - a - A M I N O ACIDS Z.J.Kaminski, M.T.Leplawy, A.Esna-Ashari, S.Kühne, S.Zivny, M.Langer, and H.Brückner

298

H P L C SEPARATION AND C O N F O R M A T I O N O F P E P T I D E D I A S T E R E O M E R S C O N T A I N I N G a , a - D I A L K Y L A T E D GLYCINES T.Yamada, M.Nakao, T.Yanagi, T.Miyazawa, S.Kuwata, and M.Sugiura

301

AN E F F I C I E N T R O U T E F O R T H E F O R M A T I O N O F Me3SiCl + P-NH-CH-C-OE I II 0

P-NH-CH-C-OE • Me3SÌ-N-CH-CO2R I II II 0 Carboxylic

silyl

esters

(2)

have

been

(1)

> P-NH-CH-C-N-CH-CO2R + E0-SiMe3 I IM I 0 considered

as

carboxylate

ion

equivalents (3). Activation with an appropriate electrophilic reagent will afford

the

activated

species

1

with removal of trimethylchlorosilane.

Using the N-trimethylsilyl derivatives 2 in the coupling step will

Peptides 1988 © 1989 Walter de Gruyter & Co., B e r l i n - N e w York-Printed in G e r m a n y

induce

23 the

removal

of

another neutral molecule. In this way it will be no more

necessary to add a racemizing tertiary base, and the what

can

be

considered

synthesis

in

with some reservations as "neutral conditions".

Morever the basicity of the amino component will decrease and

occur

by

silylation,

this can also contribute to limit the extent of racemization. Finally

trimethylsilylation will increase the solubility of segments in

non-polar

less racemizing solvents. Actually Bz-Val-0SiMe3 did not react at all and

with

chlorides

with

alkyl

chlorocarbonates

pivaloyl chloride in dichloromethane solutions. With phosphinic (B0P-C1,

corresponding

Dpp-Cl),

mixed

very

anhydrides

small

3

quantities

(1-2%)

of

the

could be characterized. However silyl

esters and phosphinic chlorides led to a reversible reaction :

0

(3)

0

The equilibrium was quantitatively shifted to the right by the

silyl

chloride

carrying

away

with a stream of nitrogen. As a result of the steric

hindrance, no reaction at all occured even after 120h at 20°C when 3a made

to

react

were isolated (CH2CI2, 40h, 20°C). This low yield now

unexpected

importance

of

the

illustrated

electrophilic

method.

the

until

activation due to the

trialkyl ammonium chloride formed at the beginning of anhydride

was

with TMS-Val-OMe. With Val-OMe, 32# of dipeptide (DL \h%)

the

regular

mixed

Accordingly addition of a catalyst should be essential

to apply scheme 1. Somewhat

similar

results

were obtained with Dpp-Cl. After carrying away

Me3SiCl with a stream of nitrogen, the reaction

of

3b

with

TMS-Val-OMe

afforded the dipeptide with a 50% yield (CH2CI2, l6h, 20°C) and W Another method was then considered between

silyl

esters

and

in

Dpp-Cl:

order

to

shift

the

DL.

equilibrium

trapping Me3SiCl with an epoxide(4).

These conditions were used with our test Bz-Val-OTMS + TMS-Pro-OMe (CH2CI2 Dpp-Cl

1

eq.,

epoxycyclopentane

2

eq.,

activation

time

lh at 20°C,

coupling time l6h). The influence of a number of inorganic additives (5,6) (1 eq.) is shown in the table:

24 Bz-Val-OTMS + TMS-Pro-OMe coupled with Dpp-Cl and epoxycyclopentane

Yield %

DL%

None

87

77

CuF2

21

Additive

Yield X

DL*

ZnF2

92

61

ZnCl2

51

Additive

CuCl2

56

1,0 1,0

ZnCl2-Et2 0

53

5.3

CuBr2

65

1.3

ZnBr2

62

0.7

C0CI2

49

3.7

Znl2

75

0.9

When Pro-OMe was used in place of the N-silylated

7.3

derivative, the figures

in presence of Znl2 were 90% and 1,7% respectively.

References 1. Kemp, D.S. 1979. In: The Peptides, Vol. 1 (E. Gross and J. Meienhofer). American Press, p. 336. 2. Miyazawa, T., T. Yamada, S. Kuwata. 1982. Peptide Chemistry, p. 69. 3a.Lednicer, D. 1972. In: Advances in Organic Chemistry, Vol. 8 (E.C. Taylor ed.). Wiley, p. 1793b.Sakaitani, M., Y. Ohfune. 1987. Tetrahedron Letters 28, 39874. Andrews, G.C., T.C. Crawford, L.G. Contillo. 1981. Tetrahedron Letters 22, 3803. 5. Jakubke, H-D, C. Klessen, E. Berger, K. Neubert. 1978. tetrahedron letters p. 1497. Berger, E., K. Neubert, H. Bang, H-D. Jakubke. 1982. Z. Chem. 22, 379. 6a.Miyazawa, T., T. Otomatsu, T. Yamada, S. Kuwata. 1984. Tetrahedron Letters 25, 771. 6b.Miyazawa, T., T. Otomatsu, Y. Fukui, T. yamada, S. Kuwata. 1988. J. Chem. Soc. Chem. Comm. p. 419-

S Y N T H E S I S O F D N A - B I N D I N G P R O T E I N II (HBs) B Y T H E U S E PROTECTED-PEPTIDE S-ALKYL THIOESTERS

S. A i m o t o ,

C. M a e g a w a ,

S. Y o s h i m u r a a n d H .

Institute for P r o t e i n Research, Suita, Osaka 565, Japan

OF

Hojo

Osaka University,

Yamadaoka,

Introduction In 1988,

w e r e p o r t e d a f a c i l e m e t h o d for the

of p o l y p e p t i d e

(1). T o s i m p l i f y

a r a t i o n of p a r t i a l l y - p r o t e c t e d an S-alkyl carboxyl

the procedure

preparation

for

peptide segments,

the we

designed

thioester m e t h o d based on our m e t h o d and the

segment

coupling

m e t h o d d e v e l o p e d by Blake protein

Results

and

synthesis

isolated

from

Discussion

synthesized S-carbamoylethyl according

terminal residue

t h i o e s t e r of p r o t e c t e d

to the p r o c e d u r e as s h o w n in Fig.

tripeptide

b e c a u s e of

on the resin. P e p t i d e - c h a i n

c a r r i e d out by the standard blocked by an acid-stable successively

Amino

acid

method

Z-ONSu,

(DMF) a n d

(3).

Asn-Pro

Zn/acetic

was

After resin

was was

acid-Ai,JV-

N-(t-butoxycarbonyloxy)succin1_ i n a y i e l d of 20

o b t a i n e d b y H F t r e a t m e n t of t h e analysis:

the

thereafter

the t e r m i n a l a m i n o group

to give peptide

material

of

elongation

peptide

carboxyl-

introducing

Troc group. The peptide

t r e a t e d w i t h HF,

(Boc-ONSu)

the crude

instability

Merrifield

c o m p l e t i o n of t h i s e l o n g a t i o n ,

dimethylformamide

1. T h e

w a s a s s e m b l e d on a r e s i n by

Boc-Arg(Tos)-Asn-Pro-SH

resin.

(HBs)

(2).

stearothermophilus.

Bacillus

imide

II

thio-

e t al

In t h i s p a p e r , w e d e s c r i b e h o w it w a s a p p l i e d to the of h i s t o n e l i k e D N A - b i n d i n g

We

prep-

Aspj 23GIU3 0 8 P r o 1

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in G e r m a n y

% based peptide

O S ^ ^ " ^ 17

on

26

CB3-C6H4CHC6B4-resin Br-CB 2 CH 2 COIIB Tos Boc-Arq-Asn-ProSH

* DIEA

Tos

CH3-C6H4CHC6H4-resin

Boc-Arq-Asn-Pro-SCHjCBjCOsa 1) T F A , 2 ) N H N , 3) B o c - a a i n o a c i d (3 e q . ( t D C C * ( B O B t ) Boc-Asp(OBzl)-Lys(Cl-i)-Val-Gin-Leu-Ile-Gly-PheGlyAsnPheGluVal-Arq(Tos) Glu(OBil)Arg(Tos)-AlaAlaArq(Tos)-Lys(Cl-i)Gly-Arq(Tos)AsnPro-SCH2CH2COMH CBJ-CJB^CBCJB^-resin

4) T F A , 5 ) N M M , 6) T r o c - O N S u , 7 ) BP treatment Troc-Asp-Lys-Val-Gin-Leu-Ile-Gly-Phe-Gly-Asn-Phe-Glu-Val-Arq-Glu-ArqAla-Ala-Arq-LysGly-Arq-Asn-Pro-SCBjCBjCOIIBj (TrocBBs(40

63)SCB2CB2COMB2)

B) S-OHSu Troc-ILys(X)41'591-BBS(40-63)-SCB2CB2COHB2 9) Zn/AcOB-DKF (1/9), 1 0 ) B o c - O N S u Boc-lLys(*)41 »59)-BBs(40-63)-SCB2CB2COIIB2

(1 )

Fig. 1 : Synthetic route of peptide 1_ Ala

1.80Val1.77Ile0.96Leu1Phe1.98Lys1.97Arg3.90- F o u n d : " / z 3212.5 ( M + H ) C a l c d for C-,44H 2 23 N 42°40 S: 3212.6. Peptide 2 was prepared according to the method mentioned in Ref. 1 in a yield of 20 % based on the crude material obtained by HF treatment of a peptide resin. Am ino acid analysis! Asp-^ Q ^ Thr

1.04Ser1.01Glu4.34Pro3.37GlY2.07Ala4.34Val1.83Met0.78Ile1

Leu

0 . 9 9 P ^ e 0 99 l Y s 4 78*

C

H

n

168 244 33°49

S:

peptide 1_ (9-8

Foun

d:

m/z 3539.8 (M+H)+. Calcd for

3540.0. Peptide 2

was

prepared by mixing

3 ymol) and peptide 2 (15.9 mg, 4.5 ymol)

in dimethyl sulfoxide (150 yl) in the presence of silver acetate (1.7 mg, 10 ymol), HONSu (3.5 mg, 30 ymol) and 4methylmorpholine

(10 yl) (Fig. 2). Peptide _3 (9.2 mg) was

obtained after isolation by reversed-phase HPLC. Amino acid analysis: A s p 4 > 0 ^ h ^ # , Q S e r 1 . 0 4 G l u 7.24 P r o 4.33 G l y4.81 A l a 5.73

27 Boc-Asp-Lys(X)-Val-Gin-Leu-Ile-Gly-Phe-Gly-Asn-Phe-Glu-Val-ArgGlu-Arg-Ala-Ala-Arg-Lys IS) -Gly-Arg-Asn-Pro-SCBjCBjCOWBj (Boc-[Lys(X)41•59)-BBS(40-63)-SCH2CH2CONH2) (1 )

CH3C02Aq

Gln-Thr-Gly-Glu-Glu-llet-Glu-Ile-Pro-Ala-Ser-Lys( Z ) - Val-Pro-AlaPhe-Lys(I)-Pro-Gly-Lys(X)-Ala-Leu-Lys(()-Asp-Ala-Val-Lys(X) ( [Lys(Z)75»80»83'86»90)-BBs(64-90) > (2)

Boc-Asp-Lys(X)-Val-Gin-Leu-Ile-Gly-Phe-Gly-Asn-Phe-Glu-Val-Arg-Glu-Arg-Ala-Ala-ArgLys(Z)-Gly-Arg-Asn-Pro-Gln-Thr-Gly-Glu-Glu-Het-Glu-Ile-Pro-Ala-Ser-LysIZ )-Val-ProAla- Phe-Lys (X)-Pro-Gly-Lys(X)-Ala-Leu-Lys(X)-Asp-Ala-Val-Lys(X) (Boc-[Lys(X)41»59,75,80,83,86,90]_HBs 5 8 M e t 0 > 9 0 I l e 1 - 7 8 L e u K 7 6 P h e 3 L y s 6 > 8 1 A r g 3 > 7 3 . In conclusion, a partially-protected peptide S-alkyl thioester was successfully prepared by a solid-phase method followed by introduction of additional protecting groups. The peptide thioester was stable during purification by HPLC or modification with protective reagents, and it was activated by silver ions to give the corresponding active ester in the presence of HONSu. The segment coupling by this method gave satisfactory results.

Acknowledgment This research was supported in part by Grant-in-Aid for Scientific Research No.62540407 from the Ministry of Education, Science and Culture of Japan.

References 1. Aimoto, S., Mizoguchi, N. and Yoshimura, S.. 1 988. In: Peptide Chemistry, 1987, (Shiba, T. & Sakakibara, S., eds.). Protein Research Foundation, pp.265-270. 2. Blake, J.. 1981. Int. J. Peptide Protein Res. VT_, 273-274. 3. Merrifield, R. B. . 1 963. J. Am. Chem. Soc. 85., 21 49-21 54.

STUDIES

ON R A C E M I Z A T I O N

PEPTIDE

SYNTHESIS

M. B e y e r m a n n ,

D. G r a n i t z a ,

A c a d e m y of S c i e n c e s DDR-1136 , Berlin M.

USING

of

FMOC AMINO

M.

ACID

CHLORIDES

IN

Bienert

the G D R ,

Institute

of

Drug

Research,

Haussner

Institute L.A.

of

Pharmacology

and T o x i c o l o g y ,

DDR-1040,

Berlin

Carpino

U n i v e r s i t y of M a s s a c h u s e t t s , A m h e r s t , MA 0 1 0 0 3

Department

of

Chemistry,

Introduction Fmoc amino reactive zation,

acid chlorides

derivatives

in t h e b i p h a s i c

homogeneous employment

Results

and

To s t u d y amines

solution in s o l i d

the

ding

phase

applicability a model

purity

by c o u p l i n g

to 1 1 1 ,

system

/3/.

have been

of r a p i d

shown

acylation,

CHClj/Na2C0j

This has prompted

to be

highly

without

/1,2/

and

racemi-

in

us to s t u d y

its

phase

tertiary

synthesis.

Discussion

as b a s e s

The o p t i c a l checked

(FAAC)

capable

of

of F A A C o n s o l i d

reaction

has been used

the e m p l o y e d

FAAC

with H-Phe-Gly-O-Me

a n d no r a c e m i z a t i o n

took

using

(see

tab.

(Fmoc-Phe-Cl)

was

in C H C l j / N a 2 C 0 j

place

( < 0.2

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

%).

1). accor-

29 Tab.

1 Racemization

base

/

studies

quantity

on s o l i d

|mmoles|

D-Phe-L-Phe-Gly (D-L-)

a) N-Methylmorpholine

phase

/0.06




concentration

5-10 minutes,

prevent

or

a tertiary

amine,

of

acylammonium

intermediates

we s u b s t i t u t e d

salt

using

the

and

a FAAC

tertiary

in

amine

30 by its salt of a weak acid (pivalic acid), c o n d i t i o n s usual forming a m i x e d a n h y d r i d e . Under these c o n d i t i o n s lation was c o m p l e t e w i t h i n 5 m i n u t e s

(f) the

for

acy-

(also with F m o c - V a l - C l ) ,

and no r a c e m i z a t i o n occured. Also with d e m i n i s h e d excess of Fmoc-Phe-Cl

(2 equiv.), but m a i n t a i n i n g

the c o n c e n t r a t i o n , a

complete a c y l a t i o n was o b t a i n e d within 5 m i n u t e s . For inverse r e a c t i o n

(Fmoc-Phe-OH/TEA+Piv-Cl)

of (f) we o b s e r v e d a rapid a c y l a t i o n tripeptide(Phe-Phe-Gly)

under the

the

conditions

(ca. 3 min.), but

was formed only in a trace

the

(formation

of P i v - P h e - G l y ) . With p r e f o r m a t i o n of the m i x e d anhydride 15 m i n u t e s at - 1 5 °C and coupling at ambient t e m p e r a t u r e a c y l a t i o n p r o c e e d e d very slowly presence of Piv-OH/TEA m a t i o n of mixed

(> 1 h). C o n s e q u e n t l y ,

for the

in

the FAAC reacts directly, w i t h o u t

for-

anhydride.

This rapid a c y l a t i o n m e t h o d might be applied especially s t e r i c a l l y h i n d e r e d amino acids or coupling

to

for

hydroxy-alkyl

resins.

Acknowledgment We wish to thank Mrs. A n n e r o s e Klose for technical

assistance.

References 1. C a r p i n o , L.A., B.J. Cohen, K.E. S t e p h e n s , Jr. , S.Y. S a d a t Aalaee, H.J. Tien, D.C. L a n g r i d g e . 1986. J. Org. Chem. 51, 3732 2. B e y e r m a n n , M., D. G r a n i t z a , M. Bienert, B. M e h l i s , H. N i e d r i c h , L.A. C a r p i n o . 1988. in: Proc. of the tenth A m e r i c a n Peptide S y m p o s i u m (G.R. M a r s h a l l , ed.). ESCOM, L e i d e n , p. 189. 3. Pass, Sh., B. Amit, A. P a t c h o r n i k . 103, 7674.

1981. J. Am. Chem.

4. Baleux, F., B. Calas, J. Mery. 1986. Int. J. Peptide Res. 28, 22.

Soc. Protein

DEVELOPMENT OF EFFICIENT FMOC SYNTHETIC METHODS AND THEIR COMPARISON WITH BOC STRATEGIES Dhirendra Chaturvedi1, James Ormberg2 and Henry Wolfe' 1

Biogen Corporation, Cambridge, MA, USA Vega Biotechnologies, Inc., Tuscon, AZ, USA 3 E.I. du Pont de Nemours & Co., Inc., Wilmington DE, USA 2

Keywords: FMOC, Peptide Synthesis, activation Application of FMOC-protected amino acids to peptide synthesis has proved to be a promising approach1. The coupling of FMOC-amino acids is commonly achieved through their symmetric anhydrides, HOBT esters or pentafluorophenyl esters. Each of these coupling procedures was carefully investigated and the first two methods of activation and coupling were further investigated using IR spectroscopy and HPLC as previously described for the BOC strategy2. Reaction conditions and results derived from these studies and the synthetic results obtained from alternate strategies led to the design of new, highly effective coupling protocols which have been incorporated into the Du Pont Coupler 2200 peptide synthesizer. Activation Studies The reactions of FMOC-De, FMOC-Gln, FMOC-Phe with DIC and HOBT were followed by IR spectroscopy5 at various concentrations, solvent ratios and temperatures. Figure 1 (curve a) shows the activation of FMOC-Phe-OH (0.14 M) with 0.5 equivalents of DIC in a 50% mixture of DCM/DMF at 25°C to generate the symmetric anhydride. Once formed, the symmetric anhydride can be rapidly converted to the HOBT ester by the addition of one equivalent each of HOBT and DIC to the newly formed FMOC-Phe anhydride (Figure 1, curve b). By comparison, formation of HOBT esters from free FMOC-amino acids takes upwards of 40 minutes (not shown), whereas they can be formed within five minutes from the anhydride. 1 1

/

—J

/

t

1 Symm An (1. . HC6T

t

—i

0

/

t

V

10

1

!

20

30

Time, minutes

Fig. 1 : Time course of activation a) FMOC-Phe symmetric anhydride formation b) FMOC-Phe HOBT ester formation from symmetric anhydride

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

32 Synthetic Strategies To test this activation strategy, several peptides were synthesized on the Du Pont Coupler 2200. Judging by its sequence, one of these (peptide 1) was particularly prone to DKP formation and the FMOC group was therefore removed with 50% piperidine and the third glycine residue incorporated as its pre-formed HOBT ester3. This strategy served admirably to produce the desired peptide in high yield; only 10% of the peptide was lost as DKP at the third residue (quantitative ninhydrin analysis). Using the Integrated Synthesis Logic (ISL) to determine the coupling protocol and frequency of double couplings, the other peptides were obtained in high yield at greater than 85% purity. Peptide 1:

Peptide 2:

VRSKIGSTENLKHQPGGG-OH

0

10

20

30

40

50

60

70

FERFEIFPKESS-OH

80

0

10

20

Time (min.)

30

40

50

60

70

80

Time (min.)

Peptide 4: Peptide 3: FGKREQAEEERYFRAR AKEQLAALK-OH

ACPF (65-74) VQAAIDYING-OH

J V j l

j l ^ J L , 0

10

20

30 40

50

Time (min.)

60

70

80

0

10

20

30

40

50

Time (min.)

Du Pont Bio Series Protein PLUS Column 300 A, 6 nm, 0.46 x 25 cm 0-80% B in 80 min. Buffer A = 0.1% TFA, H.O Buffer B = 90% CH.CN, 9% H.O, 0.1% TFA at 220 nm AUFS = 0.60

60

70

80

33 All FMOC-amino acids, resins and solvents used in this study were obtained from Du Pont Biotechnology Systems Division. FMOC-aminoacyl-p-benzyloxybenzylalcohol 4 resins and 2,4-dimethoxybenzhydrylamine resin5 were used as solid supports. The side chain protecting groups were selected as follows: Arg(Mtr); Asp, Glu t-butyl esters; Cys t-butyl thioether; His(Trt) or His(pi-Bum); Lys(Boc); Ser, Thr, Tyr t-butyl ethers. All peptide resins were dried and then treated with 100 mL/gram of cleavage solvent for the required time period. This time period was determined during a small scale cleavage (10 mg) of the resin during which aliquots were analyzed for purity by HPLC. The cleavage solvent for each was 90% TFA, 5% H20, 5-x % 1,2-ethanedithiol; where x = 1% thioanisole if any arginine residues were present.

Peptide ACPF (65-74) Somatostatin Auriculin B, rat VIP, human PTH (53-84), human

#AA 10 14 25 28 32

FMOC Chemistry # Double Couplings Purity 1 83% 62% 0 55% 5 10 32% 1 32%

BOC Chemistry # Double Purity Couplings 1 80% 4 40% 25% 15 14 33% 26 31 %

CONCLUSIONS 1) Efficient activation and coupling protocols have been developed and incorporated into the Du Pont Coupler 2200 peptide synthesizer. 2) These protocols are highly successful at avoiding DKP formation and synthesizing high purity peptides. 3) 1R studies have shown that it is more efficient to form HOBT esters from the symmetric anhydride than from the free FMOC-amino acids. 4) An efficient deprotection protocol has been developed and tested for peptides made using FMOC chemistry. 5) For the peptides tested, FMOC chemistry gave equal or better results than were obtained with BOC methods. References 1) Atherton, E. and Sheppard, R. in "Peptides; Analysis, Synthesis, Biology," Vol. 9,1987, (S. Udenfriend & J. Meien_,hofer. Eds) pp. 1-34, Acad. Press, San Diego, CA. 2) Chaturvedi, D.N., et al, 1988, in "Peptide Chemistry 1987, Proceedings of the Japanese symposium on peptide chemistry" (T. Shiba & S. Sakakibara, Eds) Protein Research Foundation, Osaka, pp. 287-290. 3) G. Barany in "Peptides; Analysis, Synthesis, Biology," Vol. 2,1980 (E. Gross & J. Meienhofer, Eds) p. 83, Academic Press, New York 4) P. Sieber, Tetrahodron Letters. 28: 6147 (1987). 5) The synthesis of this resin was improved by a proprietary modification of the method of Penke & Rivier as described in B. Penke and J. Rivier, J. Org. Chem. 52: 1197 (1987).

THE USE OF PHOSPHINYL CHLORIDES FOR CARBOXYL ACTIVATION AND Na-AMINO PROTECTION IN PEPTIDE SYNTHESIS

C.Poulos Department of Chemistry, University of Patras, Patras, Greece R.Ramage Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K.

Introduction Phosphinyl chlorides react either with amines or carboxylic acids to yield the corresponding phosphinamides and carboxylic phosphinic mixed anhydrides respectively. These aspects led to the succesful application of phosphinyl chlorides in solution (1) and solid phase (2) peptide synthesis as reagents either for Na-amino protection or carboxyl activation. To evaluate the potential of the diphenylphosphinyl chloride (Dpp-Cl) for Na-amino protection and of the 1-oxo-1-chlorophospholane (Cpt-Cl) for carboxyl activation in peptide synthesis we have used them in the synthesis of a series of analogues of the C-terminal hexapeptide of substance P where Glnb and Gly^ have been replaced by L- or D-Trp either.

Experimental The syntheses of the peptide analogues were performed in solution in a stepwise manner using the corresponding mixed carboxylic-phosphinic anhydrides in 20% excess, which were formed in CH2CI2 and/or DMF at 0°C by reacting the Na-protected amino acid with Cpt-Cl (3) in the presence of one equivalent of

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

35

Table 1. Physical constants of the synthesized peptides P E P T I D E S

M. P °C!

[«JD2

C -1

DMF

TLC(R f ) A

B

Dpp-Leu-Met-OCH^ Dpp-L-Trp-Leu-Met-•OCH3

160- 161

-62.6 0 .84 0. 94

170- 172

-86.8 0 .74 0. 83

Dpp-D-Trp-Leu-Met-•OCH3

193- 195

+11.3 0 .66 0. 78

Dpp-Phe-L-Trp-Leu-•Met-OCH3

221- 223

-61.9 0 .75 0. 82

Dpp-Phe-D-Trp-Leu-•Met-OCH3

213- 216

-13.7 0 .76 0. 86

Dpp-Phe-Phe-L-Trp-•Leu-Met-OCH3

210- 212

-81.4 0 .76 0. 85

Dpp-Phe-Phe-D-Trp-•Leu-Met-OCH3

203- 205

-76.8 0 .77 0. 89

Dpp-L-Trp-Phe-Phe- L-Trp-Leu-Met-OCH3

198- 201

-76.2 0 .66 0. 77

Dpp-D-Trp-Phe-Phe-•D-Trp-Leu-Met-OCH3

210- 213

-41.6 0 .73 0. 82

Boc-Gln-Phe-Phe-L- Trp-Leu-Met-OCH3

205- 206

-16.7 0 .70 0. 78

Boc-Gln-Phe-Phe-D- Trp-Leu-Met-OCH3

210- 213

-21.7 0 .68 0. 77

Dpp-L-Trp-Phe-Phe-•L-Trp-Leu-Met-NH2

211- 215

-70.1 0 .46 0. 63

Dpp-D-Trp-Phe-Phe- D-Trp-Leu-Met-NH2

218- 221

-38.7 0 .52 0. 75

Boc-Gln-Phe-Phe-L- Trp-Leu-Met-NH2

213- 216

-19.2 0 .43 0. 54

Boc-Gln-Phe-Phe-D- Trp-Leu-Met-NH2

218- 224

-32.7 0 .35 0. 43

A, CHC1 3 "CH 3 0H 6:1 v / v ; B , 1 - B U O H - A C O H - H 2 0 4:1:1 v/v N-methyl morpholine (NMM). After an activation time of 5 min. the amino component was introduced in the reaction mixture as the HCl-salt which was neutralised in situ with NMM, to yield the desired product (Table 1) within 3h. Deprotection of the Dpp-group was achieved with 6 equivalents of HC1 in methanol at room temperature for l-2h (4).

Results and Discussion Mixed carboxylic-phosphinic anhydrides derived from the corresponding Na-diphenylphosphinyl amino acids and Cpt-Cl react succesfully with peptide amino components to yield the desired protected peptides in high yields (72-94%) while the purity of the crude products was high as indicated by tic and microana-

36

lysis and they could be used further without any purification. It is interesting to note that with Gin no by-product formation was observed either in the activation stage with Cpt-Cl or the aminolysis, in contrast to Asn which gives rise to several products thus decreasing dramatically the yield of the desired product. The lower reactivity towards aminolysis of the mixed anhydrides RCOOCpt compared to that of RCOODpp and the solubility in water of the CptOH by—product make the Cpt— mixed anhydride more attractive for peptide chain elongation in solution as the crude product derived from RCOOCpt is generally less contaminated. The above mixed anhydrides also show the desired regiospecificity towards aminolysis and have thermal stability superior to that of carboxylic-carbonic mixed anhydrides. On the other hand the couplings are free of racemisation resulting from oxazolone formation as the latter has 31 been shown not to be formed by P n.m.r. studies. The main advantage of the Dpp-group over Boc or Z groups is its removal by mild acidic conditions in the absence of cation scavengers, thus can be safely removed in the presence of 31 Trp and/or Met in the peptide chain. It has been shown by P n.m.r. Dpp group was converted to Dpp-OCH^, which is soluble and easily removed from the reaction mixture while it incorporated in the peptide chain either by attacking le nitrogen of Trp or the sulphur of Met.

that the in ether is not the indo-

References 1. Ramage, R., D.Hopton, M.J.Parrott, R.S.Richardson, G.W. Kenner, G.A.Moore. 1985. J.Chem.Soc.Perkin Trans. I, 461. 2. Poulos, C., R.Ramage. 1984. In: Peptides 1984, Proceedings of the 19th European Peptide Symposium (Ulf Ragnarsson, ed.). Almquist and Wiksell Trycheri-Uppsala, p.161. 3. Ramage, R., C.P.Ashton, D.Hopton, M.J.Parrott. 1984. Tetrahedron Letters, 25, 4825. 4. Ramage, R., D.Hopton, M.J.Parrott, G.W.Kenner. 1984. J.Chem. Soc.Perkin Trans. I, 1357.

NEW COUPLING REAGENTS IN PEPTIDE CHEMISTRY

Reinhard Knorr, Arnold Trzeciak, Willi Bannwarth and Dieter Gillessen F.Hoffmann-La

Roche

u.Co.AG,

Basel,

Switzerland

Introduction Benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium

hexafluorophosphate

(BOP)1) was one of the first reagents for in situ formation of hydroxybenzotriazolyl esters which was successfully used in solid phase peptide synthesis2-3). The superior properties to DCC could be confirmed in a comparison with a number of different activating reagents4). A very good alternative to BOP is 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate {HBTU)5) which in our hands proved to be an excellent acylating reagent for solid phase peptide synthesis. Easy preparation, high reactivity without undesired side reactions, harmless byproducts and low costs made this compound to become our standard activation reagent. We want to describe a further optimized preparation procedure, and present a series of new analogs designed for special purposes in peptide and protein chemistry6).

Results We have successfully used the quite unknown, but excellent acylating reagent HBTU (1a) both in a shaker and in a continuous flow peptide synthesizer. Couplings proceed smoothly and without undesired side reactions like nitrite formation. The reactivity of (1) and (3) is comparable to BOP and symmetrical anhydrides6). Free aliphatic hydroxyl functions may be present. During reaction only harmless byproducts are generated which are completely soluble both in water and in organic solvents. This is essential for it's use in continuous flow systems. Beside 1hydroxybenzotriazole (HOBt) and hexafluorophosphate only tetramethyl urea (TMU) is liberated. We now have developed an improved synthetic procedure. In the preparation of the tetramethyluronium chloride (I) the dangerous phosgene was substituted by oxalyl chloride. For the direct formation of the reagent (II) a new one-pot procedure in completely waterfree organic solvents has been elaborated. We now were able to use the cheaper tetrafluoroborate as nonnucleophilic counterion which is not possible with the published procedures). Comparative experiments showed no difference in coupling rate or in racemization.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

38

»eherne 1

Scheme of Synthesis

W

CH3_N ^o OH,—N 'tHj OMU)

a'

\Q

a-

CH,-N

-COj.-CO

^ «I-N

KO-/7 , K X

y~a

21(01

CH3_N '«H (I)

*

y~o

CH— j N R «3 (II)

R=

•eo (D

(2)

(3)

-Bt

-DHBt

-OPy

bf4'

PFs

°

(4)

°

-Su

(5) -NB

(b)

We have prepared two new activating reagents which suppress racemization during fragment condensations and coupling of sensitive amino acid derivatives and compared them with other activating reagents. y confirming Murphy's law, i. e. " the principle that whatever can probably go wrong will." We report here the identification of the contaminant, then our investigations on the probable conditions having contributed to its formation. Various methods of comparison of the pure and contaminated samples are briefly summarized. No usual TLC techniques could resolve the mixture. Satisfactory resolution by analytical HPLC could be fulfilled only with phosphate buffers of pH as high as 9, quite unusual and considered to be decomposing for peptide analysis. Under this basic condition, however, repetitive preparative HPLC failed to produce the unknown substance in pure state. Amino acid analyses gave satisfactory results, and exclusively the expected residues were found. Then acidic hydrolysates were reacted with Marfey' s reagent /4/, resulting in diastereomeric products which are separable by HPLC if any epimer had been present /5/. This method showed the presence of 10 % D-Asp in the contaminated sample. At last NMR spectra taken at 400 MHz confirmed unequivocally the structure as some signals of a contaminated sample doubled, e. g. in addition to the multiplets of 095% purity by HPLC) was purified by counter-current distribution (automatic Craig apparatus solvent system: n-butanol-ethyl acetate-tetrahydrofuran-acetic acid-water (9:1:1:2:10, v/v). The product was recovered in 75% yield with 99% purity and its characterization was accomplished by TLC, HPLC, amino acid analysis, sequence determination, NMR and MS (FAB).

References 1. Woodward, R.B. et al. 1966. J.Am.Chem.Soc. 88, 852. 2. Marinier, B. et al. 1973. Can.J.Chem. 51, 208. 3. Ciardelli, T.L. et al. 1978. J.Am.Chem.Soc. 100, 7684. 4. Carson, J.F. 1980. Synthesis, 730. 5. Barany, G. and R.B. Merrifield. 1977. J.Am.Chem.Soc. 99, 7363.

N-TRITYLATED DERIVATIVES OF ais-4-HYDROXY-L-PROLINE AND THEIR APPLICATION IN PEPTIDE SYNTHESIS

D.Papaioannou, G.Stavropoulos and

K.Karagiannis

Department of Chemistry, University of Patras, Patras, Greece

Introduction Analogs of biologically important peptides incorporating

ois-

4-hydroxy-L-proline (cHyp, §_) in place of L-proline have been shown to exhibit profoundly different physiological behaviour compared to that of parent compounds (1). We report here on the synthesis of trityl derivatives of 6 and their application in the synthesis of model peptides. Key-features of the proposed synthetic methodology are: a(protection of the amino function with the trityl group, for it is labile to mild acids (2) and confers excellent resistance to racemisation (3), and b)inversion of configuration at C-4 of N-trityl-trans-4-hydroxy-Lproline (1_) via

an intramolecular Mitsunobu reaction (4).

Results and Discussion Treatment of the readily available 1 (5) with an excess of triphenylphosphane (TPP) and diethyl azodicarboxylate (DEAD) provided the key-intermediate lactone 2, in 60% yield. The structure of 2_ was unambiguously determined by X-ray crystallography. Saponification of 2 with 2N KOH in DMSO-MeOH provided trityl-cis-4-hydroxy-L-proline

N-

(3), isolated as its correspond-

ing diethylammonium (DEA) salt in 86% yield. Since lactone 2 failed to couple with amino acid esters to an appreciable degree, attempts were made to use

instead, and

activate it,in situ, with DCCI in the presence of 1-HOBt.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

92

However the formation of 2 was much faster than coupling and thus, it became apparent that protection of the hydroxy1 group of 3 was necessary- Indeed treatment of 2_ with an excess NaH and benzyl bromide gave the O-benzyl derivative A, isolated as its corresponding DEA salt in 60% yield. Treatment of 4 with DCCI and 1-HOBt afforded the active ester 5 in 90% yield.Being of the pure ester type (6), 5_ exhibited excellent acylating behaviour. R20

HO O — C O ^ H

O -

i

i

R

R

1, R=Trt

R

— 'R=H

3, Trt

0\

S

C 0 r 1

R1

OH

r2

H

4, Trt

OH

Bzl

5, Trt

OBt

Bzl

OH

H

OCH, 3 NH 2

H

6,

H

N

7, Trt

Trt

8, Trt

H

2 Transesterification of lactone 2 with excess MeOH, in presence of TPP and DEAD (7), gave cleanly the expected methyl

ester 7

(m.p. 129-30°C) in 85% yield. For comparison, its diastereomeric methyl ester (m.p. 144-45°C) was prepared from trans-4hydroxy-L-proline (11.) via

esterification followed by trityla-

tion. Ammonolysis of 2_ in THF-MeOH produced a mixture of the ester 1_ an< ^ the expected amide 8^. However clean formation of 8^ in 95% yield was realized on changing MeOH into

i -PrOH as the

hydroxylic counter-solvent. Treatment of intermediates 7 and 8^ with Ts0H.H 2 0 in

i -PrOH-THF provided the corresponding salts

9_ and 1£ in yields 85% and 90% respectively. It must be pointed out that detritylation of 2 with glacial AcOH provided 6 with m.p. and

C a l D values identical to those previously repor-

ted (la), in 30% overall yield based on 11^ Thus, the present

93

methodology also offers a simple entry to the synthesis of ais4-hydroxy-proline itself. The applicability of the so far described derivatives of 6_, in liquid phase peptide synthesis, was shown by preparation of the protected peptides Trt-cHyp(Bzl)-Leu-Gly-NH2, Trt-Cys(Trt)cHyp(Bzl)-Lys(Trt)-Gly-NH2 and Trt-Tyr-cHyp(Bzl)-Phe-cHyp-NH2. In these syntheses trityl protection was employed for the amino and sulfhydryl groups,couplings were effected by using the corresponding benzotriazolyl esters (6), and deprotection was carried out using either Ts0H.H 2 0 or 1% TFA in CH 2 C1 2 for selective N -trityl deprotection (2).

References 1. (a) Uitto, J., D.J.Prockop. 1977. Arch.Biochem.Biophys. 181, 293 and references therein, (b) Buku, A., I.L.Schwartz, N. Yamin, H.R.Wyssbrod, D.Gazis. 1987. J.Med.Chem. 3£, 1509. 2. Barlos, K., P.Mamos, D.Papaioannou, S.Patrianakou, C.Sanida, W.Schäfer. 1987. Liebigs Ann.Chem., 1025 and references therein. 3. (a) Barlos, K. , D.Papaioannou, S.Patrianakou, T.Tsegenidis. 1986. Liebigs Ann.Chem., 1950. (b) Baldwin, J.E., M.North, A.Flinn, M.G.Moloney. 1988. J.Chem.Soc., Chem.Commun.. 828 and references therein. 4. Bowers-Nemia, M.M. , M.Joullie. 1983. Heterocycles. 20_, 817. 5. Barlos, K., D.Papaioannou, D.Theodoropoulos. 1982. J.Org. Chem. £7, 1324. 6. Barlos, K., D.Papaioannou, D.Theodoropoulos. 1983. Int. J. Pept.Protein Res. 2J3 , 300 . 7. Bittner, S., Z.Berneis, S.Felix. 1975. Tetrahedron Lett., 3871.

SOLUTION - SYNTHESIS OF ENDOTHELIN

H. Immer, I. Eberle, W. Fischer and E. Moser NOVABIOCHEM AG CH-4448 Laufelfingen, Switzerland

Endothelin, an endothelium-derived vasoconstrictor peptide was isolated and elucidated by Yanagisawa et al.(l). The molecule contains 21 amino acids including two cystine moieties. Its synthesis which requires an unambiguous establishment of the two disulfide bonds presents a challenge. The solution we offer exploits some findings by Kamber et al.(2).

I

1

Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Giu-Cys-Vol-Tyr-Phe-Cys-His-Leu-Asp-lle-lle-Trp

-OH

Synthesis of the three Fragments Fragment 7-12

Fragment 1 - 6

a) DCC/HOBt b) HC1/TFE

H-fIeu|-OMe

H-jVoil-OMe

R-|Cys-Vol|-OMe

a) HA b) H^/Pd

a) R=Trt a) HA b) R=H;HC1 R-| S e r - [ e u | - O M e ) p_z b) NH(Et)2 R-J G l u - C y s - V a l l - O M e b) R-H ; HC1 t r a) MA OiBu If a) H A a) R=Fmoc b) H /Pd R-] Ser-Ser-leu - O M e b) NH(Et) b) R=H i—n a) R=Z iB'j tBu R-|lys-Glu-Cys-Vol|-OMe b) R=H;HC1 ¥—r^—i a) DCC/HOBt Boc OtBu Tii a) HA b) HC1/TFE b) NH(Et) a) R=Fmoc R-l Cys-Ser-Ser-Leu | - O M e c) NaOH in TFE/ b) R=H Tr t t B u t B u a) R=Trt a) HA water b) R=H;HC1 b) NH(Et) RrlAsp-Lys-Glu-Cys-Voll-OR, a

2

R-^ S e r - C y s - S e r - S e r - L e u l - O M e

a) ma b) l,H 2 k

ttBu1Tn 1 tBu1 îB'j

^ ctnoc aJ dR=F b) R=H

Boc-| Cys-Ser-Cys-Ser-Ser-leu |-R

Aon >Bu Trt 'Bu IE,

a) R=0Me b) R=N H 2 3

a) Fmoc-Met-OSu OtBu Boc OtBu I, a) R =pmoc;R ^ NH Et 2 >>) < >2 b) R j=H; R =0He c) Rj=H; R =0H R-(Met-Asp-Lys-Glu-Cys-Vol | - O H OtBu Boc OtBu Trt

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

a) R=Fmoc b) R=H

95

Fragment

13 -

H—file-liei—OH

21

H-fHÌsI-OMe

a)

Z-Asp(0tBu)-0Su

b)

H2/Pd a)

R—lAsp-lle-llel—OH a)

DCC/HOBt

R=z

Z-Leu-OSu

b ) HC1 1 - ] leu-Asp-lle-lle|-OH

R-|Cys-His|-OMe a

V

R

> = t b) R=H; HC1

a) Bop

a) MA b)

N

H-fîrp|-OtBu

OtBu

Tr

b)

H2/Pd R-jl.eu-Asp-lle-lle-Trp |-OtBu

A

OtBu Ddz-) Phe-Cys-His |-R Tit

a) a z i d e a) R=0Me b)

b)

coupling

HC00H/H0flc/H 2 0

R=n2h3

HPhe-Cys-His-Leu-Asp-lle-lle-Trp|-OtBu

i

a) HA b)

—

£

—

'Bu

the Molecule

and O x i d a t i v e

Boc-|Cys-Ser-Cys-Ser-Ser-Leu)-N; H 3 I I 1 I—r Acm IBu Tn iBu tBu

Ring

a)

Tit

OiBu

a)

Closures

Boc-| Cys-Ser-Cys-Ser-Ser-leu-Met-Asp-Lys-Glu-Cys-Vol | - O H I I I I I I I I I Acm iBu Trt IBu IBu OtBu Boc OtBu Trt see comment A

Boc-|Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Vof]-OH i i i i t i l Acm iBu iBu iBu OiBu Boc OtBu

Boc-Cys-: Acm tBu

I I iBu tBu

H-|Tyr-Phe-Cys-His-leu-Asp-lle-lle-Trp|-OtBu • i I iBu Tn OiBu

I I I OtBu Boc OtBu I

IBu

Trt

R=Fmoc

b) R=H

H - | Met-Asp-Lys-Glu-Cys-Vol | - O H i l l — r OiBu Boc OtBu Tit azide coupling [4]

BOP mediated coupling 13]

Ddz

b)

NH(Et)

R-ITyr-Phe-Cys-His-leu-Asp-lle-lle-lrJ-OtBu

Assembling of

a ) R=z b ) R=H

OtBu

see comment B

Boc-|Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-[ys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-A;p-lle-lle-Trp|-OlBu I I I tBu tBu tBu OtBu Boc OtBu tBu OtBu

96 Comment The

A

solvent

al. (2). reaction present

CHCI3,

rates

conversion

fold

Virtually 20

groups

of

were

MeOH

Acm

of

I2;

was

the

cleaved

of

S-Trt-

and were to

5

found

by T F A .

protected

was

FAB-MS

of

of

in

the

of

studied

the

large,

presence

allowing

the Ig

in the

was

the

the cleavage

product

way:

with

ascorbic

(yield

showed

In

the

/ 1 L; acid) .

on

of

the

selective

(1 m M o l e

The

et

in

9:1

obtained

following

Kamber

S-Acm-groups.

C^Clg/TFE

excess

by

differences

product. After chromatography

dodecapeptide

intact

Sephadex

70

%).The

t-butyl-protecting

a mass

of

1373

indica-

Cys(Acm).

of t h e s e s o l v e n t s w i t h w a t e r ,

leads to a p r e f e r e n t i a l

A d v a n t a g e of t h i s p h e n o m e n o n w a s t a k e n (2) a n d a n a l o g s

(5) t h e r e o f

12; 5 m i n . ;

r e d u c t i o n of e x c e s s

products were detected H i s a n d T r p to an

protected endothelin

The t - b u t y l - p r o t e c t i n g peptide conc. cold ether.

support

lyophi1isation

like

1.90

(2); Leu:

the a t t a c k

on S e p h a d e x

M+

of

of by-

I2 o n

MeOH

Purification

r e m o v e d by T F A / 2 - m e t h y l i n d o l e

(10 eq. o f

scavenger,

C r u d e e n d o t h e l i n w a s o b t a i n e d by p r e c i p i t a t i o n pressure

liquid c h r o m a t o g r a p h y

300i)

on V y d a c

in 0.1 % T F A a c i d w i t h a C H 3 C N pure endothelin

with

reversed gradient.

a n d r e l y o p h i 1 i s a t ion

from

obtained.

at 2 4 8 9 ;

(2); Ser: 2.01

2.45

Peptide Content (3); G l u :

(2); T y r : 0 . 8 0

(1); C y s : n o t

1. Y a n a g i s a w a , Y. Y a z a k i ,

1.07

by UV: 92 %.

(1); V a l : 1 . 0 1

(1); Phe: 0 . 8 9

determined'

M.,

H. K u r i h a r a ,

incomplete

S. K i m u r a ,

K. G o t o a n d T. M a s a k i .

B., A. H a r t m a n n ,

W. R i t t e l . 3. C a s t r o ,

pro-

of

LH 20 w i t h

(1); M e t : 0 . 9 7

(1); Lys:

1.02

(1); H i s :

h y d r o l y s i s of t h e

Ile-Ile

(1); 1.01 bond

References

2. K a m b e r ,

somato-

(5).The

(1 m M o l e / 1 L; t w e l v e f o l d e x c e s s

product. This excludes

of the f r a c t i o n s c o n t a i n i n g was

factors

Data

H P L C : 98 %; F A B - M S :

0.77

groups were

(Vydac C18, 20-30 microns,

water pure endothelin

: Asp:

G r o u p s a n d Final

It w a s p u r i f i e d by m e d i u m

phase

Analytical

reaction

oxida-

( y i e l d of 65 % ) .

2 %; 2 h r o o m t e m p . ) .

After

in M e O H

l£

residues.

of c y c l i c p e p t i d e s

natriuretic

degree. After chromatography

was obtained

of t h e P r o t e c t i n g

atrial

simultaneous

of t h e s e two

I2 w i t h a s c o r b i c a c i d ) . A g a i n v e r y s m a l l a m o u n t s

in t h e c r u d e

important

combination

in the s y n t h e s i s

and several

tected monocyclic endothelin was oxidised

AAA

were

B

t i o n of S - T r t - a n d S - A c m

Removal

extremely

oxidised

demonstrated

In M e O H , A c O H , d i o x a n e a n d m i x t u r e s

statin

be

in the c r u d e

s t r u c t u r e w i t h an

S-Acm-groups

in t h e

reduction

min. ;

was

to

disulfide

detected

cyclic

and

hexafluoroisopropy1alcohol

dodecapeptide

S-Acm-group

ting a m o n o c y c l i c

Comment

and

protected

product

the

TFE

S-Trt-groups

excess

one

with

presence

Trt

the the

reactivities

CH2C12.

of

of

case

twelve LH

dependent

In

1980.

B., J . R .

K. E i s l e r ,

He 1 v. C h i m . A c t a Dormoy,

B. R i n i k e r ,

63,

H. R i n k ,

1975. THL

P. S i e b e r

1219

1961. Col 1 . C z e c h . C h e m . C o m m u n .

results

Y.

Mitsui,

411

899

G. E v i n & C. S e l v e .

4. H o n z l , J. a n d J. R u d i n g e r . 5. I m m e r , H.; u n p u b l i s h e d

Y. T o m o b e , M. K o b a y a s h i ,

1988. N a t u r e 332,

2£,

2333

and

lie: (1);

1.62* Trp:

SOLUTION SYNTHESIS OF THYMOSIN ß«

A. Kapurniotu*, P. Link, W. Voelter Abteilung für Physikalische Biochemie des Physiologischchemischen Instituts der Universität Tübingen, Hoppe-Seyler Str. 4, D-7400 Tübingen, FRG

Introduction Thymosin 64 was first isolated from calf thymus by Goldstein et al. (1) in 1981 and has the following amino acid sequence: ac SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES Though active in several biological tests (1), its real physiological function remains still uncertain. So thesis

(solid-phase) of natural thymosin

far only one syn-

04 has been reported

in the literature (2). We describe here the first complete solution synthesis of the natural sequence of thymosin 04.

Synthesis Our synthesis is performed by condensation of the two key intermediates VII and XlVb; further details are summarized as follows (see also figures 1 and 2): -Z groups are employed for temporary protection of Ntt-amino functions with the exception for the synthesis of Met-containing fragment I for which NPS is used. -According to the orthogonal strategy applied, side chains of trifunctional amino acids are blocked by tert.-butyl protecting groups. -The subfragments are built up by stepwise coupling from the carboxy terminal amino acids using N-hydroxysuccinimide esters; in the case of Gin and Asn p-nitrophenyl esters are

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

98

w 3 ca w 3 a o

i Z O

CL. O I o â

1 U IU ca i 3 -H u

ca o

-I U

- i es i >>

« M

s

u

0

m

05 J Î U co J

« co i

s

u

m

â « s co

i

u á 3 m o

CL 1 u n i » 0 a u ¡-i N

\

m

* a

I

oi J

i

a oi

i

i

u

J i a.

u 0 a

tM

eu 1 01 t» j

CL 1 u m c. 0 Ò5 j 1 X

01

„ 3 a 0

a u

3

3 a 0

es 1

1 -i V

s a 0

1 a 01

X

0

co

a.

co u X tz >< co

u

u

0 a

3

a 0

01 >> -J a Kl




ca o

3 a

i ca •e O CS S" I a. c ft 9 •» S 3 N e o h o 9 o a:

i

H G Q

M s a 0

U O z

1 3 a a 0 1 0 u a. i u ca 0 >> a fcj 1 3 a a 0 œ


.

es

» S â . 3 5 1 0 1 3

o -H

m

S

-J

O

3

sA

3

u 1

u

o

-h es

u

m

á

* 3c a *

i CO H .>

íí 3 « 2 3 % ca

O ï -Ö O.S:

I ^ V co i o •

If a ?

• a «•

a n < 1 0 L. CL

"a a 0

a 01
, 730 .

Epimerization and Enantiomer Resolution of Tripeptides by GC on L-Chirasil-Val Bernhard Koppenhoefer, Lin Bingcheng, Volker Muschalek, Ulrich Trettin, Hans Willisch, Ernst Bayer, Institute of Organic Chemistry, D-7400 Tübingen (FRG). Introduction The possibilities and merits of dipeptide stereoisomer resolution [1,2] have been extended to the tripeptide Ala-Ala-Ala. Such investigations may circumvent, at least evaluate, the racemization problem encountered in enantiomer analysis of the amino acids formed by acidic hydrolysis of peptides [3,4 |. Resolution of Stereoisomers (Enantiomers, Diastereoisomers) The N-TFA-peptide methyl esters are formed as follows: 1 mg of peptide is reacted in a 1 mL Reactivial with 0.5 mL of HC1 in methanol (prepared from methanol and acetylchloride, v/v = 9:1) at 25 °C for 3 h. After stripping of reagents in a flow of dry ^ , traces of HC1 are removed by addition of 0.4 ml of methanol and 0.4 mL of toluene and stripping with . The dry residue is o reacted with 0.5 mL of TFA anhydride at 0 C for 5 min, stripped with ^ , dissolved in 0.3 ml of methanol, stripped with and dissolved in 0.3 mL of methanol. 0.2 piL of the solution are injected with a split ratio of 50:1 on a deactivated glass capillary (20 m * 0.25 mm), coated with L-Chirasil-Val. The chromatogram obtained for a mixture of the eight stereoisomers of TFA-A1 a-Al a-Al a-OMe at 190 °C, 0.4 bar H 2 , is depicted in Figure 1, trace C. Using two different temperatures, all eight isomers are resolved completely. The order of peak emergence is conflicting with a G-plated sheet model [5], as already observed for the dipeptide derivatives [ 2] . The ratio of K values for the interaction of the enantiomeric pair DDD/LLL with L-Chirasil-Val is at higher temperature (above T = 160 C) dictated by the entropic part T*AAS, as described by the equations (1) to (3), see Figure 2. -AAG = RT* (In K d d d - in K ^ ) -AAG = -A A H + T*A A S T g = AAH / A A S

= RT*ln ( T ^ ^

/ T ^ ^ )

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w York- Printed in G e r m a n y

(1) (2) < T!

i CFaCO-NH-CH-CO-NH-gH-CO-NH-CH-COOCHs | CHs CHs CHs |

M 174, 190 °C, L-Chirasil-Val

J

B

U LDD DDD DLL LLL DDL

U o

10

15

20

25

min Figure 1.

190°C Figure 2.

111

Such a peak reversal due to entropy control has been predicted as early as 1980 [6,7] . The point is that above T g the better fitting LLL-enantiomer, though - A H is more favorable, is less strongly bound than DDD, since the higher ordered association complex of LLL with L-Chirasil-Val also shows the more unfavorable AS value. This observation deserves particular attention in view of the nowadays widely used energy calculations and molecular graphics studies of molecular complexes, e.g. in drug design and molecular biology. Epimerization Experiments Under the proper derivatization conditions, epimerization proves negligible, see Figure 1, trace B. However, the homochiral derivative DDD-TFA-Ala^-OMe (and LLL, respectively) is found to be sensitive to prolonged storage in methanol in the presence of HCl or TFA. Almost exclusively the second stereogenic center is epimerized to form the DLD-isomer, see Figure 1, trace A. Epimerization studies on the free tripeptides with an equimolar amount of 4-dimethylaminopyridin in pyridin at 60 °C for 4 h produced similar results, i.e., LLL (or DDD) gave 13% LDL (and 13% DLD, respectively). Interestingly, the pure LDL-isomer equilibrated back to the LLL-isomer, to give solely 10% LLL. Experimental relative stabilities of these stereoisomers are of importance in view of the question of preferential prebiotic formation of particular peptide stereoisomers. Acknowledgement Dr. Lin Bingcheng is grateful Stiftung for a fellowship.

to the Alexander von

Humboldt-

References [1]

Koppenhoefer, B.; Allmendinger, H.; Bayer, E. HRC&CC, 10 (1987) 324. [2] Koppenhoefer, B. ; Allmendinger, H.; Lu, P.; Lin, B.; J. Chromatogr. 441 (1988) 89; loc. cit.. [3] Frank, H.; Woiwode, W. ; Nicholson, G.J.; Bayer, E. Liebigs Ann. Chem. 1981, 354. [4] Smith, G.G.; de Sol, B.S. Science 207 (1980) 765. [5] Frank, H.; Nicholson, G.J.; Bayer, E. Angew. Chem. Int. Ed. Engl. 17 (1978) 363. [6] Koppenhoefer, B. Thesis, University of Tübingen, 1980. [7] Koppenhoefer, B.; Bayer, E. Chromatographia 19 (1984) 123.

SLOW

CIS-TRHNS

PEPTIDES

I S O M E R I Z H T I O N OF S O M E P R O L I N E

CONTAINING

INDUCES PEAK SPLITTING DURING REUERSED PHASE

HPLC

J.C. Gesquiere, E. Diesis, A. Tartar Service de Chimie des Biomolecules, U.R.A. C.N.R.S. D-1000, Institut Pasteur and Faculté de Pharmacie,rue Calmette Lille, France

Reversed Phase H P L C is the method of choice to assess the purity of synthetic peptides. Observation of a broad peak or of more than a single peak Is usually attributed to the presence of impurities generated by side reactions that occured during synthesis. We report here several cases of medium size synthetic peptides where a slow interconversion of conformers was responsible for peak broadening or peak splitting in usual gradient reversed phase H P L C conditions. Depending on the nature of the peptide, two different kinds of observations were made. Linear Peptides :

Among the various peptides prepared in our laboratory, 4 linear sequences (MetSer-lle-Pro-Pro-Glu-Lys ; lle-Pro-Met-Ser-lle-Pro-Pro-Glu-Lys ; Leu-Ala-lle-ProPro-Lys-Arg-Leu-Asn ; s^e'ne

X=Cl,Br

[ c H . O ^ C H ^ resin halide ^ ^

A l k y l a t i o n of these r e s i n h a l i d e s ( a ) w i t h F M O C - a m i n o a c i d C s salts

(7) a n d Nal in d i m e t h y l a c e t a m i d e

solution proceeded with

ease, in h i g h y i e l d a n d w i t h o u t n o t i c e a b l e loss of the protecting ©

FMOC-

group.

+ FMOC-AA-OCs

» polystyrene j - C H 2 0 - ^ ^ - C H 2 0 - A A - F M 0 C

Table Racemization FMOC-amino

acid

tests

% D-enantiomer by a c y l a t i o n of O H - r e s i n (SASRIN)

formation by of

alkylation Cl-resin

Cys(Acm)

4.0

0.5

Cys(tBu)

4.7

0.3

Cys(Trt)

18.3

2.5

His(Trt)

26.0

0.4

He

1.1

Asn(DOD)

1.3

acylation

conditions:

alkylation cleavage

conditions

conditions:

(D-allo)

0.1

(D-allo)

0.3

1.5 e q . F M O C - A A ; 1.7 eq. DCC; 0.01 eq. DMAP ; CH-Cl,, : D M F = 3 - 20° » 0 ° C, 20 nrs 1 . 5 . e q . F M O C - A A - C s salt; 1.0 e q Nal d i m e t h y l a c e t a m i d e ; 20° C, 6 h r s a) 20 % p i p e r i d i n e in D M F b) 1 % t r i f l u o r o a c e t i c a c i d in CH„C1,

D - e n a n t i o m e r d e t e r m i n a t i o n a c c o r d i n g to

(8)

135 The data presented are uncorrected, i.e. absolute optical purity of starting FMOC-AA derivatives is assumed. When the products obtained upon cleavage from the resin were checked for chemical purity, noticeable differences were observed depending on the resin type. In all cases products recovered from SASRIN were of high purity. Thus, alkylation of FMOC-AA's by resin halides

(haloresins)

was shown to be superior than the classical acylation of hydroxyresins, such as Wang's resin or SASRIN. Furthermore, FMOC-AA's cleaved from SASRIN are obtained in higher chemical purity due to the extremely mild reaction conditions.

References 1. a) M. Mergler, R. Tanner, J. Gosteli, P. Grogg Tet. Letters 23, 4005 (1988) b) M. Mergler, R. Nyfeler, R. Tanner, J. Gosteli, P. Grogg Tet. Letters 2ji, 4009 (1988) 2. a) E. Atherton, N.L. Benoiton, E. Brown, R.C. Sheppard, B.J. Williams; J.C.S. Chem. Commun., 1981, 336 b) J.W. van Nispen, J.P. Polderdijk, H.M. Greven; Reel. Trav. Chim. Pays-Bas 104, 99-100 (1985) 3. R. Kirstgen, R. Sheppard, W. Steglich; J.C.S. Commun., 1987, 1870 4. P. Sieber; Tet. Letters 28, 6147

(1987)

5. S.S. Wang; J. Am. Chem. Soc. 95, 1328

(1973)

6. a) L. Horner, H. Oediger, H. Hoffmann; Ann. 626, 261

(1959)

b) G.A. Wiley, R.L. Hershkowitz, B.M. Rein, B.C. Chung; J. Am. Chem. Soc. 86, 964 (1964) 7. B.F. Gisin; Helv. Chim. Acta 56, 1476 8. H. Frank, G.J. Nicholson, E. Bayer; J. Chromatogr. Sci., 15, 174 (1977)

(1973)

DEVELOPMENT AND APPLICATION OF NEW ANCHOR GROUPS FOR FMOCBASED SOLID-PHASE SYNTHESIS OF AMIDES AND AMINOALKYLAMIDES

Gerhard Breipohl, Jochen Knolle, Rolf Geiger Hoechst AG, P.O. Box 80 03 20, 6230 Frankfurt/Main 80, FRG Werner Stüber Behringwerke AG, P.O. Box 1140, 3550 Marburg/Lahn, FRG

Introduction Use of the base-labile 9-fluorenylmethyloxycarbonyl (Fmoc) group (1) in solid-phase peptide synthesis (SPS) has gained in attention during recent years. A major advantage using this protecting group is, that it avoids repetitive treatment of the growing peptide chain with trifluoroacetic acid (TFA). Moreover, the peptide can be obtained by relative mild cleavage using TFA/scavenger mixtures. A number of linkers for preparation of peptides as free acid have been reported (2). However, synthesis of peptide amides was commonly performed on benzhydrylamine-type resins which are also used in Boc-chemistry. These resins have to be treated by liquid hydrogenfluoride (HF) or trifluoromethane sulfonic acid (TFMSA) to obtain the peptide as amide.

Results and discussion As we generally use a Fmoc-protocol for SPS, we decided (3) to look for anchor groups, which release peptide amids upon treatment with TFA.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , B e r l i n - N e w York-Printed in G e r m a n y

137

We now report (4) several anchor groups of structure which have the desired properties. They are easily prepared by reduction of the corresponding benzophenone to the carbinol and subsequent condensation with Fmoc-amide or Fmoc-amino acid amides in glacial acetic acid with concentrated sulfuric acid as catalyst. All linkers are fully characterized by analytical methods, which in our opinion is of advantage, as no reactions have to be performed on the resin. For peptide synthesis the anchor groups are coupled to commercially available resins, preferably to amino methylated polystyrene. Cleavage of the peptide amides proceeds smoothly with TFA/ scavenger mixtures. (Depending on the linker TFA-concentrations of 5 % to 90 % are required.) A large number of peptides have been prepared ussing this anchor groups. 0

Fmoc-X-NH R

|

R

/
90% B in 30 min. c,d,e: Nucleosil C4, 4.6x250 mm, isocratic 80% B. f,g,h: Nucleosil 5C18, 4.6x120 mm, 0% B -> 90% B in 30 min. References 1. P.A. Pietta and A.R. Marshall, (1970) J. Chem. Soc., Chem. Commun., 650. B. Penke and J. Rivier, (1987) J. Org. Chem. 52, 1197. G. Breipohl, J. Knolle, W. Stuber, (1987) THL 28, 5651. 2. Hans Rink, (1987) THL 28, 3787-3790. H. Rink, Peptide Chemistry 1987, Protein Research Foundation, edited by T. Shiba and S. Sakakibara, Osaka, pp. 279-282. 3. E. Atherton, E. Brown, G. Priestley, R.C. Sheppard and B.J. Williams, Proceedings of the 7th American Peptide Symposium (1981) pp. 163, Pierce Chemical Company, Rockford IL.

PREPARATION AND APPLICATION OF A NEW RESIN FOR SYNTHESIS OF PEPTIDE AMIDES VIA FMOC-STRATEGY B. Penke,L. Nyerges Department of Medical Chemistry, A. Szent-Györgyl Medical University, H-6720 Szeged, Hungary

INTRODUCTION Synthesis of peptide amides containing acid-sensitive amino acids (eg. tyrosine-O-sulphate, to-carboxyglutamic acid) by the Fmoc-method has been problematic. fe have recently synthesised a nev acid - labile resin (1) vhich vas based on polystyrene and could be used both in manual and automated batch systems. Our 2,4-dimethoxybenzhydrylamine - resin appears to be very promising for the synthesis of peptide amides by Fmoc-strategy. Cholecystokinin-octapeptide 0sulphate and GnRH were synthesised on this resin and the peptides vere cleaved from the polymer vith TFA-thioanisol or 95% TFA. Certain limitations are to be noted. Just the 0sulphate group has such a high acid lability that a relatively large part of the sulphate ester vas cleaved from the peptide ("desulphatation") during the final TFA cleavage of the peptide amide from the supports. More recently, a series of other anchor groups vere developed in different laboratories (2-5),some of them shoving higher acid lability than the side-chain protecting Boc, But, and 0Bufc groups. ¥e vanted to plan - on the base of theoretical considerations - a

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin N e w York- Printed in G e r m a n y

143

polymer support vhich has approximately the same acid lability as the Boc group. No* ve report the preparation of a new anchor-group and a nev resin vhich enables the synthesis o£ peptide amides under very mild conditions.

RESULTS Some years ago Juhisz and Bajusz (&) reported the use of 2, 2',4,4'-tetramethoxybenzhydryl (Tbh) group for the protection of amide groups in classical peptide synthesis. It can readily be removed by short (10-15 mln ) TFA-treatment at 20 °C. We replaced one methoxy function of the Tbh-group by electron-donating succinylamino-function; the replacement slightly increased the acid lability of the original group (2)The nev anchor molecule, the 4-succinylamino-2, 2',4-trimethoxybenzhydrylamlne (SAKBHA)vas coupled as N-Fmoc derivative vith DCC to aminomethyl-polystyrene resin. Cleavage of the Fmoc-group from anchor resulted in 4succlnylamlno-2, 2',4'-tr1methoxy-benzhydry1amine-resin (SAMBHA-polymer). The synthetic route vas already published in our previous paper (2). We tried to apply this nev resin for the synthesis of peptide amides containing acid labile amino acid using Fmoc-strategy. As a first step different Fmoc-amino acids vere coupled to the SAMBHA-polymer (DCCcondensation) and Fmoc-amino acid amides vere cleaved vlth 50% TFA in CH2CI2 from the resin. Cleavage of the amides of glycine, alanine or serine required less than 5 minutes, complete cleavage of valine and phenylalanine amide vas performed in a 10-15 minutes reaction. No side reaction occurred during cleavage. For further proof of the nev anchor, cholecystokinin-8, caerulein and cholecystolcinin-33 vere assembled on the

144

SAMBHA-resin using Fmoc-strategy. Tyrosine-O-sulphate vas introduced as Fmoc-Tyr(S03Na)-OPFP. The guanidino-function of Arg vas protected vith Pmc-group (fl). The peptide amides vere cleaved from the resin in 10 min vith 50% TFA in CH2CI2 (containing ethanedithiol, too); the reagent cleaved also the side chain protecting groups but caused only a marginal cleavage of the 0-sulphate group. These 0-sulphated peptides could be purified very easily on preparative RP-HPLC column (Yydac Cis) giving high yield. During the last year 7 different acid-labile anchor groups and polymer supports »ere synthesised in five different laboratories, all of them are suitable for the synthesis of peptide amides via Fmoc-strategy. Our nev SAMBHA-resin shovs an acid sensitivity very similar to that of the Boc, But, and OBut groups and therefore appears to be most suitable for the synthesis of peptide amides in combination vith Boc and t.butyl side chain protection.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Penke, B., J.Rivier. 1987. J.Org.Chem.52., 1197. Albericio, F., G.Barany. 1987. Int. J. Pept. Prot. Res. ¿Q., 206. Breipohl, G., J. Knolle, R.Geiger. 1987.Tetrahedron Lett5647. Sieber, P.,1987.Tetrahedron Lett.£8,2107. Rink, H., 1987.Tetrahedron Lett.¿fi, 3787. Juhäsz, A., S.Bajusz.1979.Acta Chem, Acad.Sei.Hung.102.289. Penke, B., L. Nyerges, N. Klenk, A. Asztalos.1987. In:Peptides. Proceedings of the A.Szent-Györgyi Anniversary Symposium (B.Penke and A.Török, eds.) Walter de Gruyter, p.121. 8. Ramage, R., J. Green.1987. Tetrahedron Lett .¡S., 2287 .

FACILE RELEASE OF PROTECTED PEPTIDE SEGMENT FROM Pam RESIN SUPPORT WITH TETRABUTYLAMMONIUM FLUORIDE TRIHYDRATE

M. Ueki, K. Kai, H. Horino, and H. Oyamada Department of Applied Chemistry, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan

Introduction Fluoride induced fragmentation of the ester groups with silyl substituent has recently attracted special interest as a mild method for the preparation of protected peptide segment on a solid support (1,2). However, another method would be possible. Ester groups, especially the groups of benzyl ester with electron-withdrawing substituent, can be cleaved easily with tetrabutylammonium

fluoride trihydrate (TBAF-3H20) in

methylformamide (DMF) without assistance of the silyl

N,N-digroup.

For example, when Boc-Phe benzyl esters with p-substituent of C O - N H C H Q C 6 H 5 and C H 2 C 0 - N H C H 2 C 6 H G were treated with 5 equiv. of 0.05M TBAF-3H20 in D M F at room temperature the ester groups were c l e a v e d completely in 20 and 60 min, respectively. When these conditions were applied to the synthesis of protected peptide segment on resin supports with the spacer bond of C0NH and CHQCO-NH ('Pam' resin) rapid release of the protected peptide segment was observed in the latter case ( 3 , I n

the

synthesis of [D-Ala^, Leu^]enkephalin no racemization was observed (4). From these facts this method could be expected to become one of the simplest methods for obtaining protected peptide segments. However sensitivity of the other protecting groups toward the TBAF-3H20 may prevent its versatile use. In this study side reactions in the ester cleavage reaction with TBAF-3H 2 0 were investigated in detail.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in Germany

146

Results and Discussion Recently we reported the rapid removal of 9-fluorenylmethy 1 oxycarbonyl group with TBAF-3H20 (5). Among other

urethane

type amino-protecting groups only Boc group was quite

stable.

Per cent remaining after 24h treatment of Prot.-Phe-OtBu with 5 equiv. of 0.05M T B A F - 3 H 2 0 in D M F : Z, 24%; Z(OMe), 30%; 2 C1Z, 5%. Trichloroethyloxycarbony1

(Troc) group could be re-

moved almost completely within 10 min under these conditions. In the easiness of removal the Troc group compares with the Fmoc group, but these conditions could not be applied to peptide synthesis because hydantoin formation proceeded much more rapidly even in presence of catalytic amounts of TBAF.

Troc-Gly-Gly-OBzl

0.004M TBAF•3H 2 0 in DMF (0.1 eq.) RT C=0 I CH2C02Bzl

Stability of side-chain protecting groups was then checked using Boc-AA(X)-NHBzl and the results were given in Table 1. Table 1. Instability of Various Protecting Groups toward Compound

TBAF-3H20

10

30

60

120

240

360

24(h)

101

100

97

102

—

100

103

Boc-Lys(Z)-NHBzl

98

85

81

73

62

Boc-Ser(Bzl)-NHBzl

85

65

M

41

24

Boc-Ser(tBu)-NHBzl

78

72

64

52

—

39

31

Boc-Thr(Bzl)-NHBzl

83

65

55

45

33

29

12

Boc-Tyr(Bzl)-NHBzl

74

51

32

18

11

Boc-Phe-NHBzl

Boc-Cys(MBzl)-NHBzl

79

66

60

57

—

48

38

Boc-Arg(Tos)-NHBzl

95

89

82

73

—

54

38

Boc-Arg(N0 2 )-NHBzl

98

97

95

92

—

84

70

81

68

59

Boc-Trp(For)-NHBzl Boc-Asn-Phe-NH 2

0 93

147

Aspartic (3-ester residues, regardless of the structure of the ester group, were consumed very rapidly to afford mainly 3peptide acid probably through the succinimide formation followed by hydrolysis. No effective method to suppress this side reaction has not so far been found. In order to establish the ester cleavage reaction with fluoride as a preparative method for protected segments a new more easily cleavable ester bond is necessary. Search for such a ester bond and its milder cleavage conditions are now in progress.

r0tBu

Boc-Asp-Phe-NH2

r0tBu

Boc-Asp-Phe-NH2

0.05M TBAF-3H 2 0 in DMF

(5.0 eq.)

RT -65] (1-65) and (66-104) of horse heart cytochrome c bind non-covalently to the ferric heme segment (1-25)H to form a non-productive three-fragments complex. Interestingly, when the heme is kept reduced at pH 5.6 for 48 hrs at 25°C, the peptide bond between the lactone activated Hse>-65 residue of fragment (1-65) and the Glu-66 residue of the (66104) fragment is restored to form [Hse-65] apocytochrome c with 20-40% yield (1 ). The Hse-65 apocytochrome c thus obtained forms a complex with the ferric (1-25)H segment which is indistinguishable from the analogous complex between the ferri (1-25)H segment and native apocytochrome c on the basis of the intensity of the 695 nm absorption band, the rate of reduction by lactate dehydrogenase and UV-CD spectra. Therefore, the present system, allowing the conformationally driven covalent semisynthesis of apocytochrome c, represents a useful tool for the preparation of analogs selectively modified both in the C-terminal (2, and in the N-terminal regions of this important molecule. The solid-phase synthesis, purification and characterization of the

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , B e r l i n - N e w York-Printed in G e r m a n y

170

C-terminal (66-104) fragment has been presented elsewhere (3), here we wish to report the data relative to the hexahexaconta peptide corresponding to the (1-66) N-terminal sequence.

Results Peptides corresponding to the (1-66) native sequence of horse heart cytochrome c (see scheme) were synthesized by standard solid-phase methods on a fully automated peptide synthesizer (Applied Biosystems mod. 430 A). 1 2 4 5 6 3 7 8 9 10 H - Gly - Asp - Val - Glu - Lys - Gly - Lys - Lys - lie - Phe 11 Val

12 14 15 16 19 20 13 17 18 - Gin - Lys - Cys - Ala - Gin - Cys - His - Thr - Val -

21 22 24 25 26 27 28 29 30 23 Glu - Lys - Gly - Gly - Lys - His - Lys - Thr - Gly - Pro 34 36 40 31 32 33 35 37 38 39 Asn - Leu - His - Gly - Leu - Phe - Gly - Arg - Lys - Thr 46 50 41 42 44 45 47 48 49 43 Gly - Gin - Ala - Pro - Gly - Phe - Thr - Tyr - Thr - Asp 52 54 56 60 51 53 55 57 58 59 Ala - Asn - Lys - Asn - Lys - Gly - lie - Thr - Trp - Lys 66 61 62 61 65 63 Glu - Glu - Thr - Leu - Met - Glu - OH Boc-Glu (OBzl) - 4 - (oxymethyl) - phenylacetamido methyl (PAM) resin (0.5 mmole) was used as the starting solid support. Coupling was done on a 0.5 mmole scale using the preformed symmetrical anhydrides, except Arg, Asn, and Gin which were coupled as HOBt active esters. The coupling media was DMF and DCM if the second coupling step was performed. All amino acids were protected with N-t-Boc. Side chain-protected amino acids used were: Arg (Tos); Asp (OBzl); Cys (4-MeBzl) or (Acm); Glu (OBzl); His (Z); Thr (Bzl); Trp (For), and Tyr (BrZ). Resin samples from each cycle were collected using the autosampler and the ninhydrin values were calculated to determine the extent of coupling. After completion of the synthesis, a sample of the (1-66) peptide resin was removed and submitted to quantitative Edman degradation.

171

Then, the N-terminal Gly-1 residue was acetylated by treatment with acetic anhydride and diisopropylethylamine. The peptide was deprotected and cleaved from the resin by HF treatment at -5/0 °C with the addition of anisole, p-thiocresol and dimethyl sulfide as the scavenger mixture. Preliminary syntheses in which the two Cys residues at positions 14 and 17 were protected with the HF labile 4-MeBzl group evidentiated some problems during the purification of the peptides. Better results were obtained when one ore both Cys residues were protected with the HF resistant Acm group. The purification scheme consisted of preparative HPLC on LKB TSK-CM cation exchange chromatography using 50 mM sodium dcetate, pH 5, as a buffer and a sodium chloride gradient, followed by preparative HPLC chromatography on Vydac C 18 using 0.05% TFA and a AcCN gradient as a solvent. The purified peptide elutes as a single peak in analytical HPLC and has an amino acid composition, after acid hydrolysis, consistent with the expected values. After removal of the Acm protecting group, the peptide will be submitted to CNBr cleavage in order to transform the Met-65 residue in the activated Hse>-65 lactone derivative to be used, in the presence of the (1-25)H segment, for the conformationally driven covalent semisynthesis with the synthetic (2.) (66-104) fragment.

Acknowledgements Work supported in part by the Italian National Research Council (CNR), Rome. We wish to thank Dr. Hiroshi Taniuchi, NIH, Bethesda, Md., USA, for advice and helpful discussions.

References 1. Gozzini, L., H. Taniuchi, C. Di Bello (1986), Fed. Proc., manuscript in preparation.

1617 and

2. Corradin, G., H.A. Harbury (1970), Biochim. Biophys. Acta, 221, 3036-3039. 3. Di Bello, C., M. Tonellato, A. Lucchiari, O. Buso, L. Gozzini (1987). In: Peptide Chemistry 1987 (T. Shiba and S. Sakakibara, eds) Protein Research Foundation, p. 409-412 and manuscript submitted to Int. J. Peptide and Protein Res.

METHODOLOGY AND STRATEGY IN PEPTIDE SYNTHESIS: TO THE SYNTHESIS OF UBIQUITIN

AN APPROACH

J. Green, O.M. Ogunjobi and R. Ramage University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ

Ubiquitin 1

is a polypeptide

constituted

which has a stable tertiary S-S

bonds.

It

exhibits

sequence conservation

structure, a

most

of

76

acids

without the aid of

remarkable

and the X-ray

amino

crystal

evolutionary

data

at

1.8 A

resolution 2 reveals a compact, globular small protein with a high

degree

of

secondary

structure

having

a

hydrophobic

core. UBIQUITIN 10 20 M Q I F V K T L T G K T I T L E V E P S 30 40 D T I E N V K A K I Q D K E G I P P D Q 50 60 Q R L I F A G K Q L E D G R T L S D Y N 70 I Q K E S T L H L V L R L R G G 3.5 turns cr-helix 23-24 mixed sheet of 5 strands (1-7), (64-72) parallel (10-17), (40-45), (48-50) antiparallel reverse turns involve residues (7-l'0) , (51-54) , (62-65) (3-bulges 10, 11 and 7 64, 65 and 2

Ubiquitin

has

participating

crucial

intra-cellular

in

division

cell

abnormal proteins. constituent

of the

and

biological in

the

functions 3

proteolysis

of

Also ubiquitin has been identified as a lymphocyte homing

receptor 4

and

it has

been found to be present in inclusion bodies associated with neurological disorders.

We have adopted an approach to the

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin New York-Printed in Germany

173

problem of understanding the stabilisation of ubiquitin tertiary structure which is based on the chemical synthesis of ubiquitin, and also major fragments, in order to compare the 3-D structure of these regions independent of the complete sequence and when incorporated into the intact protein. The initial phase of the programme has involved solid phase synthesis on polystyrene resin with Fmoc methodology for N a protection5, t-butyl derived side chain protection and the use of the TFA-labile linker developed by Wang 6 . Consideration of the ubiquitin sequence having four arginine residues and a labile C-terminal Gly.Gly unit suggested to us that the N G Mtr protection for Arg required to be modified in order to afford a more acid-labile guanidine protecting group. This has led to the development and introduction of the Pmc group7 which can be cleaved by TFA under similar conditions to those required to deprotect peptides having t-butyl derived side chain protection. The adopted coupling protocol involved (i) symmetrical anhydride 30 min (Die in DMA, or DMF, and CH 2 C1 2 ) followed by (ii) hydroxybenzotriazole active ester, 2 h (in DMA, or DMF and CH 2 C1 2 ), except for Asn and Gin where only HOBt/DCC activation was used. The repetitive deprotection of the a N -Fmoc groups was monitored by ultra-violet spectroscopy and was effected in 3,3,3 and 1 minute treatments of the resin-bound peptide with 20% piperidine in DMF. It was found that in the latter stages of the synthesis it would be useful to extend this protocol by a further minute. Deprotection and release of the peptide from the resin was effected using 90% TFA (containing 5% water) plus 5% thioanisole and 5% ethyl methyl sulphide scavengers. After removal of most of the TFA the residual oil was stirred with ether containing 2% mercaptoethanol and the solid was immediately dissolved in 8M urea containing 20 mmolar ammonium bicarbonate (pH 7.8). The sample was dialysed

174

peptide was lyophilised and purified graphy

and

preparative

(11-35), (36-47),

FPLC.

by G50 gel The

chromato-

sub-units

(1-35) ,

(48-76) and (60-76) of ubiquitin have been

isolated in this way and FAB mass spectrometry has proved to be most useful for characterisation.

In addition

(1-35)

has been sequenced successfully by ABI Warrington.

Acknowledgment. We thank SERC for financial support to O.M.O. and J.G. addition we

are

indebted

to Applied

Sharp and Dohme and Wendstone

Biosystems

Chemicals

In

Ltd,

Merck

for support.

We

thank K. Shaw and B. Whigham for technical support.

References 1.

Goldstein, G. , M. Scheid, Y. Hammerling, D.H. Schlesinger and D.H. Niall. 1975. Acad. Sei., 72, 11.

E.A. Boyse, Proc. Natl.

2.

Vijay-Kumar, S., C.E. Bugg and W.J. Cook. Mol. Biol., 914, 531.

1987.

J.

3.

Hershko A. and A. Ciechanover. Acid. Res. Mol. Biol., 33, 19.

Prog.

Nucl.

4.

Gallatin, M., T.P. St. John, M. Siegelman, R. Reichert, E.C. Butcher and I.L. Weissman. 1986. Cell, 44, 673.

5.

Atherton, E., D. Harkiss Bioorganic Chem., 8, 351.

6.

Wang, S.S.

7.

Ramage R. and J. Green. 1987. Tetrahedron Letters, 28, 2287; Green, J., O.M. Ogunjobi, R. Ramage, A.J.S. Stewart, S. McCurdy and R. Noble. 1988. Ibid, 29, 4341.

1973.

and

1986.

R.C.

Sheppard.

1979.

J. Amer. Chem. Soc., 95, 1328.

LARGE SCALE SYNTHESIS OF y-ENDORPHIN

W.A.A.J. Bijl, M.C.A. van Tilborg and J.W. van Nispen Organon Scientific Development Group, P.O. Box 20, 5340 BH Oss, The Netherlands

Introduction The endogenous opioid peptide y-endorphin comprises the N-terminal 17-peptide of |S-endorphin and has the following primary structure: 1 5 10 14 17 H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Se r-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH. In addition, it induces in animals behavioural effects that resemble those of neuroleptic drugs (1,2). In a number of patients y-type endorphins have shewn antipsychotic activity (3,4). A large amount, needed for further studies, was synthesized using the fragment condensation approach based on our earlier work (5).

Materials and Methods Three protected tetrapeptides (sequences 14-17, 10-13, 6-9) and one pentapeptide (sequence 1^5) were synthesized on a large scale (50-600 mmol of the starting amino acid derivatives). Tert.-butyl derived groups were used for permanent protection and Z-groups for a-amino protection with the exception of the Tyr residue in the pentapeptide; after their hydrogenolytic removal (in DMF using Pd,C as catalyst) the filtrate was used directly for the next coupling. Methyl or tert.butyl esters were used for carboxyl protection. Most of the couplings were performed in DMF using DCC (1.1-1.5 equiv.) and HOBt (1-2 eq.).

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin N e w York - Printed in Germany

176

Products were isolated by precipitation or crystallization after appropriate extractions. The purity of all intermediates was checked by TLC (Merck silica gel plates, F 254; 0.25 mm) using several solvent systems. [a]D values and melting points of solid products were determined and compared with literature values. The assembly of the 17-peptide was carried out at a scale of approx. 10, 40 and 55 mmol successively.

Results and Discussion Z-Leu-Val-Thr-Leu-OtBu was hydrogenated and the resulting compound acylated with Z-Ser-Gln-Thr-Pro-OH (obtained from the tert.butyl ester by treatment with 90% aqueous TFA) using the DCC/HOBt method; yield approx. 74%. Z-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe was treated with N 2 H 4 .H 2 0 to give in high yield the crystalline hydrazide; an azide reaction (using isopentyl nitrite for conversion of the hydrazide) with H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OtBu gave the protected segment 6-17 in 76% yield after precipitation with water. In the final coupling step between Boc-Tyr-Gly-Gly-Phe-Met-OH [obtained from Boc-Tyr-ONSu and the free tetrapeptide] and the C-terminal 12-peptide DCC and HOBt were again used; after precipitation with water, protected y-endorphin was isolated in 89% yield. Removal of the protecting groups with TFA-H20 (9-1), under N2 and in the presence of anisole at room temperature for 2-3 h was followed by isolation of the product and the exchange of TFA ions for acetate ions. Purification of 80 g crude material (see Fig.l A) was carried out in 3 portions by counter current distribution (Craig partition; solvent system l-BuOH-HQAc-H2O-toluene = 4-1-5-0.1, by vol.). We used HPLC to pool the fractions and isolated the peptide by lyophilization from water (33 g of first crop). The product was analyzed and characterized using TLC, HPLC (see Fig.l B, 98.1% main component), amino acid analysis, perchloric acid titration (89.4% peptide content), isotachophoresis (5.9% acetic acid; no other anions), Karl Fischer titration (3.9% H 2 0) optical rotation 1H-NMR spectroscopy and FAB-MS.

Referènces 1. D. de Wied, G.L. Kovàcs, B. Bohus, J.M. vein Ree and H.M. Greven, Eur. J. Pharmacol. 49, 427 (1978). 2. J.M. van Ree and 0. Gaffori, Life Sci. 33 (suppl. I), 619 (1983).

3. J.M. van Ree, W.M.A. Verhoeven and D. de Wied, Prog.

Neuro-Psychopharmacol. & Biol. Psychiat. 9, 561 (1985).

4. Organon, unpublished results.

5. W.A.A.J. Bijl, J.W. van Nispen and H.M. Greven, Reel. Trav. Chim. Pays-Bas, 98, 571 (1979).

SOLID P H A S E S Y N T H E S I S O F R H E S U S M O N K E Y

RELAXIN

P.J. Kelly, P.F. L a m b e r t , G.W. T r e g e a r , J . D . W a d e and P.D. Howard Florey

Institute, U n i v e r s i t y

3052, A u s t r a l i a ,

and * G e n e n t e c h

of M e l b o u r n e ,

Ine, San

Johnston*

Parkville,

Victoria,

Francisco, California

94080,

USA.

The peptide

hormone relaxin

t i s s u e of

the r e p r o d u c t i v e

to s o f t e n

and dilate

t h e pubis

symphysis at

the cervix

u t e r i n e contractions^-. B) w i t h

one intrachain

birth

smooth muscle

and

p r i o r to p a r t u r i t i o n .

and,

during

early

connective

A p r i n c i p a l role is It also relaxes

pregnancy,

inhibits

R e l a x i n c o n s i s t s of two p o l y p e p t i d e chains (A a n d a n d two i n t e r c h a i n d i s u l f i d e links in the m a n n e r

e x a c t l y analogous to t h a t of 1

affects the

tract d u r i n g p r e g n a n c y .

5

insulin^.

|

|

20

15

7

/

H-Gln-Leu-Tyr-Het-Thr-Leu-Ser-Asn-Lys-Cys-Cys-His-Ile-Cly-Cys-Thr-Lys-Lys-Ser-Leu-Ala-Lys-Phe-Cys-OH

H - L y s - T r p - M e t - A s p - A s p - V a l - H e - L y s - A l a - C y s - G l y - A r g - G l u - L e u - V a l - A r g - A l a - C l n - H e - A l a - H e - C y s - G l y - L y s - Ser 1

5

10

15

20

25

Thr- Leu -CIy-Lys-Ar£-Ser-Leu-OH 30

Figure 1 The

primary

structure

of

rhesus

d e t e r m i n e d in

our laboratory

from p r e g n a n t

rhesus m o n k e y

s h o w n in

F i g u r e 1.

monkey

relaxin

has

recently

b y n u c l e i c acid s e q u e n c i n g of cDNA d e r i v e d o v a r i a n tissue.

T h e amino a c i d s e q u e n c e is

T h e rhesus m o n k e y p r o v i d e s a c o n v e n i e n t m o d e l

to study t h e p h y s i o l o g i c a l effects of r e l a x i n d u r i n g pregnancy. reason, t h e

solid phase

the conventional were assembled excesses of

synthesis of

using

preformed symmetrical

system

F o r this

this p e p t i d e was u n d e r t a k e n u s i n g

Boc-polystyrene methodology. separately

been

double

Each

couplings

a n h y d r i d e s of

of t h e of

two chains

two-fold

molar

p r o t e c t e d amino acids.

Peptides 1988 © 1989 Walter de Gruyter & Co., B e r l i n - N e w York-Printed in Germany

179 All arginine, glutamine and asparagine residues were coupled in DMF after activation with amino acids, Asp and

DCC and

HOBt.

and sidechain

Glu, OBzl; Trp, For.

from the

solid support

After

scavengers (p-cresol,

purified by

protection was as follows: Ser and Thr, Bzl;

Tyr, 2-BrZ;

Bom, and

ethanedithiol for

The Boc group gave Na-protection for all

Cys, 4-MeBzl;

assembly each

reduced A-

protected peptide was cleaved

by 1 h treatment at -4°C with high H F containing thiocresol and

B-chain).

DMS for

were 10%

and 20%

and B-chains.

A-chain, and p-cresol and

The crude S-thiol peptides were separately

preparative reverse-phase

Overall yields

Lys, C1Z; Arg, Tos; His,

hplc

on

C18

respectively for

and

C4

supports.

the highly purified,

Each peptide gave amino acid analysis results

close to theory and analytical hplc in different buffer systems confirmed their high purity.

Figure 2: Hplc monitoring of combination synthetic rhesus monkey relaxin A- and B-chains. Column: Brownlee RP-300; Buffer A, 0.1% aq. TFA; Buffer B, 0.1% TFA in CH 3 CN. Flow rate, 1.5 ml/min. Linear gradient of 2040%B in 40 mins was used. Symbols: A r , S-reduced A-chain; A Q , oxidized A-chain; B r , Sreduced B-chain; R, relaxin.

10

20

30

40

10

20

30

40

Tims ( m i n i )

The results

of numerous small-scale chain combination experiments led to

the development of optimum conditions for synthetic rhesus monkey relaxin formation. pH 10.5

The S-reduced peptides were combined in a 1:1 ratio (w/w) at

in the

following manner.

oxidize in

air for

then added

the B-chain.

(as assessed after 5

18 h

to produce

product isolated

2) and

A-chain was

first

rp-hplc). the reaction

by preparative

allowed

to

a stable intermediate to which was

Relaxin spontaneously formed almost

by analytical

h (Figure

The

hplc on

No further

combination occurred

was terminated C8.

immediately

and the

The homogeneity

target of

the

180 synthetic rhesus

monkey relaxin (0.6% overall yield based on starting B -

chain-resin) was

demonstrated by

sensitivity microsequencing. expected composition

analytical hplc

Amino

acid analysis

(Figure 3), also

and high

confirmed

the

and showed that the chains were present in an equal

ratio.

Figure 3: Column, Brownlee RP300; Buffers A and B and flow rate were as described in Fig. 2. Linear gradient of 20-40XB in 50

10

20

30

40

Time (mins)

The synthetic peptide was highly active in the mouse pubic symphysis ligament assay^.

M e a n ligament length significantly increased to 0.97mm

(± 0.10) by addition of 0.5ng of peptide. 0.04).

Control length was 0.24mm (+

Further studies are in progress and will provide valuable

information on the role of relaxin in the primate.

References

1.

Steinetz, B.G., 0'Byrne, E.M. and Kroc, R.L.

In: Dilation of the

Uterine Cervix (Nalftolin, F. and Stubblefield, P.G., eds.) New York. pp.157-177

2.

Raven Press,

1980.

James, R., Niall, H.D., Kwok, S. and Bryant-Greenwood, G. 1977.

Nature, 267, 544.

3.

Steinetz, B.G., Beach, V.L., Kroc, R.L., Stasilli, N.R., Nussbaum,

R.E., Nerith, P.J. and Dun, R.K. 1960 Endocrinol., 67, 102.

COMPARISON OF FOUR APPROACHES TO THE SOLID PHASE-SYNTHESIS OF THE MAGAININS, SOME OF ITS SEGMENTS AND ANALOGUES

H. Echner, H. Voelter Abteilung für Physikalische Biochemie des Physiologischchemischen Instituts der Universität Tübingen, Hoppe-Seyler Str. 4, D-7400 Tübingen, FRG

Int roduction Magainins are peptides with a broad spectrum of microbiological activities. peptides

M.

from

Zasloff the

(1) has

skin

of

isolated

African

two

closely

frog Xenopus

related

laevis.

Both

peptides contain 23 amino acid residues and differ only in two positions

(1,2).

The

Magainins,

some

segments

and

analogues

have been synthesized in our laboratory by four different solid phase methods.

Results The details of the four different synthetic strategies are given in scheme 1. All

coupling

steps

are

monitored

using

the

Ninhydrin

method

(3). All products are purified by gel chromatography on a TSK HW-40(S) (column 1.6 x 100 cm , eluant: 5% acetic acid) and the purity

is

checked

by

analytical

18/1.25 x 4 mm, 5 |i ). The done on a p-benzyloxybenzyl resin

(4)

Ser(tBu)-OH

with

(LiChrospher

100,

RP1 is

alcohol/polystyrene/divinylbenzene

following

amino

acid

derivates:

Fmoc-

, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Ile-OH, Fmoc-

Glu(0But)-OH, OH,

the

HPLC

synthesis of native Magainin

Fmoc-Gly-OH, Fmoc-Val-OH,

Fmoc-His(Bum)-OH,

Fmoc-Leu-OH.

All

Fmoc-Phe-OH, Fmoc

amino

Fmoc-Alaacids

are

prepared according to reference (5). Fmoc-His(Bum)-OH is a pro-

peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in G e r m a n y

182

duct of novabiochem, Laufelfingen

(Switzerland). All

coupling

reactions, including the attachment of the first amino acid to the resin is carried out with BOP reagent as described recently for the synthesis of thymosin ai

(6), no double couplings are

performed. After the 11th and the 18th coupling step, peptide resin is taken out from the reactor and the Magainins 1 (13-23) and

(6-23)

are

(4:1). After

cleaved

every 5th

from

the

coupling

resin with

TFA/thioanisole

an acetylation step

is made

with Ac2O/pyridine. The final peptide is cleaved from the resin by the same procedure as already described. The

synthesis

of

(p-fluorophenylalanine

12

• 1 6 )-Magainin

1

is

performed with the PAM resin (8). Boc groups are used for temporary and

benzyl residues for permanent

protection. For the

first time a new histidine derivative is introduced for peptide synthesis:

Boc-N 1 "-2.4.6-triisopropylbenzenesulfonyl-histidine

(7). The methionine residue is coupled as Boc-Met-OSu and the final cleavage is carried out by the Sakakibara-HF procedure in the presence of anisole and ethanedithiol. The

(Aib 10 )-Magainin

1-amide

is

synthesized

on a

new

Fmoc-4(4-aminomethyl-3.5-dimethoxy-phenoxy)-valeric

resin.

acid

is

coupled with the BOP reagent to the aminomethyl-polystyrene resin (9). After Fmoc cleavage, the resin is ready for peptide coupling with the aim to produce peptide amides. The used protecting groups are the same as described in the first synthesis. Removal of the peptide amide from the resin is carried out by treatment with TFA/thioanisole (4:1). (D-Ala 3 )-Magainin with

2

is prepared

on PepSyn

4-Hydroxymethyl-3-methoxyphenoxyacetic

handle. A

combination of Fmoc- and

used

all

All

and

couplings

are

polyamide

as

a

tert.-butyl protection

is

performed

in

acid

support

DMF

(10) as

solvent.

syntheses are carried out once and are not optimized

yield or purification.

for

183 Scheme

1.

Priaary

sequences

MAGAININ Magainin

M a g a i ni n p e p t i d e s .

Residues

that

differ

between

the

two

pept ides

are

underli ned.

5 10 15 20 X : G1y-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Glv-Lva-Phe-Gly-Lya-Ala-Phe-Val-Gly-Glu-Ile-Het-Lya-Ser 11: G 1 y - 1 l e - G l y - L y s - P h e - L e u - H i s - S e r - A l a - L y a - L y a - P h e - G l y - L y s - A l a - P h e - V a l - Q l y - G l u - 1 l e - H e t - A a n - S e r

Table

1. S u r v e y

Resin

Carrier

about

the

Polysty

I'rotecl ing Groups Coupli Condi

of

Strategies

rene

used

for

the

Synthesis

of

Hagainin

(HAG)

peptides.

Polystyrene

Polystyrene

Polyamide/PepSyn

p-benzvloxybenzylalcoho1

PAH

4-(4-aainoaethy1-3.5-diaethoxy-phenoxy(valeric acid

4-hydroxyaethyl3-aethoxyphenoxyacetic

Faoc, t-butyl 8 o c i BUB

Boc, Tip7 ' , benzy1

Faoc, t-butyl Boc, Bua

Faoc, t-butyl, Boc, Bua

ng tions

BOP, Boc-Het-OSu Labortec SP 640

Labortec SP 640

Labortec SP 640

Labortec SP 640

Final CI e a v a g e

TFA/thioanisole (4:1)

HF/anisole/ ethanedithiol

TFA/thioanisole (4:1)

5X T F A / C H 2 C I 2 / thioanisole

Synthetic Peptide

native

(p-F-Phe1 HAGi

(Aib'O ) H A G i - N H 2

(D-Ala3 )-HAG]

Acetylati

on

MAGi

After

every

16

•

5th coupling

Yield after C1 eavage l'uri ty Crude

2

)-

step,

a c e t y l a t ion w i t h

of the Product

Acj O

/ p y r i d ine

90\

63%

38 . 3*

31 , 5 k

References 1 .

Z a s l o f f ,

M . .

1 9 8 7 .

2 .

C a n n o n ,

M . .

1 9 8 7 .

3 .

K a i s e r ,

E . ,

R . L .

P r o c .

N a t l .

N a t u r e

3 2 8 .

C o l e s c o t t ,

A n a l .

B i o c h e m .

3 4 »

5 9 5 .

4 .

W a n g ,

S . S . .

1 9 7 3 .

A m .

C h e m .

5 .

T e n

K o r t e n a a r ,

B . I . P r o t .

R a a b e n , R e s .

P . B . W . ,

J . H . M .

2 7 »

B . G .

A d a m s ,

v a n

(

W .

V o e l t e r :

L i e b i g s

7 .

E c h n e r ,

H . ,

W .

V o e l t e r .

1 9 8 7 .

8 .

M i t c h e l 1 ,

3 0 »

1 0 .

i

5 4 4 9 .

P . I .

1 3 2 8 .

J . M .

T e s s e r .

C o o k .

P e e t e r s ,

1 9 8 6 .

I n t .

J .

P e p t .

B . W .

G .

Z .

E r i c k s o n ,

1 9 7 6 .

J .

B a r a n y .

A m .

A n n .

( i n

N a t u r f o r s c h .

M . N . C h e m .

1 9 8 7 .

C h e m .

I n t .

R y a b t s e v , S o c *

9 8 »

J .

P e p t .

p r e s s )

4 2 b .

1 5 9 1 .

R . S .

H o d g e s ,

7 3 5 7 .

P r o t .

R e s .

2 0 6 .

S h e p p a r d , R e s .

A . R . ,

M e r r i f i e l d .

F . ,

M

3 9 8 .

H .

A l b e r i c i o ,

9 5 ,

D i j k ,

G . I .

E c h n e r ,

9 .

U S A .

B o s s i n g e r ,

S o c .

6 .

R . B .

S c i .

4 7 8 .

C . D .

1 9 7 0 .

J .

A c a d .

2 0 ,

R . C . , 4 5 1 .

B . J .

W i l l i a m s .

1 9 8 2 .

I n t .

J .

P e p t .

P r o t .

A

CONVENTIONAL AND SOLID-PHASE SYNTHESIS OF LEU-ANALOGS OF RAT MINIGASTRIN I. AND THEIR SEGMENTS L. Balàspiri. Cs. Somlai. P. E. Menvhàrt. K. Kovàcs. G. Remàk*. J. Lonovics*. V. Varrò* Institute of Medical Chemistry, *First Internal Clinic, Szent-Gyòrgyi, Medical School, Szeged, Hungary.

INTRODUCTION The gastrins, as well as the family of peptide hormones to which they belong have taken on renewed importance since they have been discovered in the brain and other nervous tissue. Several gastrins have been isolated from natural sources, then characterized and later synthetized. But the sequence of the rat gastrin I. was reported only once, in 1981 (1). No synthesis of rat gastrin I. or its Leu-analog have yet been reported. The biological existence of rat minigastrin I. is assumed but has not been proved. Until early 1980's, when the tert.butyl- and Fmoc-based synthesis has spread, few solid phase syntheses of gastrins have been reported. Synthetic gastrins and their analogs have been available mainly from solution syntheses. The main reason was the amino-acid composition of gastrins (more Glu, Trp and the Asp, Met, Tyr residues) which was unfavourable for solid phase methods involving strongly acidic conditions. RESULTS AND DISCUSSION We report solution phase and solid phase syntheses of Leu-analogs of rat little gastrin I. and that of minigastrins I. They are compared with former syntheses of human gastrin I., minigastrin I . We have synthetized the Leu-analogs of rat little (G-17) and minigastrin (G-14) I. by conventional solution phase fragment condensation from the usual 3 fragments, using minimal, Fmoc- and tert.-butyl-based, side-chain protection of the fragments.These fragments were coupled by DCC-HOPfp method. The isolated products were purified on SephadexLH-60 c o l u m n e l u t e d w i t h DMF-MeOH. The solid phase syntheses were manually produced on methyl-benzhydrylamino resin (0.23 mmole/g), using Fmoc-amino acid pentafluorophenyl activated esters (2) in two equivalent excesses (in DMF), with HOBt additive, after 30 minutes, at each coupling. All couplings were complete in maximum 75 minutes and no any extra coupling was required. After each coupling DMF was removed promptly with DCM during the washing cycles.

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin New York- Printed in Germany

185

Boc-

Glp-Arg-Pro-Pro-Leu-Glu-/Glu/,-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-PPhe «t. t „_ „ t „ t •NO. OBu OBui OBu t Bu OBu -OH Fmoc-OH H-3H_

r

rHo2

Boc-

-OH

OBut OBu* i Fmoc-j

OBu* i

Bu* i

HOPfp/DCC :

OBu* • -HH„

1. Piperidine/DMF 2. HOPfp/DCC 3. LH-60, DUF/MeOH R* O 2

Boo-

OBu* OBu*

OBu*

Bu*

OBu -HH-

1. HF/Anisole-Ethanedithiol-Dimethyl sulfide 2. Sephadex G-25 /0.4% ammonium acetate/ 3. Sephadex G-25/3.BuOH/EtOH/O.IM AcOH/ r.gastrin I. -NH„ r.minigastrin I. -iffiFigure

1.

Solution phase synthesis scheme for Leu rat gastrins I.

Glp-Arg-Pro-Pro-Leu-Glu-/Glu/j-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe Fmoc-Phe

R

OBu* 2. Fmoc-Asp-OPfp/HOBt' OBu* Fmoc-Asp-Phe

R

1. 50% Piperidine

Bu" OBu rN02 I I Glp-OPfp, Fmoc-Arg-OPfp,Fmoc-Glu-OPfp,Fmoc-Tyr-OPfp,Fmoc-Trp-OPfp rNO, OBu OBu" OBu Bu1OBu'' I 2 I , I , I I | Glp-Arg-Pro-Pro-Leu-Glu-/Glu/j-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe

15 steps

R

1. HF/AHIS0LE-DI1IETHYL SULFIDE-ETHAHEDITHIOL Glp-Arg-Pro-Pro-Leu-Glu-/Glu/j-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-NH2 2. DEAE-CELLULOSE 3. SEPHADEX G-50 Glp-Arg-Pro-Pro-Leu-Glu-/Glu/^-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-NH2 Figure

2.

Solid phase synthesis scheme for Leu rat gastrins I.

186

In both solution or solid phase syntheses protecting groups and the peptides from the resin were removed by HF treatment at 0 C with scavengers. Parts of crude peptides were purified either on twoSephadex G-25 columns (gel and partition chromatography) or on a single C-18 RP-HPLC semipreparative column, adding a linear gradient from 10% to 60% CH3CN-water-0.05% TFA. All syntheses proceeded smoothly with good amino-acid incorporation as proved by amino--acid analyses (i.e. for Leu-analog of rat gastrin I. found: Asp, 0.95; Arg, 0.98; Glu, 5.97; Pro, 2.06; Gly, 1.05; Ala, 1.06; Leu, 2.02; Phe, 0.96; Trp, 0.94; Tyr, 1.00). The purity controls of all synthetic peptides on C-18 Rp-HPLC analytical system stationary (Pharmacia-LKB) proved to be 97-99%. Acid secretion activities of all gastrin analogs in question and their pure segments were tested ( 3 doses, i.v. administration to Wistar rats) by conductometric bioassay (3). The ED50 ratios of the two gastrin analogs were very similar between 170% and 175%. Activity of the segments will be discussed separately. The overall yield varied between 45-55% in solution syntheses and between 55-62% in solid phase syntheses. These results demonstrate that syntheses of both Leu-analogs of rat gastrins and their segments were succesfull; biologically and probably immunologically (under examination) the rat gastrins and their Leu-analogs are the same as the human ones.

ACKNOWLEDGEMENT This work was supported by grants from the Hungarian Ministry of Health (ti l l ) and from the Hungarian Academy of Sciences (OTKA).

REFERENCES 1. Reeve, J.R., Dimaline, Jr.R., Shively, J.E., Hawke, D„ Chew, P., Walsh, J.H.,Peptides 2, 453 91981). 2. Kisfaludy, L., Schon, I., Synthesis (1983) 325. 3. Halter, F., Kohler, B., Smith, G.M., Helv. Med. Acta Suppl., 50,113 (1971).

FMOC- MEDIATED SOLID PHASE ASSEMBLY OF HIV TAT PROTEIN

R.M. Cook, D. Hudson, D. Tsou MilliGen/Biosearch, 2980 K e m e r Blvd., San Rafael, CA 94901 D.B. Teplow, H. Wong Department of Biology, California Institute of Technology, Pasadena, CA 91125 A.Q. Zou, E. Wickstrom Department of Chemistry, University of South Florida, Tampa, FL 33620

Introduction The human immunodeficiency virus (HIV) encodes for several regulatory proteins which are essential for expression. The Tat protein, directly or indirectly, increases the utilization of mRNA. In human cells Tat causes an increase in the level of mRNA by approximately 10 times, whereas the amount of protein produced increases 500 fold. Tat is of relatively small size (86 residues), but its unusual composition and complex sequence pose exception synthetic problems. These include the presence of a strongly basic Arg rich region which might bind nucleic acids, the presence of many Gin residues, and also of 7 Cys residues. All Cys residues exist in free SH forms coordinated to A zinc atoms in a dimer. The synthesis of even uncomplicated proteins remains fraught with uncertainties. Almost all examples have employed the classical Merrifield method of synthesis, although the harsh acid deprotection is damaging in sensitive cases. One aim of this work was to test improvements to Fmoc protocols developed in the Biosearch laboratories. Other aims were to obtain sufficient pure material to analyze the structure and function of Tat and of partially protected forms and fragments.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w York- Printed in G e r m a n y

188

Results Polystyrene was selected for the support rather than encapsulated polydimethylacrylamide. The efficient (1) and generally useful (2) BOP + HOBt coupling method was adopted. The synthesis was performed on a MilliGen/Biosearch Model 9600 using protection and coupling times as shown in the table below. Residue Protection Coupling (hrs)

Met -

Glu Pro OBut 2 2

Val 2

Residue Protection Coupling (hrs)

Pro

Gly

Gin Pro Tmob 2 2

-

-

2

2

Ser But 2

Asp Pro OBut 2 2

Lys Boc 2

Arg Leu Glu Pro Trp Lys Mtr OBut Boc 2 2 2 2 2 2 2

His Fmoc 2

Thr But 2

Cys Trt 2

Tyr But 2

Phe

lie

-

-

2

2

Lys Boc 2

Arg Mtr 2

Thr But 2

Asn

His Cys Fmoc Trt 2 2

Gin Val Tmob 2 2

Cys Trt 2

Tyr But 1

Gly

Arq Mtr 2

Lys Boc 1

Gin Gly Tmob 1 1

Ser But 1

Gin Thr His Tmob But Fmoc 2 2 2

Ser But 1

Gin Ser Tmob But 1 1

Ala -

2

Cys Trt 2

-

2

•1 Residue Protection Coupling (hrs)

Cys Acm 2

Lys Boc 2

Lys Boc 2

Cys Acm 2

Cys Acm 2

Phe

Residue Protection Coupling (hrs)

Thr But 2

Lys Boc 1

Ala

Leu

Gly

lie

-

-

-

-

1

1

1

2

Ser But 1

Residue Protection .Coupling (hrs)

Arg Mtr 2

Gin Arq Tmob Mtr 2 2

Arg Mtr 2

Arq Mtr 2

Pro

Pro

-

-

1

1

Residue Protection Coupling (hrs)

Gin Val Tmob 2 1

Ser But 1

Leu

Ser But 1

Lys Boc 1

Gin Pro Tmob 2 1

Residue Protection Coupling (hrs)

Gly -

Asp Pro Thr Gly OBut But 1 1 1 1 1

Pro 1

Lys Boc 2

-

2

4> -

1

i -

1

Thr But 1

Arg Mtr 1

Glu OBut -

Samples, arrowed, and the final product were treated with Reagent R (TFA/Thioanisole/ethane dithiol/anisole; 90:5:3:2, 8 hours) which cleanly removed Mtr protection. The products were assessed by HPLC, AAA and sequencing. All peptides gave single main peaks on HPLC after DTT reduction, and sequenced correctly. No preview resulting from incomplete coupling was detected. The figure on the following page shows the Polyacrylamide gel electrophoresis of fully reduced and tris Acm forms of materials from G50-50 Sephadex chromatography.

189

Figure: SDS- Gel Electrophoresis of 86-mer fractions

30

21.5

>6.9-» 14.4*

Hp

8.2"*

Sfl

M.3

C4

c3

„ e4 e3 Sf2

d4

d3

fs

f4

3 or 4 designates void volume or subsequent fraction respectively; d & f are tris (Acm) derivatives, c & e are after Hg(0Ac>2 treatment; c & d are cleaved with Reagent R for 8 hours, f & e for 16 hours, standards were: Stl is Sigma MWSD517, St2 Amersham "Rainbow Markers".

Conclusions The results demonstrate a highly efficient assembly of one of the most complex series of peptides yet prepared by Fmocmediated solid phase synthesis.

No data is available on bio-

logical activity as yet.

Acknowledgement Thanks are due to Susan Morrison for SDS-Page studies.

References 1.

Biancalana, S., Hudson, D., Tsou, D., unpublished results.

2.

Hudson, D., Journal of Organic Chemistry, 1988, 53, 617.

SYNTHESIS OF A PROPOSED SEQUENCE FOR THE ASPARTIC PROTEASE OF THE HUMAN IMMUNODEFICIENCY VIRUS.

D. F . Veber, R . F . Nutt, S. F . Brady, E. M. Nutt, T. M. Ciccarone, V. M. Garsky, L. Waxman, C. D. Bennett, 3. A. Rodkey, I. Sigal, P. Darke.

Merck Sharp ¿c Dohme Research Laboratories, West Point, PA.

19486.

The human immunodeficiency virus (HIV-1), like other retroviruses, is thought to require an aspartic protease for processing of polyproteins.'

The noninfectivity

of a mutant virus modified at the protease active site lends support to hypotheses that

inhibitors

of this enzyme offer

treatment of AIDS. determined.

potential

as therapeutic agents for

the

The structure of the HIV-1 protease has not been directly

However, the common Asp-Thr-Gly of aspartyl proteases along with

likely (auto?) cleavage sites at Phe-Pro have been used to propose a reasonable sequence of 99-residues (Fig. 1)^ within the known gene structure of HIV-1.^ Pro-Gln-lle-Thr-Leu-Trp-Gln-Arg-Pro-Leu-VaJ-'nir-lte-Lys-lle-Gly-Gly-Gln-Leu-LysGlu-Ala-Leu-Uu-Asp-Thr-Gly-Ala-Asp-Asp-Thr-Val-Leu-Glu-Glu-Met-Ser-Leu-Pro-Gly Arg-Trp-Lys-Pro-Lys-Mel-llo-Gly-Gtylle-Gly-Gly-Phe-lle-Lys-Val-Afg-Gln-Tyr-AspGln-lle-Leu-lle-Glu-ne-Cys-Gly-His-Lys-Ala-lle-Gly-Thr-Val-Leu-Val-Gly-Pro-ThrPro-Val-Asn-lle-lle-Gly-Arg-Asn-Leu-Leu-Ttir-Gln-lle-Gly-Cys-TN-Leu-Asn-Phe Fig. 1 To date, synthesis of peptides in this size range without benefit of the isolated natural product has been precluded because the methods have not been considered sufficiently reliable to produce products which could be independently characterized.

In view of the health hazards associated with handling live virus and the

importance of obtaining sufficient enzyme to start the search for inhibitors, we initiated a chemical synthesis of this sequence.^

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , B e r l i n - N e w York-Printed in Germany

191

Recent

advances in the speed and fidelity of solid phase peptide synthesis,

improvements in the HF deblocking procedure and development of powerful purification methods led us to choose the solid phase method. initiated with Boc-Phe-O-Pam

resin (0.74 mmole Phe/g).

temporary protection throughout.

The synthesis was Boc was used for

The following sidechain protection was used:

tosyl for Arg, cyclohexyl for Asp and Glu, p-chlorocarbobenzoxy for Lys, p-methyl benzyl for Cys, 2-bromocarbobenzoxy for Tyr, N -benzyloxymethyl ln

N -formyl for Trp and benzyl for Ser and Thr.

for His,

Boc-amino acids were activated

using dicyclohexylcarbodiimide (DCCI) and introduced either as hydroxybenzotriazole (HOBt) esters [(Arg(Tos), Asn, Gin, His(Bom)] or as symmetrical anhydrides preformed in C H j C l j followed by solvent exchange with DMF.

All operations

were performed using an Applied Biosystems peptide synthesizer Model 430A. Amino acids were introduced using a minimum of two couplings per residue and as many as five per residue for difficult couplings.

"Capping" of amino termini

with acetic anhydride was included at the end of each amino acid incorporation. Comparison of cleaved 26-peptide prepared by either the "noncapping" or the "capping" protocol indicated higher purity and more facile isolation for material synthesized by the latter route.

Sequence analysis of the 99-peptide-resin a f t e r

removal of the terminal Boc protecting group showed cumulative preview of 7% within the N-terminal hi residues.

Sequence analysis carried out for 82 cycles

also confirmed the accuracy of synthesis toward the C-terminal region.

In order

to insure complete formyl group removal from Trp in the two-step HF reaction, 1,2-ethanedithiol (EDT) was added in the S^2 step and thiocresol was replaced with 1,4 butanedithiol in the SN1 step.

Initial purification was by gel filtration

(Sephadex G-50, and G-75), using 50% aqueous acetic acid as eluent. The product was characterized at this point for structure and purity using amino acid analysis, sequence analysis before and after CNBr cleavage, UV and disc gel electrophoresis. HPLC analysis was consistent with a major component or group of closely related peptides. The product was folded to active enzyme by dialysis in the presence of 0.1% bovine serum albumin against an optimized pH 5.5 buffer (0.05M NaOAc, 10 ^M DTT, lO'^M EDTA, 10% glycerol, 5% ethylene glycol).

The folded protein was

purified on a Sephadex G-75M column under non-denaturing conditions.

All of

the enzymatically active product was eluted at a point consistent with a molecular

192 weight of about 20,000, suggesting a dimeric structure.

This observation may

support proposals relating this enzyme to much higher molecular weight acid proteases through dimerization.^ Two lines of evidence have been used to establish the specificity and activity of the synthetically produced protein. First, it catalyzes specific hydrolysis between Tyr and Pro of Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, a cleavage site in the HIV polyprotein gag p55. Secondly, studies of cleavage of gag p55, expressed in yeast, have also shown the synthetic protein to be effective and specific for the expected cleavage sites. It has not been possible to establish an absolute purity or potency of the synthetic enzyme because of the unavailability of natural protease.

How-

ever, hydrolysis of 600-800 nmoles of octapeptide substrate/min/mg of enzyme suggest a relatively high purity (possibly 10-40%).

Nonetheless, the successful

chemical synthesis of this protease and substrates has served as a practical route to the establishment of screens for the discovery of specific inhibitors, which include the standard inhibitor of acid proteases, pepstatin.

In this study we have

attempted to combine optimal methodology developed in many laboratories to execute the synthesis of an active protease never before isolated. indicates the usefulness of synthetic methods available today.

The success

The partial puritv

emphasizes the continuing challenges to peptide chemists.

References 1. Toh, H., M. Ono, K. Saigo, T. Miyata

1985 Nature 315, 691.

2. Kohl, N.E., E.A. Emini, W.A. Schleif, L.J. Davis, J.C. Heimbach, R.A.F. Dixon, E.M. Scolnick, I.S. Sigal Proc. Natl. Acad. Sei. U.S.A. 1988 85 4686-4690. 3. Nutt, R.F., S.F. Brady, P.L. Darke, T.M. Ciccarone, C.D. Colton, E.M. Nutt, J.A. Rodkey, C.D. Bennett, L.H. Waxman, I.S. Sigal, P.S. Anderson, D.F. Veber Proc. Natl. Acad. Sei. U.S.A., 1988 85, 7129-7133. 4. Ratner, L., W. Haseltine, R. Patarca, K.J. Livak, G. Starcich, S.F. Josephs, E.R. Doran, J.A. Rafalski, E.A. Whitehorn, K. Baumeister, L. Ivanoff, S.R. Petteway, Jr., M.L. Pearson, J.A. Lautenberger, T.S. Pappas, J. Ghrayeb, N.T. Chang, R.C. Gallo, F. Wong-Staal 1985 Nature 313, 277-284. 5. Pearl, L.H., W.R. Taylor

1987 Nature 329, 351-354.

TEMPLATE-ASSEMBLED SYNTHETIC PROTEINS (TASPS) CONTAINING TWO FOLDING DOMAINS

M. Mutter, R. Gassmann, R. Hersperger, L. Kiirz, G. Tuchscherer Institute of Organic Chemistry, University, CH-4056 Basel, Switzerland

Introduction The construction of new proteins has become a challenging goal in peptide and protein chemistry (1). The critical hurdle in the 'de novo design' of a desired tertiary structure lies in our limited understanding of the folding pathway of a polypeptide to a globular conformation. The concept of assembling amphiphilic secondary structures via loops to predetermined single domain folding units is severely limited by the high tendency of these polypeptides to intermolecular aggregation. We have recently developed a new strategy (Template-Assembled Synthetic Proteins, TASP), which uses the tools of organic synthesis to construct macromolecules with a much higher propensity for intramolecular folding (2-5). Here, we describe the construction of TASP molecules exhibiting two independently folded single domains.

Results A schematic representation of the target molecules, T 8 -(4a)(4P), I, and T 8 -(4a 1 )(4a 2 ), II, is depicted in Fig. 1. According to the general prinicples of TASP design, we attach amphiphilic helical (a) and (J-sheet-(P) forming oligopeptides to a specially designed multifunctional earner peptide (template) (Table I). The intrinsic tendency for selfassociation of the amphiphilic peptide blocks results in a template-enhanced intramolecular folding of the molecule to a predetermined three-dimensional conformation. Computer-assisted molecular modelling suggests a low energy conformation, characterized by a 4-helix-bundle and a (J-barrel-like conformation (TASP I) on opposite sites of the |}-haiipin forming template molecule. Similarly, two 4-helixbundles ( 4 a j and 40^) are accommodated on opposite sites of the template in TASP n. Both molecules were synthesized by solid-phase procedures, making use of orthogonal protection techniques (Scheme I)(6). TASP I and II were readily soluble in aqueous buffer solutions at

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , B e r l i n - N e w York - P r i n t e d in G e r m a n y

194

w

F^tt 7

^Ju^Jj

II

I

I

^

|

H

T

T

J ^ J T X

La

a i

Fig. 1: Schematic representation of TASPI and TASPII

1. 2. 3. 4.

Trt (Boc) removal N-acylation Boc (Fmoc) removal 15 (13) coupling cycles a (a,)

5. Fmoc (Alloc) removal 6. 9 (15) coupling cycles p (a 2 ) 7. N-acylation 8. Deprotection and cleavage

V U a l U p )

,

(T

8

-{4a,)(4a

2

))

Scheme I: Solid-phase synthesis of TASP I (upper part of boxes) and TASP H (P = polymeric support)

195

Table I : Primary Sequences of TASPI and TASPII I«: T g -(4a)(4ß)

a : Ac-E-A-L-E-K-A-L-K-E-A-L-A-K-L-G ß: AC-(V-K)4-G a n : Tg-(4a 1 )(4a 2 ) a , : Ac-W-D-A-A-T-A-L-A-N-A-L-K-K-L-G a 2 : Ac-Y-E-K-A-F-I-E-F-R-E-F-S-S T: Ac-(K)4-P-G-(K)4-G a For nomenclature of TASP molecules, see ref. (2,3). different pH values and proved to exist as monomeric species. CD- and IR-data are in support of the postulated conformations in various systems. Most notably, template enhanced secondary structure formation was observed, i.e. the critical chain length for the onset of helical- and ß-sheeted conformations were significantly shorter in template assembled molecules compared to the corresponding single blocks. Also, a mutual stabilization of the two folding domains could be verified by denaturation studies. We have experimental evidence, that the folding pathway proceeds via a nucleation process of the secondary structures (K„), followed by intramolecular association (K^s). Prospectively, the newly designed two-domain TASP molecules may be considered as prototypes for macromolecules exhibiting binding and catalytic activity as well as immunological functions.

Acknowledgement

The support of the Swiss National Science Foundation is gratefully acknowledged.

References 1. a) Mutter, M. 1988. Angew. Chem. Int. Ed. 24, 639-653. b) Mutter, M„ K.-H. Altmann, G. Tuchscherer and S. Vuilleumier. 1988. Tetrahedron 44, 771-785. 2. Mutter, M. 1988. In: Peptides Chemistry and Biology (G.R. Marshall, ed.). Escom, Leiden, pp. 349-353. 3. Mutter, M„ E. Altmann, K.-H. Altmann, R. Hersperger, K. Nebel, G. Tuchscherer, S. Vuilleumier, H.-U. Gremlich and K. Müller. 1988. Helv. Chim. Acta 21, 835-847. 4. Mutter, M. and G. Tuchscherer. 1988. Makromol. Chem. Rapid Comm. 2,437-443. 5. Mutter, M. 1988. Trends in Biochem. Sei. 12,260-265. 6. Mutter, M. and R. Hersperger: Proteins (in press).

PEPTIDE

SYNTHESIS

ON

POLYSTYRENE-GRAFTED

POLYETHYLENE

SHEETS

Rolf H. Berg,*® Kristoffer Almdal,b Walther Batsberg Pedersen,b Arne Holm,a James P. Tam,c and R.B. Merrifield c

department

of General and Organic Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark bChemistry Department, Ris0 National Laboratory, DK-4000 Roskilde, Denmark cThe Rockefeller University, 1230 York Averne, New York, N.Y. 10021, U.S.A.

Introduction The present work concerns a novel approach (1) to solid-phase synthesis of peptides. This approach is based on the provision and use of a solid support comprising polyethylene sheets to which are grafted high molecular weight and essentially noncross-linked polystyrene chains, which function as efficient carriers to support the synthesis of peptides. The sheets are well suited both to produce a single peptide via the "linear" solid-phase scheme or to produce multiple peptides via a rapid "parallel" scheme. The method applies to conventional solidphase methodology and is readily adapted to both microgram and milligram scale synthesis.

Results and Discussion

Polystyrene-grafted polyethylene sheets Polystyrene-grafted sheets were prepared by a radical-initiated reaction between the polyethylene sheet and styrene monomer present in a methanolic solution. Poor swelling of the grafted polystyrene chains during the grafting process in such relatively hydrophilic surroundings maintains the mobility of *To whom correspondence should be addressed

Peptides 1988 © 1989 Walter de G r u y t e r & Co., Berlin • New York - Printed in G e r m a n y

197

the growing polystyrene chains at a low level. Consequently, the diffusion-controlled chain-termination processes will be retarded and thus facilitate the growth of particularly

long

polystyrene chains. An attractive feature of the high molecular weight of the grafted polystyrene chains is that they may be presumed to behave as if they were in homogeneous

solution.

For the purpose of peptide synthesis a polyethylene sheet was grafted to the extent of 443% (Graft% = Q m a s s of final sheet) - (mass of polyethylene)!] x 100/(mass of polyethylene)). Peptide

Synthesis

The following describes a manual "linear" synthesis of two human parathyroid hormone fragments: pentapeptide and decapeptideCAsp 7 6 J-hPTH-(75-84).

hPTH-(80-84)

Equally sized rectangular

strips (1.5 cm x 4.5 cm) of 443% polystyrene-grafted

poly-

ethylene sheet were aminomethylated by a procedure similar to that of Mitchell et al. (2) to give a final substitution of 1.00 mmol NH2/g sheet as determined by using the quantitative ninhydrin test developed by Sarin et al.

(3). Initial

loading

of BocGln was carried out quantitatively via the preformed Pam handle described by Tam et al.

(4).

Ser(Bzl)Gln-0CH2-Pam-sheet

BocValAsp(OBzl)ValLeuThr(Bzl)-

and

BocLys(C1Z)AlaLys(C1Z)-

Lys(C1Z)AlaLys(C1Z)Ser(Bzl)Gln-0CH2-Pam-sheet

were assembled

stepwise following a standard solid-phase protocol. Each residue was coupled once as a preformed symmetrical anhydride (3 equiv., 0.05 M ) in DMF:CH2Cl2 (1:4, v/v) and except for the coupling between BocSer(Bzl) and H-Gln-0CH2~Pam-sheet

(94.0%

efficiency) the coupling efficiency was always ^ 99.7%. The free peptides were obtained by deprotection and cleavage from the sheet support when treated with HF:anisole (9:1, v/v) for 1 h at 0°C. Figure 1 shows HPLC chromatograms of the crude peptides. The yield of hPTH-(75-84) calculated from the content in the crude product was ca. 85% based on quantitative amino acid analysis and overall yields of the pure peptides were ca. 70%. Further work is in progress in our

laboratories

to investigate this approach to solid-phase peptide

synthesis.

198

Minutes Figure 1. Analytical HPLC chronatograms of (A) exude H-LysAlaLysSerGln-OH and (B) crude H-ValAspValLeuThrLysAlaLysSerGln-OH on pBONDAPAK™ Cxg (300 x 3.9 mm, 10 pin, Waters). Buffer A, H20/0.095% CF3OOOH; buffer B, 90% acetcnitrile/10% H2O/0.072% CF3OOOH; flow rate, 1.3 ml/mixi.

References 1.

P a t e n t p r o t e c t i o n has been a p p l i e d

for.

2.

M i t c h e l l , A . R . , S. B. H. Kent, B. W. E r i c k s o n and R. M e r r i f i e l d . 1976. T e t r a h e d r o n L e t t . , 3795-3798.

3.

S a r i n , V. K . , S. B. H. Kent, J. P . Tam and R. B. M e r r i f i e l d . 1981. A n a l . Biochem. 117, 147-157.

4.

Tam, J. P . , Merrifield.

S. B. H. Kent, T . W. Wong and R. B. 1979. S y n t h e s i s , 955-957.

POLYSTYRENE-POLYOXYETHYLENE GRAFTCOPOLYMERS FOR HIGH SPEED PEPTIDE SYNTHESIS

W. Rapp, L. Zhang, R. Häbich, E. Bayer Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 7400 Tübingen 1

Introduction Increasing importance of peptides in biochemistry places new demands on peptide chemistry. A large number of different peptides must be synthesized in a short time and this requirement can only be met by automated synthesis processes. With SPPS in combination with the continuous-flow process we developed a method in which reagent consumption is reduced to a minimum and on-line monitoring allows the reaction profile to be checked.

Results and Discussion In "polymer-supported reactions" the reaction takes place within the bead. The overall reaction rate is thus governed by both the peptide coupling reaction rate and the diffusion time. Parameters such as geometry of the support and composition of the resin influence the reaction behaviour. We have developed new supports consisting of a low crosslinked polystyrene-matrix grafted with polyoxyethylene spacer arms. In different solvents the graft copolymers show uniform swelling factors and the new supports are pressure stable up to 200 bar with capacities up to 0.25 meq/g. Monosized microparticular graft copolymers were developed, each polymer bead having the same diameter as a consequence of bead size uniformity. We divide the total reaction space, located in the polymer support, into small and exactly equal spaces. The effect upon the reaction is that identical conditions are encountered on each bead at any time. Microparticulate monosized graft copolymers enable us to carry out peptide coupling

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin N e w York - Printed in Germany

200

total cycletime: coupling: method: yield/HPLC:

27 min 15 min DIC/HOBt 85 %

S_Jv—l Figure 1:

HPLC: H20/ACN/0.1 % TFA

=

"

Figure 2:

HPLC: H20/ACN/0.1 % TFA

total cycletime: 22 min coupling: 8 min method: DIC/HOBt yield/HPLC: 82 %

201 within 2.5 min with cycle-times in CFPS of 10 min or less. We have synthesized peptides of various chain length up to the sequence of p-endorphin with 31 amino acids residues by the FMOC-strategy at the graft copolymer. We used a super acid-labile handle (1-5 % TFA). Generally coupling was done with 4 fold excess of FMOC-AA. Activation was carried out with DIC/HOBt. After cleavage and deprotection TFA is evaporated, the peptide dissolved in acetic acid and precipitated with ether. From this product without further purification HPLC was done. Figure 1 shows the 16-peptide H-Met-Ala-Cys(S-tBu)-Ser-Thr-Leu-Pro-Lys-Ser-ProLys-Asp-Lys-Ile-Asp-Pro-OH. Only single coupling was done, and the total synthesis time was 6 h 45 min. The time for synthesizing the 31-peptide p-endorphin was 11 h 8 min (Fig. 2). Asn and Gin were coupled as Pfp-esters, 20 min each and for lie we used double coupling.

Conclusion The polystyrene-polyoxyethylene graftcopolymers allows highspeed peptide synthesis in CF reactors with coupling times of 20 min or less. Monosized supports with reduced bead diameter decrease peptide coupling to 2.5 min and coupling cycles to less than 10 min. In spite of this short coupling times we obtain peptides with high purity.

CONTINUOUS FLOW ULTRA-HIGH LOAD POLYMER SUPPORTED PEPTIDE SYNTHESIS WITH SOFT GEL PACKINGS

A . F . C o f f e y , R. Epton and T . Johnson Wolverhampton P o l y t e c h n i c , Wolverhampton, England, WVl 1SB, U.K.

Conventional s o l i d flow

(gel)

phase supports deform when used under continuous

(CF) i n packed column r e a c t o r s .

pressures e v e n t u a l l y r e s u l t .

R e s t r i c t e d f l o w r a t e s and high back

S w e l l i n g v a r i a t i o n s , which occur as r e a p t i o n s

are performed and as r e a g e n t s o l u t i o n s are changed, exacerbate the problem. Low pressure CF with s o f t g e l s e i t h e r r e q u i r e s l o o s e packed columns o f a d j u s t a b l e volume

(1) or i s c o n f i n e d t o Fmoc chemistry

(2) .

The b e s t

developed low pressure CF method employs a r i g i d macroporous k i e s e l g u h r packing c o n t a i n i n g p o l y ( d i m e t h y l a c r y l a m i d e )

g e l w i t h i n i t s pores

(3).

Loading must be kept low because the entrapped g e l s cannot expand t o accommodate an i n c r e a s i n g amount o f

peptide.

Our i n t e r e s t i n u l t r a - h i g h l o a d methods has l e d us t o d e v i s e a new, low p r e s s u r e , manually s e r v i c e d CF system f o r use with s o f t g e l s In t h i s ,

(Figure

1).

the CF column r e a c t o r i s s e t up with a s o l v e n t f i l l e d gap o v e r

the packed g e l bed. bed f l u i d i s a t i o n .

This a l l o w s f o r g e l expansion and f o r

intermittent

Two f o u r way taps are used t o e f f e c t d e l i v e r y

r e a g e n t s o l u t i o n s and o f washing s o l v e n t s ,

of

d i r e c t from a p e r i s t a l t i c pump,

t o the top o r t o the bottom o f the r e a c t o r .

In CF, l i q u i d s l e s s dense

than the r e a c t o r c o n t e n t s are d e l i v e r e d t o the top and l i q u i d s more dense are d e l i v e r e d t o the bottom.

This causes l a y e r e d displacement o f

reactor

f l u i d s , which t r a v e l i n uniform bands through the g e l bed and across the gap.

I n t r o d u c t i o n of a r e a g e n t s o l u t i o n by l a y e r e d displacement,

followed

by back c y c l i n g o f the dispensed l a y e r to the o t h e r end o f the column r e a c t o r , causes bed m i x i n g / f l u i d i s a t i o n .

F l u i d i s a t i o n a l s o occurs during

upward r e c y c l i n g i n the d e p r o t e c t i o n and coupling s t e p s .

Switching t o

downward f l o w or e f f e c t i n g l a y e r e d displacement i n e i t h e r d i r e c t i o n w i t h an incoming s o l v e n t causes the g e l bed t o repack.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

203

Flow rate (cm3 min"1)

0

5

10

15

20

25

PiTmp speed (rpm)

F i g . 1. (a) Schematic r e p r e s e n t a t i o n o f manually s e r v i c e d CF apparatus. Key: C, 2.5 cm i . d . g l a s s column r e a c t o r ; E, PTFE end f i t t i n g ; S, PTFE s i n t e r e d f r i t ; A, a d j u s t a b l e p l u n g e r ; F, PTFE 1.6 mm i . d . t u b i n g ; R, s o l v e n t / r e a g e n t v e s s e l ; T j & T 2 , 4 way t a p s ; P, Watson Marlow 501 U/R o e r i s t a l t i c pump f i t t e d with Marprene 3.2 mm i . d . t u b i n g , (b) Packed bed f l o w r a t e v a r i a t i o n w i t h pump speed f o r HCONMe2 ( P l o t X) and 3-MeCgH^OH/ Cl3CMe (4/1) ( P l o t Y) . O i n d i c a t e s CF o p e r a t i n g range. For u l t r a - h i g h l o a d CF w i t h Boc amino a c i d s , bead-form c r o s s l i n k e d poly(N-[2-(4-hydroxyphenyl)ethyl]acrylamide)

(Core Q ) ,

f u l l y loaded w i t h

the t a r g e t p e p t i d e C - t e r m i n a l amino a c i d bound by a phenyl e s t e r was used as support

(4).

removal was by upward r e c y c l i n g with CF 3 C0 2 H/3-MeC e Hi t 0H/Cl3CMe (30 m i n ) .

linkage,

Amino a c i d l o a d i n g was 5 mmol/g o f Core Q.

Boc

(5/4/1)

P e p t i d e chain e x t e n s i o n i n v o l v e d upward r e c y c l i n g with

Boc-AA-OBt/HOBt/MeNCH2CH2OCH2CH2

(3/3/2 e q u i v . )

i n HCONMe2 (60 m i n ) .

I n t e r m e d i a t e washing s o l v e n t s were S-MeCgH^OH/CljCMe (4/1) d e p r o t e c t i o n ) , HCONMe2/Mel!lCH2CH2OCH2CH2 dipeptide stage)

(neutralisation)

and HCONMe2 ( p r e n e u t r a l i s a t i o n ,

( p r e - and p o s t (excluding

p r e - and p o s t -

coupling).

For u l t r a - h i g h l o a d CF with Fmoc amino a c i d s , a m o d i f i e d c o r e m a t r i x , 4-H0CH 2 C 5 H 4 0CH 2 C0-Pro-0-Core Q, f u l l y loaded with the p e p t i d e C - t e r m i n a l amino a c i d bound by an a c i d l a b i l i z e d b e n z y l e s t e r l i n k a g e , was used (5) . Amino a c i d l o a d i n g was 2.8 mmol/g o f m o d i f i e d c o r e .

Fmoc removal was by

upward r e c y c l i n g with Et2NH/HCONMe2 (1/4)

P e p t i d e chain

(30 min) .

e x t e n s i o n i n v o l v e d upward r e c y c l i n g with Fmoc-AA-OBt/HOBt HCONMe2 (60 m i n ) .

(3/3 e q u i v . )

in

A l l i n t e r m e d i a t e washings were w i t h HCONMe2.

Synthesis o f the dermorphin sequence,

Boc-Tyr(Bzl)-D-Ala-Phe-Gly-Tyr(BrZ)-

P r o - S e r ( B z l ) - O - C o r e Q (Assembly 1) and the neurotensin sequence,

Glp-Leu-

Tyr (But) -Glu (OBut) -Asn-Lys (Boc) - P r o - A r g (Mtr) - A r g (Mtr) - P r o - T y r (OBut) - l i e -

204 Leu-0CH2C6Hit0CH2C0-Pro-0-Core Q (Assembly 2) , illustrates ultra-high load CF using Boc and Fmoc chemistry.

The assemblies were prepared on a

1 mmole scale using a 2.5 cm i.d. CF glass column reactor. of Assembly

Treatment

with 10% BF 3 .Et 2 0 in 3-MeCgHijOH for 48 h, followed by

washing with HC0NMe2 and treatment with NH 3 saturated HCONMe2 for 1 h, liberated crude dermorphin.

Treatment of Assembly 2_ with CFsCC^H/CgH^SMe

(19/1) for 4 h liberated crude neurotensin.

Following removal of the

final reaction solvent, both peptides were isolated by precipitation from Me0H/Et20.

HPLC traces are shown in Figure 2.

Fig. 2. Reverse phase HPLC of (a) crude dermorphin and of (b) crude neurotensin on a 5n Waters C18 Novapak column eluted with 10-90% MeCN/l^O at 0.04% v/v CF3CO2H concentration. Inset (a') and (b1) are HPLC analyses for the same peptides after reverse phase MPLC purification on a Whatman LRPl column under similar elution conditions.

References 1. Krchnak, V., J. Vagner, M. Flegel and O. Mach. 1987. Tetrahedron Lett. 28, 4469-4472. 2. Frank, R., H. Leban, M. Kraft and H. Gausepohl. 1988. In: Peptides Chemistry and Biology (G.R. Marshall, ed.) ESCOM. Leiden, pp. 215-216. 3. Dryland, A. and R.C. Sheppard. 1986. J. Chem. Soc., Perkin Trans. 1. 125-137. 4. Epton, R., G. Marr, B.J. McGinn, P.W. Small, D.A. Wellings and A. Williams. 1985. Int. J. Biol. Macromol. ]_, 289-298. 5. Butwell, F.G.W., E.J. Haws and R. Epton. 1988. Makromol. Chem., Macromol. Symp. _19_, 69-77.

SIMULTANEOUS PEPTIDE SYNTHESIS USING CELLULOSE PAPER AS SUPPORT MATERIAL

J. Eichler, M. Beyermann, M. Bienert Academy of Sciences of the GDR, Institute of Drug Research A.-Kowalke-Str. 4, DDR-1136 Berlin M. Lebl Czechoslovak Academy of Sciences, Institute of Organic Chemistry and Biochemistry Flemingovo nam. 2, CS-16610 Praha 6

Introduct i on Simultaneous

solid

phase

peptide

synthesis

by

support

segmentation has been developed in order to meet the growing need of synthetic peptides as tools for the investigation of protein - ligand interactions (1,2,3). Cellulose

paper

was

esterified

with

Fmoc-amino

acid

chlorides (4) providing a mechanically and chemically stable support

material.

simultaneous synthesis

The applicability of

peptide

synthesis

was

of model peptides following

this

support

demonstrated different

by

for the

synthetic

strategies.

Results The functionalization of the paper (Vhatman 540) is outlined in

Scheme

groups

was

1. The substitution of the determined by photometric

support

with

measurement

amino of

the

Fmoc-cleavage product (dibenzofulvene-piperidine adduct) and comes to 1 to 2 ¿imol/cm2.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

206

HO-cellulose (i) , (ii> R Fmoc-NH-CH-CO-O-cellulose (iii> H 2 N-CH-C0-0-cellulose R —

""OHg

Scheme 1: Functionalization of the paper with Fmoc-amino (i>: 1 M NaOH 15 min,

24h

Fig. 1: BOP mediated coupling of Fmoc-Ala-OH to H-Phe-NH2 in DMF solution at various concentrations ofN-methyl morpholine Without base the reaction stopped at 48% yield and even after 24 h no change was observed. With a single equivalent of base the reaction levelled off at 82%. Using 8 equivalents of base to amino component the coupling reaction was essentially complete (>99 %) after only 1 min. This is far below preactivation times usually applied for anhydride formation. However, the suitable range of base concentration and reaction time is limited: At the highest concentrations tested some 0.6% (4 eq. NMM) and 1.5% (8 eq. NMM) of Fmoc-Ala-Ala-OH were found after 30 min of coupling. These values rose to 10% and 12% respectively after 24 h, indicating that coupling times have to be kept short at high base concentration. Asparagine coupling using BOP A serious side reaction of asparagine activation is dehydration to the corresponding nitrile. It also occurs during activation of asparagine by BOP, an HOBt derivative expected to avoid dehydration by rapid formation of HOBt esters. Nitrile formation could be reduced to 7% by addition of an equimolar amount HOBt. It could only be completely prevented by use of side chain protected derivatives such as Fmoc-Asn(Tmob)-OH or Fmoc-Asn(Mbh)-OH with BOP activation. The protection groups were removed by 95 % TFA/ethanethiol in 1 or 6 h respectively. Coupling of Fmoc-Asn-OPfp also led to a homogeneous product, provided the starting material was free of nitrile from preparation of the active ester. The reaction leading to nitrile formation was analyzed in detail for the peptide Met-Lys-AsnVal-Pro-Glu-Pro-Ser. Several methods of activation were compared to find suitable conditions for asparagine coupling avoiding dehydration.

243

Svnthesis conditions Fmoc-Asn-OH, BOP Fmoc-Asn-OPfp Fmoc-Asn(Mbh)-OH, BOP Fmoc-Asn-OH, HOBt, BOP Fmoc-Asn-OH, DCC Fmoc-Asn-OH, HOBt, DCC Fmoc-Ala(CN)-OH, BOP Fmoc-Asn(Tmob)-OH, BOP Fmoc-Asn-OPfp. new batch

#1 #2 #3 #4 #5 #6 #7 #8 #9

coupling vield

nitrile

correct peptide

>99 % 93% >99% >99% 52% >99 % >99% >99% >99%

50% 18% » OMe

> >

COOH.

Substrate Specificity - Table 1 shows efficiency and reaction rate for a variety of substrates. TABLE 1 - CHYMOTRYPSIN - VARIOUS SUBSTRATES (Ser-OEt 200mM; 50% DMF; 25°C; pH 8.5) Substrate

Efficiency %

Rate (min-1)

Ac-W-OMe (lOOmM) Ac-Y-OEt (lOOmM) Bz-Y-OEt (lOOmM) Z-S-Y-OEt (lOOmM) Bz-A-OMe (50mM) Z-G-D()OBzl (lOmM) + Z-L-R-W-OMe (20mM) Z-G-W-OMe (20mM) * Z-L-OMe (2OmM) Z-V-Y-OEt (2 5mM) *

41 81 73 76 80 58 30 46 70 75

+ product unstable

* product precipitates

1,800 2,600 12,600 390 ~2 ~15 245 750 ~3 4 ,800

Conclusions 1. Chymotrypsin can be used for the synthesis of most peptide bonds following hydrophobic residues. Efficiencies of 50-90% would be expected on optimisation. 2. Synthesis is optimal at high nucleophile concentrations but limited by solubility. High concentrations can be achieved by using organic solvents to 60% and detergent as required, although the enzyme is unstable under these conditions; DMF is preferable to ethanol. Temperature increases are not indicated unless a corresponding increase in nucleophile concentration compensates for the high temperature efficiency loss. 3. The preferred nucleophiles are amino acid amides (and peptides) but satisfactory results are obtained with esters.

ENZYMATIC SAFETY-CATCH

C O U P L I N G : A N A P P R O A C H TO B R O A D E N

S Y N T H E S I S P O T E N T I A L OF C* - C H Y M O T R Y P S I N A N D T O P R E V E N T H Y D R O L Y S I S IN K I N E T I C A L L Y C O N T R O L L E D

V . Schellenberger, H.-D. Dakubke

PEPTIDE

THE

PRODUCT

SYNTHESIS

U. Schellenberger, A. Kucharski

and

K a r l Marx U n i v e r s i t y , B i o s c i e n c e s D i v i s i o n , D e p a r t m e n t of Biochemistry, DDR-7010 Leipzig, German Democratic Republic

Int r o d u c t i o n Although

p r o t e a s e s h a v e been used as p r a c t i c a l

in D e p t i d e

synthesis

however, a serious

(1), the s u b s t r a t e

limitation which

p r e v e n t s the

approach

f r o m b e i n g u n i v e r s a l l y a p p l i c a b l e . We

attempts

to b r o a d e n

tically controlled

the a p p l i c a t i o n peptide

rather narrow substrate

biocatalysts

specificity

here

in

i n d e p e n d e n t l y of

specificity known from

kinethe

hydrolysis

studies. Furthermore,

the f o r m a t i o n of the p e p t i d e

bond

s h o u l d be k i n e t i c a l l y

irreversible

time

if

in the

reaction

the P^ a m i n o a c i d r e s i d u e d o e s not meet the

chymotryptic

hydrolysis

far,

enzymatic

report

of c h y m o t r y p s i n

synthesis

is so

frame

requirement

of

specificity.

Results In o r d e r

to c i r c u m v e n t

f i c i t y of c h y m o t r y p s i n

the

t i c h y d r o l y s i s of a w i d e both

relatively narrow substrate

we have f i r s t l y

studied

the

range of a c y l d o n o r e s t e r s

differing

in the P^ a m i n o a c i d r e s i d u e and in the n a t u r e of

ester moiety

(2). S e l e c t e d

In k i n e t i c a l l y c o n t r o l l e d

kinetic peptide

speci-

chymotrypthe

d a t a are

listed

in T a b l e 1

synthesis

strict

substrate

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

248

SDecificity

is not a l w a y s a r e q u i r e m e n t . T h e use of a c y l

esters with high k c a t / K p | values will orevent l y s i s of Table

the f o r m e d

peptide

product

1. K i n e t i c c o n s t a n t s of acyl donor esters

Mal-Phe-OMe

the c h y m o t r y p t i c

6 .6

Mal-Leu-OBzl

6 .9

Mal-Asp(OBzl)-OBzl

0 .8

Mal-Leu-ONb

6.7

Mal-His-ONb

9.8

Mal-Met-ONb

27 .3

Therefore,

+

0 .8

+

0 .2

+ +

+ +

the

0 .02

0 .85

0 .3

0 .20

0 .3

0 .41

0 .4

0 .15

2 shows partition constants

Table

+ +

+

103

2

2.1 *

102

0 .2

9.6 *

103

0 .08

1.0 *

io3

0 .01

3.4*

104

0 .02

2.4 *

104

0 .01

1.8*

105

+

coupling

rather narrow

7.6 *

+

0 .73

r e a c t i o n s can

primary

of

2

+

31

chymotrypsin-catalyzed

despite

hydrolysis

k cat/KM (M-l S " 1 )

+

13

6

donor

hydro-

enzyme.

Km M ( mM) +

99

Mal-Leu-OMe

extended

by the same

k cat (s-l)

S u b s t rate

secondary

be

specificity.

(1) f o r some

model

reac t i o n s . Table

2. P a r t i t i o n c o n s t a n t s for c h y m o t r y p s i n - c a t a l y z e d kinetically controlled coupling reactions Nucleophile

Acyl

Arg-- N H 2

Met-- N H 2

donor

Leu-NH2 p

Mal-Phe-OMe

3 .3

0 .11

Leu-Ala-NH2

(mM) 4.2

7 .9

Mal-Leu-OBzl

0 .24

4 .9

10 .1

5.9

Mal-Asp(OBzl)-OBzl

0 .45

14 .3

23 .9

32.7

Mal-His-ONb

0 .67

8 .4

8 .8

8.5

In o r d e r

to d e m o n s t r a t e

safety-catch

coupling

sin s u b s t r a t e

Glt-Leu-Phe-pNA

of G l t - L e u - O R z l within

the e f f i c i e n c y of (3) s t a r t i n g

and G l t - L e u - O N b ,

5 min . U s i n g G l t - L e u - O N b

increased indicate

that

enzymatic the

chymotryp-

from a 3fold

respectively,

and

the

excess

Phe-pNA

the p r o d u c t y i e l d c o u l d

from 34 to 63 % . In a d d i t i o n ,

significant

the

p r o c e d u r e we s y n t h e s i z e d

be

r e s u l t s in T a b l e

the e s t e r m o i e t y of the a c y l d o n o r c a u s e s a

acceleration

of

the s y n t h e s i s

reaction which

is

3

249 in a c c o r d a n c e

w i t h the i n c r e a s i n g

k c a t / K ^ v a l u e s as seen

in

Table

1.

Table

3. S y n t h e s i s of M a l - L e u - P h e - p N A c a t a l y z e d by c h y m o t r y p sin (0.15 mg) f r o m 20 mM a c y l d o n o r e s t e r and 5 mM P h e - p N A in w a t e r / D M S O ( 9 : l , v / v ) at pH 9 a n d 25°C

Acyl donor

ester

Mal-Leu-OMe

Reaction

time

(min)

Yield

10

3

Mal-Leu-OBzl

5

65

Mal-Leu-ONb

5

80 .5

F i g . 1 s h o w s the k i n e t i c using

p a t t e r n of the s y n t h e s i s

experiment

Mal-Leu-ONb.

TIME (MIN) F i g . 1. K i n e t i c a l l y c o n t r o l l e d s y n t h e s i s of using c h y m o t r y p s i n as b i o c a t a l y s t Mal-Leu-Phe-pNA (o-o); Mal-Leu-ONb P h e - p N A (*-*•) ; pNA ( o - o )

Mal-Leu-Phe-pNA (x-x);

References

1.

Zlakubke, H . - D . 1 9 8 7 . I n : T h e P e p t i d e s .-Analysis, S y n t h e s i s , B i o l o g y , V o l . 9 (S. U d e n f r i e n d a n d Meienhofer, eds.) A c a d e m i c P r e s s , N e w Y o r k . pD. 1 0 3 - 1 6 5

2.

S c h e l l e n b e r g e r , V . , U. S c h e l l e n b e r g e r , C o l l . C z e c h . C h e m . C o m m u n , (in p r e s s )

3.

O a k u b k e , H . - D . , H . D ä u m e r , A . K ö n n e c k e , P. K u h l 3. F i s c h e r . 1 9 8 0 . E x p e r i e n t i a 36, 1 0 3 9 - 1 0 4 0 .

H.-D. Dakubke : and

PEPTIDE SYNTHESIS CATALYZED BY PAPAIN IN ORGANIC SOLVENTS CONTAINING MINIMUM WATER

Yu.V.Mitin Institute of Protein Research, Academy of Sciences of the USSR, 142292 Pushchino, Moscow Region, USSR V.Schellenberger, HTD.Jakubke Karl Marx University, Leipzig, GDR

Peptide synthesis catalyzed by proteolytic enzymes is often accompanied by difficulties connected with low solubilities of hydrophobic components in water. Addition of organic solvents increases solubility of the starting component as well as of the final product, and thus facilitates the process. Water missable organic solvents are usually added in a 10-50% quantity. A higher concentration of organic solvents can cause an essential change in enzymic activity and specificity. But an immobilized or modified enzyme as well as an enzyme in the solid state endures higher concentrations of organic solvents without any dramatic change of its properties (1,2). We have studied the possibility of papain-catalyzed peptide synthesis in organic solvents containing a very low concentration of water. A hydrophobic Z-Ala-Val-OBut was chosen as a model peptide. The best solvent for this synthesis is acetonitrile. Four types of esters (methyl, benzyl, phenacyl and carboxamidomethyl) were tested as carboxyl components. Carboxamidomethyl esters (Cam) are the most suitable for papaincatalyzed peptide synthesis in acetonitrile. The water content in acetonitrile strongly affects the system. Peptide synthesis, together with considerable hydrolysis occurs within the limits of a 50-80% water concentration; in a range of 5-50% only

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

251

hydrolysis of the above-mentioned esters is observed. At a water content of less than 5%, peptide synthesis prevails over hydrolysis, and a maximal effect is achieved at a water concentration of 0.3-0.5%. Here the peptide yield rises to 99.5%. But the reaction rate is 50-100 times lower than that in water; Immobilized papain gives the best effect at peptide synthesis carried out in organic solvents. It is not necessary to obtain papain covalently bound to a carrier. It is enough to use the papain adsorbed on an inert carrier as it is insoluble in acetonitrile. Polyamide, silicagel, ion-exchange resins, charcoal, porous glass, etc. can be used as a carrier. We found that the most suitable carrier is polyamide used for thin layer chromatography. The optimal papain-polyamide correlation is 1:50. The papain not adsorbed on a carrier does not catalyze peptide synthesis at the used conditions. The properties of papain change somewhat in such an unusual medium as acetonitrile. Indeed, papain activity and specificity noticeably changed in comparison with those in water. Table 1 shows that the specificity of papain is significantly changed in acetonitrile. Table 1. Effect of Amino Components on Peptide Synthesis Catalyzed by Papain in Acetonitrile Using Boc-Tyr-Gly-OCam as a Carboxyl Component Amino Component

Product of the Reaction

Yield

H-Gly-Phe-Leu-OBut H B0C-Lys-Ala-0But

BOC-Tyr--Gly-Phe-Leu-OBut BOC-Tyr- G Y i t •Lys-Ala-OBu BOC-' BOC-Tyr-•Gly-NH(CH2)4-CH3

98

NH2-(CH2)4-CH3

%

99 55

Practically any aliphatic amine can serve as an amino component in these conditions. Peptides consisting not only of natural L-a-amino acids but also of D-amino acids and unnatural

252

amino components can be synthesized. Papain-catalyzed peptide synthesis carried out in water cannot give this possibility. The stepwise synthesis of DSIP nonapeptide can illustrate the suitability of peptide synthesis using papain in acetonitrile (% yields at each step are given below) BOC-Trp-Ala-Gly-Gly-Asp(OBzl)-Ala-Ser(Bzl)-Gly-Glu(OBzl)2 ? 85 77 61 58 51 98 Unfortunately acetonitrile is not a very suitable solvent for higher peptides and we could not obtain good results at the last step of condensation. Thus, we can conclude that papain can catalyze peptide synthesis in acetonitrile and that this process can be used for synthesis of several hydrophobic peptides (3).

References 1. Isowa, Y., M. Kakutani, M. Yaguchi. 1982. In: Peptide Chemistry, 1981 (T. Shioiri, ed.). Protein Research Foundation, Osaka, pp. 25-30. 2. Matsushima, A., M. Okada, Y. Inada. 1984. FEBS Lett. 178, 275-277. 3. Mitin, Yu.V., V. Schellenberger, H.-D. Jakubke. 1988. Bioorgan. Khim. 14, 5-9.

PEPTIDE SYNTHESIS CATALYZED BY a-CHYMOTRYPSIN IN ULTRA LOW WATER SYSTEMS

U. Slomczynska and T. Leplawy,

Jr.

Institute of Organic Chemistry, Technical 90924 L6dz, Poland

University,

Introducti on

A number of proteases have been used as catalysts in peptide synthesis

(1>,particularly o-f small

application in some cases

is

still

-fragments. However,

their

limited because o-f un-

favorable thermodynamic equilibrium,

low solubility of

phobic reactans in aqueous media and undesirable

hydro-

hydrolysis.

In order to overcome these drawbacks it would be profitable to carry out enzymatic peptide synthesis in organic media of water. Recently, several

instead

enzymes have been shown to function

in nearly anhydrous organic systems

(2-5).

In this paper

we

report our studies on peptide synthesis catalyzed by a—chymo— trypsin in organic systems containing

low amount of

water.

Results

In our model

study Z—Tyr—OMe,

Z-Phe-OMe or Z-Trp—OMe were used

as acyl donor and L - L e u - N H 2 or D - L e u - N H ^ as nucleophile. Bovine a-chymotrypsin, CT, obtained from Sigma Chemical

Co.

was used in crystalline form. Syntheses were studied in many organic solvents, containing various water contents

(determined

by Karl Fischer methodl) . Reactions were followed and yields determined by HPLC using the "common—chromophore" We found that the most useful

organic systems for

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

method. peptide

254 synthesis catalyzed by a-chymotrypsin are solvents with water

like C^HgCH-j, C C 1 4 ,

0.2-0.3% of water.

CHCl-j, C H 2 C 1 2

The dipeptide y i e l d s were 96-98% and

•for l—Leu—NH^ and d—Leu-NH^ respectively,

used a s

in 1.2 fold excess. No appreciable reaction w a s in organic solvents containing general,

12 h in C H C 1 3 or

TABLE

(Table

detected

in water

CH2C12. variety

1).

1. Peptide Obtained via Synthesis Catalyzed a—Chymotrypsin

Acyl donor (mmol

In

and

2 h in C,H_CI-L or CC1 . 6 5 3 4

This method was utilized for preparation of dipeptides

88-93%

nucleophiles

less than 0.2% of water.

the reaction time is longer than

d e p e n d s on the nature of solvent, and

immiscible

containing

in Organic

Nucíeophi1e (mmol)

Z -Trp -OMe(2) Ser-OEt

by

Solvents

Solvent (ml)

Product

b>

Yield %

z -Trp -Ser-OEt

90

Z -Trp -OMe(1) Leu—NH^

z -Trp-- L e u - N H 2

84

z -Trp -OMe(1)

z -Trp - D - L e u - N H 2

80

z -Tyr -Leu—NH,,

82

z -Tyr-- D - L e u - N H 2

78

z -Tyr--Sei—OMe

89

z -Tyr--D-Ser—OMe

87

z - T y r -OMe(1) z - T y r -OMe(1) z - T y r -OMe(2) z - T y r -OMe(1) z - T y r -OMe(1) z - P h e -OMe(3)

(2. 2)

C 6H5CH3(20) (1. 1) C,H_CH_ (10) 6 D D-Leu-NI-L, (1.2) C.H.-CH^dO) 6 D Leu-NH2 (1. 2) C , H c C H _ ( 1 0 ) o 3 -3 D - L e u - N H 2 ( 1 . 2) C,H„CH^(10) 6 Ü £ Ser-OMe (2. 1) CC1„ (20) 4 D-Ser-0Me(1. 2) C , H _ C H T ( 1 0 ) O J Ser-OEt (1. 2) CC1,, (10) 4 D-Leu-NI-L, H - A A 1 A A 2 - O H + H-AA-|-OH +

EtOH

a m i n o a c i d as N - c o m p o n e n t . H-AA-] - O E t + H - A A 2 - 0 H + H 2 O The

process

is b a s e d o n

lyse Not-unprotected catalyse

acyl

contrast

to

tion

amino does

amino

transfer

acids

this approach stereospecific

from esters and

poorly not

in

(3),

that

its

on

slowly its

e s t e r s of

site

(3,5). of

amino

hydro-

ability

acids

nucleophile

as we f o u n d

N^-unprotected

to

to f r e e a m i n o on

occur. Synthesis

is a l s o p o s s i b l e , for

esters

CPD-Y

is s u c h t h a t h i g h e r

bind

therefore

a b i l i t y of

acid

endoproteases),

site t o p o l o g y which ted

the

(4)

(in

binding

N^-unprotecOligomeriza-

D,L-dipeptides that CPD-Y

acid

Peptides 1988 © 1989 Walter de Gruyter & Co., B e r l i n - N e w York-Printed in G e r m a n y

to

esters

by

is

less

than

for

272

Not-protected ones. This peculiar lack of stereospecificity appears to be more pronounced with CPD-Y than with some endoproteases (6 ).

Results and Discussion Table I lists some examples of CPD-Y catalysed L,L-

and

D,L-dipeptides.

Interestingly,

the

synthesis of acyl

transfer

yields with D-amino acid esters as substrates are consistently and significantly higher than with L-amino acid esters. Table I: CPD-Y3) CATALYSED SYNTHESIS OF LL- AND DL-DIPEPTIDES Substrateb)

Nucleophile

L-TyrOEt D-TyrOEt L-LeuOiPr D-LeuOiPr L-MetOEt D-MetOEt L-TyrOEt D-TyrOEt L-TyrOEt D-TyrOEt L-TyrOEt D-TyrOEt

H-Arg-OH H-Arg-OH H-Met-OH H-Met-OH H-Met-OH H-Met-OH H-Cys-OH H-Cys-OH H-Arg NH 2 H-Arg-NH 2 H-Val-NH 2 H-Val-NH2

(0. 8 (1. 0 (0. 3 (0. 3 (0. 3 (0. 3 (1. 0 (1. 0 (0. 2 (0. 2 (0. 3 (0. 3

M) M) M) M) M) M) M) M) M) M) M) M)

pH

Product

9. 5 9. 0 9. 0 9. 0 9. 0 9. 0 8. 0 8. 0 9. 0 9. 0 9. 0 9.0

L-Tyr-L -Arg--OH D-Tyr-L -Arg -OH L-Leu-L -Met--OH D-Leu-L -Met -OH L-Met-L -Met--OH D-Met-L -Met -OH L-Tyr-L -Cys--OH D-Tyr-L -Cys -OH L-Tyr-L -Arg-- N H 2 D-Tyr-L -Arg - N H 2 L-Tyr-L -Val-- N H 2 D-Tyr-L'v a l - N H 2

Yield % 30 75 35 70 25 65 30 85 50 85 75 95

a) CPD-Y: 5-25 uM. Reaction times: 6-10 hours b ) Substrate esters were 50 mM for convenience Homodipeptides can be made simply by incubating an amino acid ethyl

(or higher) ester with CPD-Y. As the ester is hydroly-

zed, the free amino acid - which functions as the amino component - is generated in situ, with subsequent formation of the homodipeptide, e.g. L,L-MetMetOH can be formed by incubating a 0.5-1.0 M solution of H-L-MetOEt at pH 9. If a 0.5 M solution of H-L-MetOMe is incubated, a precipitate of methionine oligomers is rapidly formed!

273

When L,L-heterodipeptides are to be synthesized, the homodimer of the amino acid used as the C-component may be formed as a byproduct. This can be controlled by appropriate choice of reactant

concentrations,

and does

not

occur

with

D-amino

acid

esters as C-components, because, in this case, the hydrolysis product is a D-amino acid. Dipeptide provided

amides can also be produced by this simple process, the amino acid amide binds poorly

in the Si~site

of

CPD-Y, as is the case for arginine and valine (see table I). We have synthesized and attempted to synthesize many more

di-

peptides than shown here and conclude that the method is currently not as general as the NCA-method, since some tected more

amino

acid

seriously,

esters

acyl

are

turned

over

transfer

yields

to

N^-unpro-

very

slowly,

several

amino

and, acids

are unpractically low. We believe, however, that the simplicity of this method, both in terms of number of

reaction

steps

and reaction conditions, makes it an attractive option for industrial production of certain dipeptides. Further work to widen the scope of the method is thus warranted.

References 1. Blacklock, T.J., Hirschmann, R. and Veber, D.F. 1987. The Peptides, Vol. 9 (Udenfriend, S. and Meienhofer, eds.), Academic Press, 39-102. 2. Kitabatake, S. et al. 1987. Pharmaceutical 154-157 and reference cited therein.

Research,

In: J., 4^,

3. Thorbek, P., Aasmul-Olsen, S. and Widmer, F. Patent application: A Process of Enzymatic Production of Dipeptides. WO 88/06187, 25.08.88 (Publ.), 13.02.87 (Priority) 4. Widmer, F. and Johansen, J.T. 1979. Carlsberg Res. Commun., 44, 37-46. 5. Widmer, F., Breddam, K. and Johansen, J.T. 1980. Carlsberg Res. Commun., £ 5 , 453-463. 6. Purdie, J.E. 523-526.

et

al.

1972.

Biochim.

Biophys.

Acta,

268,

ATTACHMENT OF LINKER GROUPS TO CARBOXYL TERMINI USING ENZYMEASSISTED REVERSE PROTEOLYSIS

Keith Rose, Robert M.L. Jones, Ganesh Sundaram, Robin E. Offord Departement de Biochimie Medicale, C.M.U., 9 avenue de Champel, 1211 Geneva 4, Switzerland

Introduction

The attachment, to a polypeptide chain, of a group of interest (reporter group, radiolabel, cytotoxic agent, polypeptide chain, etc.) is useful -in many areas of research. In general, such attachments (e.g. iodination of tyrosine residues, acylation of lysine residues) are made by group-specific, but not regio-specific, reactions. As a result, even mono-reacted material is in general itself heterogeneous, being a mixture of species each with one group attached to one of several residues of the type involved in the modification reaction. We believe that much is to be gained from the homogeneity of the products of site-specific attachment of groups of interest, in contrast to the non-regiospecific procedures. We (1-4) have exploited the specificity of proteases working in reverse to fix activating groups exclusively at the carboxyl terminus of polypeptides. In a subsequent chemical reaction, a group of interest is directed specifically to the activated group of a modified polypeptide. (It is not usually possible or desirable to fix enzymatically the group of interest directly to the polypeptide chain for reasons of enzyme specificity and the large excess of amino component generally required to drive the enzymatic coupling to high yield). The specificity of the conjugation reaction may be achieved by chemical complementarity of the reacting groups (see below) or, in the case of the conjugation of two polypeptides, by conformational assistance (4). In this article, we describe the coupling, using enzyme-catalyzed reverse proteolysis, of two reactive

compounds,

carbohydrazide

and

1,3-diamino-2-propanol,

to

des(AlaB30)insulin, and we discuss conjugate formation using these new Unfortunately, space is very limited: full details will be published elsewhere.

Peptides 1988 © 1989 Walter de G r u y t e r & Co., Berlin • N e w York - Printed in G e r m a n y

LysB29

of

derivatives.

275 Experimental and Results

Coupling between des-Ala B 30-insulin

(DAI) and carbohydrazide,

catalyzed by Lysyl

Endopeptidase, was carried out in aqueous solution. Coupling between DAI and 1,3diamino-2-propanol, catalyzed by trypsin, was carried out in butane-l,4-diol/H20 9:1 (v/v). Reversed phase HPLC analysis (TFA system) of both coupling mixtures showed clean transformations of DAI into a more hydrophilic product. Yields of the couplings were approximately 70%; (yields greater than 95% of DAI-NHNHCONHNH2 could be obtained in the presence of organic solvent). After isolation, the products were characterized by electrophoresis and peptide mapping and behaved as expected for DAI to which a single group

had

been

NHNHCONHNH2

attached

specifically

to

the

carboxyl

and DAI-NHCH2CH(OH)CH2NH2.

group

of

LysB29 :

DAI-

Quantitative oxidation of

DAI-

NHCH2CH(OH)CH2NH2 to an aldehyde, DAI-NHCH2CHO, was achieved using periodate under exceptionally mild conditions (5 equivalents of periodate and 300 equivalents of ethylene glycol over DAI-NHCH2CH(OH)CH2NH2, pH 8.3). Such mild conditions (small excess of periodate and large excess of glycol to destroy unreacted periodate) are possible owing to the extreme susceptibility to periodate of -CH(OH)-CH(NH2)- groups (5).

Conjugation reactions of DAI-NHCH2CHO (100 ¿iM) with ligands (500 ^iM) carrying an aminooxy function (aminooxyacetyl-ferrioxamine,

and N -aminooxyacetyl-poly(glutamic

acid)) were found to proceed well at 22°C in 0.1M acetate buffer, pH 4.6. The conjugation reaction between DAI-NHNHCONHNH2 with a ligand carrying an aldehyde group (2,4dihydroxybenzaldehyde) also proceeded cleanly under similar conditions. All appropriate control mixtures, where unmodified insulin was substituted for modified insulin, produced no reaction in any case.

Conclusions and Acknowledgements

In previous work (6), hydrazide and aldehyde groups have been attached non-soecificallv to proteins using chemical reagents, and protein conjugates have been formed via hydrazone formation. We show here that it is possible to use enzyme-catalyzed reverse proteolysis to

276 attach the reactive groups (a hydrazide, and an aldehyde through 1,3-diamino-2-propanol) specifically to the carboxyl terminus (Lys B 29)

0f

DAI. Considerable improvements over

previous work are: the production of an aliphatic aldehyde under very mild conditions (much milder than those used to oxidize the cis-diol group); the coupling of a reactive compound (carbohydrazide) in fully aqueous solution (some polypeptides may be denatured by or insoluble in high concentrations of organic solvents, even mild ones such as butane1,4-diol), and the fact that no reductive stabilization of a Schiff base is necessary. Such reduction can be responsible for the irreversible formation of unwanted inter- and intramolecular links (through lysine side chains, for example). The use of enzyme-catalyzed reverse proteolysis to attach activating groups specifically to the carboxyl termini of polypeptides would seem to be a promising route to the preparation of well defined protein conjugates.

We thank the Ligue Suisse Contre le Cancer, the Luzerner Krebsliga, Hoffmann-La Roche A.G., Celltech Ltd., and the Schmidheiny Foundation. In addition, the Fonds National Suisse de la Recherche Scientifique provided much of the general instrumentation on which this work was carried out.

References

1.

Offord, R.E. and K. Rose. 1986. In: Protides of the Biological Fluids (H. Peeters, ed.). Pergamon, Oxford, pp 35-38.

2.

Offord, R.E., S. Pochon and K. Rose. 1987. In: Peptides 1986 (D. Theodoropoulos, ed.). W. de Gruyter, Berlin, pp 279-281.

3.

Rose, K., C. Herrero, A.E.I. Proudfoot, C.J.A. Wallace and R.E. Offord. 1987. In: Peptides 1986 (D. Theodoropoulos, ed.). W. de Gruyter, Berlin, pp 219-222.

4.

Rose, K., C. Herrero, A.E.I. Proudfoot, R.E. Offord and C.J.A. Wallace. 1988. Biochem. J. 249 83-88.

5.

Fields, R. and H.B.F. Dixon. 1968. J M 883.

6.

King, T.P., S.W. Zhao and T. Lam. 1986. Biochemistry 2 1 5774-5779.

THE USE OF PENICILLIN ACYLASE FOR SELECTIVE N-TERMINAL DEPROTECTION IN PEPTIDE SYNTHESIS

H. Waldmann Institut für O r g a n i s c h e Chemie, J o h a n n - G u t e n b e r g M a i n z , Becherweg 18-20, D-6500 M a i n z , FRG

Universität

Introduction

Numerous problems arising in natural product chemistry demand for advanced protecting group technologies. In many cases functional groups have to be liberated selectively under nearly neutral conditions and in the presence of chemically sensitive structures (1). Enzymes often accelerate chemical transformations under exceptionally mild conditions and combine high chemo- and regioselectivity with broad substrate specificity. Although these biocatalysts are increasingly used in organic synthesis they were applied in protecting group chemistry only in isolated cases. Here the use of penicillin acylase (EC 3.5.1.11), an enzyme that selectively hydrolyzes phenylacetamides and -esters (2), for the liberation of the amino function in peptide synthesis is described (3).

Results

N-Phenylacetyl(PhAc)-protected dipeptides can be synthesized at 0°C in good yields by coupling PhAc-amino acids with various amino acid esters using either the modified carbodiimide procedure (DCCI/HoBt) (method A) or EEDQ (method B) as the condensing agent (Scheme (I) (3). In both cases product formation is accompanied by ca. 5-7% racemization of the N-terminal amino acid. However, the highly crystalline totally protected dipeptides are easily obtained in enantiomerically pure form by a single

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

278

recrystallization. The phenylacetamido moiety proves to be stable under basic and acidic f ] ~ \ U

1 2 method A • — N H — A A — O H + H — AA—OPG method B

O L

— J —

, 2 PhAc — A A — A A — O P G

1

PhAc

method A:

®

DCCI/HoBt

method B:

EEDQ

PG = methyl (Me), benzyl (Bzl), allyl (All), tert-butyl (tBu).

conditions and in the presence of noble metal catalysts. Thus it is not affected during the cleavage of methyl, tert-butyl, benzyl and allyl esters (Scheme (II)). On the other hand it can be selectively removed from the peptides by the action of penicillin acylase (Scheme (HI) (3). Even in the presence of substantial amounts of organic cosolvents, e.g. methanol and N-methyl-2-pyrrolidinon (NMPD), the enzyme accepts a broad range of dipeptides as substrates and liberates the amino function without attacking the ester groups. Although the hydrolysis rates decline with increasing steric demand of the N-terminal amino acid it

PhAc-Phe-Leu-OMe

Na0H/H20/CH30H

•

PhAc-Phe-Leu-OH

•

PhAc-Phe-Leu-OH

87% H 2 /Pd-C/CH 3 OH PhAc-Phe-Leu-OBzl 99% (PPh3)4Pd(0)/Morpholln

(II)

93% PhAc-Phe-Leu-OH

PhAc-Phe-Leu-OAII 63% (PPh 3 ) 3 Rh(l)CI

CF 3 COOH PhAc-Gly-Phe-OtBu

100%

•

PhAc-Gly-Phe-OH

279

tolerates variations of both, the amino acids and the ester functions. For the synthesis of N-terminally deprotected dipeptide esters the tert-butyl derivatives are suited best since they are not prone to diketopiperazine formation (Scheme (III)). However, it should be noted that in the construction of higher oligopeptides this intramolecular cyclization does EC 3.5.1.11 PhAc-Gly-Phe-OtBu

65%

•

H-Gly-Phe-OtBu

•

H-Thr-Ala-OtBu

EC 3.5.1.11 PhAc-Thr-Ala-OtBu 75%

PhAc-Ser-Leu-OtBu

EC 3.5.1.11

(III) •

H-Ser-Leu-OtBu

EC 3.5.1.11 — •

H-Ala-Ala-OtBu

63% PhAc-Ala-Ala-OtBu

no longer occur as a side reaction. Finally the selectively deprotected dipeptides can be coupled to tetrapeptides in good yields using again EEDQ (PhAc-Gly-Phe-Ala-Ala-OtBu: 92%; PhAc-Gly-Phe-Thr-Ala-OtBu: 95%). Together with the recently developed methods for enzyme-catalyzed peptide bond formations (4) the possibility to remove protecting groups by enzymatic techniques could complement

and enhance the methodology

of peptide synthesis.

Enzyme-based

transformations could especially prove to be invaluable tools in achieving peptide syntheses in aqueous solutions.

References

1.

Greene, T. W.. 1981. Protective Groups in Organic Synthesis. Wiley and Sons, New York .

2.

Savidge, T. A. and Cole, M. 1976. Methods Enzymol. 43, 705.

3.

Waldmann, H. 1988. Tetrahedron Lett. 29,1131.

4.

For a review see: Jakubke, H. D., Kuhl, P. and Konnecke, A.. 1985. Angew. Chem. Int. Ed. Engl. 26,294.

PROTEASE MEDIATED SYNTHESIS OF THYMOPENTIN

S. Aasmul-Olsen, F. Widmer and A.J. Andersen Carlsberg Biotechnology Ltd., Tagensvej 16, DK-2200 Copenhagen

Introduction Thymopentin, H-ArgLysAspValTyr-OH, is the pentapeptide sequence 32-36 of the long chain, naturally occuring protein thymopoietin

II

(1). Like its parent molecule, the short peptide

has modulatory effects on the immune system

(2) and can thus

be used to treat disease states associated with defects of the immune system. Sound, industrial scale methods for the production of this peptide are therefore needed. We report here results of our work

(3) allowing

the production of

thymopentin

and its analogs by economically attractive chemoenzymatic synthesis strategies. Konig et al. (4), Heavner and Heinzel

(5) and Voelter

(6) have described fully chemical synthesis stra-

tegies .

Results and Discussion The sequence of thymopentin is in principle suitable for production by the stepwise enzymatic strategy whereby amino acid esters are coupled together in the C-terminal direction with a series of highly specific endoproteases (7). However, this very simple approach is unapplicable, because the arginine specific clostripain, the Asp/Glu specific S.aureus V8 protease and the valine specific protease (8) are currently unavailable for production purposes. Therefore, we have made peptides

chemically

and

then

coupled

them

intermediate

enzymatically

at

"strategic" places to ensure optically pure products even when

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin • N e w York - P r i n t e d in G e r m a n y

281

optically impure fragments are used (9), and to generally take advantage of the ability of the enzymes to select the right compounds from semipurified intermediates, a fact which significantly

reduces production

costs. We

used

available enzymes trypsin (T), thermolysine

the

commercially

(Th) and carboxy-

peptidase Y (CPD-Y). Three of the fragment coupling strategies we have examined in some detail are shown below: ZArgLysOEt + AspValOEt

T

> ZArgLysAspValOEt CPD-Y ZArgLysAspValOEt + TyrNH2 > ZArgLysAspValTyrNH2 ZArgLysOEt + AspValTyrNH2

T

(la) ZArgLysAspValTyrNH2

(2)

Th ZArgLysAsp(OBzl)0H + ValTyrNH2 — > ZArgLysAsp(OBzl)ValTyrNH2 The

tryptic

coupling

in

reaction

1a

proceeds

(3) surprisingly

well, since, previously, we only got poor yields with nucleophiles

having

a free

Asp

at

the N-terminal

(10). The

high

yield in the above reaction is probably due to the hydrophobic valine in P 2 ', since trypsin has a high affinity for hydrophobic

residues

in the S 2 '-site

(11). Coupling

of

TyrNH 2

with

CPD-Y to the tetrapeptide ester (reaction 1b) proceeded slowly due to poor turnover at valine, resulting

in some subsequent

product hydrolysis and tyrosine oligomerization. Another satisfactory fragment coupling was obtained with trypsin in reaction 2. Again, the hydrophobic residues in P21 and P31 of the nucleophile fragment are responsible for good, productive

interaction of the nucleophile with the trypsin acyl

enzyme intermediate in spite of the free Asp-residue

in P-|".

No transpeptidic coupling of AspValTyrNH2 to arginine was observed, since acylation at the lysine ester bond is much faster than at the Arg-Lys peptide bond (7). The thermolysine coupling

in reaction 3 is difficult to con-

trol as several transpeptidic byproducts are formed making purification

difficult.

Very

interestingly, we found

that

the

same reaction with ZArgLysGlu(OBzl)OH - to synthesize the thy-

282 mopentin

analog

spleninopentin

- as

the C-component

is

more

easily controlled. The additional CH2~group must be responsible for giving a clean product precipitation in good yield. After

enzymatic

coupling overall

deamidation

product, yield

thymopentin

with

analysis: Asp

and

the

(0.95); Arg

hydrogenation

is obtained

typical

of

final

in good purity

characteristics:

(1.06); Tyr

the

(0.95); Val

Amino

and acid

(0.98); Lys

(1.07). No foreign or free amino acids. Sequence analysis phase

sequenator):

(corrected

Arg->Lys->Asp->Val->Tyr.

for water

and

acetate

Optical

content),

(Gas

rotation

: -22.1%

D

(1%

w/w in 0.1 N HOAc). The above sample showed an HPLC purity at 220 nm of more

than

98%

in several

RP-systems

and

contained

acetate and water in amounts of 9% and 5%, respectively.

References 1. Schlesinger D.H. and Goldstein G. 1975. Cell, 5^, 361-365 2. Goldstein G. et al. 1979. Science, 204,

1309-1310

3. Aasmul-Olsen S. and Andersen A.J. Patent Application: Enzymatic Process for Producing Immunomodulating Pentapeptides and Intermediates. DK 158/88, filed 14.01.1988 4. König W. et al., DE 3421614 AI, Patent Application, 1984 5. Heavner, G. EP 0042291 B1 Patent

(1987)

6. Voelter W. and Heinzel W. 1987. Chemiker-Zeitung, 111, 82 7. Widmer F. and Johansen J.T. 1985. In: Synthetic Peptides in Biology (Alitalo K. et al., eds.), Elsevier, 79-86 8. Abbasi A., Voelter W. and Hoppe Seyler, 367, 441-445

Zaidi

Z.H.

Biol.

Chem.

9. Thorbek P. and Widmer F. 1985. In: Proc. 9th Am. Symp (Deber C.M. et al., eds.), Pierce, pp. 359-362

Pept.

10. Widmer F. et al. 1985. In: Peptides ed.) Almquist, pp. 193-198 11. Christova 626-629

E.

et

al.

1982. Arch.

1986.

1984

Biotech.

(Ragnersson Biophys.,

U.

218,

PROTEIN ENGINEERING OF CYTOCHROME C: SUBSTITUTIONS OF TYR", THR78, AND ALA83 OF THE HORSE PROTEIN BY SEMISYNTHESIS

Carmichael J. A. Wallace, Amanda E.I. Proudfoot Biochemistry Department, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 and Biochimie Médicale, Université de Genève, CH-1211 Genève 4. Paolo Mascagni and Stephen B.H. Kent Division of Biology, California Institute of Technology, Pasadena, CA 91125

Introduction Cytochrome c has proved to be a good model for protein engineering by semisynthesis (1): analogues produced thereby have been useful in studies of structure-function relations general to proteins (2) or specific to the electron transfer process (3). In the course of these studies, many tactics have been employed for fragment modification. We have used sequential degradation and resynthesis at both N and C-termini (4), fragment-specific chemical modification (5), substitution by natural fragments derived from another species (6), or de novo synthesis by solution methods (6). Others have employed total synthesis by solid-phase methods (3). In view of recent advances in the technology of this latter method, we were hopeful that the yield and quality of fragments, and consequently of semisynthetic proteins could be improved. We have made analogues of the 39-residue CNBr fragment 66-104 of the horse sequence, which may easily be reincorporated in the complete protein through conformationally-catalysed coupling to the haem-containing fragment 1-65 (6). Three sites have been chosen for modification, to examine the functions of the strongly conserved residues Tyr 61, Thr?», and Ala 83 . Tyr has been replaced by Phe at position 67 to scrutinise the role of the phenolic hydroxyl group, conserved in all but one known species. The only known natural replacement for Thr 78 is Asn: we have made this same substitution in the horse sequence, and also inserted aminobutyric acid, thus eliminating the hydroxyl group of threonine. Residue 83 is alanine in animal species but proline in all plant cytochromes^. We have inserted proline in the horse sequence to learn more about the differences between the two groups. A double mutant, [Asn78, Pro83], was constructed and, as a procedural control, a synthetic fragment of the natural sequence.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

284 Experimental and Results Solid phase peptide synthesis was performed by methods described elsewhere (7) and the structure of the HPLC-purified products checked by mass spectrometry. Recombination of the synthetic pepdde and natural fragment was achieved by published methods (5). Yields of crude coupled products were 52-58%, except in the case of [Aba 78 ] cytochrome g, 33%. Ion-exchange on Trisacryl SP, first in the ferri, then in the feiro-cytochrome form, was used to obtain products 100% pure by UV-Vis spectroscopic criteria. Spectroscopy also revealed significant deviations in the properties of some of the analogues: a red-shift in the major bands of the [Phe67] protein; and a more or less marked weakness in the 695 nm absorption in analogues modified at position 78 that proved to be due to a drop in stability of the haem crevice, of which the loss of the 695 band is a sensitive signal. The crevice strength was probed by both thermal and pH titrations of this absorption band, which is due to the haem iron-Methionine 80 sulphur co-ordination bond. The results are set out in table 1. Also shown in that table are redox potentials determined by the method of mixtures (6) and specific activities in a succinate oxidase assay system (4). Three analogues show a drop in potential; only the synthetic product of natural sequence has a normal biological activity. Table 1. Physicochemical and Biological Properties of Cytochrome £ Analogues. Cytochrome

pK695

Tm695[°C]

Eo'(mV)

Native Horse Synthetic Native [Phe67] [Aba78] [Asn78] [N78.P83] [Pro83]

9.25 9.25 10.65

0.

III

/=\ 0.

ZtrpNHT

C l N H j " ^COjMe

IV

(B6X)

COjMe I61X)

Lawesson's reagent =

Ztrp [thzl] P h e O M e

OMe

H

Pro

V

(66X)

trp[thzlJPhe

II

-OH

trp

Leu

H DCCI/ HONSu

(73%)

|HF/anisole ; N3HCO3 (95X1 BOC- -ONSu

HVIII

Phe

VI

VII

NH,

NH,

NH,

I THF 17IX) BOCI TFA/H,0 ; NaHCOj (70X)

IX

(B6X)

NH,

NH,

294 assumed to be the oxo-thioamide. Hydrolysis of the ester yielded the acid II which was coupled with the tripeptide amide V I using DCCI/HONSu to give an excellent yield of V I I as a single diastereoisomer (N.M.R.). Deprotection with HF gave V m in excellent yield as a single diastereoisomer. The material was chromatographically identical to the less polar diastereoisomer obtained from the Hantzsch product. These data indicate that our thiazole synthesis proceeds without significant racemisation (4) ethyl acetate solution was evaporated, Z-L-Alay [CN4] -L-Ala-OBzl was separated from unreacted starting material by flash chromatography (solvent system dichloromethane/acetone, 30:1, v/v) and isolated (24.9%) as

white crystals; m.p. 142-143° C; [a] 25 o - 51.2° (c=l, MeOH); FABMS

m/e 410 (MH+), calcd. for C21H23O4N5 409. The Z group was removed with HBr/AcOH and Boc-Phe coupled using isobutyl chloroformate to give BocPhe-L-Alay [CN 4 ]-L-Ala-OBzl. corresponding

to

Hydrogenolysis

Phe^-Ser^-Pro' of the

gave

[Ala®]-BK

incorporated by solid phase synthesis to give both Ala 7 ]-BK

and

[L-Ala 6 y [CN 4 ]-D-Ala 7 ]-BK

after

HF

the

tripeptide

analog.

This

was

[L-Ala®y[CN 4 ]-Land

purification.

Racemization of the tetrazole dipeptide has been shown on exposure to 10% triethylamine in methylene chloride, and probably accounts for [LAla6V|T [CN4 ] -D-Ala 7 ] -BK due to multiple exposure to this reagent.

Z-Proy[CN4]-L-Ala-OBzl was prepared in a similar manner in 68% yield; m.p.

97.5-98° C; [a] 2 5 D - 15.9°

calcd. for C23H25O4N5

(c=0.5, MeOH); FABMS m/e 436

(MH+);

435. Single crystals of Z-Prof[CN4 ]-Ala-OBzl

(C23H25N5O4) were grown from an ethylacetate/petroleum ether mixture by slow evaporation. The crystals are monoclinic, space group P2i with cell constants a = 22.176(3)A, b = 6.141(1)A, c = 8.275(1)A, P = 98.31(1)°, Z = 2 and r c = 1.297 g/cm3. The structure was solved by direct methods and showed

that

the

chiral

centers

of the Pro

and Ala a-carbons

had

identical chirality. The most remarkable feature of this structure is the similarity of the tetrazole ring system to that observed (2) in the diketopiperazine, c-[L-PheGroup

A

>Group

B

)Group

C

6 (ppm)

Fig. 1. Separation Factors (a) and Chemical Shift Differences of Val-NH between the L - L and D-L Isomers of 1. o b v i o u s l y c o r r e l a t e d with chemical diastereomers CD

spectra

sents L-L near

in

1,2-dichloroethane

each

group

also

exhibit

of ^ a

(X-Y

= Aib-Gly)

nm,

X - Y = Alb-Gly

(A6) of Val-NH

between

in CDCl^.

isomer 240

shift differences

(A6)

indicating (a = 1.78)

the

(DCE),

of d i a s t e r e o m e r s of _]_ which

differences of

folded

group

of A

three

conformation.

Alb-Alb

groups

(Fig.

shows d i s t i n c t

(a = 1.27)

Fig. 2. CD Spectra of Diastereomers of 2 i n

On

the

2).

Cotton

other

X - Y = Gly-Alb

repreThe

effect

hand, (a = 1.10)

1i2-Dichloroethane.

CD

303 pattern of the suggesting

D-L isomer of Iji well

random

conformations.

resembles the ones of other groups,

The

remarkable

contrast

between CD spectra of the L-L isomer of 1_a in two solvents:

was

observed

Cotton effect

near 240 nm observed in DCE (Fig. 2) is negligibly small in MeOH. More

detailed

investigation

by NMR

measurements

reveals

the

significant,

conformational differences between diastereomers of 1_a as follows: 1 13 (1)

H and

C

chemical

shifts

of

some

groups

besides

Val-NH

in

CDC1 ^

differ remarkably between diastereomers, while those in DMSO-dg are almost same.

(2)

are small

The

temperature

coefficients

of

NH

chemical

6,6,-tetramethylpiperidine-1-oxyl a

in

for Gly-NH of the L-L isomer and Val-NH of the D-L one,

ing that those NH may be fairly exposed to solvent.

in

shifts

significant

broadening

CDCl^

indicat-

(3) Addition of 2,2,

(TEMPO) to CDC1 ^ solution of J_a resulted

of Aib-NH

of

the

L-L

L-L

isomers

isomer

and

Aib-NH

and

Val-NH of the D-L one. The

above

6-turn

observations

conformations

suggest

(Fig.

that

the

3) in such nonpolar

of

aprotic

group

solvents

A

prefer

as

CDCl^

and DCE, but their conformations become rather random in such strong hydrogen

bond-accepting

L-L

solvents as DMSO-dg and MeOH.

not

to

be

isomers

of

group

appears

time while

of

contact

favored

the

A appear

with

conformations

by

of

the the

D-L

to prefer the

hydrophobic D-L

isomers.

ones

HPLC

folded

surface remain

The folded In

of

conformations

ODS

random.

conformation

conditions,

stationary Therefore,

at

the

phase, the

isomer seems to be much more strongly retained than the D-L one in HPLC.

\

the

Fig. 3. Proposed Conformay* C H 3 tion for the L-L Isomer of la (X-Y = Aib-Gly) in

0

Reference

1. Yamada, T., M. Nakao, K. Tsuda, S. Nonomura, T. Miyazawa, S. Kuwata, M. Sugiura. 1988. Peptide Chemistry 1987, 97.

L-L

AN EFFICIENT ROUTE FOR THE FORMATION OF PEPTIDES

«£-/W-AMINQADIPYL/-

B. L i b e r e k , R. Kasprzykowska, K. Wisniewski I n s t i t u t e of Chemistry, U n i v e r s i t y of Gdansk, PL-80-952 Gdansk, Poland

Introduction A l l organisms t h a t produce p e n i c i l l i n s and cephalosporins s y n t h e s i z e and u t i l i z e the common p r e c u r s o r H 1) TEA

O. TEAHO,SO

n

R: BzlOCH,

AQ-.I

2) N a H C O i

COOSu NHAc [ RO

R: BzlOCHa Pd(OH?i/HCOONH4 ^

SOjNa

NaOjSO

MeOH

Bulgecin A

20% (5 steps)

RO 3

the

ho —r

OAllyl

i—UBZ1 J-O

|[/dabco

to

n

NHAc

NHAc

"Sff

was applied

NHAc

77%

BzlBr

OH

method

H.OAc|^

Ac( NHj-HCI

PhCH(OMe)i TsOH Ph

r—OAC OAc )— O

DeCf/CHCi,

Ac,0 Py

H,OH •

Oxazoline

R: BzlOCHj

Fig. 2. Synthetic scheme of bulgecin A.

yCOOMe

345 coupling

of

N-acetylglucosamine

and

bulgecinine

bonyl (Troc) group for the protection of residue w a s then ther coupling

replaced

parts.

5-hydroxymethyl

with benzyloxymethyl

reactions.

A promising

order

Trichloroethoxycargroup in

bulgecinine

(Bom) group to proceed

of

subsequent

reactions

construction of bulgecin A molecule w a s s u g g e s t e d

as follows, i.e.,

tion

succinimidyl

of

carboxyl

sulfation; coupling imino

product

group

c o u r s e of

Pd

in

3

in

thus

bulgecinine

reaction

residue

However,

at

this

hydrogenation

not

defect with

be

could only

was

residue

presumably

might

N-Methylation

catalysts

prepared

bulgecinine

the reaction

undesirable group.

group

3) coupling with taurine residue.

subjected

was via

a

by

caused

was

overcome and

slowness even

in

shown

2)

O-

during

below.

hydrogenolysis

a

This

of

Bom

in use of various kinds of but

also

an

application Synthetic

of

reduced

pressure.

catalytic

bulgecin

A

transfer thus

ob-

respects.

the present study, we could confirm the proposed s t r u c t u r e of Furthermore,

study

to syntheses

be

the

hydrogénation,

methylated

as

HCOONH^.

bulgecin A synthetically. may

ester;

catalytic

tained was c o m p l e t e l y identical with the natural product in all A s a result of

for

1) a c t i v a -

in f a c t , when the final

unexpectedly

pressure

by

to

mechanism

not be avoided atmospheric

PdlOH^

as

However,

fur-

applicable

the synthetic s t r a t e g y of

other

bulgecin-like

realized

in this

glycopeptides

as

well as bulgecin B.

References 1. A. Imada, K. Kitano, K. Kintaka, M. Muroi, M. Asai. 1981. Nature 289, 590. 2. A. Imada, K. Kintaka, M. Nakao, S. Shinagawa. 1982. J . Antibiot. 35, 1400. 3. S. Shinagawa, F. K a s a h a r a , Y. Wada, S. Harada, M. Asai. 1984. Tetrahedron 40, 3465. 4. R. Cooper and S. Unger. 1986. J . Org. Chem. 51, 3942. 5. T. Wakamiya, K. Yamanoi, M. Nishikawa, T. Shiba. 1985. Tetrahedron L e t t . 26, 3465. 6. T. Wakamiya, K. Yamanoi, K. Kanou, T. Shiba. 1987. Tetrahedron L e t t . 28, 5887.

ANTIBACTERIAL AND IMMUNOSTIMULATORY PROPERTIES OF CHEMOTACTIC N-FORMYL PEPTIDE ANTIBIOTIC-CONJUGATES.

P.M. Lockey, B.W. Bycroft, R.J. Grout, A.J. Penrose, P. Williams Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, NG7 2RD, U.K.

Introduction N-formyl oligopeptides such as formyl-methionyl-leucyl-phenylalanine (FMLP) stimulate chemotaxis and chemokinesis as well as cytotoxic activities such as production of superoxide anion and hydrogen peroxide in leukocytes (1). These effects are induced by binding of the peptides to the chemotactic peptide receptor (CPR) on the plasma membrane of the cells . Four N-formyl dipeptide conjugates of ampicillin and amoxycillin were synthesised. Formyl-methionyl-leucyl-ampicillin

1

Formyl-methionyl-R-leucyl-ampicillin

2

Formyl-methionyl-R-leucyl-amoxycillin

3

Formyl-methionyl-a-aminoisobutyric acid-ampicillin

4

The conjugates were assessed for antibacterial activity and affinity for the CPR on differentiated human promyelocytic leukemia (HL-60) cells. HL-60 cells can be induced to express functional receptors for N-formyl peptides providing an in vitro model for investigation of the effects of the conjugates on leukocytes (2).

Results All of the conjugates exhibit good antibiotic activity against Gram positive and Gram negative bacteria. The most active compounds have approximately one fourth the activity of the parent ^-lactam antibiotics.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin New York - Printed in Germany

347 In competition binding assays using [3H]-FMLP the conjugates displayed affinity for the CPR. Binding activity was related to the structural resemblance of the conjugates to FMLP. The order of potency being 1 > 2 > 3 > 4. The conjugates were found to be potent agonists, stimulating both chemotaxis (Boyden chamber technique) and release of superoxide anion and hydrogen peroxide (assessed using lucigenin- and luminol- enhanced chemiluminescence) by HL-60 cells. The rank order of potency in stimulating functional responses was identical to that for receptor binding activity. The influence of the conjugates on phagocytosis and intracellular killing of bacteria by leukocytes is currently being investigated. Thus these compounds combine potent antibacterial and immunostimulatory activity in the same molecule and merit investigation of their in vivo effectiveness.

References 1.

Rot, A., L.E. Henderson, T.D. Copeland, E.J. Leonard. 1987Proc. Natl. Acad. Sci. U.S.A. 84, 7967-7971-

2.

Harris, P., P. Ralph. 1985. J. Leukocyte. Biol. 37, 407-422.

ANTIBACTERIAL PEPTIDES CONTAINING 2-AMINOPIMELIC ACID

P. Le Roux, D. Blanot, D. Mengin-Lecreulx and J . van H e i j e n o o r t U.A.

1131 du C . N . R . S . ,

U n i v e r s i t à de P a r i s - S u d ,

91405 Orsay, France

Introduction

G i l v a r g and co-workers have shown t h a t L-2-aminopimelic acid f a l s e substrate f o r t e t r a h y d r o d i p i c o l i n a t e which p a r t i c i p a t e s lysine

(2).

succinylase

(Apm) i s a

( 1 ) , an enzyme

i n the b i o s y n t h e s i s o f diaminopimelic a c i d

(DAP) and

C e r t a i n p e p t i d e s c o n t a i n i n g Apm are a n t i b a c t e r i a l ;

b a c t e r i o l y s i s occurs only i f

however,

l y s i n e i s included to the c u l t u r e medium

In order to o b t a i n p e p t i d e s which might be b a c t e r i o l y t i c pe.fl i t ,

(1).

we have

s y n t h e s i z e d d i - and t r i p e p t i d e s c o n t a i n i n g both l y s i n e and Apm: Lys-DL-Apm (1),

DL-Apm-Lys ( 2 ) , Lys-Ala-DL-Apm ( 3 ) and DL-Apm-Ala-Lys

(4).

Reference

compounds Ala-DL-Apm ( 5 ) and DL-Apm-Ala ( 6 ) have a l s o been prepared. o v e r , we have a s s o c i a t e d Apm to g - c h l o r o - L - a l a n i n e r i a l amino a c i d

(3),

More-

(fSClAla) , an a n t i b a c t e -

i n d i p e p t i d e s 3ClAla-DL-Apm.(7) and DL-Apm-BClAla

(8).

Syntheses

7, 3, 5 and 7 were s y n t h e s i z e d by a method s i m i l a r and 8

to G i l v a r g ' s

2, 6

were obtained from Z-DL-Apm(0Bzl)-0Su; d e p r o t e c t i o n was achieved by

H 2 /Pd (2 and 6)

or HBr (8).

4 was s y n t h e s i z e d by c o u p l i n g

Ala with L y s ( Z ) - O B z l by the Weygand-Wiinsch method ( 4 ) ,

Minimum i n h i b i t o r y

MIC v a l u e s minimum

(1).

(Table

Z-DL-Apm(OBzl)-

f o l l o w e d by H 2 /Pd.

concentrations

1) were determined on Eic.hexic.hia

medium (M63 supplemented w i t h g l u c o s e and

CoLL

K12 HfrH i n

thiamine).

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany

liquid

349 Table 1 .MIC Values of the Synthetic Peptides Peptide

MIC without lysine

I 2 3 4 5 6 7 S

(yg/ml) with 0.5 mM lysine

> 1024 > 1024 128 256 > 1024 1024 64 32

> 1024 > 1024 128 128 32 16 64 16

Effects on growing cells E.coLL

cells were grown in the same medium as above, with or without sup-

plementation of certain amino acids (0.5 mM each). The peptides were added at 1.5 mM to mid-exponential phase cells. Reference peptides 5 and 6 were bacteriostatic alone and bacteriolytic in the presence of lysine, as described by Gilvarg (1). Dipeptides 7 and 2 had no effect, regardless to the medium. Tripeptides 3 and 4 were ineffective alone, bacteriostatic with lysine and bacteriolytic with Lys, lie, Leu and Phe. 7 and 8 were bacteriostatic with or without lysine, but

bacteriolytic

with

Lys, lie, Leu

and Val. The effect of peptide 6 on the intracellular pools of nucleotide peptidoglycan precursors was tested. It was added at 100 yg/ml to E. C.0ÍÁ. cells growing exponentially in the above medium supplemented with lysine. Cells were harvested 30 min afterwards and the nucleotide pools were quantitated according to Mengin-Lecreulx QjL CLÍ. (5) . An important accumulation of UDPMurNAc-dipeptide was observed (2080 nmol/g dry weight, instead of 6 nmol for untreated cells), whereas the concentrations of the subsequent nucleotide precursors (UDP-MurNAc-tri- and

-pentapeptide) were strongly dimi-

nished. In non-harvested cells, lysis started a few minutes later.

Discussion Lysine has two effects : it restaures protein synthesis, and it acts as a feedback inhibitor of DAP pathway (1). Both effects bring about lysis of

350 c e l l s treated by 5 or 6. The weak or n i l a n t i b a c t e r i a l a c t i v i t i e s and the absence of b a c t e r i o l y t i c e f f e c t of peptides 1-4, which contain both lysine and Apm, are surprizing ; they might be due to poor transport by permeases or/and poor cleavage by cytoplasmic peptidases. I t i s noteworthy, however, that the tripeptides display b a c t e r i o l y t i c a c t i v i t y in the presence of amino acids ( l i e , Leu and Phe) which are synergistic with lysine f o r the i n h i b i t i o n of DAP pathway ( 2 ) . gClAla inhibits alanine racemase (peptidoglycan synthesis) and transaminase B (branched amino acids l i e , Leu and V a l ) . Peptides 7 and 8 are moder a t e l y antibacterial

; as expected, a b a c t e r i o l y t i c e f f e c t i s observed i n

the presence of amino acids which restaure protein synthesis. The strong accumulation of UDP-MurNAc-dipeptide in c e l l s treated by pept i d e 6 is quite consistent with the i n h i b i t i o n of DAP pathway by Apm. Lugtenberg showed that this nucleotide precursor is accumulated in temperature-sensitive mutants of E. coti possessing very low DAP-adding a c t i v i t y ¿n V-itAO ( 6 ) . Both methods can be used f o r the production of UDP-MurNAcdipeptide in large amounts .

References 1. Berges, D.A., W.E. DeWolf, J r . , G.L. Dunn, S.F. Grappel, D.J. Newmann, J.J. Taggart, C. Gilvarg. 1986. J. Med. Chem. 29, 89. 2. Patte, J.C. 1983. In: Amino Acids, Biosynthesis and Genetic Regulation (K.M. Hermann and R.L. Sommerville, e d s . ) . Addison-Wesley, p.213. 3. Manning, J.M., N.E. M e r r i f i e l d , W.M. Jones, E.C. Gotschlich. 1974. Proc. Nat. Acad. Sci. USA 71, 417. 4. Weygand, F . , D. Hoffmann, E. Wiinsch. 1966. Z. Naturforsch. 21b, 426. 5. Mengin-Lecreulx, D., B. Flouret, J. van Heijenoort. 1982. J . B a c t e r i o l . 151, 1109. 6. Lugtenberg, E . J . J . , A. van Schijndel-van Dam. 1972. J. B a c t e r i o l . 41.

110,

TRICHOLONGINS B I AND B H : ISOLATION AND AMINO ACID SEQUENCE DETERMINATION

Sylvie REBUFFAT and Bernard BODO Laboratoire de Chimie, Muséum National d'Histoire Naturelle, UA 401 CNRS, 63, rue Buffon, 75231, Paris, Cedex 05, France.

Introduction From a culture broth of the fungus Trichoderma longibrachiatum (M 3431), we isolated new peptides which exhibited an antibiotic activity against the Gram + bacteria Staphylococcus aureus (strain 209 P). The peptidic fraction was separated into two main groups we named tricholongins A (LA) and B (LB). LA and LB consisted in microheterogeneous mixtures as shown by reversed-phase HPLC analysis. Tricholongins B belong to the peptaibol class as they contain an acetylated N-terminal residue, a high proportion of a-amino isobutyric acid (Aib) and an amino alcohol. The present communication refers to the isolation and primary sequence determination of the two main tricholongins B, LB I and LB II. Materials and methods FAB mass spectra were recorded on a VG analytical MM ZAB-HF mass spectrometer fitted with an Ion Tech saddle field primary atom gun, with 8 KeV xenon atoms as ionizing beam and cc-monothioglycerol as matrix. NMR experiments were performed on a Bruker WM 500 spectrometer equipped with an Aspect 2000 computer using Dis NMR P830601 software. Results and discussion The culture filtrate of T. longibrachiatum was shaken for 24 h with the adsorbing resin XAD 4. The resin was then poured into a column and washed with a gradient from water to MeOH. The crude LA and LB peptides were desorbed selectively: LA eluted first (from 10% to 40% MeOH) followed by LB (from 80% to 100% MeOH). The LB fraction was chromatographed on Sephadex LH 20 (MeOH) and Si0 2 (CH 2 Cl 2 /MeOH gradient) successively, to afford the pure LB group which was then submitted to reversed-phase C l g HPLC (Fig. 1) to isolate the main components LB I and LB H.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin New York - Printed in Germany

352

Fig 1: HPLC Chromatogram of tricholongins B (C18 Spherisorb ODS2; 7.5 mm x 30 cm; Me0H/H 2 0 83/17; 2 ml/min. Det. UV220nm).

0

20

40

60

80 ttmln)

The amino acid analysis of the complete acid hydrolysates showed them to contain both Gly (1), L-Glu (3), L-Leu (1), L-Leuol (1), L-Phe (1), L-Pro (1), L-Ser (1), L-Val (1). The presence of Aib (9) in LB I and Aib (8), D-Iva (1) in LB II distinguished the two peptides. As they did not give reaction with diazomethane, the 3 Glu of the hydrolysates arose from 3 Gin. Gly

•Val

Pht

Aib

Aib

Aib

332

Alb

Gin

Gin

Gin

417

Aib

Alb

Aib

Set

Leu

Aib

Leuol

> [m + N a J + 1933

Hi

972

1170 leasl

Fig.2 Positive ion FAB mass spectrum of LB I showing the fragmentation pattern The positive ion FAB mass spectrum of LB I (Fig. 2) exhibited the cationated (M + Na) + pseudomolecular ion at mlz 1933 in agreement with the molecular formula C g j H ^ ^ i O ^ derived from the amino acid and amino alcohol composition (H=1.007). As expected for peptaibols (1,2), the preferential cleavage at the Aib-Pro bond occurred, resulting in the formation of two complementary oligopeptides which underwent independent sequential cleavages that superimposed in the spectrum. The sequence of LB I was thus determined as shown in Fig. 4. The sequence of LB H was assigned in a similar way but an ambiguity remained related to the reciprocal position of the isomeric residues Val/Iva Thus, the Val/Iva location was specified by a

NMR study of LB II.

353

T f ^ l

. 0a

i Iva I Ûe ,

Val G Ì y # - G 0 C0-- a r o, o G®- a--

Fig.3 RELAY spectrum of LB II (500.13 MHz, CD 3 0D) showing the connectivités for Iva, Val, Gin, Gly.

l n i

0>2 A two dimensional relayed coherence transfer spectroscopy (RELAY) experiment performed in CD3OD (Fig. 3) provided the connectivities between the lateral chain of each residue and the a-proton. The assigned signals were further correlated to the amide protons by a H.H-COS Y in CD3OH. The sequence elucidation (Fig. 4) resulted then from the observation in the 2D NOESY spectrum (CD3OH, 273°K) of NOE effects between the amide protons of contiguous residues. 1 2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18

19

LB I : Ac Aib Gly Phe Aib Aib Gin Aib Aib Aib Ser Leu Aib Pro Val Aib Aib Gin Gin Leuol LB II: Ac Aib Gly Phe Aib Aib Gin Aib Aib Aib Ser Leu Aib Pro Val Aib Iva Gin Gin Leuol Fig. 4: Primary sequence of LB I and LB n

Acknowledgments : We are indebted to Dr D.Davoust (Laboratoire de Chimie Organique Structurale, Université P. et M. Curie, Paris) for the NMR spectra and to Dr D. Fraisse (Centre de Spectrométrie de Masse, CNRS, Lyon) for the FAB mass spectra.

References: (1) Pandey, R.C., Meng, H„ Cook, J.C., Jr and Rinehart, K.L.Jr., 1977, J.Am.Chem.Soc. 99, 5203-5205. (2) El Hajji, M„ Rebuffat, S„ Lecommandeur, D., and Bodo, B„ 1980, Int.J.Peptide Protein Res. 29, 207-215.

HELICAL CONFORMATION OF TRICHORZIANINES IN SOLUTION

Bernard BODO, Sylvie REBUFFAT, Mohamed EL HAJJI, Laboratoire de Chimie, Muséum National d'Histoire Naturelle, UA 401 CNRS, 63, rue Buffon, 75231, Paris, Cedex 05, France. Daniel DAVOUST Laboratoire de Chimie organique structurale, Université P. et M. Curie, UA 455 CNRS, 4, place Jussieu, 75230, Paris, Cedex 05, France.

Introduction Trichorzianines are 19-residue hydrophobic peptides of the peptaibol class, isolated from the mould Trichoderma harzianum (1). They consist in two major groups, neutral trichorzianines A (TA) and acidic trichorzianines B (TB), which are complex microheterogeneous mixtures as shown by HPLC analysis (2). The main peptides from the two groups (9 TA and 7 TB) were isolated and their sequence determined (2). The only difference between the homologous TA and TB consists in the replacement of a Gin at position 18 for a Glu. 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18

19

TA mc: Ac Aib Ala Ala Aib Aib Gin Aib Aib Aib Ser Leu Aib Pro Val Aib lie Gin Gin Trpol TA VH: Ac Aib Ala Ala Aib Iva Gin Aib Aib Aib Ser Leu Aib Pro Val Aib De Gin Gin Pheol TB IIIc: Ac Aib Ala Ala Aib Aib Gin Aib Aib Aib Ser Leu Aib Pro Val Aib lie Gin Glu Trpol TB VII: Ac Aib Ala Ala Aib Iva Gin Aib Aib Aib Ser Leu Aib Pro Val Aib lie Gin Glu Pheol Trichorzianines were shown to interact with phospholipid bilayers and to increase the membrane permeability. They also induced growth inhibition and cell lysis of the amoeba Dictyostelium discoideum. Biological activities appeared to be related to the membrane properties; the TB exhibited weaker effects than the homologous TA (3). A conformational analysis of trichorzianines in solution was thus undertook by

and

NMR, in order to delineate if a possible conformational change would contribute to the different activities noticed for TB, as compared to TA.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin N e w York-Printed in G e r m a n y

13

C

355 Materials and methods NMR spectra were recorded on a Bruker WM 500 spectrometer. CD spectra were obtained on a Jobin Yvon Mark EI dichrograph.

Results and discussion The CD spectra of trichorzianines in various organic solvents (TFE, methanol...) were characteristic of a high proportion of a right helix. The conformation in such solvents was considered to be rather relevant to the conformation of trichorzianines in phospholipid membranes and the NMR study was realized for methanol solutions. Dynamic information on TA IHc was obtained from longitudinal relaxation times T1 of the protonated a carbons: the homogeneous values proved the peptide to be fully helical (Table 1). 1

T1

J

NHC(XH

Ac Aib 1 Ala 2 Ala 3 Aib 4 Aib 5 (Iva) Gin 6 Aib 7 Aib 8 Aib 9 Ser 10 Leu 11 Aib 12 Pro 13 Val 14 Aib 15 lie 16 Gin 17 Gin 18 Trpoll9 (Pheol)

13

A5/AT

AS/AT

13

CaH

tcbound

0.4ns-»>2.6ns. This clearly indicates that this part

at least of the side-chain interacts with the S^' subsite of the active site of collagenase. It must be stressed that such a result could not be obtained by proton relaxation even though deuteration

is carried out at Cg-Cf, because of the

overlapping of different proton resonances in this part of the spectrum even at 500MHz. This direct implication of the norvaline side-chain is compatible with the 10 fold difference in the activity observed between Suc-Pro-Ape (Ki= 20jiM) and Suc-Pro-Ala (Ki=210;iM) .

401

Conclusions The present work shows that tritium relaxation in a specifically -CHT-CHT- moiety located in selected parts of a small ligand is a powerfull aid in the interpretation of relaxation in terms of structural and dynamic parameters for the ligand either in the free state or when bound to a macromolecule. He Ha,

Hc H ».

"OOC-C-C-CO-Pro-Ala, "OOC-C-C-CO-Pro-Ala, II II

Suc-Pro-CH-COO" I CH-2 I CHTy CH2T$

compound I

Compound II

compound III

Table I Proton ( a ) and Tritium ( b ) Relaxation Rates (sec -1 ) at 298°K of compounds I, II and III.(a) 500MHz, (b) 320MHz (I)

«•f=+0. 02 b

(Ii>

(III)

=-11. 6

R^0.44Ha,0.42Hc

R^ S =0.48 H a ,0.45 H c

R

R

b=30Ha'

31

HC

bS=8-5Ha'12Hc

« f =+0.10

R^=1.47 Tb ,1.70 Td

R^ s =1.57 T b ,1.60 T d

«•b=-14

R

b=17

ns R - 3 K b

« f =0.08

R

f=1-42T,

«•=-9.3 b

R

b=

11

Tb' 1 8

Td

Tb'

4

Td

-3T,

References 1. Valensin, G., Sabatini, G., Tiezzi, E. 1986. In: Advanced Magnetic Resonance Techniques in Systems of High Molecular Complexity (N. Niccolai and G. Valensin Eds). Birkhauser Boston, p.69 2

Valensin, G., Kushnir, T., Navon, G. 1982 J.Magn.Reson. £6, 23.

3

Yiotakis, A., Dive, V. 1986. Eur.J.Biochem. 160, 413.

SUICIDE INHIBITION OF PROLYL 4-HYDROXYLASE BY PEPTIDES CONTAINING 5-OXA- OR 5-AZAPROLINES S.Henke, D.Brocks, H.Gaul, R.Geiger, V.Günzler, H.Plankenhorn Hoechst AG, D-6230 Frankfurt am Main 80, West Germany

K.I. Kivirikko, R. Myllylä Collagen Research Unit, Dept. of Medicinal Biochemistry, University of Oulu, SF-90220 Oulu, Finland

Introduction The prolyl 4-hydroxylase (PH; EC 1.14.11.2) is a key-enzyme in the biosynthesis of collagen and one of the primary targets for the

development of antifibrotic compounds (1). It exclusively

catalyzes

the hydroxylation of peptide-bound proline residues

attached to the N-terminus

of glycine (2). The hydroxyproline

residues are of eminent importance

for

the thermal stability

of the triple helix and the active secretion of collagens (3). Inhibitors of PH are valuable compounds for the therapy of diseases involving excessive deposition of collagen. This include fibrosis

of

the lung and liver, scleroderma and arterioscle-

rosis . We report here tors

of

the synthesis of novel mechanism-based inhibi-

PH specifically acting

at the peptide binding site.

We designed peptides which mimic the of procollagen and which

are

hydroxylatable

sequence

converted to reactive molecules

by means of the enzymatic hydroxylation. The

synthetic

peptides

irreversibly inactivate PH in a time

dependent manner.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in G e r m a n y

403

Results and discussion Based

on

the

peptides contain

have

V*

reaction catalyzed by PH been

synthesized

which

the unphysiological amino acids

isoxazolidine-3-carboxylic acid (oxaproline) (4) and

Opr =X-0 Apr = X= NH Apr(Boc) X-N-Boc

pyrazolidine-3-carboxylic

acid (azaproline) (5) instead of proline in a hydroxylatable position. Their syntheses are outlined in scheme 1. The

oxaproline

peptides proved to be specific and highly ac-

tive mechanism-based

inhibitors

of PH complying with all re-

quirements necessary for a suicide the

analogues

inactivation

(6)

whereas

azaproline peptides showed only a slight acti-

vity. A detailed

structure-activity-relationship has been performed

by various alterations

done

at the N- and C-terminus of oxa-

proline. The C-terminal glycine proved inactivation.

led to inactive compounds. ved

using

isomere.

to

be

necessary

for

Substitution of glycine by L- and D-amino acids No suicide inactivation was obser-

peptides containing D-oxaproline instead of the LTripeptides

R^-AA-Opr-Gly-OI^

containing

aromatic

amino acids (AA) and aromatic substituents (RJ,R2> were

found

to be most effective. Z-Phe-Opr-Gly-OBzl

was the most active compound tested giving

50 %

of

inactivation

284 M * s

.

PH

in

one hour at

0.8 (iM

^obs^ 1

=

The activity of other enzymes was not affected.

404

AA

-

Z-fOH

DCC / HOBt

Opr -

Gly

H - ~ 0*Bu

TFA / Ani sol e

-

Z--OH

Apr H

DCC / HOBt

-o'Bu •OH

AA

H-|-OBzl

DCC / HOBt

-OBzl

NaOH / MeOH

-

Gly

Boc OMe ,Boc ¿OMe .Boc ¿OH H—f-OBzl Boc

DCC

TFA / Anisole

/ HOBt

•OBzl

-OBzl

Scheme 1 : Syntheses of the peptides

References 1. Hanauske-Abel, H.M., Gtinzler, V. 1982. J. Theor. Biol. 94, 421-455 2. Kivirikko, K.I., Myllylä, R. 1986. Ann. N.Y. Acad. Sci. 460, 187-201 3. Ramachandran, G.N., Ramakrishnan, C., (1976) in Biochemistry of Collagen (Ramachandran, C.N., Reddi, A.H. eds) 45-84, Plenum Press, New York 4. Vasella, A., Voeffray, R. 1981, J.C.S., Chem. Comm. 97-98 5. a) Attwood, M.R., Hassall, C.H., Lambert, R.W. Lawton, G., Redschwa, S., Ger. Offen. DE 3317290 Al b) Henke, S., Gaul, H., manuscript in preparation 6. Criteria for mechanism-based inactivation described in: Walsh, Ch.. 1982. Tetrahedron. ¿8, 871-901

P E P T I D Y L F L U O R O M E T H A N E S . THIOL R E S I S T A N T P R O T E A S E AFFINITY L A B E L S .

Herbert Angliker Friedrich Miescher Institut, P.O.Box 2543, CH-4002 Basel, Switzerland.

Introduction Peptidyl chloromethanes are quite specific inactivators of serine and cysteine proteases In vitro. Their usefulness for cellular studies appears compromised by low molecular weight thiols and thiol groups of proteins. To overcome this, a functional group less reactive than chloromethanes is being studied: fluoromethanes.

Synthesis Fluorine was introduced by two methods: A: Phthaloyl-aminoacid-dlazomethane

(1) was treated with 5 2 % HF in pyridine to yield

fluoromethane (2) and hydroxymethane (3) (ratio 1:1 to 2:1) which were chromatographicaliy separated. Deprotection with NaBH 4 and acetic acid led to the aminoalcohol (4). Coupling with the aminoacid

or peptide yields the hydroxy-peptide

(5) which Is oxidized

by

Cr03/pyrldine or DMSO/SOg/pyridine to the peptide (6).

HF-pyr

Pht=NCHCCHN,

I R

OH I H,N-CHCHCH,F I R

1. NaBH, 2. AcOH

Ay i H

0

II Pht=NCHCCH,F

Pht=NCHCCH 7 OH I

R

Coupling

OH I R '-NHCHCHCH,F I R

O

M R '-NHCHCCH,F 2 I

R

R' = Z, prot. amino acid, prot. peptide R = side chain

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

406 B: The final peptide (7) is converted by the method of Dakin-West with fluoroaceticanhydride and triethylamine to the fluoromethane (8) in which the C-terminal aminoacld is fully racemized.

O II R -NHCHCOH I R 1

O II R' -NHCHCCH..F I R

(FCH 2 C0) 7 0 = = NEt,

7

8 R1 R

= Bz, prot. amino acid, prot. peptide = side chain

Deprotectlon of the peptldyl fluoromethane, where needed, was carried out in trifluoroacetic acid or liquid HF. In case of Z-protected dipeptidyl fluoro (or chloro and bromo) methane (9) the major product was a pyrazine derivative (10) which had lost HF (or HCI and HBr) and water, for example:

Z-Phe-Ala-CH 2 F

TFA -Z, -HF, -H 2 0

XX

C e H s CH,

CH,

10

Results Derivatives of phenylalanine, alanine [1], lysine [2] and arginine [3] have been synthesized which satisfy the specificity of the enzymes. Thiol reactivity is much reduced (tested towards glutathione), whereas the reactivity to a cysteine protease Is largely retained [4], Peptidyl fluoromethanes are irreversible inhibitors which alkylate the enzymes: the imidazole ring In the active center of a serine protease and the thiol group in the active center of a cysteine protease.

407 Kinetic Comparison of Peptidyi Fluoromethanes with Chloromethanes as Enzyme (E) Inhibitors (I) +

E

complex

l-E

K, (M)

k 2 (min 1 )

kj/Kj (M" 1 min 1 )

Ala-Phe-Lys-CH 2 F

4.0 • 10 ®

0.0024

6.0 • 10 2

Ala-Phe-Lys-CH 2 CI

5.6 • 10 ®

0.18

3.2 • 10"

1.4 • 10"5

3.3

2.4 • 105

s

12.5

5.4 • 10 s

Plasmln

Cathepsin B Z-Phe-Phe-CH 2 F Z-Phe-Phe-CH 2 CI

2.3 • 10

In case of the serine protease plasmin, Ala-Phe-Lys-CH 2 F shows a slightly higher affinity towards the enzyme, but a much smaller alkylatlon rate than the corresponding chloro-compound, which makes the fluoro-compound fifty times less effective than the chlorocom pound. In case of the cysteine protease cathepsin B, Z-Phe-Phe-CH 2 F shows also a higher affinity towards the enzyme than the corresponding chloro-compound but not such a reduced alkylation rate as with the serine protease, which makes the fluoro-compound only two-fold less effective than the chloro-compound. However, if the fluoromethanes are less toxic than the chloromethanes they may be more useful for physiological experiments.

References 1.

Rauber, P., H. Angliker, B. Walker and E. Shaw. 1986. Biochem. J. 23?, 633-640.

2.

Angliker, H., P. Wikström, P. Rauber, P. and E. Shaw. 1987. Biochem. J. 241, 871875.

3.

Angliker, H„ P. Wikström, P. Rauber, S. Stone and E. Shaw. 1988, Biochem. J. in press.

4.

Shaw, E., H. Angliker, P. Rauber, B. Walker and P. Wikström. Biochim. Acta 45, 1397-1403.

1986.

Biomed.

SYNTHETIC PEPTIDES RELATED TO THE CELL-BINDING DOMAIN OF FIBRONECTIN

J. S. Davies, J. Orchison Department of Chemistry, University College, Swansea, SA2 8PP. U.K. G. E. Jones, Department of Anatomy and Human Biology, King's College London (KQC) London WC2R 2LS, U.K.

Fibronectin (1), a cell surface glycoprotein which interacts with other extracellular matrix molecules is of interest because of the information it can generate on how cells interact with their surroundings.

Cell-

binding to fibronectin appears to depend on the tetrapeptide sequence Arg-Gly-Asp-Ser- located in a

fi-turn

conformation in Fibronectin, and,

peptides of this sequence act as competitive reversible inhibitors in assays of cell adhesion (2).

Our interest in the active domain sequence

was initiated by a desire to study eel 1-substrate interactions (3) using model sequences bound to glass or polymer matrices, and to mimic the active domain in a form representative of a £-turn conformation. Present Work. The active domain sequence has been synthesised using a solution phase approach as outlined in Scheme 1.

The route up to compound II in the

scheme proved to be routine but deprotection gave a very complex mixture, presumably due to the many rearrangements possible especially the

aspartyl migration (4).

Alkaline hydrolysis (2 eq NaOH in IMF)

removed both benzyl and methyl esters.

Improvement in purity could be

achieved by starting with the C-terminal benzyl ester of serine to synthesise Boc-Arg(N0 2 )-Gly-Asp(OBz1)-Ser(OBz1)-OBz1. and hydrogenation led to the deprotected sequence.

Removal of Boc

Recycling of the

crude product on a C -reversed phase column (eluant 0.01M NH„OAc/CH,CN o 4 o 90:10) gave a pure product.

Peptides 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin N e w York-Printed in G e r m a n y

409 Scheme 1 Arg

Asp

Gly

Ser

Bzl ¿—OMe

Bzl

OH H— DCCl/HOBt Bzl

Boc-

OBzl —OMe

BocTFA ,Bzl Boc-

OBzl

HDCCI/HOBt

-OH

OBzl

, Bzl

N0_

Boc-

z.

OBzl

-0H HMixed Anhydride

Boc-

^-OMe

OBzl OMe

z

Bzl

Boc-

The H-Arg-Gly-Asp-Ser-OH w a s biologically tested

Bzl OMe

(5) by seeding

of purified fibronectin on glass slides, with human skin

II 'lawns'

fibroblasts.

For concentrations of fibronectin < 10Mg/ml, concentrations of tetrapeptide as low as 0.5 m M were sufficient

to reduce

eel1-fibronectin

attachment by >80%, and in studies on inhibition of cell spreading

lnM

concentrations of tetrapeptide were sufficient to cause > 7 0 % of cells to retract over a 60 m i n period.

Control peptides H-Gly-Lys-Gly-Asp-OH

and H-Arg-Gly-Glu-Ser-OH did not respond

in the tests.

This again is

added proof for the requirement of a -Arg-Gly-Asp- sequence for attachment. to kill

the cells, so the counterion had to be exchanged.

a C-h.p.l.c. o

specific

The control peptide as the trifluoroacetate salt w a s

found

Recycling on

reversed phase column using CH.CN/0.01M NH.OAc as eluants o 4

has proved a satisfactory w a y of exchanging

counterions.

The active domain sequence has also been attached successfully Sepharose using activated CH-Sepharose 4B.

to

Analysis showed a loading

value of 5 umol tetrapeptide per 1ml of swollen gel.

A 1 ml column of

the linked tetrapeptide was tested for its 'affinity' for cell membrane preparations but no conclusive evidence of binding has yet been detected. The tetrapeptide coupling

linked to Sepharose via soluble

(2) has shown activity.

carbodiimide

The contrasting results from the two

410 coupling approaches needs further exploration. A successful approach

for linking the tetrapeptides on to glass

surfaces (6) is shown in the scheme. In preliminary tests human skin fibroblasts will adhere to these surfaces and in some cases cells have OMe [7 CMe 1 | I OHC(CH ) CHO | I S i-O-S i(CH ) NH — > S i-0-Si (CH ) N=CH(CH ) CH=Arg-G1y-Aspe J I I Arg-Gly-Asp-Ser I I ' J SerOH J OMe OMe 'Silylated Glass Surface'

LI

been observed to spread in a normal manner on such surfaces. quantitative studies are in progress.

Further

Our ultimate aim was to insert

the -Arg-Gly-Asp-Ser sequence into a tf-turn conformation in model cyclic peptide mimic cyclo-(Lys-Arg-Gly-Asp-Ser) with the side-chain of Lys being available for linking to a polymer or glass matrix.

This work is

currently progressing but has not reached a sufficiently mature stage for a detailed report to be made.

Acknowledgement. We are very grateful to the S.E.R.C. for financing this project.

References. (1)

Hynes, R.O., 1986. Scientific American. (6), 32.

(2)

Pierschbacher, M. D. and E. Ruoslahti. 1984. Nature. 309. 30; Ginsburg, M. , M.D. Pierschbacher, E. Ruoslahti, G. Marguerie and E. Plow, 1985. J. Biol. Chem. 260, 3931; Akiyama, S.K. and K. Yamada, 1985. ibid. 260, 10402; Singer, I.I., D.W. Kawka, S. Scott, R. A. Mumford and M.W. Lark, 1987. J. Cell. Biol. .104, 573.

(3)

Pizzey, J. A., J. Witkowsky and G.E. Jones. 1984. Proc. Natl. Acad. Sei. U.S.A. 81„ 4960.

(4)

Perseo, G. , R. Forino, M. Galantino, B. Gioia, V. Malatesta and R. de Castiglione. 1986. Int. J. Peptide Protein Res. _27, 51.

(5)

Jones, G.E., R.G. Arumugham and M.L. Tanzer, 1986. J. Cell. Biol. 103, 1663.

(6)

Aplin, J. D. and R. C. Hughes, 1981. Anal. Biochem. ¿13, 144.

INHIBITION OF PSEUDOPEPTIDES

CARBOXYPEPTIDASE

A

WITH

KETO-METHYLENE

G. Shoham, D.A. Oren, A. Ewenson and C. Gilon Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, ISRAEL

Summary Carboxypeptidase A (CPA) is an exopeptidase that exhibits preferred specificity towards peptides and esters bearing large hydrophobic C-terminal residues (1). Neither the detailed binding mode nor the exact mechanism of hydrolysis are fully determined. We investigated the specific binding of CPA with a series of substrates and their non-cleavable analogs, for the purpose of isolating the binding effects of substrates from other catalytic effects. We chose a special family of peptide analogs in which the scissile peptide bond is replaced with the non-cleavable keto-methylene unit. A series of these keto-methylene pseudopeptides were synthesized (2) and tested as inhibitors of CPA (3) . Two pseudopeptides were found to inhibit the esterase activity of CPA in the lower micromolar range. These inhibitors were crystallized with CPA to form a stable enzyme-inhibitor complexes which were studied by x-ray crystallographic methods. Results and Discussion Table 1 summarizes the inhibition constants of the compounds assayed as CPA inhibitors. The pseudopeptides 2, 3 and 5-8 were assayed as their diastereomeric mixture resulting from their synthesis. Table 1. shows that only compounds 2 and 5 are potent inhibitors of CPA with K^ in the range of 0.5-5 (1M. Our results confirm that at least the last three residues at the C-terminus of a peptide substrate are participating in the binding interactions with the enzyme. The results also indicate that the last three side chains are preferred to be aromatic or branched aliphatic. A comparison of the inhibition constants of 2 and 5 relative to 7 and 8 demonstrates the importance of

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

412

Table 1. Inhibition of CPA Esterase Activity by Keto-Methylene and Dehydro-Keto-Methylene Pseudopeptides and their Analogous Peptides No.

Compound

K^ [|i.M]

1 2

Boc-Phe-Phe-OH Boc-(RS)Phe-\y(COCH2)(RS)Phe-OH

0.7

3

Boc- (RS)Phe-\|f(COCH2)A(E)Phe-OH

>1000

4

pGlu-Phe-Phe-OH

5

pGlu- (RS)Phe-\|/(COCH2) (RS)Phe-OH

4.1

6

pGlu-(RS)Phe-\|f(COCH2)A(E)Phe-OH

>1000

7

Boc- (RS) Phe-\(i (COCH2) Gly-OH

8

Boc-Gly-\|/(COCH2) (RS)Leu-OH

>1000 900

Figure 1. Stereoview of the CPA/5 complex. Pertinent active-site residues are indicated (thin line - native enzyme, double line - bound enzyme). Note the specific interactions of the gem-diol with the Zn ion (center), Glu270 (left) and the Argl27 (bottom). Note also the large conformational change around Ile247- Tyr248 (top).

413

hydrophobic

interactions

at

the

S1 ^

binding

subsite.

A

comparison of the inhibition constants of the pseodopeptides with their dehydro counterparts (2 with 3 and 5 with 6) demonstrate the conformational flexibility required from this hydrophobic side chain for proper interaction with the enzyme. with 8 demonstrate the A comparison between 2 and 5 importance of specific hydrophobic (or aromatic) interactions at the S^ binding subsite. These conclusions which are based on kinetic studies in solution were further supported by structural studies of CPA in the crystal. A 1.75 A resolution data was collected from CPA/2 crystals and was used for model building (4). Even though the native CPA crystals were subjected to four diastereomers of 2, the enzyme specifically binds only one isomer, namely the S,S stereoisomer. Moreover, the species observed to bind to CPA is the hydrated form where the ketone group appears as gem-diol. This interesting result indicates that the enzyme-inhibitor complex is most stable with a species resembling a structure along the hydrolytic reaction coordinate rather than a species resembling a reactant or a product. A 2.0 A resolution data collected from the CPA/5 crystals resulted in a similar structure. A stereoview of the important interacting groups in the CPA/5 complex is shown in Figure 1. Especially noted are the specific interactions of the hydrate with the Zn ion, Glu270 and Argl27. The structure of both CPA/2 and CPA/5 complexes confirms the presence of two hydrophobic pockets at the S 1 and S^ subsites and enables the exact determination of the boundaries and interactions in these sites. References 1.Ludwig, M.L., W.N. Lipscomb. 1973. In:Inorganic Biochemistry (G. L. Eichhoren, ed.). Elsevier, Amsterdam, Vol 1, p. 438. 2.Ewenson, A., R. Cohen, D. Levian, Z. Selinger, M. Chorev, C. Gilon. 1988. Int. J. Pep. Prot. Res. ¿1, 296. 3.Latt, S.A., D.S. Auld, B.L.Vallee.1972. Anal Biochem. 56. 4.Shoham, G., D.W. Christianson, D.A. Oren. 1988. Proc. Natl. Acad. Sci. USA 684.

r-GLUTAMYL D E R I V A T I V E S OF 1 0 - P R 0 P A R G Y L - 5 , 8 - D I D E A Z A F O L I C AS LIGANDS FOR THYMIDYLATE SYNTHASE A F F I N I T Y

K. P.

Pawelczak, Wieczorelc,

M. B.

Kempny, L . K r z y 2 a n o w s k i , Rzeszotarslca

I n s t i t u t e of Cliemistry, -15-052 O p o l e , Poland.

J.

W.

CieSla,

ACID

CHROMATOGRAPHY

Pedagogical

University

of

Opole,

Rode

Nencki I n s t i t u t e of Experimental B i o l o g y , of S c i e n c e s , 02-093 Warszawa, Poland.

P o l i s h . Academy

I n t r o d u c t i on

Thymidylate gous

synthase

and a n t i v i r a l

deoxyurldylate

to

hydrofolate

its

zyme.

The

or

tive and

studies

to

bind

fication

of

order

create

to

TS

glutamic (II,

acid

Scheme

of

requiring

the

the

liver

very

poorly proved

viral,

Starting

coen-

affinity to

a

compara-

Hymenolepis

found the

diminuta,

enzyme

from

both

10-formyl-5, 8-dldeazafolate-

earlier

to

bacterial

a more u n i v e r s a l

as the

TS b i n d i n g

coenzyme.

to

of

5,10-methylenetetra-

s u c c e s s f u l l y by

tapeworm,

we

antifun-

conversion

derivative

most

the

rat

of

catalyzes

t h e dUMP-dependent

TS f r o m

aminoethyl-Sepharose,

matography,

on

analogue

of

in anticancer,

-r-oligoglutamate was p u r i f i e d

regenerating

sources

chemotherapy,

based

Sepharose-bound

a target

thymidylate,

enzyme

chromatography

(TS),

be

effective

by

puri-

and mammalian o r i g i n .

ligand

for

TS a f f i n i t y

In

chro-

10-propargyl-5,

S-dideazapteroyl-L-glutamyl-T-L-

(I,

and

1)

Scheme were

1)

synthesized

its

protected

and

their

tested.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin N e w York-Printed in G e r m a n y

GABA

analogue

applicability

415 Results

eiu

pAB P

MA HO Z—OtBu

rGlu

I: Q^pAB-Glu

I^OtBu

¿OtBu

Z—|— CI

z-

H

•OtBu

P: HC=C-CH 2 — pAB:

-OtBu

PBr

4-H 2 NC 6 H4C00H

•OtBu

QBr

P rGlu(0tBu)2 QJ-pAB-Glu-GABA

II:

h2

•OtBu

TFA-gJ

SCHEME 1

Synthesis

of

I

(1)

ported

to belong

potent

inhibitor

generating

rat

spectively.

is presented to

of

the the

liver,

on

(i)

with the

using

(both

and h a v i n g

free

based

a f f i n i t y adsorbent

ml

of

aminoethyl-Sepharose)

of

both the

from

its

ment

of

the

preparations,

crude

extracts,

as p r e v i o u s l y

polymer-based

affinity

11

1 per ml) for

ii

and ( i i i )

and i i i

amino

by

immo-

to

coupling.

copolymer

macroporous

cross-linked

groups).

The

followed

adsorbents

by (2).

1 per

TS ( T a b l e

ammonium Both

1)

treatsulfate

macroporous

(synthesized using way ( n o t

di-

binding

obtained by s t r e p t o m y c i n described

pol-

Sepharose-

0 . 5 mg o f

liver

of

with

e x p r e s s e d dUMP-dependent

bound TS i n a s i m i l a r

was s y n t h e s i z e d

available

macroporous

(synthesized using

fractionation of

(ii)

tapeworm and r e g e n e r a t i n g r a t

crude

re-

inhibition I was

a

re-

5 nM and 9 nM,

w e r e 6 PM and 0 . 1 jjM.

and . n - b u t y l a e r y l a t e

acrylonitrile

vinylbenzene

re-

was a l s o

l-ethyl-3-(3-dimethylaminopropyl)carbodiimide,

aminoethyl-Sepharose,

of

150 v a l u e s

The c o r r e s p o n d i n g v a l u e s d e s c r i b i n g

acrylonitrile ymer

The p r o d u c t ,

TS i n h i b i t o r s ,

enzyme f r o m b o t h t h e tapeworm and

lO-formyl-5, 8-dideazafolate bilized,

i n Scheme i .

strongest

0 . 2 mg

shown).

obtain a ligand with only

one

carboxyl

Immobilization

the

macro-

on e a c h o f

416

TaUle

l.

Affinity

Regenerating

Chromatography of

Rat L i v e r

(R.r.l.)

Amlnoethyl-Sepharose-Immobilized Enzyme Source Preparation

R.r.l.

Synthase on the

I CI ml column)*.

Volume ml

Crude

H.d.

tue Tapeworm C-ff.d-) ana

Thymidylate

Total a c t i v i t y nmol^mln

Yield *

4

1. 7

100

A f t e r chr omat ography

84

1. 3

78

Crude

13

15. 9

lOO

695 63 A f t e r chromatography 10.. O *TS d i d not hind t o the column i n the absence of dUMP. To adsorb the enzyme, i t s p r e p a r a t i o n c o n t a i n i n g 20 pM dUMP was passed through the column. A f t e r washing the column w i t h O.2 M phosphate b u f f e r pH 7.5 c o n t a i n i n g O.1« T r i t o n X-lOO, 10 mM 2-mercaptoethanol and 20 pM dUMP, the enzyme was e l u t e d w i t h the same b u f f e r without dUMP. polymers mentioned above was a c h i e v e d by the mixed an-

porous hydride

method and

fluoroacetic adsorbents but of tion

a c i d as bound the

the r e s u l t s the column of

11

the

t - b u t y l groups were removed w i t h

elsewhere d e s c r i b e d mammalian enzyme

(3).

tri-

Both a f f i n i t y

i n dependence on dUMP

were not r e p r o d u c i b l e w i t h d i f f e r e n t batches material.

enabling the

b e f o r e immobilization,

Further

studies,

removal of

the

aimed at m o d i f i c a protecting

groups

are i n p r o g r e s s .

T h i s work was supported by g r a n t s CPBP 01.01-2.03 and - 2 . 0 4 .

References 1. Pawelczak, K., T . R . Jones, M. Kempny, A . L . Jackman, D.R. N e w e l l , L. Krzy:tanowski and B. R z e s z o t a r s k a . J.Med.Chem. ( i n p r e s s ) . 2. C i e ä l a , J . , Z. Z i e l i i i s k i , B. Machnicka and W. Rode. Acta B i o c h i m . P o l . 31, 291-298.

1987.

3. Pawelczak, K., L. Krzy±anowski, B. Rzeszotarska, M. Kempny, P. Wieczorek, J. C i e ä l a and W. Rode. C o l l . Czech.Chem.Commun. ( i n p r e s s ) .

C O N F O R M A T I O N A N D A C T I V I T Y O F S H O R T IgAl P R O T E I N A S E

S t e p h e n G. W o o d , J a m e s

INHIBITORS

Burton

E v a n s D e p a r t m e n t of C l i n i c a l R e s e a r c h , U n i v e r s i t y H o s p i t a l , Boston University Medical Center, Boston, MA 02118, U.S.A. A r t u r P e d y c z a k , I g n a c y Z. S i e m i o n D e p a r t m e n t of C h e m i s t r y , U n i v e r s i t y of W a r s a w , W a r s a w ,

Poland

Introduction: A b o u t 70% of the b a c t e r i a l

i n f e c t i o n s in the d e v e l o p e d

world

are c a u s e d by m i c r o o r g a n i s m s w h i c h s e c r e t e a p r o t e i n a s e selectively

i n a c t i v a t e s h u m a n s e c r e t o r y IgAl.

d u c i n g IgAl p r o t e i n a s e

Pathogens

I n a c t i v a t i o n of

is c a u s e d b y c l e a v a g e at s p e c i f i c p r o l y l r e s i d u e s (Fig 1).

inhibit neisserial

Octapeptide substrate analogs

GalNAc

I

I

I

GaMAc

|

Fig.l.

T S. sanguis S. pneumoniae S. mitior

here.

Gal

I

Gal

GalNAc

T H. influenzae H. aegyptius

Gal

I

GaNAc

-Cys-Pro-Val-Pro-Ssr-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thrt

which

IgAl p r o t e i n a s e s as m u c h as an o r d e r of

Gal

B. melaninogenicus

were

Structure-

for a s e r i e s of t e t r a p e p t i d e s

m a g n i t u d e m o r e e f f e c t i v e l y are p r e s e n t e d

220

IgAl

in the h i n g e

p r e v i o u s l y s h o w n to i n h i b i t the IgAl p r o t e i n a s e s . activity relationships

pro-

i n c l u d e : N. g o n o r r h o e a e , N. m e n i n g i t i -

d i s , H. i n f l u e n z e a e , and S. p n e u m o n e a e .

r e g i o n of IgAl

that

I

I

GaNAc

I240

Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cyst

N. meningrtidis-2 N. gonorrhoeae-2 H. influenzae-2

C l e a v a g e s i t e s of h u m a n IgAl by the IgAl

P e p t i d e s 1988 © 1989 W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w York - P r i n t e d in G e r m a n y

t N. meningititis-1 N. gonorrhoeae-1

proteinases.

418 Results Six s e r i e s of u n i q u e t e t r a p e p t i d e s and t h e i r b l o c k e d

N-acetyl,

C - a m i d e , and N - a c e t y l C - a m i d e a n a l o g s w h i c h s p a n the

hinge

r e g i o n of h u m a n IgAl w e r e s y n t h e s i z e d by s o l i d - p h a s e

tech-

niques.

P e p t i d e s w e r e c l e a v e d from the s u p p o r t by HF/10%

a n i s o l e , gel f i l t e r e d o n S e p h a d e x G - 1 5 , and p u r i f i e d to h o m o g e n e i t y by r e v e r s e d p h a s e H P L C .

The 24 p e p t i d e s w e r e

for t h e i r a b i l i t y to inhibit the T y p e 2 n e i s s e r i a l proteinase using previously described techniques v a l u e s for the v a r i o u s t e t r a p e p t i d e s FREE PEPTIDE

STPP

ACETYL

TPPT

PPTP

tested

IgAl

(1).

PIC5Q

are s h o w n in Fig 2. AMIDE

PTPS

ACETYL, AMIDE

TPSP

PSPS

HINGE REGION PEPTIDE

Fig,2» P I C 5 Q v a l u e s for the t e t r a p e p t i d e inhibitors.

* = IC cr , > 1 m M .

IgAl

proteinase

419 Discussion: E x a m i n a t i o n of the i n h i b i t o r y p r o p e r t i e s of p e p t i d e s s h o w n Fig.2 s h o w s t h a t the b e s t i n h i b i t o r s of the p r o t e i n a s e s p a n e i t h e r the

o r the

neisserial regions.

P J - P J '

For

the first s e r i e s , the free p e p t i d e a n d the a c e t y l p e p t i d e the m o s t e f f e c t i v e

inhibitors.

For the P ^ ' P j

1

series,

a c e t y l p e p t i d e and the p e p t i d e a m i d e p r o v i d e the b e s t tion.

T h e b e s t i n h i b i t o r is P r o - T h r - P r o - S e r - N H 2

w h i c h has an ICgg v a l u e of 5.3 juM.

inhibitor

(1).

the inhibi-

T h i s is an o r d e r of IgAl

P h y s i c a l s t u d i e s u s i n g N M R and C D

t e c h n i q u e s i n d i c a t e that m a n y of the p e p t i d e s have able solution conformations.

identifi-

No c l e a r r e l a t i o n s h i p

between

i n h i b i t o r y p r o p e r t i e s a n d s o l u t i o n c o n f o r m a t i o n is s e e n .

Acknowledgment T h i s r e s e a r c h w a s s u p p o r t e d by NIH g r a n t D E - 0 7 2 5 7 P r o t e i n a s e and D e n t a l C a r i e s " a w a r d e d b y the I n s t i t u t e of D e n t a l

"IgAl

National

Research.

Reference: 1.

are

(HRP-48)

m a g n i t u d e b e t t e r t h a n the b e s t p r e v i o u s l y r e p o r t e d proteinase

in

B u r t o n , J . , S . G . W o o d , M. L y n c h , A . G . P l a u t . J . M e d . C h e m . 31, 1647.

1988:

EFFECT OF ARTIFICIAL DDT-BINDING POLYPEPTIDES ON DDT D E G R A D A T I O N BY A C Y T O C H R O M E P - 4 5 0 M O D E L S Y S T E M

T. H e h l g a n s , H. L a n g e n , M. L i n d e n , T. E p p r e c h t , a n d B. G u t t e Biochemisches Switzerland

S.

Klauser,

I n s t i t u t der U n i v e r s i t ä t Zürich, C H - 8 0 5 7

Zürich

Introduction We found that a m i x t u r e of h e m i n a n d e x c e s s c y s t e i n e

(1), a

m o d e l s y s t e m of c y t o c h r o m e P - 4 5 0 e n z y m e s , w a s a b l e to d e g r a d e the i n s e c t i c i d e D D T

(1,1,l-trichloro-2,2-bis(p-chlorophenyl)

e t h a n e ) p a r t i a l l y . H e r e we r e p o r t the e f f e c t of a d e s i g n e d 2 4 - r e s i d u e D D T - b i n d i n g p o l y p e p t i d e a n d s e v e r a l of

its

a n a l o g u e s o n the rate of the h e m i n - c y s t e i n e - m e d i a t e d

DDT

degradation.

Results and

Discussion

D D T d e g r a d a t i o n b y the h e m i n - c y s t e i n e m o d e l s y s t e m

yielded

m a i n l y three n o n - t o x i c c o n j u g a t e s of DDT m e t a b o l i t e s c y s t e i n e w h o s e s t r u c t u r e s w e r e e l u c i d a t e d b y gas graphy - mass spectrometry

with

chromato-

(manuscript submitted).

Cysteine

or h e m i n a l o n e was i n a c t i v e . The rate of this d e g r a d a t i o n at least 8 x 10^ t i m e s h i g h e r t h a n that of the reaction.

uncatalyzed

In the p r e s e n c e of a d e s i g n e d 2 4 - r e s i d u e

binding polypeptide

(2)(Fig.

i n c r e a s e was o b s e r v e d c o u l d be m a i n t a i n e d c y s t e i n e at

1) a n a d d i t i o n a l

was

DDT-

four-fold

rate

(Fig. 2). The initial d e g r a d a t i o n

rate

for at least 25 h b y a d d i n g

f r e s h DDT a n d

intervals.

The propose'd 3 - s h e e t c o n t e n t of the d e s i g n e d p e p t i d e

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

was

421

jgja

V *

F i g u r e 1. A m i n o a c i d s e q u e n c e a n d p r o p o s e d f o l d i n g of the d e s i g n e d 2 4 - r e s i d u e D D T - b i n d i n g p o l y p e p t i d e (bottom) a n d s p a c e - f i l l i n g m o d e l of the l i g a n d (top; the a r r o w s p o i n t to the five c h l o r i n e a t o m s of DDT). A b i n d i n g site of h i g h c o m p l e m e n t a r i t y is f o r m e d b y the side c h a i n s of Phe 14, His 16 a n d lie 21 for the a r o m a t i c ring a, Phe 14, Met 11 a n d lie 4 for the t r i c h l o r o m e t h y l g r o u p , a n d His 16 a n d lie 4 for the a r o m a t i c ring b. D o t t e d lines are h y d r o g e n b o n d s . quantitatively confirmed by CD measurements, and N M R s t u d i e s a l s o s h o w e d the p r e s e n c e of o r d e r e d

preliminary structure.

The d e s i g n e d 2 4 - r e s i d u e p o l y p e p t i d e b o u n d D D T ^ 1 0 0 0 more strongly than serum albumin complex,

0.8 x 1 0

-6

( K D of the

times

polypeptide-DDT

M).

The D D T - b i n d i n g p r o p e r t i e s of a n a l o g u e s of the d e s i g n e d

pep-

tide s u p p o r t e d the p r o p o s e d s t r u c t u r e of the p o l y p e p t i d e complex

(Fig. 1). R e p l a c e m e n t of T y r l 5 a n d T y r l 7 b y

r e s u l t e d in a 3 - f o l d i n c r e a s e of the d i s s o c i a t i o n w h e r e a s e x c h a n g e of P h e l 4 b y v a l i n e w e a k e n e d the D D T i n t e r a c t i o n 2 5 - f o l d . T h e r e was a n o t i c e a b l e

DDT

threonine

constant polypeptide-

correlation

b e t w e e n D D T a f f i n i t y a n d rate a c c e l e r a t i o n of the h e m i n c y s t e i n e - c a t a l y z e d D D T d e g r a d a t i o n b y the d e s i g n e d

poly-

p e p t i d e s . The p e p t i d e s h o w n in Fig. 1 i n c r e a s e d the y i e l d of the d e g r a d a t i o n p r o d u c t s

in 6 h 3 - f o l d , the T h r l 5 ,

a n a l o g u e 2 . 3 - f o l d , a n d the V a l l 4 - a n a l o g u e

1.5-fold.

Thrl7-

422

Time (min)

F i g u r e 2. R e s o l u t i o n of the p r o d u c t s of h e m i n - c y s t e i n e - p o l y peptide-catalyzed DDT degradation by reversed-phase h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y . F i r s t s o l v e n t , 10% CH3CN i n 0 . 1 % a q u e o u s T F A ( t r i f l u o r o a c e t i c a c i d ) ; s e c o n d s o l v e n t , 75% CH3CN in 0 . 1 % a q u e o u s T F A . T h e r e a c t i o n c o n d i t i o n s w e r e a s f o l l o w s : 2.5 m M D D T , 0 . 3 4 m M h e m i n , 6 8 m M c y s t e i n e a n d 0 . 1 m M d e s i g n e d 2 4 - r e s i d u e D D T - b i n d i n g p o l y p e p t i d e in 0 . 0 5 M NH4HCO3, p H 7 . 7 / e t h a n o l (5:6, v / v ) , 6 h , 3 7 ° C . P e a k s 2 - 4 a r e D D T m e t a b o l i t e - c y s t e i n e c o n j u g a t e s , p e a k 6 is u n r e a c t e d D D T . It is a n a t t r a c t i v e

n o t i o n to c o n s i d e r

the

hemin-cysteine-

p o l y p e p t i d e m i x t u r e a m o d e l of a p r i m i t i v e

enzyme

the p o l y p e p t i d e

the s u b s t r a t e

i m p r o v e d t h e s o l u b i l i t y of

the h e m i n - c y s t e i n e - c a t a l y z e d

in

which for

reaction.

Acknowledgement This work was supported

in p a r t b y the

Schweizerische

Nationalfonds.

References 1. S a k u r a i ,

H.

1980. Chem.

Pharm.. B u l l .

2. M o s e r , R., R . M . T h o m a s a n d B. G u t t e . 247-251.

28,

3437-3439.

1983. FEBS Lett.

157,

BASIC

J.P.

PEPTIDES ACCELERATE

Pelerin,

Centre

B. B a r b i e r ,

de B i o p h y s i q u e

Recherche

THE HYDROLYSIS

A.

ACIDS

Brack

Moléculaire,

Scientifique,

OF R I B O N U C L E I C

C.N.R.S.,

45071 Orléans

cedex

1A, a v e n u e

2,

de

la

France

I n t r o d u c t ion

Sequential

polypeptides

drophobic ture

residues

in s a l i n e

aqueous

amino-acids

consist

polypeptides

accelerate

ribonucleotides dipeptides, synthesized

Synthesis

The

with

have been

shown

solution

the b a s e

(3,4) whereas

to d e t e r m i n e

dipeptide

or

lysine,

the threshold

of

hydrophilic

the

alternating of

oligo-

is i n a c t i v e .

n = 1,

hy-

struc-

the

hydrolysis

lysine

with

and

a B-sheet

When

induced

free

Ac-(Leu-Lys)n-NHEt

hydrophilic

to a d o p t

(1,2).

in a r g i n i n e

and C o n f o r m a t i o n

Boc-Leu-OH

alternating

3 and

Oligo-

5 have

of h y d r o l y t i c

been

activity.

Oligo(Leu-Lys)

has been prepared

+ HC1,H-Lys(2C1Z)-NHEt

HC1

as f o l l o w s

M. A . • Boc-Leu-Lys (2C1Z)-NHEt Ac?0 — A

• H C l ,H-Leu-Lys(2ClZ)-NHEt

:

c

- Leu-Lys (2C1Z)-NHEt

HBr •

The

hexa-

se

procedure

N^-2C1Z

and

decapeptide using

acetic

was

achieved

Acetylation

anhydride. with

hav« been prepared

a phenylacetamidomethyl

protections.

with

Ac-Leu-Lys(HBr)-NHEt

Cleavage

ethylamine

was

by

solid pha2 N -Boc and

resin,

carried

out

of t h e p e p t i d e

in m e t h a n o l

the

from

(1.5/1

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

on the

in

the

resin resin

volume)

424

which a f f o r d e d C - t e r m i n a l cleaved

ethylamides.

by HBr in t r i f l u o r o a c e t i c

p u r i f i e d by HPLC on L i c h r o s o r b trile/water/0.2 were All

CD

spectra

significant

of

A

of

mixture

monomer run

dichroism

spectra

a random c o i l

NaClO^.

conformation

f 3 - s t r u c t u r e s c o u l d be

and

no

detected.

oligoribonucleotides oligo(A)s

7 days at

Lys+/P0^

complexation

containing

all

the o l i g o m e r s

(3,4).

t o ensure a c o m p l e t e

A precipitate

mixed t o t h e o l i g o ( A ) s

the were

appears when t h e

indicating

a

remains c l e a r

with the d i p e p t i d e .

translucent

gel

is

A similar

with

a

decapeptide

the f o r m a t i o n of formed but

The I . R .

show t h e f o r m a t i o n of

with the d e c a p e p t i d e .

8

oligoribonucleotide

and t h e o l i g o n u c l e o t i d e s .

hexapeptide,

t h r e e complexes

from

Hydrolysis

50°C in Gly-Gly/NaOH b u f f e r pH

ratio

p l e x between t h e p e p t i d e s

Lys)26

Circular

were

acetoni-

in 0 . 2 M NaCl and in 0.05 M

indicated

were

and t h e p e p t i d e s

t o t h e 20-mer was used as s u b s t r a t e .

for

2.5:1

acid

side-chains

RP18 Merck column w i t h

TFA g r a d i e n t s .

amounts o f

Hydrolysis

is

%

run in pure w a t e r ,

Lysyl

the

spectra

a |3-sheet

a

com-

With

the

solution run on t h e

structure

only

b e h a v i o u r was found f o r

(Leu-

(5).

Figure Variation

of the r e l a t i v e

hydro-

l y t i c a c t i v i t y (obtained by r e f e r ring the percentages of hydrolyzed phosphodiester

bonds to that

ob-

tained in the case of the polypeptide) length. in the peptide chain

as

a

function

of

chain

425

Control

Dipeptide

Hexapeptide

Decapeptide

Polypeptide

3.4

6.2

11.8

64.3

69.4

% of Hydrolyzed Phosphodiester Bonds Influence of the peptide chain length on the hydrolytic a c t i v i t y . mental conditions : -3 5.10 M expressed

50°C,

7 days,

Experi-

polypeptides

01igo(A)s -2 1.25.10 M

h y d r o l y t i c a c t i v i t y has been e x p r e s s e d as t h e

percentage

in phosphate,

Gly-Gly b u f f e r 0.1 M pH 8, Oligo- and

expressed in l y s i n e . The of the

hydrolyzed phosphodiester

d i m e r and hexamer b u t i n c r e a s e s

tide

(from

The a c t i v i t y

the

formation

hydrolytic activity of a g - s h e e t

the

in

the

p r o p e n s i t y already observed with

(leucyl-lysyl) The

present

taining phobic

and t h e

g-sheet

alternating

poly (5).

short oligopeptides

con-

r e s i d u e s are able to adopt a 6-sheet conformation to oligoribonucleotides.

t h e h y d r o l y s i s of t h e

Brack, A., L.E. Orgel.

2.

Brack,

A.,

A.

They a c c e l e r a t e

when

markedly

oligoribonucleotides.

1.

1975. N a t u r e 2_56 , 383.

Caille.

1978.

Int.

J.

Pept.

Protein

Res.

128.

3.

Barbier,

B . , A. B r a c k .

1987. O r i g i n s of L i f e 17,

4. 5.

Barbier,

B . , A. B r a c k .

1988. J . Am. Chem. S o c . ( i n

Barbier,

B . , A. B r a c k .

1988.

(in

This

s a m p l e s c o n t a i n i n g b o t h L and D r e s i d u e s

study demonstrates that

to

complex.

o n l y t e n a m i n o a c i d s w i t h a l t e r n a t i n g b a s i c and h y d r o -

complexed

U,

decapep-

polypeptide).

a p p e a r s t o be r e l a t e d

structure

c o n f i r m s t h e r e l a t i o n s h i p between t h e a c t i v i t y formation

i s weak f o r

sharply for the

17 t o 92 % of t h e a c t i v i t y of

Therefore, the

bonds.

press).

Forum P e p t i d e s ,

381. press).

Nancy 1988

C O N F O R M A T I O N A L D I F F E R E N C E B E T W E E N h A N P AND

S. K o y a m a , A. Sato, M.

Met(0)12-hANP

Kobayashi

R e s e a r c h L a b o r a t o r i e s , F u j i s a w a P h a r m a c e u t i c a l Co., L t d . , K a s h i m a , Y o d o g a w a - k u , O s a k a 532, J a p a n Y. K o b a y a s h i , T. O h k u b o , Y.

Kyogoku

I n s t i t u t e for P r o t e i n R e s e a r c h , O s a k a U n i v e r s i t y , S u i t a , 565, J a p a n

Osaka

N. Go F a c u l t y of S c i e n c e , K y o t o U n i v e r s i t y , K y o t o 606,

Japan

Introduction We have d e t e r m i n e d the s t r u c t u r e of h u m a n A N P (hANP) in 1 s o l u t i o n t h r o u g h the c o m b i n e d use of 'H-NMR s p e c t r o s c o p i e s (1 1 a distance geometry algorithm.K

' The r e s u l t s h o w e d t h a t h A N P

h a d three r e g i o n s a l o n g the s e q u e n c e t a k i n g some o r d e r e d 1

7

ary structures; S e r - C y s , A r g

11

"-He

1 5

and

Gln

18

-Tyr

These p a r t s w e r e c o n n e c t e d w i t h two hinge r e g i o n s ; 1

and G l y ^ - A l a

17

28

second-

.

Gly^-Gly1®

. V a r i o u s a n a l o g u e s have b e e n s y n t h e s i z e d to

i n v e s t i g a t e the r e l a t i o n s h i p b e t w e e n c h e m i c a l s t r u c t u r e b i o l o g i c a l a c t i v i t y of h A N P . A m o n g these s t u d i e s , the on of M e t 1 2

and

into M e t C O ) 1 2 has b e e n s h o w n to reduce the

of h A N P into a l m o s t n o n e T h e r e

and

oxidati activity

have b e e n no d i s c u s s i o n

such a d i f f e r e n c e of the a c t i v i t y of A N P c a u s e d by

on

chemical

m o d i f i c a t i o n s f r o m a s t r u c t u r a l a s p e c t so far. H e r e , we

deter-

m i n e d the s t r u c t u r e of this d e r i v a t i v e a n d a n a l y z e d the

struc-

t u r a l d i f f e r e n c e b e t w e e n hANP a n d

Met(0)

12

-hANP.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

427 Results and Discussions

Sequential resonance assignments of the two dimensional spectra and interpretations of the intensities of NOE peaks in the NOESY spectra of Met(O) 1 2 -hANP to the corresponding atomic distances were carried out following to the case of hANP. Using the distance geometry algorithm proposed by Braun and Go, the three dimensional structure of Met(0)^ 2 -hANP was elucidated by minimization of a target function which consists of the square-sum of the differences between atomic distances in the calculated structure and the corresponding distances obtained by the interpretation of the NOEs. One hundred trials of the minimization were carried out from hundred individually given initial conformations which were constructed in the computer using random values of dihedral angles. The resulting conformers were evaluated in terms of residual values of the target function and root mean square of distances among individual conformers. In Fig. 1, the profiles of the convergences of six conformers with small residual values of the target functions were demonstrated. The backbones of the peptides were superimposed to minimize the r.m.s.d. values. Such a profile of hANP is

shown in Fig. 2. Comparing these profiles of the whole mole-

cules in tops of each figure, it was obvious that the convergence of Met(O) 1 2 -hANP was worse than that of hANP, and that large conformational changes were induced by such a chemical modification beyond expectation. However, rather good resemblance between their structures was localy conserved especially in the C terminal region i.e. Gin 18 -Tyr2ft . These facts revealed 1? that the oxidation on Met' of hANP caused a local conformation changes interfering the bend formations in the regions of Ser''Cys^ and Arg''^-Ile^ ^ respectively. The studies of the relationship between chemical structure 11and the activity of hANP have indicated that the region of Arg lie''^ are very important because substitutions of amino acid residues including lie''5 were quite effective on the loss of the activity.(3)

428

Thus we c o n c l u d e d t h a t the o r d e r e d s e c o n d a r y s t r u c t u r e of h A N P is n e c e s s a r y to p r o v i d e it w i t h b i o l o g i c a l a c t i v i t y a n d

inter-

f e r e n c e on the o r d e r e d s t r u c t u r e r e s u l t s in the loss of the activity.

Ser 1 Ser 1 Cys 7

Cys 7 Arg 11

lie 15 Arg 11

lie 15

Gin 18

Gin 18

Tyr 28 Tyr 28 Fig. 1 M e t ( 0 ) - a - h A N P

Fig. 2 a - h A N P

Reference

(1) K o b a y a s h i , Y. et a l . , (1988) J.. B i o c h e m . U^., 3 2 2 - 3 2 5 . (2) H a y a s h i , Y. et a l . , (1986) Peptide Chem.1985, 27-32. (3) K o n i s h i , Y. et al., (1988) P e p t i d e s , 4 7 9 - 4 8 1 .

COMPARISON OF BIOLOGICALLY ACTIVE CONFORMATIONS OF OLIGOPEPTIDES WITH THEIR STRUCTURES IN SOLUTION

G . V . N i k i f o r o v i c h , M.D.Shenderovich, B.G.Vesterman, J . B e t i p s I n s t i t u t e of Organic S y n t h e s i s , Latvian SSR Academy of Sciences, Aizkraukles 21, 226006, Riga, USSR

Introduction I n studying conformation-function r e l a t i o n s h i p s

i n oligopeptides i t i s im-

portant to know whether b i o l o g i c a l l y active structures are present

among

conformers i n s o l u t i o n and i f so, what i s their approximate s t a t i s t i c a l weight. This communication i s an attempt to apply t h i s approach to c y c l i c analogues of bradykinin and substance P as well as to enkephalin and s p i n labelled angiotensin by estimating the geometrical s i m i l a r i t y between their possible conformers in s o l u t i o n and the models proposed e a r l i e r for b i o l o g i c a l l y active conformations ( 1 - 4 ) . The s i m i l a r i t y of conformer A and B pairs was evaluated with the aid of an algorithm described i n ( 5 ) , the s i m i l a r i t y c r i t e r i o n being expressed as: D = l/Ni^*}-

x?)2+ (y?- y ^

+ ( z

A_

z»)2]>. a

S

where N i s the number of atom p a i r s being superimposed ( a l l C - and C atoms i n t h i s study), x , y , z are cartesian coordinates. Conformers A and B are regarded geometrically s i m i l a r i f D i s below a given threshold value

Results Cyclobradykinin (LYS 1 -Pro 2 -Pro 3 -G1y 4 -Phe 5 -Gly 6 -Pro 7 -Phe 8 -Arg 1 9 ,

CBK).

The spatial structure of CBK i n DMSO s o l u t i o n has been examined i n (6) by constructing a l l s t e r i c a l l y acceptable backbone structures s a t i s f y i n g the

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

430 r e s t r i c t i o n s imposed by NMR data. Their comparison with the b i o l o g i c a l l y active conformation of bradykinin for

B,, receptors (1) gives high

2

D values (the lowest value D n = 9.7 8 ) indicating the absence of the biol o g i c a l l y active conformer i n solution. This conclusion i s consistent with the r e s u l t s of biological testing of CBK, showing a high and longl a s t i n g depressor a c t i v i t y in vivo but lack of a c t i v i t y with respect to smooth muscle (7). Cycloanalogue of substance P (Glu 6 -Phe 7 -Phe 8 -Gly 9 -Leu 1 0 -Met 1 1 1 , CSP6). 1 nh-(ch2)3-nh—I I t has been reported also i n (6) that a cycloanalogue of the C-terminal hexapeptide of substance P (CSP6) can assume a large number of low-energy backbone structures p a r t i a l l y consistent with NMR data obtained i n DMSO s o l u t i o n . They include structures s i m i l a r to the b i o l o g i c a l l y active conformation of the C-terminal hexapeptide of substance P proposed i n (2) for rabbit mesenteric vein receptors, e . g . , CAECAA (CSP6) and EDECEE ( SP6), Dq= 1.2 ^

(designations the same as in ( 8 ) ) . Unfortunately, the

pharmacological i n v e s t i g a t i o n of CSP6 was r e s t r i c t e d g i s t r a t i o n of i t s s l i g h t hypotensive effect

t i l l now to the re-

in rats (9).

Enkephalin (Tyr 1 -Gly 2 -Gly 3 -Phe 4 -Leu 5 , EK). The s t a t i s t i c a l weights of EK backbone conformers in aqueous solution have been obtained using

a priori

calculations after the Monte-Carlo technique in (10). On the other hand, mutual comparison of sets of low-energy backbone structures performed for i —) j—o 1c three preferential ju-receptor agonists (Tyr-DOrn-Phe-Asn, [DCys ,L/DCys ] f~2 1 5 EK) and two preferential 6-receptor agonists ([DPen ,L/DPen ]-EK (3)) revealed that b i o l o g i c a l l y active structures for both receptor types can be divided after the DQ= 0.6 ft2 c r i t e r i o n : FD*AEC for jj- and FH*GAC for 6type with the respective s t a t i s t i c a l weights 0.318 and 0.430 in s o l u t i o n . I t i s obvious, though, that such d i v i s i o n for the f l e x i b l e EK molecule appears formal . Angiotensin ( S L - A s n 1 - A r g 2 - V a l 3 - T y r 4 - V a l 5 - H i s 6 - P r o 7 - P h e 8 , SL-AT). The s t a t i s t i c a l weights of SL-AT backbone conformers i n aqueous s o l u t i o n were evaluated by combined use of computational

and physico-chemical data

(11). Each conformer was represented by a s t a t i s t i c a l set of conformations, which allowed to construct histograms of interatomic distances estimated for the C^-atoms in SL-AT. As the most c h a r a c t e r i s t i c features of b i o l o g i c a l l y active conformations of AT proposed in (4) are the r e s t r i c t i o n s

431 cjjg < 8 . 3 8 and Cjy< 9.2 K, i t can be seen from the histograms that s t r u c tures with a d i s t i n c t B-type turn i n the region Tyr^-Val^ s a t i s f y t h i s r e quirement. The total weight of such structures i n s o l u t i o n amounts to 0.716-0.830.

References 1. N i k i f o r o v i c h , G.V., L.V.Podips. 1982. Bioorgan.Khim. 8, 453. 2. N i k i f o r o v i c h , G.V., Yu.Yu.Balodis, G.Chipens. 1981. In:Peptides 1980 (K.Brunfeldt ed.) S c r i p t o r , Copenhagen, p.631. 3. N i k i f o r o v i c h , G.V.,

J . B a l o d i s . 1988. FEBS L e t t e r s , 227, 127.

4. Bai o d i s , Yu.Yu., G . V . N i k i f o r o v i c h . 1980. Bioorgan.Khim. 6 , 865. 5. Nyburg, S.C. 1974. Acta C r y s t . B30, part I , 251. 6. Shenderovich, M.D., G . V . N i k i f o r o v i c h , J . S a u l i t i s , G.Chipens. 1988. Biophys.Chem. ( i n p r e s s ) . 7. Mutule, I . , F . M u t u l i s , 0.Landò, A.Asmanis, V.Grigorieva, N.Myshlyakova, V.Klusha, G.Chipens. 1984. Bioorgan.Khim. 10, 891. 8. Zimmerman, S . , H.A.Scheraga. 1977. Biopolymers, 16_, 811. 9. M u t u l i s , F . , I.Mutule, G.Maurops, I . S e k a c i s , V.Grigorieva, E.Kukain, V.Golubeva, N.Myshlyakova, V.Klusha, G.Chipens. 1985. Bioorg.Khim. 11, 1276. 10. B è t i p s , J . , G.V.Nikiforovich, G.Chipens. 1986. J .Mol .Struct.-THE0CHEM, 137, 129. 11. N i k i f o r o v i c h , G.V., B.Vesterman, J . B é t i p s , L.Podips. 1987. J.Biomol. Struct.Dynam. 4, 1119.

CONFORMATIONAL ANALYSIS OF TWO EPIMERIC CYCLIC HEXAPEPTTDES RELATED TO SOMATOSTATIN Dale F. Mierke, Christian Pattaroni and Murray Goodman Department of Chemistry, University of California, San Diego La Jolla, California 92093 USA

In this paper, we present the results from NMR studies and molecular dynamics simulations of two epimeric cyclic hexapeptides related to somatostatin (1): c[gSar-(R and S)-mPhe-D-Trp-Lys-Thr-Phe] Studies on these and other peptidomimetic containing cyclic hexapeptides are part of a collaborative effort in the development of a structure-activity relationship of somatostatin (2). A complete report covering the efforts of our collaborations will be reported elsewhere. The assignment of the proton resonances was accomplished with 2DHOHAHA, employing different mixing times, and phase sensitive COSY spectra at 500 MHz. The observed NOE's for the analogs from ROESY experiments using mixing times between 75 - 300 msec are shown in figure 1. oc The observation of strong NOE's between mPhe C H and D-Trp NH (S isomer) and mPhe C a H - gSar NH (R isomer) allowed for the unambiguous assignment of the chirality of the mPhe residue. The S isomer adopts a (311' turn at the D-Trp and Lys residues stabilized by an intramolecular hydrogen bond between the Thr NH (-A8/AT = 1 . 1 ppb/K) and mPhe CO. The strong NOE between the alpha protons of Phe and gSar indicate a cis orientation of the gSar-Phe amide bond. In DMSO there is an interconversion between two C9 turns with hydrogen bonds between gSar NH - Thr CO (-A5/AT = 1.9 ppb/K) and Phe NH - mPhe CO (-A8/AT = 1.6 ppb/K), the

Peptides 1988 © 1989 Walter de G r u y t e r & Co., Berlin • N e w York - Printed in G e r m a n y

433

Figure 1. Measured Nuclear Overhauser Enhancements gSar

R-mPhe 7

D-Trp

Lvs

Phc 1 1

Thr

N ajO^Me a p $ N a p $| N a p N a p

y

N a

M

Q Q / 4>

•

0 0 0 0/

0

O

e

0 0 /

0 /

0

0

0 /

0 •

/ e

/ o

0

0/

/

0/

e 0 / © / 0 e /

0

e / 0 e / 0e

/

0

0

15

a N

1

P a N p a N

0

0

^ a N

e •

0

Phc»

T*

Lys

D-Trpr

$ P S-mPhe 7 a Me

/ 00 •

gSar

• N

latter of which is disrupted upon addition of either water or CDC13. The R isomer adopts a y turn about the D-Trp-Lys-Thr stabilized by hydrogen bonds between Thr NH - Lys CO (-A5/AT = -1.3 ppb/K) and D-Tip NH - Thr CO (-A5/AT = 2.3 ppb/K). The NOE between Phe C a H and gSar C a H indicates that the gSar-Phe amide bond is in the cis orientation. In the bridging linkage all of the amide protons are solvent exposed, (-A8/AT > 4.0 ppb/K). Nevertheless, conformational constraint is indicated by the large difference in the chemical shift of the alpha protons of gSar (A5 = 1.35 ppm), compared with a difference of 0.30 ppm observed for the S isomer. Molecular dynamics were carried out for 20 psec at 300 K employing the Discover program modified to allow for a half harmonic forcing potential to be applied as constraints for the observed NOE's. The force constants were varied with the strength of the NOE and applied throughout the simulations.

434

In figure 2 the structures from the simulations are shown.

4 pa

8 pa

12 pa

16 ps

20 pa

Figure 2. (Top) S Isomer rotated for clarity, (Bottom) R Isomer.

The S isomer maintains a type II' P turn conformation throughout the dynamics. In the bridging region there is an equilibrium between the two C9 structures found from the NMR analysis. There is a significant interaction between the Phe 11 and D-Trp side chains. The simulation of the R isomer displays an equilibrium between two 7-membered y turns. As for the side chains the DTrp and Lys side chains are in close proximity, an average distance of 5.5 A. In the in vitro inhibition of the release of growth hormone the S isomer is half as active as the parent compound, [Pro-Phe-D-Trp-Lys-Thr-Phe] (3). The R isomer shows no activity at all (3). The differences in the biological activity of these epimers are most likely due to their differences in conformational preferences. 1. 2.

3.

Veber, D. et al. Nature 1981 292, 55-58. Bovermann, G.; Moroder, L.; Wünsch, E.; Tancredi, T.; Motta, A.; Temussi, P.; Mierke, D. F.; Lucietto, P.; Goodman, M. in "Peptide 1986: Proceedings of the 19th European Peptide Symposium," D. Theodoropoulos, eaT, Walter de Gruyter, Berlin, pp. 311-314. Assays carried out by W. Vale and G. Yamamoto of the Salk Institute.

CONFORMATIONS OF VASOPRESSIN ANTAGONISTS IN SOLUTION DERIVED BY NMR SPECTROSCOPY AND MOLECULAR DYNAMICS SIMULATION REFINEMENT

J. Schmidt, H. Rüterjans Institut für Biophysikalische Chemie, Theodor-Stern-Kai 7, Universitätsklinikum Haus 75A, 6000 Frankfurt 70, FRG Z. Grzonka, E. Kojro, F. Fahrenholz Max-Planck-Institut für Biophysik, Kennedyallee 70, 6000 Frankfurt 70, FRG

Introduction The following chemically modified analogues of the neurohypophyseal hormone vasopressin are of pharmacological interest because of their exceptionally high vasopressor-antagonistic potency and selective V^receptor affinity 1

2

3

4

5

6

7

8

(1-3):

9

Mca -Xxx -Phe -Gln -Asn -Cys -Xxx -Arg -Gly -NH2 I I with Mca = 6-Mercapto-S,6-cyclopentamethylenepropionic acid and Xxx2/Xxx7 = Tyr2/Pro7 (i) , Tyr2/Sar7 (2), or D-Phe2/Sar7 (3). The structural features of the compounds were studied in DMSO solution by means of two-dimensional NMR spectroscopy.

Material and Methods The trifluoroacetate of 1 was dissolved in dry DMSO to yield a final concentration of 30 mM; samples of compounds 2 and 3 were prepared as acetate salts. The homonuclear coupling networks were assigned using DQF-COSY, E.COSY, and HOHAHA spectra. NOESY spectra were recorded with mixing times ranging from 50 ms to 200 ms. The chemical shift values of carbons bearing at least one proton were derived from a ^

13

C-DEPT-COSY experiment. A ^ " C - C O L O C spectrum covering

the carbonyl shift region, and a 1H-detected-1H, 13C-multiple

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

436

bond correlation (HMBC) spectrum (4) covering the complete carbon spectral width yielded information about dihedral angle rotations. The hydrogen bonding pattern was derived from the temperature-dependent determination of the NH shift values.

Results and Discussion In DMSO solution, each of the examined vasopressin antagonists occurs in at least two different conformations in the slow exchange regime, thus, distinct subsets of NOE spectra are present. Deviations occur in the endocyclic type II B bend made up of residues Tyr2 to Asn5 as well as in the C-terminal tripeptide region, which is folded in the Pro7 derivative 1 to form another type II R bend between residues Cys6 and Gly9. The conformationally less restricted Sar7 derivatives 2 and 3 show two distinct correlation patterns usually found for cis/transisomer mixtures of proline compounds. In the favoured conformation the a> 6-torsional angle adopts the trans orientation, which is obvious from the short internuclear distance between the Cys6-Ha and the Sar7-N-methyl protons while correlations between the Cys6-Ha and both of the Sar7-!!" are clearly absent. In contrast, the chemical shift differences found for compound 1 are attributable to local side chain dihedral transitions rather than to cis/trans-isomerization. By comparison of the Sar7-NH-Haa' cross peak intensities, the ratio of the two dominant conformer populations in peptide 2 was calculated to be approximately 4:1. This ratio is valid only for the exocyclic part of the molecule; a conformational change in this region does not necessarily involve an accompanying reorientation of the endocyclic 6 bend. The conversion of the rate constants of the initial magnetic cross relaxation of pairs of protons yielded interhydrogen distances restricted to values smaller than 3.5 A for the given molecular size; the only long-range NOEs observed connect the sequentially non-neighbouring residues Mca1 and Cys6. The presence of multiple conformation sites within the molecules

437 necessitated weighting all NOE cross peak intensities of affected proton pairs by an appropriate factor to account for the population distribution. In this way, unambiguous internuclear distances were determined, and values too large for geometrical reasons have been avoided. Approximately 50 distance values and four dihedral angle restraints have been obtained. They were applied in the Molecular Dynamics refinement in order to reduce the accessible conformational space. The converged MD structure of 1 resembles that of deaminooxytocin in crystalline environment (5). Neither the crystal structure of pressinoic acid (6) nor the simulated vasopressin models (7,8) fit our experimental data obtained by twodimensional NMR investigations. For the vasopressin derivatives 2 and 3 two sets of conformations were obtained.

Acknowledgements Access to the programs GROMOS (W.F. van Gunsteren), HYDRA (R.E. Hubbard), and 2DNMR (R. Boelens) is gratefully acknowledged.

References 1. Krusezynski, M., B. Lammek, M. Manning, J.Seto, J. Haldar, W.H. Sawyer. 1980. J. Med. Chem. 23, 364. 2. Gazis, D., I.L. Schwartz, B. Lammek, Z.Grzonka. 1984. Int. J. Peptide Protein Res. 23., 78. 3. Fahrenholz, F., R. Boer, P.Crause, G.Fritzsch, Z. Grzonka. 1984. Eur. J. Pharmacol. 100. 47. 4. Bax, A., M.F. Summers. 1986. J. Am. Chem. Soc. 108. 2093. 5. Wood, S.P., I.J. Tickle, A.M. Treharne, J.E. Pitts, Y. Mascarenhas, J.Y. Li, J. Husain, S. Cooper, T.L. Blundell, V.J. Hruby, A. Buku, A.J. Fischman, H.R. Wyssbrod. 1986. Science 232. 633. 6. Langs, D.A., G.D. Smith, J.J. Stezowski, R.E. Hughes. 1986. Science 232. 1240. 7. Hagler, A.T., D.J. Osguthorpe, P. Dauber-Osguthorpe, J.C. Hempel. 1985. Science 227. 1309 8. Somoza, J.R., J.W. Brady. 1988. Biopolymers 27, 939

RESTRAINED MOLECULAR DYNAMICS SIMULATIONS OF CYCLIC PEPTIDES

J. Lautz Biosym Technologies GmbH, Schatzbogen 54, 8000 München 82, FRG H. Kessler Inst. f. Org. Chemie Johann-Wolfgang-Goethe-Universität, 6000 Frankfurt a. M., FRG W.F. van Gunsteren, H.J.C. Berendsen, R.M. Scheek Dept. of Phys. Chemistry, University of Groningen, 9747 Groningen, The Netherlands R. Kaptein Dept. of Org. Chemistry, University of Utrecht, 352 6 Utrecht, The Netherlands J. Blaney E.I. du Pont, Wilmington, USA

Restrained

molecular

dynamics

(MD) is a powerful

tool

for

dynamic modelling of solution conformations using NMR data, especially NOE data. The NOE information is translated

into

distances, which are used as constraints during the simulation. To achieve this, an additional harmonic distance

restraining

term is added to the standard force field potential functions [1] . The results of the simulations of two cyclic

peptides

Cyclosporin A and Anatamanide will be presented here. Cyclosporin A is an undecapeptide, with potent suppressive

immuno-

action. In the case of CPA a set of 58 distance

constraints was used throughout the simulations. As starting structures we used the experimental X-ray structure (X-ray) and a model built structure (SMS). Starting from these two initial conformations, which are substantially

different

(0.14 nm) ,

they converge nicely during the restrained MD simulation to the same final structure (0.05nm, and the distance constraints are nicely satisfied (MDS1 starting from X-ray; MDS2 starting from

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

439 SMS) . The MDS1 simulation was performed for 40ps and the MDS'2 simulation for 30ps. The simulations were performed in vacuum [2] .

In addition distance geometry calculations (DG) [3] were performed using the same set of 58 distance constraints to search a larger conformational space. The DG calculations resulted in 27 structures which could be grouped into 9 general conformational classes. From each class one structure was singled out for further MD refinement. None of the DG structures satisfied the constraints as well as the previously described MDS1 structure and they also differ from the latter by 0.16-0.39nm. It turned out that only two of the nine selected conformations could be refined to satisfy the constraints and to be energetically reasonable at the same time. These two structures are similar to the MDS1 conformation. But the DG structure satisfying the constraints (sum of violations: 0.08nm) became one of the worst structures after the refinement, the violations increased to 0.81nm. Apperantly the DG structure was highly strained in order to satisfy the constraints. This shows the necessity to refine DG structures by MD, due to the missing energetic term in the DG method and also points out that MD can be very sensitive to the choice of the starting structure and may not be capable of making large conformational changes [4]. To check the quality of the obtained MDS1 conformation, a free MD simulation (without constraints) in apolar solution CCI4 starting from the MDS1 structure was performed for 50ps and the conformation was nicely retained. This shows that the structure

obtained

in vacuum

by

restrained

dynamics

is a

reasonable and stable conformation of CPA in apolar solution. In the case of CPA the obtained conformation by restrained MD

represented

the

solution

conformation,

however

if

the

obtained structure does not fit all the NMR parameters, this may be due to errors in the experimental parameters or to the occurence of multiple conformations. Such a situation, where the available NOE- and J-coupling data cannot be satisfied by

440

one conformation of the molecule, has been found in the case of Antamanide, a cyclic decapeptide. When performing the restrained MD simulation starting from the X-ray structure of An tamanide using 40 NOE constraints, one was not able to find a conformation satisfying all of them (sum of violations: 0.24nm). Homo- and heteronuclear coupling constants provided the first entry into the discovery of the conformational inhomogeneity, which results from an amide bond flip around the 4 Ala-5 Phe and the 9 Phe-10 Phe peptide units. Using the NOE data and the coupling constants, one was able to describe the experimental data with an average of four conformations, obtained from four MD-simulations, which differed only in the combination of the flips in the peptide units of the starting structures. The overall backbone conformation is not severely influenced by the flip of the amide units [5]. Due to the fact that distances obtained by NOE measurements are sensitive to short range effects, one must be aware of multiple mutually incompatible conformations to the set of NMR parameters, used for conformational analysis. MD simulations can be a tool to tarce such situations and to get a crude estimate of the conformations involved in a process of fast equilibrium compared to the NMR time scale.

[1] van Gunsteren, W.F., Kaptein, R. and Zuiderweg, E.R.P (1983) in: Nucleic Acid Conformation and Dynamics, ed. Olson, W.K. (CECAM, Orsay), p. 72-92 [2] Lautz, J., Kessler, H.,Kaptein, R. and van Gunsteren, W.F. (1987) J. Comp. Mol. Design 1219-241 [3] Havel, I., Kuntz, I.D. and Crippen, G.M. (1983) Bull. Math. Biol. 45 665-720 [4] Lautz, J, Kessler,H., Blaney, J.M., Scheek, R.M. and van Gunsteren,W.F., Int. J. Peptide Protein Res., (in press) [5] Kessler, H., Griesinger, C., Lautz, J., Müller, A., van Gunsteren, H.J.C. (19889) J. Am. Chem. Soc. 110 3393-3396

CONFORMATIONAL MOBILITY IN CYCLIC PEPTIDES

C.A. D'Ambrosio, K.D. Kopple* L-940, Smith Kline & French Laboratories, P.O. Box 1539, King of Prussia, PA 19406, U.S.A. Y.-S. Wang Illinois Institute of Technology, Chicago, IL 60616, U.S.A.

Conformation exchanges with rates 10-100 times faster than those producing obvious NMR line broadening can be detected by their contributions to the rate of nuclear magnetic spin6X

lattice relaxation in the rotating frame, 1 / T ^ .

Although

conformational interconversions of a peptide may be kinetically complex, the contributions to relaxation are dominated by the processes producing the largest chemical shift fluctuations.

We define an effective site-site chemical shift

difference Av(eff) and exchange lifetime x e (eff) as values estimated using the two-site, equal population treatment of relaxation measurements made over a range of spin-locking fields (1,2).

At readily accessible spin-locking fields (up

to 10 kHz) there is a window for this analysis, limited by line broadening at x g > 50 ps and by absence of detectable field dependence at t < 5 >is. Below 5 jis exchange lifetimes, Tg(Av)2 may be estimated. The effective site-site chemical shift differences we observe (1) in a series of diastereomeric cyclic octapeptides, up to 1 ppm for backbone N-H and a-protons, are in accord with the chemical shift ranges that occur in (conformationally stable) proteins.

It is reasonable to hypothesize that larger values

of Av(eff) correspond to larger conformational excursions.

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin-New York-Printed in Germany

442

From the data in the Table below, the octapeptide c(D-Ala-GlyPro-Phe) 2 may be presumed to be more flexible than c(AlaGly-Pro-Phe) 2 # and both to be more flexible than the tetrapeptide c(D-Phe-D-Pro-Ala-Pro) (2). Results for these are compared with results for two cyclic hexapeptides. The hexapeptides show no spin-locking field dependence but important exchange contributions to relaxation. Lower limits to Av(eff) are near 1 ppm, indicating significant internal motion. We have begun to investigate the temperature dependence of the internal mobility reflected by the 1 / T ^ contributions, with the idea that T,

lp

measurements could be

replaced by line shape analyses at lower temperatures (3). The processes being observed appear to have very low activation enthalpies that require further investigation.

For

example, the effective exchange lifetime of cyclo(Ala-Gly-

Table 1. Effective Exchange Lifetimes and Chemical Differences for Cyclic Peptides. 1/T®*, s

1

Field Dep.?

Shift

x e ( e f f ) , ys

Av(eff), ppm

c(Ala-Gly-Pro-Phe) 2

(Note 1)

DMSO/CDCI3, 15°, NH

3.1-3.8

Yes

8-10

0.7-0.8

5.9-7.5

Yes

9-10

0.9-1.1

2.3-4.0

No (Note 2)

0.7-0.9

DMSO, 20°, NH

2.5-3.2

No (Note 2)

0.7-0.8

c(D-Phe-D-Pro-Ala-Pro) CDC13, 30°, C«-H

0.3-0.7

Yes

c(D-Ala-Gly-Pro-Phe)2 DMSO, 20°, NH

c(Pro-Phe-D-Trp-Lys-Thr-Phe) DMSO, 20°, NH c(Gly-D-Leu-Leu>2

12-14

0.2-0.3

Note 1. 1/T®* for 300 MHz, spin-locking field 8300-8600 Hz. Note 2. Limiting values for x g ( e f f ) and Av(eff) for < 10% change in 1/T® X in the range 2 kHz < (s.-l. field) < 9 kHz.

443

Pro-Phe)2 appeared to be constant over the range 0-45°, so that the Arrhenius activation energy for the exchange process involved was estimated as less than 2 kcal/mole (1), although the values of xe(eff) suggest that the free energy of activation is 10-12 kcal/mole. The 400 MHz spectrum of cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) in methanol shows line broadening by exchange to be < 5 Hz at -80°. Using a lower limit of effective chemical shift differences, 0.7 ppm, (see Table), an upper limit to -te(eff) at -80° is estimated at about 20 ps, suggesting an apparent activation energy of < 2 kcal/mole. A low activation energy is similarly indicated for the tetrapeptide cyclo(D-Phe-D-Pro-Ala-Pro), which also shows little exchange broadening at -80°. An upper limit to x (eff) at -80 is probably about 110 jis; x&(eff) is about 13 vis at 30°. The corresponding Arrhenius activation energy would be slightly greater than 2 kcal/mole, while the activation free energy from Tg(eff) at 30° is estimated as 11 kcal/mole. Deber, Fossel and Blout (4) reported an exchange process in c(Pro-Gly)2, probably amide plane rotation, which is clearly visible in spectral changes between 30° and -60° and has an activation free energy about 13 kcal/mole at ca. -15°'. Their spectra suggest that this process has an activation energy in excess of 6 kcal/mole, however. References 1.

Kopple, K.D., Y.-S. Wang, A.G. Cheng, K.K. Bhandary. 1988. J. Am. Chem. Soc., H H , 4168-4176.

2.

Kopple, K.D., Y.-S. Wang. 1988. Int. J. Peptide Protein Res. (in press).

3.

Mlynarik, V. 1987. Collect. Czech. Chem. Commun. 52, 541-546.

4.

Deber, C.M., E.T. Fossel, E.R. Blout. 1974. J. Am. Chem. Soc. ££, 4015-4017.

CONFORMATIONAL FLEXIBILITY OF CYCLIC TRIPEPTIDES

M. Rothe, K.-L. Roser Lehrstuhl Organische Chemie II, University of Ulm, 79 Ulm, FRG

Cyclic tripeptides (CTPs) containing 9 ring atoms and at least one unsubstituted peptide bond hold a unique position in peptide chemistry. Due to their ring size and a surprising conformational flexibility, two of the peptide groups can get into close proximity. Hence, they can undergo a transannular reaction with each other forming highly reactive tetrahedral adducts, cyclols. In addition to the CTPs c-(Pro-X-Pro), the tautomeric cyclols are stable in some cases, or there exists an equilibrium between the two tautomers in solution (1). Now we have synthesized the first CTPs of the type c-(Pro-X-X) containing one substituted and two unsubstituted peptide bonds, c-(Pro-Val-Val) and c-(Pro-Ile-Ile) . Studying the sequences Pro-X-X, however, we found sometimes not only two, but surprisingly four isomers, two cyclols and two cyclotripeptides. Their structure and conformation could be determined by NMR and IR spectroscopy. Cyclotripeptides can be easily distinguished from cyclols in the "^C spectrum by the occurrence of 3 carbonyl C atoms at -170 ppm, whereas the corresponding cyclols show a characteristic signal of the tetrahedral C atom at ~ 95 ppm. The elucidation of the two cyclols was performed by means of the sequence Pro-Ala-Ala. They turned out to be structural isomers in which the amino acids have changed their positions around the ring. The cyclol formed first at 0°C is called primary cyclol; addition of tert. amine gives the isomeric "secondary" cyclol. Both cyclols differ from each other in the pathway of dehydration at elevated temperatures. Prim, cyclols are converted into ketene aminals for which the NH band of the remaining peptide bond is maintained at >3200/ cm• Sec • cyclols easily

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

445

dehydrate as well, leading, however, to acylamidines for which ijoth the NH and OH band of the cyclol >3200/cm have disappeared. According to the

H-NMR spectrum sec. cyclols possess two kinds

of protons exchangeable by deuterium, but no amide protons in contrast to prim, cyclols. Moreover, the positions of all amino acids could be determined by the coincidence of the 13 analogous C signals with those of the well-known cyclols Pro-Ala-Pro resp. Ala-Phe-Pro. All sec. cyclols X-X-Pro (X = Ala, Phe, Val, Leu, lie) have the same conformation. Among the CTPs only those with bulky side chains proved to be stable, c-(Val-Val-Pro) is formed under the same conditions as the prim, cyclol of Ala-Ala-Pro. Characteristic of its structure is a sec. trans-peptide bond which follows from the occurrence of an amide II band in the IR spectrum at 1525/cm as well as of two NH bands at 3260 and 3210/cm for trans and cis amide bonds, resp.. It can be concluded from the coincidence of the

C signals of a Pro and a Val residue with those in

c-(Pro-Val-Pro) that both peptide backbones have identical screw-like conformations. In fact, there are two diastereotopic screw conformations differing in the positions of the proline. The values of the a,B couplings indicate the presence of the screw I form where the trans-peptide bond is situated

446

between Pro and Val. According to the ^ C spectrum in polar solvents the CTPs adopt an all-cis conformation with a threefold axis, the well-known crown. These and further NMR studies have furnished a complete picture of CTP and cyclol chemistry including their structural isomerism, tautomerism, and conformational isomerism. Accordingly, their synthesis starts from N-prolyl diketopiperazines Pro-c-(X-X) which undergo intramolecular aminolysis yielding first the prim, cyclols as reactive intermediates which yet could be isolated as Ala derivative. They are destabilized by bulky substituents and hence transformed into the CTPs with transannular ring opening leading to the screw I conformation with a trans-peptide bond between Pro and X. In spite of their small ring size they showed a surprisingly high flexibility of the peptide backbone. In polar solvents the all-cis crown conformation is formed due to trans •+• cis isomerization. If the formation of CTPs is sterically hindered, as in the case of the sequence Pro-Aib-Aib, the reaction stops at the prim, cyclol. In all cases the most stable end product of the cyclol rearrangement is the sec. cyclol. It follows from its structure that it can be formed only by transannular reaction from the diastereotopic screw II conformation of the CTP, in which the X-X and the Pro-X peptide bond lie opposite each other. This requires a cis -» trans isomerization in the crown conformation, this time at the X-X bond. The cyclol isomerization represents a new type of reaction in peptide chemistry due to reversible reactions between peptide groups and subsequent cis-trans isomerizations of the peptide bonds leading to an inversion of the peptide sequence. This paper is dedicated to Prof. Th. Wieland on the occasion of his 75th birthday.

We gratefully acknowledge financial

support from the Fonds der Chemischen Industrie. 1. Rothe, M., M. Fahnle, S. Wermuth. 1984. In: Peptides 1984 (U. Ragnarsson, ed.). Almqvist & Wiksell, Stockholm, p. 573.

CYCLIC REGULARLY ALTERNATING L,D PEPTIDES

V. Pavone, E. Benedetti, B. Di Biasio, C. Pedone, A. Lombardi Dipartimento di Chimica, Università' di Napoli, Napoli, Italy G. P. Lorenzi Institute fur Polymere, ETH-Zentrum, Zurich, Switzerland

Introduction The structural and conformational analysis of cyclic

peptides

has been recently the object of many investigations

(1,2,3).

Most

of

the

peptides,

interest

in

account

of

on

this

field

their

arises

because

particular

these

conformational

properties, are useful models for biological compounds. In

fact,

cyclic

peptides

constraints,

that

making

suitable

them

reduce to

are

their

characterized

by

conformational

better

understand

activity of more flexible, linear analogues.

steric

flexibility,

the

biological

Furthermore,their

ability to interact w i t h metal ions is useful in the study of ionophores

and

enzymes,

that

require

metal

ions

for

their

biological function.

Results As

part

of

properties crystal

of

our

continuing

cyclic

analyses

of

we

on

the

report

two cyclic hexapeptides

[cHV] and c-(L-Phe-D-Phe) 3 oligopeptides

effort

peptides,

presenting

conformational

the

X-ray

single

c-(L-Val-D-Val) 3

[cHP], as model compounds of cyclic regularly

alternating

residues.

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin New York-Printed in Germany

L

and

D

448

cHV crystallizes in the hexagonal system, space group R3, with a=19.52(6)A, c=14.57(5)A and Z=18. Density measurements indicate that the independent unit is represented by a single Val residue and one trifluoroacetic acid molecule. The cyclic peptide, then, retains in the solid state the Sg crystallographic symmetry. cHP crystallizes in the triclinic system, space group PI with a=12.102(6)A, b=12.822(9)A, c=15.609(6)A, a=104.35(4)° , (5=111.31(5)°, i =67.82(6)° and Z=2. The independent unit is represented by half cyclic hexapeptide molecule (3 Phe residues) and four independent trifluoroacetic acid molecules. Both structures have been solved with MULTAN (4) and at the present they have been refined to R factors of 0.068 and 0.10 for cHP and cHV, respectively. Both peptides show a conformation which falls in the "so called" p-region of the -v map. For each structure the observed values of the V and "> are given in the figure.

Figure. Molecular models of the cyclic hexapeptides: cyclo(L-Val-D-Val)3

(left) and

cyclo(L-Phe-D-Phe) 3

(right). The independent units and the relevant conformational parameters of the backbone are

indicated

449

The almost disk

-

regular succession

like

molecule

of $ ,

Ca

with

-

pairs gives

C^ bonds

in the

rise to

a

equatorial

position w i t h respect to the backbone atoms of the ring.

Both

peptides strongly interact w i t h trifluoroacetic acid molecules in

the

C=0

crystal,

with

the

H - 0 and the C=0

distances

are

respectively.

in

ranges

formation

of

H-bonds

of

both

H-N types. The 0----0 and 0- • • -N o o - 2.6 A and 2.9 - 3.0 A,

2.5

In both peptides

the planes

of the amide bonds

are perpendicular to the average plane of the disk-like cyclic molecule. In this conformation groups

of

each

cyclic

the H - b o n d donor and

peptides

are

acceptor

linked

to

the

trifluoroacetic acid molecules. Six (3 o n each side) or eight (4

for

each

respectively,

side)

solvent

molecules

for

in both cases essential for the

cHV

and

cHP,

crystallization

of the peptide, "sandwich" the hexapeptide molecules.

References 1. Tolle, J.C., M.A. Staples, E.R. Blout. 1982. J. Am. Soc. 104, 6883.

Chem.

2. Staples, M.A., J.C. Tolle, E.R. Blout. 1983. In: Conformation in Biology (R. Srinivasan and R. H. Sarma, eds). Adenine Press, New York, p. 147 and references therein. 3. Campbell, B.E., K.R.K. Easwaran, G. Zanotti, M.A. Staples, E.T. Fossel, E.R. Blout. 1986. Biopolymers 25, S47. 4. Germain, G., P. Main, M.M. Woolfson. 1971. Acta Crystallogr. 1971. A-27, 368.

SYNTHESIS AND ION BINDING OF AN HETERODETIC BICYCLIC DECAPEPTIDE

Giancarlo Zanotti, Francesco Pinnen, Gino Lucente Centro di Studio per la Chimica del Farmaco del CNR and Dipartimento di Studi Farmaceutici, Università "La Sapienza", Roma, Italy 00185

Maria D'AI agni Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive del CNR and Dipartimento di Chimica, Università "La Sapienza", Roma, Italy 00185

Livio Paolillo, Gaetano Barbato, Gabriella D'Auria Dipartimento di Chimica dell'Università, Napoli, Italy 80134

Introduction Bicyclic peptides represent a new class of medium sized ion binding cyclopeptides whose synthesis and conformational

investigation have been deve-

loped in recent years with the purpose to obtain useful models of the three dimensional structure of proteins as well as novel ionophores (1). In this paper we report the synthesis and the ion binding properties, as determined by CD and NMR techniques, of the heterodetic bicyclic decapeptide cyclo(Glu^-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7-Ala8)cyclo(ly-5j3)Phe9-Gly10

( 5 ) . In bicyclic

peptide (5)an homodetic eight-membered ring is fused to an heterodetic seven-membered ring which contains a lactone bond connecting the carboxyl 10 5 function of Gly to the alcoholic function of Ser .

Results and Discussion All the linear precursors as well as the monocyclic and bicyclic peptides (4)and (5 )have been prepared in solution by adopting the MA and/or the DCCI coupling methods. The final synthetic steps are depicted in Scheme 1. The

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

451 fragment condensation of peptides(l_) and (2) afforded the branched linear decapeptide lactone(3) This is characterized by the presence of two pairs of selectvely and compatibly protected carboxyl-amino functions. Stepwise deprotection of each pair of functions followed by condensation to form Pro^-Ala^ and Pro^-Gly^ bonds respectively, afforded in turn the heterodedic monocyclic eptapeptide(4)and the bicyclic title compound 5. H-Gly — 0 I Bcc-Gly-Ser-Ili-Pro-OBil (1) 0-i

1r-Phe-OH t • Z-Ala-Glu-Leu-Pro-0 Bu I 1 hydrogen bond (0.281 nm) (S), which can not explain the strong effects observed in solution. We evaluated vicinal, heteronuclear coupling constants and calculated relative distances from a ROESY-experiment. To assign the C^-signals of Aib and to proof the effect of turn structure on the chemical shift, we compared the C B -signals of Aib in the peptide with Boc-Ala-(R)-Aib[D,]-Ala-OMe in solution and in the solid state. Boc-Ala-Aib-Ala-OMe (l) and Boc-Ala-(R)-Aib[D,]-Ala-OMe (2) were prepared from the protected amino acids as described before (6), however, using Aib-OEt and AibfD,]-OEt, which was prepared according to Scheme 1 (7, 8). The configuration of the deuterated AibfDiJ was controlled with Na[Eu m (R- and S-propylenediaminetetraacetate)(H 2 0) 3 ] x 2H 2 0 (R- and S-LSR). The induced shifts correspond to those obtained with Aib and Aib[D3] (9). The S-CH 3 -protons exhibit a shift of 1.2i ppm per mol ratio S-LSR/AibCDi], and the R-CH 2 D-protons of 1.17 ppm per mol ratio R-LSR/Aib[D,L We took vicinal coupling constants 3 J from the 'H- and coupled l 3 C- spectra. We also performed a coupled "N-DEPT in order to estimate the angle. From the 3 J-values for the $! and angles a p-turn type II and III can be derived. Because of the different f 2 angles in ideal fi-turns type II and III a decision has to be made between these conformations. The high uncertainty about the T,-value estimated from 3 J H C-4

none none 4

X : unspecified

H,

5—>2

solvent molecules

Results and discussion Single

crystals

of

from

water

b =

13.191

R

0.047 ). The

=

2

solution A,

Leu-enkephalin (space

group

c = 21 . 350 A, molecules

3

3

trihydrate P2^2^2^,

Z = 4, 2551

are tightly

were a

=

obtained o

10.967

reflections

folded

by two

A,

used, fused

4

B U I (Gly -Gly ) and 61 (Gly -Phe ) bends with two intramolecular hydrogen bonds (N...0 = 2.98 A (4 Each

molecule

is

in

direct

contact

Fig. 1. Stereoviews of the morphine phalin (lower) molecules.

->-1) and 3.11 A (5 ->-2)). with

four

(upper)

neighbouring

and

Leu-enke-

497 ones and 8 water molecules, lar

NtTyr1)

4

0(Leu5)

to

Phe

aromatic

cycles

this

arrangement

bis-folded

structure

lin.

similarity

the to

6

in

fact

leads

rest

and

with

The

recognize

folded

sites

the

should

the

the

prefer

diverse

encourage

n

probably receptor

and

contact

and

tyramine

and

mobility

new

crystal

Leu-enkephaexplains

site,

sites

extended

polymorphism

more

of

enkephalin

an

Tyr1

This

|j r e c e p t o r

conformational

such

of

molecule.

morphine by

intermolecu-

The

orthogonal

c o n f i r m s the f l e x i b i l i t y

preferred.

to

2.66 A.

that

is

that

tide

to

the m o r p h i n e

recognized

6-receptor

of

rather

are

selectively

while The

site

in

similar

cycles

Its

are

is

cyclohexyl

enkephalins

a n d t h e r e is a short

distance

are

known

conformations, structure

of

how

although

this

is of

pentapep-

general

systematic work

on

(5). inte-

crystallo-

genesis.

Acknowledgement We This

thank work

foundation

Prof. was

G.

Ourisson

supported

for

by C . E . C .

"Leonidas Zervas"

stimulating (ST2J-0184)

( s c h o l a r s h i p to

discussions. and

the

Greek

N.B.).

References 1. S c h i l l e r , P.W. 1984. In: The Peptides (S. Udenfriend a n d J. M e i e n h o f e r , eds.). A c a d e m i c P r e s s , O r l a n d o , F l o r i d a , U . S . A . Vol. 6, p. 219. 2. S m i t h ,

G.D.,

J.F.

Griffin.

1978.

Science

199,

3. K a r l e , I.L., J. Karle, D. Mastropaolo, A. N. C a m e r m a n . 1983. A c t a C r y s t a l l o g r . B 3 9 , 625.

1214.

Camerman,

4. G r i f f i n , J . F . , D.A. L a n g s , G.D. S m i t h , T.L. Blundell, I.J. T i c k l e , S. B e d a r k a r . 1986. Proc. Natl. A c a d . Sci. USA 83, 3272. 5. H a n s e n , P.E., B.A. M o r g a n . 1984. In: T h e P e p t i d e s f r i e n d a n d J. M e i e n h o f e r , e d s . ) . A c a d e m i c P r e s s , F l o r i d a , USA. V o l . 6, p. 269.

(S. UdenOrlando,

17

0

NMR

AND

FT-IR

STUDY

OF

HYDRATION

OF

C. Sakarellos, I. Gerothanassis, T. Karayannis, M. Sakarellos-Daitsiotis Department of Chemistry, 45110 Ioannina, Greece

University

N.

of

LEU-ENKEPHALIN

Birlirakis,

Ioannina,

Box

1186,

B. Vitoux, M. Marraud LCPM-ENSIC-INPL, Cedex, France

1,

rue

Grandville,

B.P.

451,

54001 Nancy

Introduction 17 17 2 5 0 NMR studies of [ O-Gly ,Leu ]-

We have recently reported and

[ 1 7 0-Gly 3 ,Leu 5 ]-enkephalins

in CH,CN/DMS0 (4:1 v/v) 17 and aqueous solutions (1). The 0 chemical shifts were found to be very similar for both compounds and practically pH independent. In CH 3 CN/DMS0 both Gly 2 and Gly 3 exhibit a significant, comparable shift to higher frequency ( A 6 ^2830 ppm) . This was attributed to the breaking of the solvation, mainly by one water molecule occurring in water solu2 3 tion for both Gly ing both the

the

solvents. hydration

solvent

and Gly

formation

of

In

a

peptide oxygens, and thus excludspecific

order

to

of

these

state

composition

B-turn

obtain

bond

in

further

positions,

chemical

hydrogen

shifts

of

information 1 on 7 we performed 0

N,N'-dimethylacet-

amide (DMA) and temperature dependent chemical shift measure17 2 5 17 3 5 ments for [ O-Gly ,Leu ]-, [ O-Gly ,Leu ]-enkephalins, N-methylacetamide (NMA) and DMA. The two latter compounds are

appropriate

two

molecules

models of

B^O

of

a

peptide

hydrogen

functional

bonded

on

the

group

amide

with

oxygen

in aqueous solution, with one molecule of water in t^O/CH^CN mixture

(26:74,

molar

ratio)

and

free

of

hydrogen

bonding

in CH^-CN solution, as shown from our extensive FT-IR studies.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

499

Fig. 1. IR d i f f e r e n c e spectra with r e f e r e n c e to L e u - e n k e p h a lin for [ 1 3 C - G l y 2 , L e u 5 ] (a) and [13C-Gly3,Leu5]-enkephalin (b) in CH3CN/DMSO (4:1 v/v) m i x t u r e s (left) a n d in w a t e r (right). Results and discussion 17 O-NMR

spectra

trometers

were

operating

in

concentrations

in

CH^CN/DMSO

1 M

for NMA

IFS-85

run at

of

(4:1

shift

in

of

to

lower

by

% 55

(c

=

frequency ppm

FT-IR

1M) by

Since

the

and

for

25|im of

at x 20

5

cell mM.

MHz

respectively, 4 and

scanned

The

ppm

I^O/CH-jCN

differential

to

chemical 17

2

was

shift

and CaF-

chemical shifted

mixture

that

mM

Bruker

with

O-NMR

abundance

in

on a

e q u i1p7p e d

relative

10

enkephalins

natural

solution,

spec-

both

both

spectra w e r e a

and A C - 2 0 0 P

27.13

mixtures

with

in a q u e o u s

and

in w a t e r

concentrations

DMA

solution.

mM

v/v)

a n d DMA.

Bruker AM-400

54.48

10

spectrometer

windows,

on

in

and CH^CN

in

5

1^0 17

a n d 3 C H , C5N / D M S 0 m i x t u r e of b o t h [ 0 - G l y , L e u ] - and [ 0 Gly ,Leu 2] - e n k e p h a l i3n s is only 28-30 ppm, we can c o n c l u d e that Gly a n d Gly peptide oxygens are not s o l v a t e d to the

same

of

the

model ture be

extent lesser

amides

with

the

DMA

hydration

was

dependence

linear,

as

obtained studies. the

value

carbonyl.

these 17 from 0

Further

of

two

The

relation A 6 / A T

was

found

+128

NMA

and

of

sites

confirmation

chemical ppb/K

for

relative

shift

to

temperato +113

500 ppb/K

for

DMA,

as

opposed

to

+81

and

+84

ppb/K

for

Gly

2

a n d Gly"* r e s p e c t i v e l y . With

r e f e r e n c e to L e u - e n k e p h a l i n , the IR d i f f e r e n c e s p e c t r a 2 5 13 3 5 13 [1- C - G l y ,Leu ] a n d [1- C - G l y ,Leu ] - e n k e p h a l i n s

for

the

in C H , C N / D M S 0 cy

shift

vibrations a

very

Gly

25

cm

of

both

(2).

and

Gly

carbonyls of

the

The

a

suggests

with

C=0

larger

probably 0-NMR

does

A

frequency

provides

p h e n o m e n a of small p e p t i d e

show

both no

not

have

intramolecular water,

shifted

by

15

that

involve two

shift,

of

that

the

new

sites In

are

by

frequen-

stretching

results.

confirming

solvation

experiments

FT-IR

and

absorptions

probably

that

the

frequencies,

oxygens.

above and

This

environment

lower

induce

O-NMR

1).

agreement 3

to

orbitals would

(Fig. in

2

a

36

similar

contact, the

m i x t u r e s are a l m o s t i d e n t i c a l , w i t h -1 12 13 cm b e t w e e n the C=0 and C=0

of

both

lone

water about on

pair

molecules 30-40

combined

information

to

hydration

cm ^

use

of

hydration

hormones.

Acknowledgement This

work

was

supported

Greek

General

Greek

Foundation

FEBS

(Summer

f e l l o w s h i p to

Secretary

by for

"Leonidas

fellowship

to

C.E.C.

(Grant

Research Zervas" N.B.),

and

ST2J-0184),

the

Technology,

the

(Scholarship and

EMBO

to

N.B.),

(short-term

I.P.G.).

References 1. S a k a r e l l o s , C., I. Gerothanassis, N. Birlirakis, T. Karayannis, M. Sakarellos-Daitsiotis, M. Marraud. 1 989 . B i o p o l y m e r s 2J3 (in p r e s s ) . 2. T ê t e , F., B. Vitoux, M. Sakarellos-Daitsiotis, I. Gerothanassis, N. Birlirakis, T. Karayannis, C. S a k a r e l l o s . 1988. In: P e p t i d e s 1988 (E. B a y e r and G. J u n g , eds.). W a l t e r de G r u y t e r , B e r l i n (this v o l u m e ) .

iH-NMR OF LEU-ENKEPHALIN IN CRYOPROTECTIVE MIXTURES. T.Tancredi, A.Motta, ICMIB del CNR, via Toiano 6, 80072 Arco Felice, Italy D.Picone and P.A.Temussi Dipartimento di Chimica, Università di Napoli, via Mezzocannone 4, 80134 Napoli, Italy.

Determination of the relationship between conformation and biological activity of opioid peptides is made difficult by the fact that all natural peptides

and most of their

analogs,

synthesized

for

classical

structure-activity studies (SAR), are small and linear. Accordingly, their conformational flexibility is so high that they show no tendency to adopt a single stable conformation, nor even a small number of low energy conformations.

This situation has led some authors 1 to the rather

extreme statement that it is an elusive goal to try to determine any relationship between conformation and activity in small linear peptides with biological activity.

Actually, it is possible to gather valuable

conformational information, at least in the form of conformational tendencies, by exploiting the strongly non linear dependence of NOE's on internuclear distances, and by using several forms of conformational confinement. We have already shown that it is possible to induce stable folded conformations in the cationic forms of enkephalin amides 2 and of the N-tetrapeptide fragment 3 of dermorphin by binding the NH3+ group with a crown ether and dissolving the resulting complexes in CDCI3, an environment that mimics some of the features of the receptors. Another form of efficacious conformational confinement proved to be the combination of high viscosity and low temperature in a polar environment 4 , made possible by a mixture of DMSO and water, one of the so-called cryoprotective mixtures 5 » 6 .

We have now undertaken a

systematic study of the conformational tendencies of Leu-enkephalin (LE) in several solvent systems.

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin - N e w York - Printed in G e r m a n y

502

Here we present the preliminary results of an NMR study of LE in dimethylformamide (DMF) and in cryoprotective mixtures of DMF/water and ethylene glycol (EG)/water. LE was purchased from Sigma (St.Louis, MO, USA) as acetate salt, purified with

a Sep-pak C^g cartridge and

converted into trifluoroacetate salt. NMR experiments were performed on a Bruker AM-400 spectrometer, using standard Bruker software.

RESULTS Residue type and sequential assignments were made ex novo, on the basis of standard 2D experiments, since no literature data existed for the solvent systems employed. Table I summarizes chemical shift data and temperature coefficients for the labile protons, that usually yield the most direct information on the conformation of the backbone. Table I. Chemical Shifts (ppm) and Corresponding Temperature Coefficients (pph/K) of the Labile Protons of Leu-enkephalin in Several Solvent Systems.

_ _____ _ _ 8 A8/AT Gly2 Gly3 Phe 4 Leu 5

___ _ 8 AÔ/AT

_______ 8 A8/AT

ÉG/H2O 8 A8/AT

8.76

-5.5

9.02

-5.7

9.04

-6.3

8.60

-4.8

8.09

-4.9

8.14

-3.6

8.26

-5.1

8.09

-3.8

8.00

-5.8

7.90

-3.3

8.05

-2.8

8.02

-4.5

8.47

-7.1

8.25

-5.3

8.50

-6.7

8.39

-6.5

All coefficients are too high to suggest the presence of stable intramolecular hydrogen bonds. However, the NOESY experiments, both in neat DMF and in the two cryoprotective mixtures examined, show well developed cross peaks corresponding to intrachain and sequential NOE's that hint a non random distribution of conformera. In fact the spectrum of Figure 1 shows that in DMF there is also a diagnostically useful NH-NH effect, involving the two Gly residues, consistent with the presence in solution of a detectable population of a folded conformer.

503

3 J[ 1 bàIna

i

G

2

0

0

9.5

F

,5 L

8.5

7.5 ppm

F i g u r e 1. Low field region of a 400 MHz phase sensitive NOESY spectrum of 2.4 mM Leu-enkephalin in DMFjy at 283 K with a mixing time of 500 ms. The shown spectral region contains a cross-peak between G^ and G^ amidic protons.

This result confirms our previous study in a DMSO/ water cryoprotective mixture 6 . References 1.

Rose, G. D., Gierasch, L. M. and Smith, J. A.. 1985. Adv. Protein Chem. 37,1-109.

2.

Temussi, P.A., Tancredi, T., Pastore, A., and Castiglione-Morelli, 1987. Biochemistry 26, 7856-7863.

3.

Castiglione-Morelli, M. A , Lelj, F., Pastore, A., Salvadori, S., Tancredi, T., Tomatis, R., Trivellone, E. and Temussi, P. A.. 1987. J.Med.Chem. 30, 2067-2073.

4.

Motta, A., Picone, D., Tancredi, T. and Temussi, P.A.. 1987. J.Magn.Reson. 75, 364 -370.

5.

Douzou, P. and Petsko, G.A.. 1984. Adv. Protein Chem. 36 , 245-361.

6.

Motta, A., Picone, D., Tetrahedron 44, 975-990.

Tancredi,

T.

and

Temussi,

P.A..

M.A..

1987.

FT-IR STUDY OF THE TYR HYDROXYL VIBRATIONS IN LEU-ENKEPHALIN IN AQUEOUS SOLUTION.

N. Birlirakis, I. Gerothanassis, C. Sakarellos Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece. M. Marraud LCPM-ENSIC-INPL, Cedex, France.

1,

rue

Grandville,

BP

451,

Box

1186,

54001

Nancy

Introduction The Tyr hydroxyl group has been shown to play a fundamental role

in the

opioid

Gly-Phe-Leu)(1).

properties

However,

the

of

the

enkephalins

spectroscopic

(Tyr-Gly-

studies

of

this

phenol group reported so far do not permit clear conclusions about

its

eventual

structural

functions

(2).

In this

work,

we have assigned the in plane C-O-H bending and C-0 stretching vibrations

for Leu-enkephalin and its prototype molecule 1g• using the 0 isotopic effect. It is shown that

p-cresol the

Tyr-OH

group in Leu-enkephalin

is fully exposed

to the

aqueous environment.

Results and discussion 18

O-enriched

p-cresol

was

obtained

by

hydrolysis

of

the

18

corresponding

diazonium

salt

by

H£

0

at

97 %

enrichment.

FT-IR spectra were run at room temperature on a Bruker IFS-85 apparatus for

using

enkephalin

length

25

pm,

aqueous and

near

solutions saturation

resolution

2

cm

of

20

mM

concentration

for p-cresol, and

cell path

accumulation

scans.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

of

512

505

1300

1250

1300

120

1250

120

WAVENUMBER, CM"

WAVENUMBER, CM"

Fig. 1. IR spectra of Leu-enkephalin (a,1) and Gly-Gly-PheLeu (a,2) in water (pH 5.5, concentration 20 mM). Self deconvoluted spectrum of Leu-enkephalin (b) showing the two^ contributions attributed to the Tyr C^-0 stretching (1265 cm ) and C-O-H bending vibration (1247 cm ). The

IR

spectrum

exhibits

a

at

cm \

1250

two main this and

of

Leu-enkephalin

strong

and

and

C-O-H

bending

However, lacking

vibration

and

1247

modes. similar cm is

is

Leu-enkephalin

— 1

cm

can

Under IR

the

expect

much be same

1).

than to

conditions,

contributions,

which

18

band

Tyr

C-0

with

the

the

reveals the

Therefore

assigned

neutral

the

1). In

stretching amide the

observed bands C-O-H

p-cresol

are

into

III

Gly-Gly-Phe-Leu that

the Tyr

pH

centered

1247 cm ^ (Fig. the of

residue

weaker

(Fig.

at

self-deconvolution

together

examination Tyr

water

absorption

by

1265 and

we

vibrations

tetrapeptide III

at

domain,

absorption.

in

composite

decomposed

contributions

frequency

in

shifted

absorption at

1265

vibration

exhibits by

amide

quite

nearly

15

— 1

by O-enrichment. Moreover, the 1265 cm component retained upon deprotonation of the Tyr-OH group in Leu-

enkephalin,

whereas the

1247 cm

component vanishes.

These

observations allow us to assign the 1265 and 1247 cm ^ contri-

506

butions tion, and

to

the Tyr C-0 stretching

respectively.

p-cresol

equally

The

in water

exposed

to

similar suggest

the

not

seem

to

be

probably

aqueous

involved

solvated

in

by

data

that

both

the

very

fact

that

sensitive

to

the

phenol

intramolecular

two

p-cresol

aggregation,

vibra-

Leu-enkephalin groups

are

Contrary

to

(3), the Tyr-OH group does

water

as

but

in

the

(4). This is confirmed

C-O-H and

contacts,

molecules,

crystals of Leu-enkephalin trihydrate by

for

environment.

the proposition of Khaled et al. more

and C-O-H bending

IR

bending

absorbs

vibration

near

1 240

is cm

when the hydroxyl group acts both as a donor and an acceptor group (5).

Acknowledgement This

work

was

supported

Greek

General

Greek

Foundation

Secretary

by for

"Leonidas

C.E.C.

(Grant

Research Zervas"

and

ST2J-0184), Technology,

(scholarship

to

the the N.B.)

and EMBO (fellowship to I.P.G.).

References 1. Morley,

J.S.

1980.

Ann.

Rev.

Pharmacol.

Toxicol.

20,

21 .

2. Schiller, P.W. 1984. In : The Peptides (S. Udenfriend and J. Meienhofer, eds.). Academic Press, Orlando, Florida, USA. Vol. 6, p. 219. 3. Khaled, M.A., D.W. Urry, R.J. Soc. Perkin Trans. II, 1963. 4. Aubry, A., N. Birlirakis, C. Sakarellos, M. Marraud. Commun. 96 3.

Bradley.

1979.

J.

Chem.

M. Sakarellos-Daitsiotis, 1988. J. Chem. Soc., Chem.

5. Birlirakis, N. , I.P. Gerothanassis, C. Sakarellos, M. Marraud. 1988. In: 2eme Forum Peptides (A. Aubry, M. Marraud and B. Vitoux, eds.). John Libbey Eurotext, London (in press).

THE

ASX-TURN

STRUCTURE

V. P i c h o n - P e s m e , A.

IN

ASN

AND

ASP-CONTAINING

Aubry

CNRS-UA-809, University Cedex, France

of

Nancy

I, BP

A. A b b a d i , M. M c h a r f i , G. B o u s s a r d , M. CNRS-UA-494,

PEPTIDES

ENSIC-INPL,

BP

451,

239,

54506

Vandoeuvre

Marraud

54001

Nancy

Cedex,

France

Introduction

A

few e x a m p l e s

terized and in

by

a

involving position

to

short

contact

C^O

group

i and

the

N-H

the

Asx-turn,

diffraction

reported

the

determine

called

of the t u r n s

closing of

we

have

carried

or

r e s i d u e i+2 out

the

2,

peptides

are

charac-

ten-membered (Asp

stability

on

B o c - A s n - X - S e r - ( O M e , NHMe) ,

a

an Asx

b o n d of

intrinsic

experiments

in p r o t e i n s

of

Asn) (1).

In

this

^-NMR,

cycle residue order

structure

IR

and

X-ray

Boc-Asx-Pro-NHMe, (X = Pro

and

and

Ala), the

l a t t e r c o n t a i n i n g the s e q u e n c e c o d i n g for N - g l y c o s y l a t i o n

(2).

Results The

short

in

and medium-range

thirteen

bond

crystallized

typical

of

However,

when

accurate

crystal

Asx-X Type by

far

with the

Asx-turn

selecting

sequences I

the

the

most

interactions proteins is

ten

of

structures,

it

adopt Asx

of the A s x

reveal

that

largely these

hydrogen

predominant

proteins

appears

side-chain

the

that

with the

(1). very

folded

t h r e e t y p e s of A s x - t u r n s (Table 1 ) . ct 8 C - C p bond, in the t c o n f o r m a t i o n is

frequent,

and

type

n e a r l y 80° m a i n l y c o n c e r n s A s x - G l y

II

with

sequences.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

the

X-i> a n g l e of

508 Table I. A v e r a g e C o n f o r m a t i o n a l A n g l e s (°) for the Types of A s x - t u r n s , with Occurrence in the Folded S e q u e n c e s of T e n C r y s t a l l i z e d P r o t e i n s . Type

Occurrence

Asx

(%)

*

x

X

1

*

$

X*

I

70

120

180

180

-80

0

II

22

0

60

180

80

0

8

180

60

180

-80

0

III

In all

the

peptides

a n d 2,

the

existence

of

i n t e r a c t i o n is d e m o n s t r a t e d by the f o l l o w i n g the

stretching

NH(Me) by

bond

nearly

-

the

and

in

frequency

1,

cm" 1

100

temperature

the

CHCl^

weak

and

of

reference

to M e ^ S O )

of

the

stretching

Asn-c"^0

bond

the

(up

to

or

70 g

+

results

%)

of

type, that

tially

assume of

coupling larger

(8

Me2S0

2

a

shifted

free

(2.6

(

X*

168 - 1 7 0

99

$

67

151

-

66

-23

-101

172 - 1 7 4

-

59

-27

-

87

1

-112

24

-

79

172

-176

-179

-

86

if

-

-164

-20

Ser

-

x1

X*

1

-

55

-179

130

-

61

127

-172

72

-

-

75

2a : Boc-Asn(Me)-Pro-Ser(Bzl)-NHMe; 2b : Boc-Asn-Pro-Ser (Bzl)-NHMe; 2c : Boc-Asn(Me)-Pro-Ser-NHMe; 2d : Boc-Asn(Me)Ala-Ser-OMe. residues. However, the Ala-$ angle in 2d differs significantly from the current value for the Pro residue in the other three tripeptides. This study demonstrates the intrinsic stability of the Asx-turn which assumes three conformational states in the proteins. The different occurrences of these three conformations suggest that they do not have the same stability, which can also be modulated by long-range interactions.

References 1. Baker, E.N., 44» 97.

R.E.

Hubbard.

1984.

Prog.

Biophys.

Mol.

Neel.

1974.

Macromolecules

Boussard,

M.

2. Bause, E. 1983. Biochem. J. 209, 331. 3. Cung, M.T., 7, 606.

M.

Marraud,

4. Aubry A., A. Abbadi, New J. Chem. JJ_, 739.

G.

J.

Marraud.

1987.

5. Pichon-Pesme, V. , A. Aubry, A. Abbadi, G. Boussard, M. Marraud. 1988. Int. J. Peptide Protein Res. (in press).

THE CONFORMATIONAL PREFERENCES OF THE PARTIAL SEQUENCES OF HUMAN IMMUNOGLOBULIN IgAl HINGE REGION

I.Z. Siemion, A. P^dyczak Institute of Chemistry, Wroclaw University, 50-383 Wroclaw Poland S.G. Wood, J. Burton Evans Department of Clinical Research, University Boston, MA USA

Hospital,

Introduction The hinge region of human IgAl immunoglobulin molecule consists of the sequence:

..ProValProSerThrProProThrProSerProSerThrPro..

This fragment of the peptide chain is sensitive to the action of extracellular proteases produced by several human bacterial pathogens. The cleavage of the peptide chain inactivates the major component of the secretory immunoglobulin and is thought to allow the first step of the bacterial

invasion.

In order to investigate the local conformational

preferences

within the peptide chain the following tetrapeptides were investigated by CD and ThrProProThr ThrProProThrNH 2

13

C - N M R methods:

I II

ThrProSerPro ThrProSerProNH 2

XII XIII

AcThrProProThr AcThrProProThrNH 2

III IV

AcThrProSerPro AcThrProSerProNH 2

XIV XV

ProProThrPro ProProThrProNH, AcProProThrPro AcProProThrProNH 2

V VI VII VIII

ProSerProSer ProSerProSerNH, AcProSerProSer AcProSerProSerNHo

XVI XVII XVIII XIX

ProThrProSerNH« AcProThrProSer AcProThrProSerNH 0

IX X XI

Peptides 1988 © 1989 Walter de Gruyter & Co., B e r l i n - N e w York-Printed in G e r m a n y

511

Results The measurements were performed in water, at three different pH values, i.e. in acidic, neutral, and basic media. The positions and intensity of Cotton effects appeared in CD spectra of peptides were analysed in terms of the domination in the equilibrium of definite regular peptide chain conformation (poly-Pro II, -turn, unordered form). ^ C - N M R data enabled to determine: i. The total amount of cis-Pro amide bond isomers. The increase of cis-Pro isomer content evidences the increase of conformational freedom of the peptide. ii. The values of mean conformational angles

for the succe-

ssive amino acid residues. For this purpose our hydantoin scale (1) was utilized, i.e. the c P the set of

Ah

resonances expressed as iC

coefficient values ( V

For the calculations the following

CP

pept." ^^ydantoin5• chemical shifts of

the corresponding hydantoins were used: Pro - 26.65, Ser 60.19, Thr - 65.13). A detailed analysis of

13

C - N M R and CD

data is given in the separate papers (2,3). On the ground of our experiments the following conclusions can be formulated: 1. In the case of peptides I-IV the central moiety ProPro favorizes a local conformation of poly-Pro II type. Such a conformation seems to be most stable in acidic solution; increase of pH as well as acetylation and/or amidation of the peptide leads to unordering of the structure. 2. For the peptides V and VI the predominance of

y?-turn confor-

mation is clear. The acetylation (peptides VII and VIII) destabilizes the ^ - t u r n . Thus, it may be concluded that* after including of ProProThrPro segment into the peptide chain its preference for ji-turn should diminish. 3. For the series IX-XI and XVI-XIX of tetrapeptides the predominance of unordered forms was established. 4. In the case of peptides XII-XV a strong preference

ofyi-turn

structure was observed. Contrary to ProProThrPro sequence the

512 acetylation of the peptide does not destabilize the

^?-turn

structure. 5. The lowest amounts of cis-Pro isomers appeared for the peptides I and XII, indicating the lowering of the conformational freedom in these fragments of the peptide chain. The obtained results may be illustrated by following scheme: unordered I 1 poly-Pro II unordered j 1J ...SerThrProProThrProSerProSerThr.. . (I -turn

r

t u T n

Thus, the conformational preferences change very rapidly along IgAl molecule peptide chain. Assuming that the conformational preferences obtained for the short fragments are preserved in the intact molecule we can predict the greatest probability of the appearance of

^ - t u r n in the fragment ThrProSerPro. The N-

terminal segment ThrProProThrPro may exist in poly-Pro II type conformation. Acknowledgement This research was supported by Polish Academy of Sciences grant CPBP 01.13 and by NIH grant DE-01257.

References 1. Siemion, I.Z.. 1985. In: Natural Products Chemistry

(R.I.

Zalewski and J.J. Skolik, eds.). Elsevier North Holland, p.335. 2. Siemion, I.Z., A. P^dyczak, J. Burton: Biophys. Chem. (in press). 3. Burton, J., S.G. Wood, A. P^dyczak, I.Z. Siemion: Biophys. Chem. (submitted for publication).

NMR CONFORMATIONAL STUDIES O F GUEST-HOST PEPTIDE ANALOGUES IMMUNOO F T H E TORPEDO A C E T Y L C H O L I N E RECEPTOR a 67-76 M A I N GENIC REGION

M.T. Cung, M.

Marraud

CNRS-UA-494,

ENSIC-INPL,

I.

Hadjidakis,

E.

BP

Bairaktari,

D e p a r t m e n t of C h e m i s t r y , 45110 Ioannina, Greece I. P a p a d o u l i ,

451,

54001

V.

Tsikaris,

University

S. P o t a m i a n o s ,

S.

Hellenic Pasteur Institute, 11521 A t h e n s , G r e e c e

Nancy Cedex,

of

C.

France

Sakarellos

Ioannina,

Box

1186,

Sofias

Av. ,

Tzartos 127

Vassilissis

Introduction The

main

receptor

immunogenic

(AChR), responsible

localized quence

in

(1).

and

antigenic

due

of

ted

step

have

the by

a-subunit,

In

order

role

of

step

by

(MIR) and

to

the

mainly

in

investigate

this

decapeptide

on

acetylcholine

and these

1D

the

the

a 67-76

has

and

was se-

conformational

sequence,

(WNPADYGGIK)

alanine, out

of

for t h e myasthenia gravis d i s e a s e

sequence

carried

each

been

2D-NMR

guest-host

resi-

substitu-

experiments

analogues

in

solution.

Results and Strong

and

fragment

discussion multiple

of

conformation the

the

Torpedo

been

DMSO

region

D

, G

Torpedo

short

and

AChR

argue

stabilized and

K

amide

by

long-range in

three

protons

NOEs

favor

of

in a

interactions (2).

the a 67-76

rigid

involving

Substitution

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w Y o r k - Printed in Germany

folded effects

514

of alanine guest residue are represented in Fig. 1 by plotting the difference between the chemical shifts of a given

oi

NH or C H proton of the analogues with reference to those of the Torpedo decapeptide. The perturbations observed illustrate the modification of the peptide backbone conformation induced by the substitution or the magnetic shielding change due to the original side-chain. Figure of

the

1 clearly

shows

that

peptide

backbone

are,

the at

conformational least

locally,

properties strongly

HOST V—

LU 3 A 1 0

1 wv ^^ A2

O "O

862 with I>o(I) were used for structure

determination and refinement. The structure was solved using the direct program M I T H R I L (7) and has only been refined to an R value of 0.14. A disordered diisopropyl ether seems to be associated with each peptide molecule. Further refinement is in progress. The perspective view of the molecular structure is shown in Figure 2. D-Pro

Fig.2. Molecular structure of Ace-D-Pro-D-Ala-L-Leu as viewed along the normal to the mean plane of all atoms.

527 The conformation observed for Ac-D-Pro-D-Ala-L-Leu does not agree with the expected result. The crystal structure shows an extended conformation with the Ala and Leu side chains situated on the same side of the peptide backbone and Pro on the opposite side. In the crystal, the peptide backbones are

situated

approximately in the be plane. Translation of the molecules along a results in the formation of parallel ^-pleated sheets held together by two hydrogen bonds between the classical atom pairs NH...O. The linear peptides are quite flexible and can adopt several energetically favourable

conformations. The

present

structure

shows

only

one

of

the

conformations accessible to the peptide. In order to get more definitive insight into the conformational preferences of such linear peptides, it is essential to study other analogues or longer peptides including the same sequence.

References 1. Boger, J., Lohr,N.S., Ulm,E.H., Poe,M., Blaine,E.H., Fanelli,G.M., Lin, T.Y., Payne, L.S., Schorn, T.W, Lamont,B.I., Vassil, T.C., Stabilito, I.I., Veber, D.F., Rich, D.H. & Bopari, A.S. (1983), Nature, 303, pp 81-84. 2. Bott,R.R & Davies,D.R. (1983), in Peptides: Structure and function (Hruby,V.J. & Rich,H. eds), Pierce Chemical Company, 111. pp 531-540. 3. Foundling, S.I., Cooper, J., Watson, F.E., Cleasby, A., Pearl, L.H., Sibanda.B.L., Hemmings, A., Wood, S.P., Blundell, T.L., Valler, M.J., Norey, C.G., Kay, J., Boger, J., Dunn, B.M., Leckie B.J., Jones, D.M., Atrash, B., Hallett, A. & Szelke, M. (1987), Nature, pp 349-352. 4. Precigoux,G., Ouvrard,E. & Geoffre,S. (1985), in Peptides: Structure and function (C.M.Deber, V.J.Hruby, K.D.Kopple, eds.). Pierce Chemical Co., Rockford,111., pp 763-766. 5. Precigoux, G., Barrans, Y., Geoffre, S., Picard, P & Hospital, M., (1987), in Peptides 1986 (D. Theodoropoulos ed.) Walter de Gruyter. Berlin-New-York, pp 323-326. 6. Mellado,J.M. & Geoffre,S. (1983), Micro Bulletin. Cook,P.I. (Bolliet.L. ed.), 9, pp 123-128, G.I.S., Saint Martin d'Heres. 7. Gilmore,C.J. (1984), MITHRIL. An integrated direct-methods computer program. J. Appl. Cryst. 17, pp 42-46. 8. Precigoux,G., Geoffre,S. & Ouvrard,E. (1986), Acta Cryst. C42, pp 721-724.

CONFORMATIONAL STUDY OF PEPTIDE-PROTEIN INTERACTIONS IN COLLAGENASE INHIBITOR SYSTEMS

M. Sakarellos-Daitsiotis, S. Tsiga, C. Sakarellos Department of Chemistry, 45110 Ioannina, Greece

University

of

Ioannina,

Box

1186,

M.T. Cung CNRS-UA-494, ENSIC-INPL, B.P. 451, 54001 Nancy Cedex, France

Introduction Collagenases the

are

native

bond

acid, while of

sequence

have

and

containing

or

sites

and

to

uses.

study

(NOESY)

of

These

results

are

restrictions

in

these

a

appropriate

or

less

(3) the

series

of

new

potent on

analyzed

the

NMR

bound in

to

conformation

tions .

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

and

in

order

suitable and

subfor for

conformational

terms

into the collagenase

synthedi-

enzyme

chromatography

tripeptides

tripeptide

the

inhibitors

affinity report

of

potent

sulfhydryl,

collagenase

specificity

briefly

the

insight

of

Clostridium

now

an

reported

the

by

We

with as more

ligating groups like

for

develop

purification

some

properties

investigate

therapeutic

obtain

reported

zinc

carboxyl

further

enzyme

oligopeptides been

inhibition

at

alanine or hydroxyproline. A great

(1,2). We have recently

tripeptides to

amino

already

usually

cleaving X-Gly

synthetic

collagen,

of

the

the

hydroxyl

of

capable

sequence X-Gly-Pro-Y, where X may be any Y is usually

variety

sis

metalloproteases

triple-helix

of the

inhibitors

zinc

collagenase. of in

inhibitor

mobility order

to

interac-

529 Results and The

Discussion

NOESY

The

experiments

sequence

formed of

with

effects

time

in

by

all

m~

run 90

phase

sensitive

"-acquisition

w a s

to

minimize

achieved

Jump-Return

experiments

in

250 ms. A

applied

to

was

-T

time of

was

in order

suppression pulse

90°-t 1 - 9 0 °

a mixing

mixing

were

cancel

the

by

t^

scalar

noise.

20

mM

The for

Per_

variation

correlation

The

B^O

resonance

of

the

last

substitution

sequence.

were

10 % random

mode.

concentrations

the

inhibitors

90° used

and

0.6

mM for the collagenase in D2O or F^O. The

free

usually rates.

the

be

of

observed.

showed latter, proline in NOE

the

(K^ =

more

strongest

only

weak

ring bound

former H~0/D„0

ring L-Ser

cross-peaks observed, on

the

for

are

the

more

analogue.

(90 : 10 v / v )

mobility

For

D-Ala-D-Pro-L-

77 |aM,

same In

showed

8/B

in of

increase the

K^

the

in

the

and

case 8/y

of

the

of

the

indicates

inhibitor

backbone

(K^ = 77 pM) to

NOESY

a cross-peak

65 (jM)

D-Ala-D-Pro-L-

the

8/Y

the

potencies,

(K^ =

8/(3 and

compared

addition,

and

solution

the

D-Ala-D-Pro-Gly

intense

80 nM

nevertheless

of

of

presence

In

between which

None

in

and

ones.

the

experi-

aqueous

following

cross-peaks

in

in

(D-Ala-D-Pro-L-

D-Ala-D-Pro-L-Cys

weakest

state.

cross-peaks

proline

NOE

state.

and

progressive

less

the

in

However,

absent

work

116 nM,

NOEs

a

or

the

are

restrictions the

showed

values

in 2D-N0ESY

bound

this

(K^)

enzyme.

Indeed,

116 (aM)

often

L-Ala-L-Pro-L-Cys

collagenase,

intensities,

are

the

in

constants

the

NOE

to

studied

respectively)

absence

NOEs

(Tc)

time

negative NOEs

attributed

inhibition

was

some

the

D-Ala-D-Pro-Gly, of

correlation

positive

tripeptides

Clostridium

Ser

small

Therefore,

should four

65 nM, the

the

peptides,

Ser, Cys

to

state.

ments the

< 1 ) and bound (iot > 1 ) states of a ligand c c exhibit opposite signs for the cross-relaxation

Due

small free

(wt

the

protons

of

the

those

of

the

experiments

between

the

in

Gly-NH

530

and the g-methyl group of alanine. This fact argues for the existence of a folded structure of the tripeptide in the presence of collagenase. For D-Ala-D-Pro-L-Cys (K^ = 65(iM) the most intense cross-peaks were observed between a/3/ 3 /3 , 3/y a n d y/ 6 proline protons and the 3/3 cysteine protons. Moreover, long-range NOEs between the two side-chain 3 protons of cysteine and alanine demonstrate the structuration of the peptide backbone. For L-Ala-L-Pro-L-Cys (K^ = 8 0 |jM) the NOE cross-peaks are less intense compared to the latter D-analogue, which indicates a lower proportion of the folded form. In conclusion, the above experiments in the presence of collagenase reveal some distance restraints between pairs of bound ligand protons, and some insight into the collagenase inhibitor interactions might be deduced. Work is now in progress to quantify these data.

Acknowledgement This

work

Research

was

supported

Committee

of

the

by. the

C.E.C.

University

of

(ST2J-0184), Ioannina

and

the the

General Greek Secretary for Research and Technology.

References 1. Vencill, C.F., D. Rasnick, K.V. Crumley, J.C. Powers. 1985. Biochemistry 24_, 3149.

N.

Nishimo,

2. Yiotakis, A., A. Hatgiyannacou, V. Dive, F. Toma. Eur. J. Biochem. 172, 761.

1988.

3. Tsiga, S. , M. Sakarellos-Daitsiotis, E. Papamichael, C. Sakarellos. 1988. In: Peptides (B. Penke and Torok, eds.). Walter de Gruyter, Berlin, p. 57.

FTIR

AND

OXYGEN-17

NMR

STUDIES

C A S E FOR N - A C E T Y L A T E D A M I N O A C I D

OF

PEPTIDE

HYDRATION.

THE

DERIVATIVES

F. Tête, B. V i t o u x Laboratoire ENSIC-INPL,

de C h i m i e - P h y s i q u e 1 rue G r a n d v i l l e ,

M. Sakarellos-Daitsiotis, I. T. K a r a y a n n i s , C. S a k a r e l l o s D e p a r t m e n t of C h e m i s t r y , 45110 I o a n n i n a , G r e e c e

M a c r o m o l é c u l a i r e , C N R S - U A 494 BP 451, 54001 N a n c y , France Gerothanassis,

University

of

N.

Birlirakis,

Ioannina,

Box

1186,

interactions

in

model

Introduction An

understanding

compounds to

the

is

study

cules

of

of

of a q u e o u s

( 1 ).

In

this

transform

infrared

to

the

examine

bond of

(i)

side

the

work,

chain

Results and

hydration

pure

DMSO -1

1700

cm

the

v(C=0)

self of

carboxyl

NMR

(iii)

Fourier

experiments

X-preceding

Leu),

ionization

and

biomole-

deconvoluted

the

(X = G l y , A l a ,

prerequisite

on larger

oxygen-17

behaviour

a

as a

state,

the

H20/dimethylsulfoxide

(ii)

the

of

the

nature (DMSO)

amide

function

mixtures).

discussion

experiments

bonds

or

and

effects

used

and

hydrophobicity, (D2O

FTIR

tivity

we

(FTIR)

adjacent

interest

solvation

in A c - X - O H m o l e c u l e s

environment

of

solute-solvent

fundamental

to

pure

) was

D,0.

carried

out

group

as

a

(2).

amide

However,

ranging

I vibration

because

of

interactions.

proton

characteristic

vibration

mixtures

investigated

intermolecular

carbonyl

in

The v ( C = 0 )

thoroughly

towards induces

were

low due

acceptor

its

the

within

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin-New York-Printed in Germany

broad

(1600sensi-

Involvement

frequency-shifts to

from

hydrogen of

the

intrinsic

532

1600 lBbo 1600 isko WAVENUMBERS (cm"1) WAVENUMBERS (cm"1) Fig. 1. Influence of D 2 0 molar fraction on the self-deconvoluted Amide I absorption of Ac-Leu-OH in the protonated (A) and deprotonated (B) states.

profile

of

procedures from

the

amide

(3) to

discrete

progressive

I

band,

narrow

hydrated

increases

the

we

species. in

the

used

overlapped Starting water

self-deconvolution components from

content

arising

pure

DMSO,

resulted

in

two successive splittings of the amide I contour by respectively of

% 20 and mono-

12 cm

and

(Fig.

di-hydrated

1), indicating the occurrence

carbonyl

complexes

(2).

Among

them only the latter remained detectable in pure D^0, suggesting here an identical in

both

during

neutral the

content) ted

and

first

where

samples

and

full solvation

ionic

hydration

comparison revealed

a

states. steps

between

This

was

not

(mixtures with protonated

decreased

of the C=0 in the former species

for all

solvent

(Fig.

of oxy-analog of the y-turn conformer

and

compounds the low

case

water-

deprotona-

accessibility

1). Residual

amounts

(4,5) or lower overall

basicity of the unionized species can be invoked as possible causes for this temporary shielding. In aqueous solution, the protonated molecules studied exhibited

very

small

oxygen-17

chemical

shift

differences

accor-

533 ding

to

the

identical

nature

safely

conclude

small.

On

resulted cies

by

nated

the in

latter

case.

DMSO

the

unionized

a

series

gave

previously results

differential to

be

form

and

of

the

values

of

amides

in

similar

evidence

proposed

will

for

especially be

from

a

FTIR

the s p e c t r o s c o p i c d a t a

a

group

frequen-

DMSO

molar

the

proto-

pronounced shift of

for

in

the

between 40-43

water

ppm,

the

ionized

ppm

observed

(5).

Such

for one, for

results

accessibility

protonated

experiments.

by

carboxyl higher the

solvents the

very

both

50-55

hindered

for

improved

ppm of

are

of

range

46-47

the

could

for

more

chemical

in

than

oxygen,

shift

species,

found

the to

increases

we

effects

of

shift

high-frequency

less

further

amide

The

was

somewhat

chemical

Assuming

studies,

substituent

Successive

a

( A 6 < 2 ppm). FTIR

deprotonation

oxygen-17

ppm.

chain from

Ca-alkyl

deprotonated

and

side

deduced

contrary,

induced

and

the

as

that

an

8-9

fraction

of

hydration

of

the

species,

These

quantitative

as

preliminary

treatment

of

obtained.

Acknowledgement We

are

General

indebted

to

Secretary

Foundation

the

for

C.E.C.

Research

"Leonidas Zervas"

(Grant and

ST2J-0184),

Technology

and

the

Greek

the

Greek

( s c h o l a r s h i p to N.B.)

References 1. P a c k e r , L. 1986. M e t h o d s in E n z y m o l o g y , m i c P r e s s , O r l a n d o , pp. 1-416. 2. S y m o n s , M . C . R . a n d G. E a t o n . T r a n s . 1 81_, 1963-1 977.

1985. J.

Vol.

Chem.

127.

Soc.,

AcadeFaraday

3. K a u p p i n e n , J.K., D.J. Moffatt, H.H. Mantsch C a m e r o n . 1981. A p p l . S p e c t r o s c . . 3 5 , 2 7 1 - 2 7 6 .

and

D.G.

4. T o n i o l o

9,

1-44.

C.

1980.

C.R.C.

Crit.

Rev.

Biochem.

5. H u n s t o n , R.N., I.P. Gerothanassis and 1985. J. A m . C h e m . Soc. 107, 2 6 5 4 - 2 6 6 1 .

J.

Lauterwein.

MOLECULAR PROPERTIES OF RECEPTORS FOR NEUROHYPOPHYSEAL HORMONES

F. Fahrenholz, M. J u r z a k , I . Pàvó, E. K o j r o , M. Hackenberg, J . Z s i g ò , D. J a n s . Max-Planck-Institut

für Biophysik,

Kennedy A l l e e

70, 6000 F r a n k f u r t

70

F.R.G.

Introduction The e x i s t e n c e of o x y t o c i n r e c e p t o r s has been demonstrated i n the

animal

and human myometrium. V 2 ~ v a s o p r e s s i n r e c e p e t o r s mediate the v a s o p r e s s i n induced

antidiuresis

in

collecting

ducts.

We report

a f f i n i t y chromatography f o r receptor p u r i f i c a t i o n monoclonal

Material

use

of

and the g e n e r a t i o n

of

a n t i b o d i e s to v a s o p r e s s i n

the

receptors.

and Methods

Myometrial from

anti-idiotypic

here

membranes

bovine

affinity

kidney

from guinea were

chromatography

pig

prepared were

as

prepared

at

late

pregnancy

described by

solid

(1,2). phase

coupled through t h e i r f r e e amino group to the carboxyl

and

membranes

Ligands

for

synthesis

and

group of the

gel

matrix. The

monoclonal

anti-vasopressin

antibody

mAb

A113

was

generated

by

immunizing BALB/c mice with the conjugate of the p h o t o r e a c t i v e [2-(p-azidophenylalanine), The

affinity-purified

monoclonal

8-arginine ] vasopressin

mAB

anti-idiotypic

113

served

as

and

antigen

antibodies.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w York- Printed in G e r m a n y

to

thyroglobulin. produce

mouse

535 Results and Discussion

The oxytocin receptor from guinea pig and the kidney

retained

after

affinity for their

solubilization

by

receptor

nonionic

from

detergents

ligands. Therefore affinity chromatography

bovine binding

could

be

used for purification of the receptor proteins. For the isolation of the oxytocin receptor, analogues were synthesized which allow of the ligand to the affinity gel

matrix

either

at

the

the side

coupling chain

of

ornithine or of 2,4-diaminobutyric acid (Dab) (Table 1).

Table 1: Ligands for affinity chromatography: Affinity for the

oxytocin

(OT) receptor in the guinea pig myometrium

No

Peptide

KQ(M)

^(analogue) K n (oxytocin)

1

OT

(3.7 + 0.6) xlO"

2

[Mpa\sar7,0rn8]0T

(3.2 + 0.4) xlO"

3

[Mpa1,Dab4,Sar7]0T

(4.0 + 0.4) xlO"

11

(2.9 + 0.4) xlO"

78

4. [ M p a 1 , T h r 4 , S a r 7 , 0 r n 8 ] 0 T

0.86

Mpa: 3-mercapto propionic acid

By two affinity chromatography steps using the ligands 3 and 4 (Table 1) followed

by

isolated

from

hydroxy!apatite guinea

pig

chromatography

myometrial

a

membranes.

protein The

fraction

specific

was

binding

activity of this protein for [^hJot was three orders of magnitude higher as compared to the membrane-bound oxytocin receptor.

536 For the p u r i f i c a t i o n of the renal

V^ r e c e p t o r i n bovine kidney membranes

a f f i n i t y columns with [ 8 - a r g i n i n e ] v a s o p r e s s i n

(AVP) and [ Mpa 1 ,Dab^] AVP

were used. A v a s o p r e s s i n - b i n d i n g p r o t e i n f r a c t i o n was h i g h l y e n r i c h e d by this

The

procedure.

monoclonal

experiments

anti-vasopressin

a similar

ligand

antibody

specificity

c r o s s r e a c t i v i t y was found with e i t h e r C-terminal

mAb as

113

vasopressin

oxytocin,

ELISA

a c i d or

No the

part of AVP, P r o - A r g - G l y - N H ^ . A f t e r immunization of mice w i t h

V^ r e c e p t o r

in

rat

liver

1:100. Three monoclonal influenced

inhibited

in

receptors.

pressinoic

mAB 113, two of f i v e s e r a i n h i b i t e d the b i n d i n g of

fluids

displayed

the

the

binding

membranes

to

anti-idiotypic hormone of

roughly

3

[ H ] AVP

50%

antibodies

receptor to

at

a

dilution

i s o l a t e d from

interaction: rat

t o the

[3H]AVP

liver

two

and

of

ascitic of

bovine

them kidney

membranes, one enhanced the b i n d i n g o f [^H]AVP t o the r e n a l V 2 r e c e p t o r . In experiments w i t h the LLC-PK1 p i g kidney c e l l antibodies

showed

agonistic

properties;

l i n e , the

similar

induced the p r o d u c t i o n of u r o k i n a s e i n t h i s c e l l These

results

provide

evidence

that

these

to

anti-idiotypic

vasopressin

line.

monoclonal

a n t i b o d i e s i n t e r a c t with the b i n d i n g s i t e of v a s o p r e s s i n

anti-idiotypic receptors.

Acknowledgement T h i s r e s e a r c h was s u p p o r t e d by a g r a n t of the Deutsche gemeinschaft

(SFB

Forschungs-

169).

References

1. F a h r e n h o l z , F . , M. Hackenberg, M. M l i l l e r . 1 9 8 8 . E u r . J . Biochem. J 7 4 , 81 2. C r a u s e , P . , F . F a h r e n h o l z ,

they

1982. Mol. C e l l . E n d o c r i n o l .

28, 529

VASOPRESSIN AND OXYTOCIN ANALOGS WITH HYDRAZIDE-CONTAINING CHAINS IN POSITION 4

SIDE

Diana Gazis, John Glass, I. L. Schwartz, G. Stavropoulos*, and D. Theodoropoulos*

Department of Physiology and Biophysics and Center for Polypeptide and Membrane Research, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029 and Laboratory of Organic Chemistry*, University of Patras, Patras, Greece

Introduction

The carboxamide of the Gin side chain in position 4 of oxytocin and vasopressin is not needed for activity and so minimal changes in this side chain should cause little, if any, loss of activity. The present report describes the synthesis and biological

activities of

two analogs, [4-P-glutamic hydrazide] oxytocin and [4-f-glutamic hydrazide] lysine vasopressin

with hydrazide substitutions on this

side chain.

Methods

Analogs were prepared by solution synthesis through stepwise elongation using the mixed anhydride and OSu ester methods*. Glutamic hydrazide was formed from the DCHA salt of N*-Boc-methylglutamate by the addition of Z-hydrazide and subsequent hydrolysis of the methyl group. The following bioassay preparations were used: antidiuretic activity on water-loaded 2 anesthetized rats , pressor activity on urethane-anesthetized, phenoxy3 benzamine-treated rats , milk ejection on post partum rats in their 10th 4 5 to 20th day of lactation , and uterine activity vitro in solutions with or without 0.5 mM M g + + .

Peptides 1988 © 1989 Walter de Gruyter&Co., Berlin-New York-Printed in Germany

538 Results and Discussion The biological activities of the hydrazide analogs and four related compounds are shown in Table 1:

Table 1. Biological Activities (in Internationsl Units/mg) of Hydrazide-substituted Oxytocin and Lysine Vasopressin Analogs and Related Compounds. Analog

Uterus in

Milk

Anti-

vitro.no Mg

Ejection

diuresis

450

5

5

0.5±0.2

0.18

n.d

n.d.

450

Oxytocin^

6.9±0.5

[4-r-Glutamic hydrazide]OT

38±6 300

Pressor

[4-Asparagi ne]OT^

108

Lysine Vasopressin^

5

[4-f-Glutamic hydrazide]LVP

0

2.4±0.3

44±12

19±2

[4-Asparagine]LVP 6

n.d.

n.d.

25

56

63

285

260

The hydrazide substitution has a similar effect to the asparagine substitution on the vasopressin-like activities, antidiuresis and blood pressure increase, but an opposite effect on the oxytocin-1ike activities, milk ejection and uterine contraction. The side chains of glutamine,

-glutamic hydrazide, and asparagine are as follows:

C-C-C-C0NH 2 , C-C-C-C0NHNH 2 and C-C-C0NH 2 . One may assume from these structures and from the biological activities of the compounds in Table 1 that the length of the side chain in position 4 is important for oxytocin-like but not for vasopressin-like activities.

539 Acknowledgements This research was supported by Grant DK-10080 from the National Institute of Diabetes and Digestive and Kidney Diseases and Grant HD-19517 from the National Institute of Child Health and Human Development.

References 1. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M., 1963, J. Am. Chem. Soc. 85, 3039. 2. Sawyer, W. H., 1957, Endocrinology 63, 694. 3. Dekanski, J., 1952, Brit. J. Pharmacol. 7, 567. 4. Bisset, G. W., Clark, B. J., Haldar, J., Harris, M. C., Lewis, G. P., and Roche e Silva, M. Jr., 1967, Brit. J. Pharmacol. 31, 537. 5. Munsick, R. A., 1960, Endocrinology 66, 451. 6. Berde, B., and Boissonnas, R. A., 1964, In: Handbook of Experimental Pharmacology, Vol. 23, Neurohypophyseal Hormones and Similar Polypeptides, Ed. B. Berde, Springer-Verlag, NY, pp. 802-870.

STRUCTURE-FUNCTION STUDIES IN A SERIES OF ARG ININE-VASOPRESSIN ANALOGS SUBSTITUTED IN POSITION 1 AND 2

Z. Grzonka, L. -tankiewicz, F. Kasprzykowski I n s t i t u t e of Chemistry, U n i v e r s i t y of Gdansk, 80-952 Gdansk, Poland J. Trojnar, P. Melin Ferring Pharmaceuticals, 200 62 Haimo, Sweden M. Hackenberg, F. Fahrenholz Max-Planck-Institut für Biophysik, 6000 Frankfurt am Main F.R.G.

Introduction I t i s well known that the nature of the residues in the p o s i t i o n 1 and 2 of arginine-vasopressin strongly influences either the a g o n i s t i c or antagonistic properties of AVP analogs. The present study was performed to examine the role of the peptide bond between residues 1 and 2 and the p o s i t i o n of t y r o s i n e side chain in the i n t e r a c t i o n of a hormone with i t s receptors. A s e r i e s of analogs was designed in which the cysteine residue in p o s i t i o n 1 was substituted by mercaptoacetic acid (Maa) or by 1-mercaptocyclohexylcarboxylic acid (Mcc) r e s i d u e s , and the tyrosine residue in p o s i t i o n 2 was substituted by (3-homotyrosine (Hty),

p-homo-

tyrosine(0-methyl) Hty(Me) or (3-homophenylalanine (Hph) residues, r e s p e c t i v e l y . Therefore, the only difference in the structure of these compounds, in comparison to the structures of highly active analogs of AVP, such as deamino-AVP ([Mpa^AVP) (agonist) (1) and l-(p-mercapto-p ,pcyclopentamethylenepropionic acid) AVP ([Cpp^AVP) (antagonist)

(2)

i s that they have a s h i f t e d peptide bond between residues 1 and 2 as well as a side chain of the residue 2.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin-New York-Printed in Germany

541 Results and Discussion

All analogs were prepared by solid-phase peptide synthesis previously (3). Biological

as described

activities of these peptides were estimated

on phenoxybenzamine-treated rats under urethane anaesthesia

(pressor

activity) and on water-loaded rats anaesthetized with ethanol

Table 1

Biological

activity and relative binding affinities of AVP analogs

substituted in position 1 and 2

No

Peptide*

Biological

activity

in IU/mg

Relative binding affinity ^(analogue) KD(AVP)

Vasopressor

Antidiuretic

V-j receptor

receptor

1. [Maa , H t y 2 ] AVP

0 038

0.212

2. [Maa , D - H t y 2 ] AVP

0 13

0.07

3. [Maa , H t y ( M e ) 2 ] AVP

0 02

4. [Maa ,D-Hty(Me 2 )] AVP

0 012

5. [Maa , H p h 2 ] AVP

0 4

2 6. [Maa , D - H p h ] AVP

0 14

7. [Mcc , H t y 2 ] AVP

0 014

no activity

40 000

5 000

8. [MCC , D - H t y 2 ] AVP

0 025

no activity

120

3 200

2

9. [MCC , H t y ( M e ) ] AVP

0 04

0.05

3 900

> 5

10. [MCC , D - H t y ( M e ) 2 ] AVP

0 12

0.0006

2 200

> 5 000

11. [MCC , H p h 2 ] AVP

0 024

no activity

1 100

0 37

no activity

3 500

12.

[MCC , D - H p h 2 ] AVP

000

*Abbrevations: Maa, mercaptoacetic acid; Mcc, 1-mercaptocyclohexylcarboxylic acid; Hty, ß -homotyrosine; Hty, ß-homo-(0-methylJtyrosine; Hph, ß-homophenylalanine.

542 ( a n t i d i u r e t i c a c t i v i t y ) . Binding studies were performed on membrane preparations from rat l i v e r (V-j receptors) and from bovine kidney (V2 receptors). The AVP analogs containing either Maa or Mcc residues in p o s i t i o n 1 and homo-amino acids in p o s i t i o n 2 are characterized by the very low a g o n i s t i c a c t i v i t i e s (Table 1) and none of them showed any antagonistic property both in vasopressor and a n t i d i u r e t i c responses. Compounds containing Mcc residue in p o s i t i o n 1 were checked for V-| and

receptor

binding a f f i n i t i e s . As can be seen from Table 1 t h e i r a f f i n i t i e s to V-j and V^ receptor are of 2-3 orders of magnitude lower than for the parent hormone. All analogs of AVP with both the peptide bond between residues 1 and 2 and the side chain of amino acid residue at p o s i t i o n 2 shifted by one methylene group are deprived of c h a r a c t e r i s t i c b i o l o g i c a l a c t i v i t y . This r e s u l t s could be explained by the differences in the interaction of these peptides with V-| and

receptors in comparison either to C M P a l

AVP or to [Cpp] AVP in which the appropriate peptide bond i s exposed to the surrounding molecules, including receptors

(4).

Acknowledgement This work was supported by the grant no. CPBR-3.13.4.3.2 from the P o l i s h Academy of Sciences and by the grant from the Deutsche Forschungsgemeinschaft (SFB 169) References 1. Manning, M., L. B a l a s p i r i , J. Moehring, J.H. Haldar, W.H. Sawyer 1976, J.Med.Chem. 19, 842 2. Kruszynski M., B. fammek, M. Manning, J. Seto, J. Haldar, W.H. Sawyer 1980, J.Med.Chem. 23, 364 3 Grzonka, Z., B. Lammek, F. Kasprzykowski, D. Gazis, I . L . Schwartz 1983, J.Med.Chem. 26, 555 4. Liwo, A., A. Tempczyk, Z. Grzonka: J.Computer-Aided Mol.Design (in press)

SYNTHESIS AND PROPERTIES OF ANTIPARALLEL DIMER OF DEAMINO-1-CARBA-OXYTOCIN

J.Slaninova, M.Lebl Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia J.Eichler Institute for Drug Research, Academy of Sciences A.Kowalke Str. 4, Berlin, German Democratic Republic

of

GDR,

Introduction Dimer forms of neurohypophyseal hormones were studied already at the very beginning of synthetic activities in this field. Parallel

and

antiparallel

dimers

of

oxytocin,

dimers

trimers of lysine-vasopressin, and dimeric forms of other analogs were described

(1). In all cases

and

several

significant

biological activities were found (0.25 - 4 % activity of the parent their

hormone), further

but

usually

evaluation.

little

attention

The question

was

paid

remains whether

to the

dimeric form can really fulfill the requirements of the receptor and evoke the response or whether the activity found is the result of monomeric form either contaminating the dimer or formed during the biological evaluation due to dimer-monomer equilibrium established by the transsulfidation reaction. Synthesizing such analog of neurohypophyseal hormones that would model the dimeric form stabilized against transsulfidation reaction and evaluating its biological properties would throw more antiparallel

light dimer

chosen. Monomeric

into of

this problem. For

this purpose, the

deamino-1-carba-oxytocin

form of dCOT-1

(dCOT-1) was

is known to have at least

three times higher uterotonic activity than oxytocin (2).

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

544

Experimental The dimer of dCOT-1 was synthesized using two methods: (i) it was isolated from the reaction mixture after the synthesis and cyclization on the polymer matrix using repeated gel filtration on Bio-Gel P-4 and further purified by reversed-phase HPLC. The cyclization on the polymer affords higher or lower amount of higher-molecular weight products, depending on

the

solvent which

we use

for

the

last

synthetic

step.

Dichloromethane, dimethylformamide or their mixtures gave us comparable

results,

but

the

use

of

trifluoroethanol,

as

recommended by Schiller (3) provided higher monomer to dimer ratio. The structure of dimer was proven by FAB-MS. (ii) The independent synthesis was based on the assembling of the octadecapeptide (dimer precursor) on the polymer carrier, its cyclization and cleavage from the resin

(Scheme 1). The

first three residues were coupled as Boc-amino acids and from the fourth step the synthesis was performed with Fmoc-o

Xr"

H H

X = H, F

a)DPPA,NEt 3 ,tBuOH; b)LDA, 4X-PhCH 2 Br; c)6N HC1; d) Boc 2 0. The reduced Pro-Phe analogues were prepared by reductive amination of the aldehyde (4)(Scheme 3), followed by either Z-protection or N-methylation. Epimerization during these steps was monitored by gas chromatography (5). SCHEME 3

B o c - N — — B o c - N ^ a

I

H N

Y

COOMeBoc-N b C '

O

R k^fK/ CH

CH 2 Ph

C 0 0 H

2

ph

R=Z,Me a)NaBH 3 CN, L- or D-Phe-OMe; b)Z-Cl or CH 2 =0, H 2 /Pd; c)NaOH Bradykinin analogues were obtained by solid phase synthesis using the Bocstrategy and HF cleavage. Biological

activities

The effect of the Bk-analogues on RBP and on GPI contractions are summarized in Table 1.

Substitution at the 4-5 or at the 5-6 position results in a

drop in potency without prolongation of action. g Phe

Substitution of the Pro^-

dipeptide by the alkene or reduced isostere results in analogues which

are at least as active as Bk itself and displaying prolonged activities, sometimes for over one hour. ting effect on GPI. 3.8 times that of Bk.

A different influence is seen in the contrac7 8 Only one of the Pro -Phe Bk isomers 4 has a potency The selective action of the pseudopeptides in the

two tests suggest different receptor requirements, which is quite apparent for the reduced isosteres.

The prolongation of activity in the RBP test is

in agreement with the stabilization of Bk against enzymatic cleavage.

564 TABLE 1 : Effect of Bk-analogues on Rat Blood Pressure (RBP) and on Guinea Pig Ileum (GPI) Contractions time to %BP dose regai n lowenormal ring 1 /kg) BP(min) 1

Bk

40 -60 70

1 10

8 12-15

2

[Gly 4 =Phe 5 ]Bk

20

10

20

3

[Phe 5 =Gly 6 ]Bk

50

10

30

4

5

7

8

[Pro =Phe ]Bk(isoml)

60 65 70

0.32 * 1 '3.2

6 10 30

(isom2)

0 60

1 10

10

0 70

1 5

[Gly 4 =4F-Phe 5 , 7

8

Pro =Phe ]Bk 6

[4F-Phe 5 =Gly 6 , 7

8

Pro =Phe ]Bk 7 8 9 10 _n

7

[Pro (CH 2 -NH)Phe 8 ]Bk [Pro 7 (CH,-NH)D-PheV L.

[Pro 7 (CH„-NMe)Phe 8 ]Bk C.

0 70

x

1 10

_ -

-

50 1 70 "10

8

60 1 70 *10

12

70 1 70 "10

6

_

1.00

1.0

-

ND

ND

-

ND

.ND

_

3.857

1.1

-

0.049

1.2

_

CH;. a) Cys(MBzl) X=$-SCH2-£}-CH3

A

BocNH'

CH-CHJ-NH

b) Ser

>

CH 2

CH2 CH—CH 2 —N / / '

BocNH

/

I

CH COOH

Z

Figure 2. Synthetic Scheme for the preparation of protected pseudodipeptides incorporated into ANF analogs.

582 Results and Discussion The syntheses of protected pseudodipeptides Cys-Phe and SerPhe are outlined in Figure 2. Using method A, the pseudodipeptide 9 was prepared by borane/THF reduction

(3). The yield

in this reaction was typically less than 30%. In method B, the synthesis of 9 was performed by sodium cyanoborohydride mediated reductive amination of phenylalanine methyl ester with protected cysteine or serine aldehydes

(4). The aldehydes were

prepared by lithium aluminum hydride reduction of the respective amino acid N,0-dimethylhydroxamates. The yield by this method is higher, in the 50-80% range, the purification is less tedious and the results are more reproducible. The results demonstrate that the preferred method for the preparation of the pseudodipeptides is method B. The synthesis of pseudodipeptide-containing AP analogs was performed by the Merrifield method using Pam resin. The structures of the products were verified by FAB-MS, amino acid and sequence analysis. The analogs were potent in vivo blood pressure lowering agents in rats. However, the duration of the effect was similar to that observed for unmodified APs. It is therefore unlikely that the specific endoprotease is the principal determinant of circulating lifetime and duration of AP action.

References 1. Needleman, P., S.P. Adams, B.R. Cole, M.G. Currie, D.M. Geller, M.L. Michener, C.B. Saper, D. Schwarz, and D.G. Standaert. 1985. Hypertension 1_, 469. 2. Olins, G.M., K.L. Spear, N.R. Siegel, H.A. Zurcher-Neely, and C.M. Smith. 1986. Fed.Proc. _45, 27. 3. Roeske, R.W., F.L. Weitl, K.U. Prasad, and R.M. Thompson. 1976. J.Org.Chem. 41, 1260. 4. Martinez, J., J.-P. Bali, M. Rodriguez, B. Castro, R. Magous, J. Laur, and M.-F. Lignon. 1985. J.Med.Chem. 28, 1974.

THE IONIC MECHANISM OF NK-3 RECEPTOR IN MYENTERIC NEURONS

C. Gilon, , M. Chorev, Z. Selinger Departments of Organic, Pharmaceutical and Biological Chemistry The Hebrew University of Jerusalem, Jerusalem 91904 ISRAEL M. Hanani Laboratory of Experimental Surgery, Hadassah University Hospital Mt. Scopus, Jerusalem 91240, ISRAEL

Introduction Tachykinins are a group of peptides from mammalian and non-mammalian origin which share the common c-terminal sequence -Phe-X-Gly-Leu-Met-NH2. The mammalian tachykinins: Substance P (SP), Neurokinin A (NKA) and Neurokinin B (NKB) activates preferentially the three tachykinin receptors NK-1, NK-2 and NK-3 respectively. In the present work the effects of SP and the selective analogs Ac-Arg-Senktide (NK-1 selective) and Senktide (NK-3 selective) on single myenteric neurons were studied by intracellular recordings.

Results and Discussion

The existence of multiple tachykinin receptor subtype was strongly corroborated by the introduction of receptor selective analogs. We have found that backbone modifications in the cterminal hexapeptide of SP confers receptor selectivity (1) Thus introduction of Me-Phe instead of Phe in position 8 confers selectivity towards the NK-3 receptor whereas replacement of Gly 9 by Pro (2) confers selectivity towards the NK-1 receptor. Improvement in selectivity was further obtained by the introduction of charged amino acids at the N-terminal part of the hexapeptide and by blocking the terminal amino group to protect the peptides from amino peptidases.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin New York - Printed in Germany

584

k

SP

•mill

40mV

A

Senk.

Fig.l. Changes application of are responses large increase

in SP to in

the input resistance (Rin) in response to the (A) and Senktide (B). The downward deflections hyperpolarizing currents (0.2 nA) . Note the Rj^ during the depolarization.

Response (mVJ

|20mV 40»

-90 f

EmlmVI -50

-70 -5

Fig.2. The reversal potential for Senktide response. (A) Responses induced by 600 ms pulses of Senktide (arrow). The membrane potential was displaced by passing currents through the recording electrode to the level indicated on the left of the recordings. The resting potential was -66mV. (B) A plot of the results obtained in the experiment shown in (A). Peak depolarizing or hyperpolarizing responses were plotted as function of membrane potential. The reversal potential was at about -85 mV.

585 The most selective analogs which were obtained in these studies are: Ac [Arg®, Pro 9 ] SPg.-j^ (Ac-Arg-Septide) and succ[Asp 6 ,Me-Phe 8 ] S P g _ n (Senktide) which acts on the NK-1 and the NK-3 receptors respectively at nM concentrations while their activity on the other two receptors required 3-4 order of magnitude higher concentration. In depth studies were directed at the characterization of the NK-3 receptor. A radioligand of Senktide was prepared and the presence of NK-3 receptors in the rat cortex was demonstrated by binding studies and receptor-autoradiography (3) . Most important, analysis of the myenteric NK-3 receptor in the guinea pig ilium by functional assay and the rat cortical receptor using binding studies showed that these two receptors have virtually identical pharmacological properties (3) . In view of the excitatory action of the NK-3 receptor in myenteric neurons, the ionic mechanism which underlies this excitation was studied by intracellular recording. Both SP and Senktide cause prolonged depolarization (10-20 sec) which was accompanied by an increase of the input resistance. In most cases the depolarization by Senktide was accompanied with firing of action potential. (Fig. 1.). To gain further information on the ionic mechanism mediated by the NK-3 receptor, the reversal potential of the response was measured. Analysis of the effect of Senktide showed that the reversal potential is in the range of -85 to -105 mV which is close to the K + potential (Fig. 2.). Taken together with the increase in input resistance these results suggest that the depolarization caused by Senktide is mediated by closure of K + channels.

References

1.Wornser, U., R. Laufer, Y. Hart, M. Chorev, C. Gilon, Z. Seiinger. 1986. EMBO J. 5 2805. 2. Laufer, R., C. Gilon, M. Chorev, Z. Seiinger. 1986. J. Med. Chem. 23, 1284. 3. Laufer, R., C. Gilon, M. Chorev, Z. Seiinger. 1986. J. Biol. Chem. 2£1, 10257.

Neuropeptide Y analogs with high agonistic activities A. Beck, G. Jung Institut für Organische Chemie, Universität Tübingen, D-7400 Tübingen, FRG G. Schnorrenberg, H. Koppen, W. Gaida Boehringer Ingelheim KG, D-6507 Ingelheim, FRG R. Lang Institut für Pharmakologie, Universität Heidelberg, D-6900 Heidelberg, FRG Introduction Neuropeptide Y (NPY) is a 36-peptide amide, which has been isolated from porcine brain and sequenced 1982 by Tatemoto (1). NYP belongs to the family of pancreatic polypeptides (PP) which have the same length, high sequence homology and C-terminal tyrosine amide. Together with noradrenaline, vasopressin and angiotensin NPY belongs to the strongest natural vasoconstrictors respectively blood pressure increasing components.

NPY acts as a

neurotransmitter/neuromodulator and binds to highly specific and selective receptors. Preliminary studies suggested the C-terminal part of NPY to be essential for biological activity (2-3). We want to report on the synthesis, hypothetical 3D-structure, conformational studies, receptor binding, pre- and postsynaptic activities of synthetic NPY segments and analogs. Results and Discussion A hypothetical 3D-structure of NPY has been deduced from the X-ray structure of the homologous APP (4) by exchange of side chains of differentiating amino acids, and minimization by force field calculations (Discover, Biosym Techn.) Using molecular dynamics simulation we could distinguish between the conserved helical parts (polyproline helix type II 1-8, ahelix 15-32), the flexible /3-turn 1-10 and the C-terminal tetrapeptide 33-36 (Fig.l). Starting with the minimized structure of NPY, the 30 psec simulation was performed with neutral residues and without cutoff-radius. Trajectories every 2 psec have been minimized by force field calculation. Deduced from the hypothetical structure (Fig.l) we synthesized analogs (Tab.l) via Fmoc strategy using 5-(4'-Fmoc-aminomethyl-3', 5'-dimethoxy-phenoxy)-valeryl-alanyl-resin (5)

Peptides 1988 © 1989 W a l t e r de G r u y t e r & C o . , Berlin • N e w York - Printed in G e r m a n y

587 Table 1. Receptor binding, postsynaptic and presynaptic activities of short synthetic NPY analogues in comparison to NPY.

NPY-Segment NPY NPY NPY NPY NPY NPY

Biological Activities

Receptor Binding IC 50 [M]

postsynaptic A30mm Hg [M] >10~ 5 8.0 • l O - 7 9.0 • 10- 7 >UT5 9.0 • 10" 8 2.0- lO" 10

1.1 • io-6

Ac-27-36 Ac-25-36 Ac-25-36 P 5 K] Ac-25-36 p ^ K ] l-4-eAca-25-36

7

1.6 • 1(T 1.6 • 10- 7 2.0 • 1 0 - 5 2.9 • 10~9 5.0 • lO- 1 0

presynaptic 50%inhib.[M]

>io-5

4.4 • 10~6 1.0 • 10" 5

>io-5

1.8 • lO" 7 9.0 -10" 8

Y \ R

o P—» R

30

I RH

OL

H

T3

s

\ 1

7 T

Y

O -

•

[deg

p ^ — -r

°1

\

R

Y

T/

\ \1

U

(l

VS1L 1

/

20

D

/

L

o s

A

1 o I-t «

\\

N P Y A c - 2 5 - 36

/

o _ •

0 CM 1

-

1

1 200

1

1 210

1

NPY

1-4- t A c a - 2 5 - 3 6

1

'

220

1 230

1

I 240

~ A

lnmJ

Fig.l Scheme of the highly active analog NPY l-4-eAca-25-36 according to the simulated structure (MDS) of neuropeptide Y and CD spectra of analogues NPY AC-25-36 and NPY l-4-eAca-25-36 in trifluoroethanol/water 9:1.

588 (0.25 m M N H 2 ) on a peptide synthesizer (Applied Biosystems 430 A). T h e side chains of Arg and His were protected with P m c or M t r and T r t , others with tBu and Boc. For the first coupling we used B O P / H O B t activation in N M P / D M F (1:1) adding D I P E A ( N H 2 : F m o c - a m i n o a c i d : B O P : H O B t : D I P E A = 1:4:4:4:6). T h e second coupling was performed with D I C / H O B t activation in N M P / D M F / C H 2 C 1 2 (3:2:1) adding D I P E A to t h e reaction vessel after 50 min. T h e peptide amide was splitt off using T F A / t h i o a n i s o l e / 2 - m e t h y l t h i o p h e n o l e (95:3:2) for 3h. For receptor binding assays we used t h e displacement of rabbit kidney m e m b r a n e preparations.

125

I - N P Y by NPY-segments in

Biological activity was tested using postsynaptical

(mean arterial blood pressure response of anaesthesized, pithed rats to intravenous injections of NPY-segments) and presynaptical (inhibition of contractions induced by electrical field stimulation in isolated rat vas deferens) systems. Both assays revealed dose response curves in accordance to t h e receptor binding (Table 1). We conclude t h a t t h e C-terminal p a r t of N P Y is essential for receptor binding and biological activity which are already induced by N P Y Ac-27-36. Replacement of influence whereas additional replacement of

33

A r g and

35

25

A r g by Lys has no

A r g by Lys abolished both receptor

binding and biological activity. N P Y 1-4-eAca-25-36 which is linking N-and C-terminal segments via flexible spacer £-aminocaproic acid (Aca) exhibits receptor binding almost as high as native N P Y . We suppose t h a t the N-terminal segment 1-4 is stabilizing t h e C-terminal a-helical structure 25-36. This interaction is suggested by CD d a t a : [A] 2 07/ 2 22 • 10 ~ 3 [deg x cm 2 x drool" 1 ] for N P Y Ac-27-36 13.9/8.4, for N P Y Ac-25-36 16.7/13.1, for N P Y Ac-2536[ 25 K] 16.8/13.7, for N P Y 1-4 eAca-25-36 29.5/24.3 and for N P Y 87.5/83.5 (c = 1 0 " 3 mol/1 in trifiuoroethanol/water 9:1) ( F i g . l ) .

Acknowledgement This research was supported by t h e Bundesministerium fur Forschung und Industrie.

References

1. Tatemoto,K. 1982. Proc. Natl. Acad. Sci. USA 79, 5485-5489. 2. Danger,J.M., M.C.Tonon, M.Lamacz, J . C . M a r t e l , St.Pierre, G.Pelletier, H.Vaudry. 1987. Life Sci. 40, 1875-1880. 3. Wahlestedt,C., N.Yanaihara, R.Hakanson. 1986. Regul. P e p t . 13,307-318. 4. Glover,I., I.Haneef, J . P i t t s , S.Wood, D.Moss, I.Tickle, and T.L.Blundell.1983. Biopolymers 22, 293-304. 5. Albericio,F., G.Barany. 1987. Int. J. P e p t . Prot. Res. 3Q, 206-216.

BOMBESIN RECEPTOR ANTAGONISTS

R. de Castiglione, L. Gozzini, M. Ciomei, I. Molinari

R. Mena,

M. Brugnolotti,

Farmitalia Carlo Erba/R. & D. - Erbamont Group - Milan, Italy P. Comoglio Biomedical Sciences and Oncology Dept., Turin Univ.,

Italy

D. Parolaro Pharmacology Dept., Faculty of Sciences, Milan Univ., Italy.

Introduction Since the discovery that bombesin (BBS) can act as an autocrine growth factor in human small cell lung carcinoma (SCLC), search for bombesin antagonists has been actively pursued by many research groups. In the present paper the results of a series of C-terminal bombesin nona- and decapeptide analogues, characterized by amino £.cid deletion, inversion or substitution, are presented.

Experimental Analogues have been obtained by coupling an N-terminal BBS(6-11)hexapeptide with a C-terminal di- or tripeptide. All couplings have been carried out in solution by the mixed anhydride procedure. Lys and Arg residues have been introduced on the preformed nonapeptide in order to increase water solubility.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

590

Receptor affinity has been evaluated as [I125]GRP binding inhibition on Swiss 3T3 fibroblasts. Mitogenic effects have been monitored in the 3T3 cells as [H3]thymidine incorporation and as phosphorylation of a 115 KD protein associated with the BBS receptor complex (Table 1) . BBS analogues, displaying good receptor affinity and no appreciable effects in these two assays, were tested as antagonists of the thymidine incorporation induced by 25 nM BBS. Peripheral and central effects have been assayed in the rat as urinary bladder contraction in vitro and as grooming behaviour. Spantide ([D-Arg1, D-Trp 7 - 9 , Leu11]substance P) and [D-Phe12, Leu^jBBS, known from literature as BBS antagonists, were chosen as standards.

Results and Discussion On Swiss 3T3 fibroblasts the His residue can be replaced by Phe, Ser and Ala with retention of receptor affinity and agonistic activity, whereas its deletion gives rise to analogues which still bind to the receptor but are practically devoid of mitogenic effects and, given in combination with BBS, display weak antagonistic activity. Other modifications are followed by a more or less marked drop in receptor affinity. In particular, inversion of the Trp residue or deletion of the C-terminal tripeptide results in compounds which no longer bind to the BBS receptor. None of these peptides antagonizes BBS induced thymidine incorporation. In these cells, and in the same conditions, spantide and [D-Phe12,Leu14]BBS are not BBS antagonists. In the rat urinary bladder most of the analogues tested are either agonists or weak agonists/antagonists according to the doses. Contrary to what occurs in the mitogenic test, replacement of His by D-Phe (No 6) apparently gives rise to pure antagonists, comparable to spantide (also antagonist

591

in this are

test).

observed

Similar

also

deletion)

is

antagonist

in the

D-Trp),

a

for

weak

inactive

contrasting other

compounds.

agonist

3T3 cells, in

these

results

in

the

in the

Peptide

urinary

cells,

is

No

tests

4

(His

bladder

whereas peptide N o

same

two

10

a weak

and

(Trp

—>

agonist

in

the rat test in vitro. Few

compounds

have

been

only antagonist found

assayed

in

the

grooming

is compound No 3 (Leu — >

test.

an activity similar to spantide. These observations, preliminary and incomplete, suggest the presence of receptor subtypes in the different experimental

The

D-Leu), with although different

systems.

Table 1 Effects of selected BBS analogues on mouse Swiss 3T3 fibroblasts

»

V

„

,

H-Thr

2

H-Thr

C

.2

R-Asn-Gln-Trp-Ala-Val-Gly-His- Leu--Met-NHa (Bombesin)

1

B

A

P E P T I D E S

No

pisDnp

6.1

6,.6«

-

3

-NH(CH a )„CH,

430

1

1

NH(CH a ),CH,

130

4..4

7..4

7800

1

1

3000

1

1

1..4

3 .6

1

1

4 .9

50 200

•leu-

>10000

2500

3

H-Thr

4

H-Thr

5

H-Thr

Phe-

360

6

Boc-Thr

phe-

16000

7

H-Thr

Ser-

200

2..3

8

H-Arg-Thr

Ala-

180

2..0

3..0

9

H-Thr

His6np

2500

1

1

10

Boc-Thr

- Q

-trp

[phe 1? ,Leu "*)BBS spantide

>100000

12000 6000

10

.7

>5000 50 10000

fi

500

1

-

-

1

-

1

1

120

A= Inhibition of II'"]GRP binding |ID„„ (nM)) B= [H'JThymidine incorporation. Fold increase over basal value [ 50nM and 500nM] C= Phosphorylation of pll5 protein. Minimal active dose (nM) * Value obtained at 25 nM concentration R = H-Pyr-Gln-Arg-Leu-Gly leu= D-Leu; phe= D-Phe; trp= D-Trp; open box= deleted amino acid; - = not tested

DESIGN OF LUTEINIZING HORMONE RELEASING HORMONE ANTAGONISTS W I T H REDUCED POTENTIAL FOR SIDE EFFECTS

J.J. Nestor, Jr., R. Tahilramani. T.L. Ho, J.C. Goodpasture, B.H. Vickery Institutes of Bio-Organic Chemistry and Biological Syntex Research. Palo Alto. CA 94304 U.S.A.

Sciences,

P. Ferrandon Recherche Syntex France. S.A.. Leuville-sur-Orge. B.P. 40. 91310 Montlhery, France

The advent of offered

6 "D-Arg "

the

compounds

with

class

(1)

increased

potency and greatly prolonged

of

LHRH antagonists

gonadotropin

suppressive

duration of action.

We hypoth-

esized that this was due to "hydrophilic depoting" in the body by

electrostatic interaction

phosphate

head groups

1 cationic Arg withg g the cell membranes (2). The N .N -

in

of

the

dialkylhomoarginines were designed interaction actions

(2).

Several members

detirelix. 1) were caused

is the

(4) in tance

of

the

"D-Arg

inter-

class"

(e.g.

chosen for clinical studies (3), but edema

(3).

correlated with

charges and In

stabilize this

by mast cell degranulation (MCD) was detected in toxi-

cology studies MCD

to further

by combining electrostatic and hydrophobic 6

the

hydrophobicity LHRH

MCD

multiple

several

neuropeptide

in

antagonist

reducing the between

presence of series,

success

potency

by

was

positive series. achieved

increasing the dis-

the introduced

position 8 (e.g. 6 10 D-Tyr ,D-Ala JLHRH).

Arg residue and that native at 1 2 3 5 [N-Ac-D-Nal(2) .D-pCl-Phe ,D-Trp .Arg , However.

a

common structural

feature

of neuropeptides which cause M C D is the presence of an Arg-Pro seguence

(5).

suggesting

that

Arg

8

is

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . . Berlin • N e w York - P r i n t e d in G e r m a n y

also

a

critical

593

Table 1. Biological Data for LHRH Antagonists

Cpd 1

Histamine Release EC (ug/mL)

X detirelix

Arg

2a

0., 18

hArg(Et 2 )

b 3a

0.. 6 0..43 (4/6)

24

0..3

1., 5

hArg(CH2)3 hArg(Et 2 )

Antiovulatory ED (ug)

0.. 6

200

b

hArg(CH2)3

20

0., 68

c

hArg(Bu)

15.,7

1..2

hArg(Et 2 )

13

0..29

là b c

hArg(CH2)3

1.,33

0..6

hArg(Bu)

0. 64

0., 9

[N-Ac-D-Nal(2)1.D-pCl-Phe2.D-Pal(3)3,6.Arg5.X8.D-Ala10]LHRH 1 ? OC Q 1A 3 [ N - A c - D - N a 1 ( 2 ) , D - p C l - P h e .D-Pa1 ( 3 ) ' . X ,D-Ala ]LHRH

2

1 2 3 6 8 in 4 [N-Ac-D-Nal(2) .D-pCl-Phe .D-Pal(3) ,D-X ,X .D-Ala ]LHRH feature. analogs

We

studied

the

effect

of

substitution

with increased steric hindrance (hArg(Et 2 ).

hArg(CH2)3)

in

the guanidine function

by

Arg

hArg(Bu),

in position 8

on the

antiovulatory (AO) and M C D potency of LHRH analogs. In

series 2.

hArg(Et ) (2a) was superior to hArg(CH ) (2b). 8 and had 10-fold less M C D potency than the Arg parent ( E C 5 Q 2.9 ug/mL, ED 10 times that of GRF(1-29)-NH2. 8

12

Interchange of Asp 8 2

8

12

with Lys 1 2

15

resulted

cyclo(Lys -Asp )-[D-Ala ,Lys ,Asp , Ala ]-GRF(1-29-NH 2 i.e.

in an

analog,

cyclo 8 ' 1 2 [D-Ala 2 ,

L y s 8 , A s p 1 2 , A l a 1 5 ] - G R F ( 1 - 2 9 ) - N H 2 , with nearly the same potency as cyclo 8 ' 1 2 [D-Ala2,Asp8,Ala15]-GRF(1-29)-NH2.

Molecular modeling and circular dichroism

studies of the interchanged analog revealed that the helical content was unchanged.

Conclusions Molecular dynamics calculations of GRF(1-29)-NH 2 , [Ala 1 5 ]-GRF(1-29)-NH 2 and cyclo 8 ' 1 2 [Asp 8 , Ala 15 ]-GRF(1-29)-NH 2

in 75%

methanokwater (pH 6)

and water

603 (pH 3) are summarized by ribbon representations shown in Figure 1. All three peptides are nearly fully helical in 75% methanoliwater. In aqueous solution the two linear peptides, GRF(29)-NH 2 and [Ala 15 ]-GRF(1-29)-NH 2 , have short regions of irregular helical segments. The cyclic analog, cyclo 8 - 12 [Asp 8 ,Ala 15 ]-GRF(1-29)-NH2, has a long, regular central helical region. The high in vitro and in vivo biological activity of the cyclic analogs seems to indicate that the bioactive conformation has a central helical segment.

Acknowledgment The authors thank Ms. Sarah Maines, Dr. Robert Campbell, Ms. Bogda Wegrzynski, Mr. D. Greeley and Dr. Z. Berkovitch-Yellin for valuable technical assistance.

References 1.

Lance, V.A., W.A. Murphy, J. Suerias-Diaz and D.H. Coy. 1984. Biochem.Biophys. Res.Commun. 119. 265-272.

2.

Ling, N., A. Baird, W.B. Wehrenberg, N. Ueno, T. Munegumi and P. Brazeau. 1984. Biochem.Biophys.Res.Commun. 123. 854-861.

3.

Grossman, A., M.O. Savage, N. Lytras, M.A. Preec, J. Suerias-Diaz, D.H. Coy, L.H. Rees and G.M. Besser. 1984. Clin.Endocrinol. 21, 321-330.

4.

Clore, G.M., S.R. Martin and A.M. Gronenborn. 1986. J.Mol.Biol. 191. 553-561.

5.

Brunger, A.T., G.M. Clore, A.M. Gronenborn and M. Karplus. Engineering 1, 399-406.

6.

Felix, A.M., E.P. Heimer, T.F. Mowles, H. Eisenbeis, P. Leung, T. Lambros, M. Ahmad, C.-T. Wang and Brazeau. 1987. In: Peptides 1986 (D. Theodoropoulis, ed.). W. de Gruyter, Berlin, pp. 481-484.

7.

Heimer, E.P., M. Ahmad, T. Lambros, T.McGarty, C.-T. Wang, T.F. Mowles, S. Maines and A.M. Felix (in press). In: Synthetic Peptides: Approaches to Biological Problems (J. Tam and E.T. Kaiser, eds.). UCLA Symposia on Molecular and Cellular Biology S£.

8.

Felix, A.M., C.-T. Wang, E. Heimer, A. Fournier, D.R. Bolin, M. Ahmad, T. Lambros, T. Mowles and L. Miller. 1988. In: Proc. 10th Amer. Pept. Symp. (G. Marshall, ed.). Pierce Chemical Co., Rockford, IL., pp. 465-467.

1987.

Protein

SYNTHESIS OF A NOVEL GRF ANALOG

I. Mezo, B. Szoke, Zs. Vadasz, I. Teplan 1st Inst, of Biochemistry, Semmelweis Univ. Med. School, 1444 Budapest 8. P.O.Box 260 G.B. Makara Inst, of Experimental Medicine, Acad. Sci. Hung., Budapest M. Kovacs, J. Horvath, B. Flerko Inst, of Anatomy, Univ. Med. School, Pecs

Introduction Growth hormone releasing hormone

(GRF), may play an important

role in human medicine and in veterinary applications. Soonly after the isolation of GRF it was demonstrated that the hGRF/l-29/-NH 2 was almost as active as hGRF/l-44/-NH 2

(1). This

fact initiated an extensive investigation of GRF/I-29/-NH2 analogs. We have found that pure raw product with good yield was obtained, if Boc-Gaba-OH was attached to the benzhydrylamine resin as first amino acid then followed by the 1-29 sequence of hGRF.

Results 27 Nle

30 , Gaba

-hGRF/l-30/-NH 2

(I) was synthesized by the usual

solid phase peptide synthesis methodology

(2). For couplings

carbodiimid method was used with the exception of Asn and Gin, when their activated esters were applied. After HF cleavage crude _I was purified by gel-filtration, then by MPLC methodology on reverse phase column. Content and purity of fractions were estimated by TLC and analytical HPLC (2). Our GRF analog was tested using primary monolayer cultures of the anterior pituitary, and a superfusion system of anterior pituitary cells, as well an in vivo method for the estimation of GH release.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

605

These methods were described elsewhere

(2,3).

It is known that 27-Nle substitution in the hGRF/l-29/-NH 2 sequence doubles the relative potency of the resulting compound compared to the parent compound using in vitro bioassay (4). Our compound with the combination of 27-Nle and 30-Gaba substitution shows a markedly increased potency relative to hpGRF/l-44/-NH 2 in the culture assay test

(Fig. 1). In the superfusion

gave a lower GH peak, but its effect lasted longer,

therefore the integrated GH release was higher than that of the standard hGRF/l-44/-NH 2 . In an in vivo bioassay it was found that -NH 2

had enhanced potency compared to the standard hGRF/1-29/(Fig. 2).

These in vitro methods measure slightly different characteristics of the peptide. In the culture technique integrated 2 hr secretion is measured whereas the results from the superfused cell column show minute - to - minute changes in GH secretion rate. Both techniques suggest that compared to the native peptide the new analogue possesses slightly different

GH stimulatory

charecteristics. The fact that the new analogue is only slightly more potent than the native GH-RH and has a similar time course suggests that the in vivo stability of the new peptide is similar to that of the parent molecule. To summarize the advantage of 30-Gaba-NH 2 substitution in hGRF sequence we can conclude: 1. N l e 2 7 , Gaba 30 -hGRF/l-30/-NH 2 possesses increased GH-releasing potency in both in vitro and in vivo bioassays, 2. C-terminal Gaba substitution serves as a good spacer resulting in a good yield in the synthesis, 3. Gaba is a component of normal tissues therefore this substitution may result in a peptide, which is likely to be eliminated and metabolized via physiological processes.

Acknowledgement This research was supported by the Hungarian Academy of Sciences grant No. OTKA 1-6 00-2-8 6-1-49 2.

606

600

n = 18 « p(0.01

27

Nle,30Goba-hGRF(1 - 3 0 ) - N H

Fig. 1. Dose-dependence of GRF-44 and on GH release in anterior pituitary cell culture.

2

a> S 500\ o> c 99% p u r i t y , w i t h a s p e c i f i c

o f 12 C i / m m o l In the

RP-HPLC to

3

from human insulin. The

radiochemical

examined by isocratic RP-HPLC, using

H monitoring

(10) . T h e

3

simultaneous

H peak coincided with the

i n s u l i n U V p e a k , a n d a c c o u n t e d for >98% of t h e

radioactivity.

I i

*. Wk ie

is

¿8 :5 (lirutis

L 30

35

tfl

45

50

15

£0

Figure 1 HPLC

(9) of c r u d e

30

Minutes

35

Figure 2 product

HPLC

(9) of

H-insulin

663 Being uncomplicated, the synthesis is reproducible and easy to scale

up. For the first time, specifically mono-tritiated

insulin is accessible in quantities allowing a broader application in

biochemistry. The specific radioactivity obtained,

10-15 Ci/mmol depending on be sufficient

for the

the scale

majority of

of preparation, should investigations where the

use of an authentic insulin tracer is required.

Acknowledgements We thank H.O. Voigt for carrying out the iodo-insulin, L.

purification of the

Snel and L. Gotfred for doing the HPLC ana-

lyses, and A.R. Sorensen for performing the bioassay.

References 1. Halban, P.A. and R.E. Offord. 1975. Biochem. J. 151. 219. 2. Davies, J.G. and R.E. Offord. 1985. Biochem. J. 231. 389. 3. Kaufmann, K.D., J. Oehlke, A. Hansicke, M. Beyermann, M. Bienert. 1985. 9th American Peptide Symposium, abstract P-55. 4. Grant, K.I. and Seyler 368, 239.

C. von Holt. 1987. Biol. Chem. Hoppe-

5. Dingman, J.F., W.W. Meyers, Y. Agishi, A.P. Wysocki. 1963. Federation Proceedings 22., abstract 1345. 6. Fromageot, P., L.T. Hung, J.L. Morgat. 1973. Ger. Offen. 2,247,760. Chem. Abstr. 79, 489, abstract 19128a. 7. Halstrom, J., K.H. Jargensen, L.A. Savoy. 1987. 10th American Peptide Symposium, abstract P-38. 8. Savoy, L.A., P. Vuagnat, K. Rose, J. Halstr0m, K.H. JOrgensen, K. Kovacs: Protein Engineering (in press). 9. Snel, L. , U. Damgaard, I. Mollerup. 1987. Chromatographia 24, 329. 10. Reeve, D.R. and A. Crozier. 1977. J. Chromatogr. 137. 271.

DIMERIZATION OF CYCLIC HEXAPEPTIDES: STRONG INCREASE OF BIOLOGICAL ACTIVITY

H. Kessler, M. Schudok, and A. Haupt Inst. f. Org. Chemie, Johann Wolfgang Goethe-Universtät, D-6000 Frankfurt 50, FRG

Introduction Cyclic retro analogues of somatostatin-14 show remarkable "cytoprotective" effects in vitro and in vivo (1-3). Rat liver cells are protected from phalloidin poisoning; in animal models beneficial effects, too, on induced lesions in several organs and tissues are observed. The mode of action of a cytoprotective agent is not known in most cases; however, cytoprotection in rat liver cells is a transport inhibition: phalloidin (4) and other substances make improperly use of the cholate carrier system which is usually responsible for cholate metabolism (5); competitive or sometimes noncompetitive (6) inhibition of the active uptake of cholate or phalloidin is caused by these peptides among which the "008(Z)", cyclo(-D-Pro-Phe-Thr-Lys(Z)-Trp-Phe-), and the "VDA-008(Z)", cyclo(-D-Ala-Phe-Val-Lys(Z)-Trp-Phe-) (6), are the most active. For the design of even better inhibitors without changing conformational prerequisites that have to be fulfilled for efficient binding to carrier protein binding sites (3), the 008-peptide was further modified in the lysine and phenylalanine side chains (7).

The Concept of Dimerization In the course of these works a dimeric compound was synthesized with succinic acid as a linkage between the lysins of two 008-molecules (8). This dimer was surprisingly ten times more active than the monomeric 008. Phe - Thr - Lys - NHCO(CH2)2CONH

r

i

D-Pro-Phe-Trp

- Lys - Thr - Phe

i

t

Trp-Phe-D-Pro

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in G e r m a n y

665

So the synthesis of a great number of dimeric derivatives of the 008 and other related peptides was carried out; three different types of bridges were used: (A)

aliphatic dicarboxylic acids

(B)

unsaturated dicarboxylic acids

(C)

ethylene glycol derived dicarboxylic acids.

Remarkable effects on the biological activity were found.

Synthesis The linear precursors were synthesized by the classical Merrifield solid phase peptide synthesis. Hydrazinolysis and final BOC-cleavage was followed by azide cyclization (yield of cyclic peptide 70 - 90 %). After purification (sephadex L H 20) and Z cleavage

usual

dimerization gelpermeation

methods

for peptide

coupling

turned

out to be

suitable

for

as well. Purification of the dimers could easily be achieved by chromatography

and

rp-HPLC.

FAB-mass

spectra

and

NMR-

spectroscopy furnished unambiguous structural proof. N o conformational change within the peptide rings were detectable.

Biological Activity The uptake inhibion of radioactive labelled cholate into isolated rat liver cells is indicated by IC 50-values (smaller values - higher activity). Determination: cf. lit. (1). Standard deviations lie within + /- 5 % .

Table 1: IC 50-Values of some monomeric and dimeric cyclic Peptides (nmol/ml): somatostatin

220

008(Z)

1.5

VDA-008(Z) 2x008 and linkage:

0.1 acetylene dicarboxylic acid

0.001

adipic acid:

0.011

malonic acid:

0.018

3,6,9-trioxaundecanedioic acid

0.025

hexadecanedioic acid

0.05

666 Results and Discussion

The concept of covalent dimerization, which was for the first time applied for cyclic peptides, showed remarkable effects on the biological activity in a cytoprotection assay; the best dimeric compound was 3000 ( ! ) times more active than the monomeric parent peptide. So the cholate carrier system in liver cell membranes is nearly completely blocked. A double binding to two binding sites of the carrier system could be assumed because of the high activity and a certain dependence of the activity from the chain length since dimers with long chains lack activity. Energetic and kinetic benefits would underline this together with retarded proteolytic

decomposition.

Further detailed studies are in progress.

Acknowledgement For performing the great number of biological tests the collaboration with Prof. K. Ziegler, Inst, of Pharmacology and Toxicology/University of Giessen, is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. M. S. thanks the Fonds der Chemischen Industrie for a scholarship.

References 1.

Ziegler, K., M. Frimmer, H. Kessler, I. Damm, V. Eiermann, S. Koll, J. Zarbock. 1985. Biochim. Biophys. Acta 845, 86-93

2.

cf. Klin. Wochenschr. 1986. pp. 59-63, 74-78, 79-86.

3.

Kessler, H., M. Klein, A . Müller, K. Wagner, J.-W. Bats, K. Ziegler, M. Frimmer. 1986. Angew. Chem. 98, 1030-32.

4

Wieland, T. 1986. Peptides of Poisonous Amanita Mushrooms. Springer, Heidelberg, pp. 129-154.

5.

Frimmer, M , K. Ziegler. 1988. Biochim. Biophys. Acta 947, 75-99.

6.

unpublished results; K. Ziegler, personal communication.

7.

Kessler, H., A . Haupt, M. Schudok. 1988. Int. J. Peptide Protein Res. (in press).

8.

A . Haupt, doctoral thesis. 1987. Frankfurt am Main, F R G .

SUBSTITUTION OF PHE-5 AND ILE-9, AMINO ACIDS INVOLVED THE ACTIVE SITE OF PHOSPHOLIPASE A2 (PLA), AND MODIFICATION OF ENZYMATICALLY GENERATED

IN

CHEMICAL

(LYS-6)-PLA.

Jan van Binsbergen. Arend J. Slotboom and Gerard H. de Haas. Lab. of Biochemistry, University of Utrecht, Padualaan 8, Utrecht, The Netherlands. Key words: enzymatic peptide synthesis, phospholipase A2, protein engineering. Pancreatic phospholipase A2 (PLA) is a 14 kD enzyme which (stereo)specificially hydrolyzes the 2-acyl ester linkage in 3-sn - phosphoglycerides (1). Monodisperse substrate is bound and slowly hydrolyzed in the active site. Aggregated substrate, like e.g. loosely packed micelles, is hydrolyzed 3 - 4 orders of magnitude faster. Most likely this enhanced activity is due to optimization of the active site upon binding of the enzyme to aggregated substrate. Phosphoglycerides present in bilayer structures or in natural membranes are a very poor form of substrate for pancreatic PLA in contrast to snake venom PLA, because the former PLA is not able to penetrate into these more densely packed structures. From the 1.7 A X-ray structure of bovine PLA it is known that the active site (His4®, A s p 4 9 - C a 2 + , A s p " ) is located in a cleft of the protein. The wall of the active site cleft is composed of a number of hydrophobic residues, amongst them the absolutely conserved Phe^ and lie 9 . Around the edge of the active site cleft a number of hydrophobic and positively charged residues, located in a plane, have been shown to be involved in the binding of the enzyme to aggregated substrate (lipid binding domain, LBD). This LBD consists of a number of hydrophobic and positively charged residues located at the N-terminal a-helical region (Ala 1 , Leu 2 , Trp 3 , Arg®, Lys1®) together with Leu 1 9 , Met2®, Leu 3 1 , Tyr®9 and Lys 11 ® (porcine PLA). In our structure-function studies on PLA we substituted Phe^ and Arg® (porcine PLA), and lie 9 (bovine PLA) by various amino acids using semisynthesis (cf.Table 1). Selective tryptic cleavage of the N-terminal hexapeptide of the e-amidinated porcine PLA (=AMPA) (Ala 1 .Leu 2 .Trp 3 .GIn 4 .Phe 5 .Arg 6 JJSer 7 —-Cys 1 2 4 ) and CNBr cleavage at the unique Met® residue followed by one Edman degradation of bovine AMPA (Ala1 .Leu 2 .Trp 3 .GIn 4 .Phe 5 .Asn 6 .GIy 7 .Met 8 % 9 .Lys 1 gave the

required

N-terminally shortened

protein fragments.

Peptides 1988 © 1 9 8 9 Walter de G r u y t e r & Co., Berlin -New Y o r k - Printed in G e r m a n y

-Cys 1 2 3 ), The

desired

668 N-terminal hexapeptide analogues were prepared by SPPS. Coupling of the A l a 1 - A r g 6 ( L y s 6 ) peptides (5-fold molar excess) to porcine des(Ala 1 -Arg®)AMPA was done by trypsin (10%) at

pH 6 for 24 hr, furnishing the desired AMPA

analogues in 75% yield both in the absence or presence of 25% DMF . In the absence

of organic cosolvent,

alkylphosphocholine)

need to

no product trapping be added.

agent

Noncovalent

(e.g.

micellar

complexes

of

the

hexapeptide and the proteinfragment do not result in the restoration of the original PLA activity in contrast to the presence of full enzymatic activity of the noncovalent RNase S' complex. It is very interesting to note that the trypsin catalyzed resynthesis of PLA in the absence of organic cosolvent proceeds to the extent of 7 5 % within 24 hours, whereas the conformationally favoured resynthesis of RNase-A by subtilisin yields only 4.3% in the absence of organic cosolvent and 50% in the presence of 90% glycerol (2). It has to be remarked that the porcine D - P h e 5 PLA mutant could not be prepared enzymatically,

probably because of the presence of the unnatural D-Phe at the

penultimate position. Therefore this mutant was prepared by chemical coupling using the HOSu method. For the

preparation

tripeptide

PLA

of the bovine AMPA

Gly 7 .Met®.X 9

was

V m a x a (%) micellar L-di-octanoyl -

coupled

mutants the desired

chemically

c a t / K M b (%) monomeric di-thiohexanoyl-

phosphocholine

k

(HOSu ester)

phosphocholine

K

d

protected to

a

(mM)

micellar oleoyl phosphocholine

100

100

0

0

5

4

65

6

42

100

0.66

273

133

0.02

Leu 9

20

24

0.38

Nva9

4

6

0.11

AMPA D-Phe5 Leu Lys

Pal-Lys*

5

Table 1. Kinetic

and direct

binding

bovine

0.16 5.0 0.07

properties of different e-amidinated

PLA

analogues. AMPA is the e- amidinated native porcine PLA. Nva is norvaline, Pal is e-NH 2 -palmitoylated Lys 6 -AMPA. a) determined at pH 6 in the presence of C a 2 + and b) determined spectrophotometrically at pH 8.5 in the presence of C a 2 + .

669

des(Ala 1 -lle®)AMPA, prior to the trypsin catalyzed coupling at pH 6 of the N-terminal hexapeptide in which Asn® was replaced by Arg®. Porcine Lys® AMPA was

synthesized

by

coupling

BocAla 1 . L e u 2 . T r p 3 . G l n 4 . P h e ^ . L y s ®

to

des(Ala^-Arg®) AMPA with trypsin. The unique e-NH2 group of Lys® was reacted with palmitoyl-N-hydroxysuccinimide ester. As can be seen from Table 1 porcine D-Phe 5 AMPA has no enzymatic activity and a considerably lower affinity for binding to the

micellar

substrate

oleoylphosphocholine compared to "native" AMPA. Most likely stereochemical form of Phe^ disturbs the

analogue

the opposite

N-terminal a-helix of PLA and as a

consequence the active site as well as the LBD. In contrast, L-Leu® AMPA and also L-Nle^ and L-Met® AMPA's (data not shown) do possess a functionally active site (cf. k c a t /K|v| value, Tablel) and a good affinity for binding of micelles. The low activity of these three mutants towards micellar substrate is most likely due to conformational changes in the active site upon micelbinding. Substitution of the absolutely conserved lie® by Leu and Nva in bovine AMPA has only a limited effect on binding to micelles because it is located in the interior of the protein. Leu® bovine AMPA still has some catalytic activity left, in contrast to the Nva 9 mutant (Table 1). Apparently a branched amino acid residue at this position is absolutely required for catalytic activity. So far we do not have a reasonable explanation why the Arg®=>Lys® substitution in porcine AMPA decreases the V m a x value on di-octanoyl-lecithin micelles to 42%. Upon specific introduction of a long palmitoyl chain to Lys® the V m a x value increases almost 7-fold. Most interestingly the presence of the membrane anchor enables Pal-Lys® AMPA to efficiently attack phospholipids in densely packed bilayer structures and even natural membranes in contrast to native AMPA, rendering this enzyme analogue snake venom PLA-like properties.

References. 1. Waite, M. 1987. Handbook of Lipid Research 5, The Phospholipases. Plenum Press. New York, p.155. 2. Kullmann, W. 1987. Enzymatic Peptide Synthesis. CRC Press. Florida, p.95.

Boca

Raton,

Fundamental roles for ethanolamine and choline lipids in cell excitation and transmembrane signalling: anaesthesia

A.N. Fonteh, K. McBride and W.A. Gibbons.

Department of Pharmaceutical Chemistry,

School of Pharmacy, University of London, London, WC1. Abstract Lipid methylases are identified as primary receptors for general anaesthetics and as both channel-associated and receptor complex- associated proteins. A lipid-enzyme-channel model is presented that accounts for primary processes in cell excitation (action potentials, voltage and ligand gating of ion-channels, synaptic transmission), transmembrane signalling (cyclic AMP generation) and the effects of both local and general anaesthetics on these and other biological processes. General anaesthetics have been used clinically since 1846 but, despite an immense amount of biophysical, biochemical, physiological and clinical research, their mechanism of action has not been established (1). This lack of a unitary theory of anaesthesia has been attributed to their diverse structures and electronic configurations and their wide spectrum of biological actions includes changes in action potentials, ion-channel conductance, synaptic transmission, cyclic nucleotide levels and circulating neurotransmitter levels. Specific enzymes are also inhibited: glutamate dehydrogenase, adenylate kinase, ATPases, microtubular enzymes and firefly luciferase. Halothane has been shown by NMR to bind to brain membrane proteins and to the nucleoside binding site of adenylate kinase crystals. Proposed theories include: (a) lipid theory, (b) protein theory, and (c) synaptic theory (2). No coordination of these three theories exists. That the potency of general anaesthetics is proportional to their lipid solubility led us to study the effect of anaesthetics on plasma-membrane bound lipid metabolising enzymes and on protein components of transmembrane signalling complexes. Anaesthetics of different structures were found to inhibit the P E to P C converting enzyme from brain and liver in a dose-dependent manner (3).

This reaction is not the major pathway for choline lipid

formation but is involved in agonist and antagonist stimulated events including cell growth and differentiation, stimulus-secretion coupling, chemotaxis, motility, Ca release and cAMP formation (4). Coincidentally it is just these processes, cell excitation and transmembrane signalling that are involved in general anaesthetic action. The lipid-enzyme-channel (LEC) model (3) proposed by us as a unifying mechanism of local and general anaesthesia could therefore be generalised to explain the fundamental biological processes of cell excitation and transmembrane signalling.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

671 The LEC model readily accounts for the electrophysiological effects of anaesthetics, both local and general, as follows: (a) methylated lipids have active dual roles of endogenous gating molecules and enzyme substrates as well as the passive role proposed by the Meyer-Overton lipid theory. The increase in anaesthetic potency with membrane solubility is accounted for if the membrane concentration affects the activity of the membrane bound methylases, (b) inhibition of the methylases decreases local methylated lipid concentration and hence should open channels.

The effect of lipid degrading enzymes e.g.

phospholipases should reverse

the channel gating effects of the methylases? (c) physical processes involving lipids can also explain voltage-gating of channels, and the many different rate constants and mechanisms of channel gating. These are conformation changes of the methylated head groups which occur spontaneously, or induced by voltage changes or by competition between metal cations and the alkylammonium head groups. The rates of internal motion of phospholipids detected by NMR and lateral mobility by ESR are of the correct order to explain gating dynamics. Anaesthetics therefore can affect channel gating indirectly by regulating the channel-associated enzymes (most general anaesthetics) or directly (many local anaesthetics) by binding to the lipid site on the channel protein, (d) the effect of anaesthetics on Na, Ca, K and CI channels is readily accounted for if there is a specific methylated lipid and associated specific enzymes for each channel. Thus plasmalogens, glycerophospho-lipids, sphingomyelins etc and isoenzymes of the respective LMTases can be associated with specific channels, (e) although nitrous oxide seems to be an exception to this theory recent evidence that it inhibits enzymes in the SAM recycling pathway supports its indirect effects on lipid methylases via the cofactors, (f) structureactivity relationships can now be constructed for anaesthetics based upon their resemblance to SAH, a potent methylase inhibitor a n d / o r to the lipids. Support for this comes from the binding of halothane to nucleotide requiring enzymes which have the nucleoside in- common with SAM and SAH and the many local anaesthetics and channel-acting drugs that contain alkyl ammonium moieties that resemble the head groups of lipids e.g. lidocaine, verapamil, tetraethyl ammonium. In our laboratory iontophoretic application of SAH inhibited synaptic currents at GABA and GLY synapses. SAH is a potent inhibitor of lipid methylases and causes sedation in animals. We have proposed that SAH is the endogenous benzodiazepine. To date insufficient experimental evidence exists to rationalise the effects of general anaesthetics on second messenger generation. Axelrod and coworkers (4) have shown that cAMP and lipid methylation occur simultaneously in mast cells and proposed they are linked processes. We propose here that the products of the methylases act as messengers controlling the conformational changes and hence the generation of second messengers; alternatively SAH could also compete with GTP, GDP, ATP or cAMP for binding to G-proteins, adenylate cyclase or phosphodiesterase. We have established that general anaesthetics also inhibit both

672 GTPases and ATPases albeit at higher concentrations than LMTases. Additionally, in our experiments tri, di, mono and cyclic nucleotides inhibit lipids methylases. The full importance of these findings remains to be explored but one explanation is that the products of methylases control cAMP levels and that lipid methylases are related to adenosine receptors. References 1. "Pharmacology" by H.P.R. Rang and M.M. Dale, Ch. 20, 471-473, Churchill-Livingstone Press (1987). 2.

K.W. Miller, Int. Revs. Neurology, 27, 1-61 (1985).

3.

W.A. Gibbons, A.N. Fonteh, K. McBride, pp. 265-268, in "Chemistry and Biotechnology of Biologically Active Natural Products": Proceedings of the 4th Int. Conference, Budapest 1987, Ed. C. Czantay, Hungarian Publishing Co., and Elsevier (1988).

4.

F. Hirata, J.F. Tallman, R.C. Henneberry, P. Mallorga, W.J. Strittmatter, J . Axelrod, pp. 91-97 "Regulation of the Adrenergic Receptors by Phospholipid Methylation" in Neurotransmitters and Peptide Hormones. Ed. by G. Pepon, M.J. Kahar, S. Jenna, Raven Press (1980).

S Y N T H E S I S OF H U M A N LOGICAL EFFECTS

A.N. Eberle,

'ANTI-SENSE'

R. D r o z d z ,

ACTH

W. S i e g r i s t ,

( ' H T C A ' ) AND

ITS

J.B.

J.

Baumann,

BIO-

Girard

L a b o r a t o r y of E n d o c r i n o l o g y , D e p a r t m e n t of R e s e a r c h ( Z L F ) University Hospital and University Children's Hospital H e b e l s t r a s s e 20, C H - 4 0 3 1 B a s e l , S w i t z e r l a n d

Introduction Synthetic

peptides

plementary sense'

to the m R N A

peptides)

because

they

thus could

fic

receptor

be u s e f u l

induce

repeated

human four

(HTCA^)

was

antibodies

positions

This paper

experiments

with

peptide

and

a n d MSH

bioassays

whose

respect

is a s h o r t

synthetic

the A C T H using

may

be

^^ a n d ,

speci-

their

(1). We

the a n t i - s e n s e peptide

to

have

sequence

sequence

four

com-

anti-sense

as a n t i g e n ,

receptor

in

of

originates

protein-protein

bovine

and protein

tested

receptors

'informational

on the b i o l o g i c a l

when

or b i n d i n g

in

and p r o t e i n s

DNA

interest

preparation

that

com-

('anti-

the

The h y p o t h e s i s

to the b o v i n e

report

its a n t i b o d i e s

hormones

respective

to b i n d ACTH^ for

an RNA

the

concluded

acids

said

(HTCA^)

for

by

considerable

of s p e c i f i c i t y

example,

specific

these

ACTH^^^

received

antigens

(4) w h o For

of p e p t i d e

(1, 2, 3).

of a m i n o

coding'.

is e n c o d e d

to r e s e m b l e

analysis

by B i r o

complementarity ACTH^^^

portion

thought

a comparative

plementary

structure

recently

antibodies

interactions

now

have

were

and from

whose

differ

(Fig.

effects

1). of

different

the ACTH

assays.

Results The

synthesis

of H T C A ^

was carried

methy1acry1 amide-kiese1guhr Fmuc-strategy

(5).

The

support

peptide

was

out

on a c o m p o s i t e

using

the

purified

by

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin • N e w York - P r i n t e d in G e r m a n y

polydi-

conventional classical

of in

674 1 ACTH 24 SerTyrSerMetGluHlsPheArgTrpGlyLysProValGlyLysLysArgArgProValLysValTyrPro 5'...TCCTACTCCATGGAGCACTTCCGCTGGGGCAAGCCGGTGGGCAAGAAGCGGCGCCCAGTGAAGGTGTACCCT.. .3' coding ('sense') reverse ('anti-sense') 3' ... AGGATGAGGTACCTCGTGAAGGCGACCCCGTTCGGCCACCCGTTCTTCGCCGCGGGTCACTTCCACATGGGA. . .5' 5'...AGGGTACACCTTCACTGGGCGCCGCTTCTTGCCCACCGGCTTGCCCCAGCGGAAGTGCTCCATGGAGTAGGA.. .3' ArgValHisLeuHisTrpAlaProLeuLeuAlaHisArgLeuAlaProAlaGluValLeuHisGlyValGly 1 Human HTCA 24 GlyValHisLeuHisArgAlaProLeuLeuAlaHisArgLeuAlaProAlaGluValPheHisGlyValArg 1 Bovine HTCA 24 F i g u r e 1. A m i n o a c i d s e q u e n c e o f h u m a n ACTH-j > H T C A h and HTCAj-! a s w e l l a s o f t h e c o r r e s p o n d i n g h u m a n c o d i n g a n d r e v e r s e DNA s t r a n d s . S e q u e n c e d i f f e r e n c e s b e t w e e n H T C A h and H T C A b are underlined .

chromatography coupled raised

HTCA^

to by

in

not

bind

complex.

receptor were

pletely

devoid

antibodies

assay

sence

of

HTCA

not

addition its

the

of

effect

Anolis

antibodies

to

but

seen

in

the

via

MSH

interfere

with

with

alpha-MSH

in

cells.

which

10^-fold

less the

alpha-MSH

absence

cell

potent

or

ACTH of

photoaffinity

calcium

that

ACTH

melanin than

HTCA

or did

pre-

anti-

Anolis

mela-

labelling

cross1inking,

of

or

melano-

simultaneous

stimulation

after

com-

receptors

and

the

Similar

were

B16

by

HTCA-

not

melanophore

by

when

covalent

weak

indicating

receptors.

as

ACTH/MSH

MSH-receptor

noradrenaline,

derivative

or

H T C A ^h e x h i b i t e d

not

was

were

^

melanoma

long-lasting

that

or

blocked

HTCA^

antibodies

antibodies

to

was

Unlike

suppressed

ACTH^

towards

form.

rabbits.

plates

human

10^-fold

produced

(6).

into

assay

HTCA

However,

activity

similar

receptors was

in

pure

HTCA^ did h

cell

using

with

activity 2).

This

HTCAh

nophores,

it

Furthermore,

(approximately

bodies.

MSH

of

activity

alpha-MSH).

microtiter

assay

in

high-affinity

complex

a monoiodinated

obtained

(Fig.

obtained

and

the

adrenocortical

results

tropic

of

HTCA-coated

the

MSH/ACTH

and

thyroglobulin

in

Sepharose ACTH

HPLC

injection

did

assayed

and

however,

by

the

not

exert

of

675 'sense' ACTH

-

•

AbACTH

\ Figure 2. Interactions and lack of interactions between ACTH and HTCAu, their antibodies (Ab) and ACTH/MSH receptors (R).

*

'

HTCA

^

R

ACTH

1

*

•

AbHTCA

'anti-sense'

Conclusion We believe

that, at least

of anti-sense supported receptor

peptides

is of very

by a sequence with

insulin

for ACTH/MSH

receptors,

limited

comparison

value.

by Biro

the

approach

This notion

(7) of the

and various pituitary

peptide

hormones

where short complementary

gene sequences were

found

frequency

and non-interacting

proteins.

for interacting

theless, anti-sense biological

peptides may

sometimes

produce

is

insulin

at

equal Never-

unexpected

effects.

Re ferences 1. Bost, K.L., E.M. Smith, J . E . Blalock. Sci. USA 82, 1372.

1985 . Proc.Na11.Ac ad .

2. Gores, T.J., P.E. Gottschall, D.H. Coy, A. Arimura. Peptides 7, 1137. 3. Shai, Y., M. Flashner, 669.

I.M. Chaiken.

k. Biro, 3.

Hypotheses

1981. Medical

5. Dryland, A., R.C. Sheppard. 125. 6. Eberle, A.N.

1987. Biochemistry

7, 969, 981

and

26,

995.

1986. J . C h e m . S o c . P e r k i n . T r a n s . _1_,

1984. J.Recept.Res . k,

7. Segersteen, U., H. Nordgren, p h y s . R e s . C o m m u n . 139, 94.

1986.

315.

J.C. Biro.

1986.

Biochem.Bio-

USE OF SYNTHETIC PEPTIDES FOR MAPPING OF THE IMMUNOREACTIVITY OF A PUTATIVELY IMMUNOSUPPRESSIVE REGION OF HIV-GP 41: A POTENTIAL PROGNOSTIC TEST

R. Pipkorn, E. Bernath Zentrum für Molekulare Biologie, 6900 Heidelberg, FRG J. Blomberg, P-J. Klasse Section of Virologie, Dep. of Medical Microbiologie, Lund University, Sweden. Keywords: HIV, Aids, Antibodies, p!5E. Introduction

The transmembrane protein of human immunodeficiency virus (HIV), gp41, contains an evolutionarily conserved region which shares six amino acids with a stretch of 17 amino acids which probably is responsible for the immunosuppressive activity of at least some retroviruses. This region of gp4l may then be one of the factors behind HIV-induced immunosuppression, and antibodies binding to it may counteract its effect. We have found antibodies which bind to this sequence in HIV infected persons, have correlated their presence with the state of health, and have investigated the structural prerequisites for their binding. Peptides were derived from the amino acid sequences of the HIV isolate HTLV III B, clone BH 10 (1). Table 1 shows the sequences of the peptides. The synthesis followed the stepwise solide-phase strategy using the base-labile N-a-9-fluorenylmethoxycarbonyl (FMOC-group) for N-terminal protection. The side chains were blocked in the following ways: tert.-butyl for Asp,Glu,Ser and Tyr; 4-methoxy-2,3,6-trimethylbenzenesulfonyl for Arg; tert.butyloxycarbonyl for Lys and trityl for His and Cys. A p-alkoxybenzylalcohol resin (2) was used throughout the synthesis. The couplings were performed in DMF as HOBT esters. The acylation were checked by quantitative ninhydrin test and the coupling was repeated when required. We cleaved the peptides from the resin by treatment with trifluoroacetic acid/thioanisol for 4 h.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin N e w York-Printed in G e r m a n y

677

Then we purified the peptides to homogeneity by preparative HPLC, using a reverse-phase column (Nucleosil c-18, 250 x 10 M) with different gradients (depending on peptide) of 10-90% solution B (acetonitrile:water/60:40) mixed with solution A (0.1% trifluoroacetic acid in water) at a flow rate of 6 ml/min. Detection at 215 nm. We ascertained the amino acid proportions by the procedure of Moore and Stein. The purity checked by HPLC was always at least 98%.

Fig. 1. RP-HPLC of HIVenv 583-599.

Results

We tested sera from 17 HIV seronegative and 68 HIV seropositive subjects in an enzyme immunoassay. The enzyme immunoassay was carried out according to a previously described method (3). No HIV antibody negative serum reacted with any of the peptides. Antibodies to the peptide HIV-env 583-599 (hereafter also referred to as pHIVlS) were detected in 27 of the 35 sera from healthy HIV positive persons, but only in one of the 3 3 sera from patients with HIV related disease (the single ill pHIVIS positve subject had Kaposi's sarcoma and thereby AIDS). A few of the sera reacted with pHIVIS reacted with the inverted peptide. This shows that the pHIVIS-reactive antibodies do not just recognize amino acid residues in a certain order. Another 17-mer (HIV-env 579-595 ,displaced four amino acids N-terminally from pHIVIS) reacted with fewer of the sera from healthy seropositives than pHIVIS but with no serum from ill seropositive patients. HIV-env 587-603 displaced four amino acids C-terminally from pHIVIS reacted with the sera from nearly all subjects, regardless of clinical status. In conclusion, antibodies which bind to the putatively immunosuppressive sequence of gp41 were demonstrated. Such antibodies were more common in healthy than in diseased HIV infected persons. A shift towards the N-terminus of four amino acids diminished antibody binding, and replacement of them with an irrelevant sequence abrogated it. Thus, both primary and secondary structure of the peptides probably are important for binding of these antibodies.

678 Further studies ought to elucidate the role of pHIVIS in HIV pathogenesis and whether antibodies to pHIVIS can abrogate its immunosuppressive effects; if they can, they may even be therapeutically useful. Prospective studies are needed to elucidate the usefulness of pHIVIS as a prognostical reagent.

HIV—any aaino position

Saquanca reactivity

Traquancy of $ +H +D 1/16 n.d."

HrV-«nv 583-595LQARILAVERYLXSSGG SSGG HXV-«nv 381-599 KQLQAJULAVERYLKDQQL 13/16 HIV-am 579-395 GIK QLQJUULAVERYLX 12/35 HlV-sai 583-599 L Q A S I I A V E R X L X D Q Q L 2 7/35 (pHIVIS) Hrv-ani inv 599-58] LQQOKLYREVALIKAQL 1 1 /35 HXV-anv 587-603 ILAVERYLKDQQLLCIW 31/35 HIV-ani 586-606 XILAVERYLKDQQLLGIHGCS 31/35

n.d.' 0/33 •1/33 3/33 21/35 29/33

Tha nuabaring systaa of Ratnar at al (1) vaa uaad. $ "*"H • haalthy HIV aaropoaitiva parsona. 5 - disaaaad HIV aaropoaitva patiants. • Tha singla ill pHIVIS poaitiva subjact had Kapoai's sarcoma and tharaby AIDS. * n.d. - not dona References

1. Ratner, L., W. Haseltine, R. Patarca, K.J. Livak, B. Starcich, S.F. Josephs, E.R. Doran, J.A. Rafalski, E.A. Whitehorn, K. Baumeister, L. Ivanoff, S.R. Petteway Jr., M.L. Pearson, J.A. Lautenberger, T.S. Papas, J. Ghrayeb, N.T. Chang, R.C. Gallo and F. Wong-Staal. 1985. Nature 313, 277-284. 2. Wang, S.S. 1973. J. Am. Chem. Soc. 95, 1328-1333. 3. Klasse, P.-J., R. Pipkorn and J. Blomberg. 1988. Proc. Natl. Acad. Sei. in press, Vol 85.

USE OF CARRIER-BOUND OLIGOPEPTIDES AS ANTIGENS FOR HIV-1 AND HIV2 SPECIFIC DIAGNOSTICS Susanne Modrow, Hans Wolf Max von Pettenkofer Institute, Pettenkoferstr. 9a, D-8000 München 2, F R G Brigitte Höflacher Landstr. 1, D-8042 Neuherberg, F R G GSF, Ingoldstädter

Synthetic peptides are usefull as specific antigens in diagnostic test systems for the detection of antibodies in patient sera and for the elicitation of monospecific and monoclonal

antibodies

in animal systems.

In the conventional

method,

side-chain

protecting groups are removed after synthesis, simultaneously the peptide is cleaved from the carrier. The product has to be purified and is recoupled to plastic or protein supports (BSA, KLH) according to application. By combination of Boc and Fmoc chemistry sidechain protecting groups can be removed without simultaneous cleavage of the synthesized peptide from polystyrene resin. This method is applicable to most automatic peptide synthesizers and allows a very fast reaction to strain variation of new pathogenic isolates, since synthesis and characterization of the peptide antigen can be achieved very quickly. We applied this method to the development of a diagnostic test system for distinction between HIV-1 and HIV-2 specific antibodies. Using chloromethylated polystyrene with the first amino acid bound in ester linkage to the resin, peptides were synthesized using orthogonal solid-phase peptide synthesis with Fmoc-protected amino acids. In order to stabilize the linkage to the carrier for treatment with secondary amine to remove Fmoc-protecting groups and with trifluoroacetic acid to cleave side-chain protection groups, we selected alanine as the first residue, which has no functional side-chain and should give an approximately equal distribution of electron density in the linkage. The synthesized epitopes were selected f r o m the amino acid sequences of HIV-1 (1) and HIV-2 (2) according to their potential antigenicity using a computer program for amino acid analysis, which combines parameters for secondary structure with values for local hydrophilicity, flexibility and surface probability (3). HIVHIVHIVHIVHIVHIVHIVHIV-

1 2 1 2 1 2 1 2

p24 : p26 : pl7 : pl6 : gp41A: g p 3 6A: gp41B: gp36B:

226-237 228-242 109-123 111-126 562-575 556-569 588-604 582-598

GQMREPRGSDIA QMREPRGSDIAGTTSA NKSKKKAQQAAADTGA ETGTAEKMPSTSRPTA RAIEAQQHLLQLTVA DWKRQQELLRLTVA VERYLKDQQLLGIWGCSA IEKYLQDQARLNSWGCA

Table 1: Selected epitopes

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin N e w Y o r k - P r i n t e d in G e r m a n y

680

Serum

HV-1

HIV-1

HIV-2

HV-2

Antigen

HIV-1

HIV-2

HIV-2

HIV-1

p24/p26 ^

l

\

gp41/ gp36A

\ gp41/ gp36B

\

p17/p16

k Slis Figure 1: Examples of reaction

of

peptides

with H I V - 1 and HIV2

sera;

hatched:

negative sera

In the transmembrane proteins gp41/gp36 regions were selected wich are located in the protein part probably positioned outside the membrane (4). From this external loop region two corresponding antigenic sites were selected, which contain the lowest possible content of equal amino acids (ca 50 %). In the core protein p24/p26 the epitope is highly conserved in H I V - 1 and H I V - 2 . The most variable epitope between H I V - 1 and H I V - 2 was selected from an antigenic region near the carboxyterminus of p l 7 / p l 6 (Tab.l). The synthesized peptides were analyzed by gasphase amino acid sequencing. ELISA-tests were performed in Milititer plates sealed at the bottom with a membrane of low proteinbinding capacity, using 500 ng of carrier-bound peptide, which represents about 200 ng of antigen. The reactivity of the antigen was shown in by ferritin-labelled antibodies at

1 SO

12SO

P17/MIV-1

pUj/lllv.;

HIV-1 ami*,

HtV-1 anlisoi

1SOO

pi7/m\ i i i i v . : .nil..,

Figure 1: Examples of reaction

of

peptides

with

HIV-1

HIV-2

sera;

and

pl7/pl6

hatched: negative sera

• ':;V.«'.M::!i! iikiikv.;;',;;.;'.' 1 SO

1JSO

1 soo

i *SOO"

In the transmembrane proteins gp41/gp36 regions were selected wich are located in the protein part probably positioned outside the membrane (4). From this external loop region two corresponding antigenic sites were selected, which contain the lowest possible content of equal amino acids (ca 50 %). In the core protein p24/p26 the epitope is highly conserved in HIV-1 and HIV-2. The most variable epitope between HIV-1 and HIV-2 was selected f r o m an antigenic region near the carboxyterminus of p l 7 / p l 6 (Tab.l). The synthesized peptides were analyzed by gasphase amino acid sequencing. ELISA-tests were performed in Milititer plates sealed at the bottom with a membrane of low proteinbinding capacity, using 500 ng of carrier-bound peptide, which represents about 200 ng of antigen. The reactivity of the antigen was shown in by ferritin-labelled antibodies at

682 the surface of the polystyrene beads. Sera of H I V - 1 - , HIV-2-positive and -negative individuals were tested in dilutions up to 1:2500 in parallel experiments on HIV-1 and H I V - 2 specific antigen (Fig.l). Using p24/p26 derived peptides, all H I V - 1 - and H I V - 2 positive sera showed a high degree of cross-reactivity, all sera were positive on both peptides with a slightly elevated reaction on the corresponding antigen (HIV-1 sera tested on HIV-1 specific antigen). Positive reaction was assumed when values for optical density exceeded 2.1 times the values of negative sera in the respective dilution. Using gp41/gp36 peptides as antigen, sera showed an elevated positive reaction when tested on the strainspecific corresponding antigens. Due to the content of similar and equal amino acid residues in combination with isolate-specific sequence variation, some degree of crossreactivity was observed when HIV-1 sera were used with HIV-2 antigens and vice versa (Fig.l). The reactivity of the individual sera on both gp41/gp36 peptides A and B was different. One H I V - 2 serum did not react at all to gp36/B, the reaction to gp36/A however was rather good. From our tests we cannot decide whether the non-reactivity to gp36/B is due to the genetic constellation of the patient or to amino acid sequence variation in the infective HIV-2 isolate. The best distinction between HIV-1 and HIV-2 infection allowed the ELISA tests done with p l 7 / p l 6 peptides. Based on the highly d i f f e r e n t sequence in this region, reaction was observed on the strain-specific peptides, especially with serum dilutions 1:250 and 1:500. p l 7 / p l 6 antigen can however not be used solely, since not all patients develop antibodies to those proteins in the course of infection. In conclusion, we have shown that carrier-bound synthetic peptides are an excellent tool f o r a specific and quick ELISA test system and can be used for distinction between HIV1 and HIV-2 infections. Due to the optimal presentation of antigens covalently bound via the carboxy terminal ends, artefacts resulting by unspecific adsorption to plastic support at high pH values are avoided. The simultaneous testing for antibodies directed to a set of peptides allows a clear and easy distinction, also when sera fail to react with one of the epitopes.

References 1. Ratner, L., Haseltine, W„ Patarca, R., Livak, K.J., Starcich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Withehorn, E.A., Baumeister, K „ Ivanoff, L „ Petteway, S.R.Jr., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C., Wong-Staal, F. (1985) Nature 313:277-284. 2. Guyader, M., Emeran, M „ Sonigo, P., Clavel, F., Montagnier, L. Alizon, M. 1987. Nature_32&662-669. 3. Wolf, H., Modrow, S., Motz, M „ Hermann, G., FSrtsch, B. 1987. CABIOS 4:187-191. 4. Modrow, S., Hahn,B.H„ Shaw, G.M., Gallo, R.C., Wong-Staal, F„ Wolf,H. 1987. J. Virol. 61:570-578.

CHARACTERIZATION OF IMMUNOLOGICAL FUNCTIONS LOCALIZED IN VARIOUS REGIONS OF THE HIV-1 ENVELOPE PROTEINS USING A SERIES OF SYNTHETIC OLIGOPEPTIDES

Susanne Modrow. Hans Wolf Max von Pettenkofer-Institut, Pettenkoferstr. 9a, D-8000 München 2, F R G Brigitte Höflacher, Andreas Willer, Volker Erfle GSF, Ingoldstädter Landstr.l, D-8042 Neuherberg, F R G Britta Wahren The National Bacteriological Institute, Lundagaten 2, S-10521 Stockholm, Sweden

The envelope protein complex gpl20/gp41 of the human Immunodeficiency virus (HIV) is responsible for adsorption of the particle to the CD4-receptor on the surface of Thelper cells (1), for penetration and for fusion activity of HIV-infected cells (2). Most virus-neutralizing antibodies are directed to this protein complex (3); a major problem for the use of

the outer membrane protein gpl20 as a vaccine is the

extensive

heterogenicity of HIV-isolates, especially in antigenic sites (4,5), which may result in the induction of pre-dominantly virus-type specific antibodies (6). In addition to humoral immune response cytotoxic T-cells have been shown to be directed to gpl20 (7,8). To locate the diverse functions in the amino acid sequence of gpl20/gp41, we synthesized a series of oligopeptides from variable and conserved protein regions and tested those for their capacity to be reactive with the humoral and cellular immune system and to bind to the surface of T-cell lines. According

to computer analysis (5), the glycoprotein complex of HIV-1, which is

synthesized as a precursor of 160 kDa and subsequently cleaved into the outer membrane protein g p l 2 0 and the transmembrane part gp41 consists of highly variable and conserved regions. Avoiding potential N-linked glycosylation sites, we selected sequences f r o m variable (V), intermediately conserved (I) and conserved (C) regions and synthesized those using Fmoc-chemistry (Tab.

1). Peptides derived f r o m conserved and

intermediately

conserved regions were selected that possibly occuring flexible residues with high values for ß - t u r n s were located in the centre of the synthesized region; these sequences may represent relatively good epitopes with respect to the surrounding unflexible residues. All peptides were purified by HPLC and subjected to amino acid and sequence analysis. To test the reactivity of the peptides with the humoral immune system of HIV infected persons, 200 ng peptide / well were coupled to ELISA-plates. Serum collections of HIVpositive individuals with and without indications of ongoing disease divided in AIDSand ARC-patients and those, who were still without symptoms were used for testing. To avoid unspecific rections relatively frequent in HIV-positive sera, dilutions of 1:500 were used. Negative sera showed occasionally unspecific reactions up to dilutions of 1:100 with

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

684 the exception of peptide 213-225, which reacted with all negative sera also in higher dilutions. Localisation on g p l 2 0 / g p 4 1

Region

80-92

A-C-V-P-T-D-P-N-P-Q-E-V-V-G

CI g p l 2 0

100-112

F-N-M-W-K-N-N-M-V-E-Q-M-C

CI g p l 2 0

170-181

T-S-I-R-D-K-V-Q-K-E-Y-A-L-C

V2 g p l 2 0

213-225

P-K-V-S-F-E-P-I-P-I-H-Y-C

C2 g p l 2 0

254-266

C-T-H-G-I-R-P-V-V-S-T-Q-L-G

C2 g p l 2 0

315-326

S-I-Q-I-G-P-G-R-A-F-V-T-C

V3 g p l 2 0

349-361

L-K-Q-I-V-T-K-L-R-E-Q-F-G-C

13 g p l 2 0

378-389

I-V-T-H-S-F-N-C-G-G-E-F-G

13 g p l 2 0

435-446

Q-E-V-G-K-A-M-Y-A-P-P-I-G-C

C3 g p l 2 0

512-523

V-V-Q-R-E-K-R-A-V-G-I-G-C

C4/5 gpI20/gp41

653-664

I-E-E-S-Q-N-Q-Q-E-K-N-E-C

C6 gp41

Table 1: Sequences of synthesized peptides

The reactivity of the peptides showed a very variable degree, probably based on the high amount of sequence variation in the individual isolates; none showed 100 % reactivity. A relatively good reaction with 63 % of AIDS-patients sera showed peptide 315-326 derived f r o m a most antigenic region V3 and peptide 100-112 f r o m constant region C I . All other peptides reacted only with 50 % or less of sera tested (Tab. 2). For most peptides, AIDSpatients showed a lower degree of reactivity with respect to sera f r o m HIV-positive s y m p t o m - f r e e individuals. For further analysis we titrated consecutive sera of the same patients,

who

were

without

symptoms

when

tested

initially

and

developed

AIDS

subsequently. Also here it could be shown that antibody titers to peptides 100-112 and 316-326 were almost constant during progressive disease; the other peptides, however, showed decreasing reaction. In order to test the recognition of the peptides by the cellular immune system, T-cell proliferation was measured by incorporation of

^H-thymidine

after incubation of T-cells with irradiated B-cells, whose surface was adsorbed with the purified peptides. One peptide showed a realtively good reactivity with 50 % (435-446) of the tested T-cell preparations of HIV-infected persons (Tab. 2). These two peptides located in highly conserved rather hydrophobic regions of gpl20 showed only limited reactivity with antibodies; these findings also reflect the different structural conditions, which have to be assumed f o r B- and T-cell specific epitopes. To test if some of the peptides show preferential binding to the surface of T-cells and may be involved in the adsorption of gpl20 to the CD4-receptor, we labelled tyrosine-containing peptides with

the

using chloramine T methodology. A EBV-specific

peptide derived f r o m open reading frame BNLF1 was used as control. These peptides were allowed to react with the surface of various B- and T-cellines; peptide 435-446

685 showed preferential binding to T-cell surfaces (Fig.l). The same region was identified to be involved in virus adsorption by different methods (6). Peptide 435-446 had shown a rather weak antibody reaction, T-cell recognition however was satisfactory. We conclude that this region may be located in a cleft between two highly variable antigenic regions, which is not accessible for antibody binding.

Peptide AA - AA

humoral imraunrespoose % of s e r a w i t h positive r e a c t i o n positive carrier

ARC

cellular proliferative % of p a t i e n t s with positive r e a c t i o n

response

AIDS 22.

80-92

33

0

12

33

100-112

50

50

38

33

170-181

33

0

12

38

213-225

100

100

100

0

254-266

50

50

0

29

315-326

50

50

63

-

349-361

50

50

12

0

378-389

33

50

0

-

435-446

50

0

0

50

512-523

15

50

12

29

653-664

33

0

12

0

20.18 16 14 12. 10 8 4 . ?

0 . 170-181

Table 2: Reactivity of peptides

213 - 225

435-446

'.ytjma E8V

Fig 1: Reaction with cell surface

References 1. McDougal J.S., A. Mawle, S. Cort, J. Nicholson, D. Cross, J. Scheppler, D. Hicks Sligh. 1986. J Immunol JL2S, 3151-3157.

J.

2. Sodroski J., W.C.Goh, C. Rosen, K. Campbell, W.A.Haseltine. 1986. Nature 222, 470474. 3. Weiss R.A., P.R. Clapham, R. Cheingson-Popor. A.G. Dagleish, C.A. Carne, J.V.D. Weller, R.S. Tedder. 1985. Nature 316, 69-72. 4. Starcich B.R., B.H. Hahn, G.M. Shaw, P.D. McNeeley, S. Modrow, H. Wolf, E.S. Parks, W.P. Parks, S.F. Josephs, R.C. Gallo, F. Wong-Staal. 1986. Cell 45, 637-648. 5. Modrow S., B.H. Hahn, G.M. Shaw, R.C. Gallo, F. Wong-Staal, H. Wolf. 1987. J Virol 6 i , 570-578. 6. Lasky L.A., G.E. Groopman, C.W. Fennie, P.M. Benz, D.J. Capon, D.J. Dowbenko, G.R. Nakamura, W.M. Nunos, M.E. Rens, P.W. Berman. 1986. Science 232, 209-212. 7. Plata, F., B. Antran, L.P. Martins, S. Wain-Hobson, M. Raphael, C. Maynard, M. Denis, J.M. Guillon, P. Debre. Nature 1987, 328,721-724. 8. Walker B.D., S. Chakrabarti, B. Moss, T.J. Paradis, T. Flynn, A.G. Durno, R.S. Blumberg, J.C. Kaplan, M.S. Hirsch, R.T. Schooley. Nature 1987, 328.345-348.

Distinction of HIV-1 and HIV-2 infection using novel synthetic lipopeptide-conjugates as antigens in ELISA

R.-P.Hummel, W.Tröger, G.Jung Institut für Oranische Chemie, Universität Tübingen, D-7400 Tübingen, FRG T.Böltz, W.Bessler Institut für Immunbiologie, Universität Freiburg, D-7800 Freiburg, FRG L.Biesert, H.Rübsamen-Waigmann Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, D-6000 Frankfurt, FRG Assays using synthetic peptides have several advantages: no need for virus cultivation and purification and simple preparation of chemically unambigously defined peptide-antigens in any desired amount. To avoid problems of attachment of the peptides to ELISA plates we used lipopeptide-conjugates with the analogue of the N-terminus part of E.coli lipoprotein N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-serine (Pam 3 Cys-Ser). For identification of the immundominant epitopes of the viral proteins we used the epitope prediction performed by our program HYCON (W.Troeger et.al. unpublished results).

The

version 88 of this program combines the predictions obtained from the parameters of hydrophilicity' 1 ', hydropathy' 2 , 3 ', acrophilicity' 4 ', antigenicity' 5 , 6 ' and

flexibility'7'

together with

conformational predictions of alpha-, beta-, turn and coil conformation probabilities' 8 , 9 , 1 0 '. The parameters of antigenicity depend on the type of the protein. We distinguished globular and membrane proteins and found new parameters for calculation of the antigenicity. All predictions are weighted and multiple positive prediction for alpha, beta, turn and coil probabilities were eliminated.

These results are summed up called FAZIT demonstrating the most probable

antigenic site. Two peptides as a result of the predictions, known epitopes of the gp-41 envelope proteins' 1 1 ' of HIV-1 and HIV-2, were synthesized on p-alkoxybenzylalcohol anchor on PS-1% DVB using Fmoc(t-Bu) strategy. Pam3Cys-Ser was coupled to the N-terminus of both peptides (Fig. 1 and Fig.2)

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

687

3.0

2.8 2.6 2.4

2.2 2.0 1.B

1.6 1.4

1.2

1.0 0.8

0.6 0.4

0.2 Fig. 1 Results of sera tested with an ELISA (OD 4 0 5 ) employing Pam 3 Cys-Ser-[HIV-1(598-609) cyclic disulfide] as immunoadsorbent antigen • = HIV-1 positive •

= HIV-2 positive o = controls. The cutoff for positivity (mean of negative controls + 3

SD) is shown as a horizontal line. 3.0

2.8 2.6 2.4

2.2

2.0 1.8

1.6 1.4

1.2

1.0 0.8 0,6 0.4

0.2

Fig.2 ELISA values (OD 4 o 5 )obtained with sera from HIV-1 infected patients • HIV-2 infected patients

and controls o by using Pam 3 Cys-Ser-[HIV-2(593-603) cyclic disulfide] as solid

phase antigen. horizontal line.

The cutoff for positivity (mean of negative controls + 3 SD) is shown as a

688 The attachment of small peptide antigens to microtiter plates can be considerably improved by this method due to the highly amphiphilic properties of the three fatty acids containing lipopeptide moiety. In further representative test series we compared the reactivity of human sera in HIV-1 and HIV-2 ELISA tests, firstly using linear cysteinyl and secondly cyclic cystinyl lipopeptide conjugates as solid phase antigen. The results clearly indicated considerable improvement of discrimination using the cyclic lipopeptide conjugates for coating the ELISA plates

as

shown in Fig.l and Fig.2. In the HIV-1 ELISA test sera from 117 of 121 HIV-1 infected patients reacted with the cyclic Pam 3 Cys-Ser-[HIV-l(598-609) cyclic disulfide]. None of the 142 uninfected controls had detectable antibodies against this peptide. Sera from HIV-2 infected patients showed a marginal positive response. In the HIV-2 ELISA test 5 out of 5 HIV-2 sera of HIV-2 infected patients reacted with the cyclic Pam3Cys-Ser-[HIV-l(593-603) cyclic disulfide]. None of the 48 uninfected control sera reacted with this peptide. References 1. Hopp T.P., K.R. Woods. 1983. Mol.Immun. 20, 438-489. 2. Kyte J., R.F. Doolittle. 1982. J.Mol.Biol. 151, 105-132. 3. Sweet R.M., D. Eisenberg. 1983. J.Mol.Biol 171, 479-488. 4. T.P. Hopp in:Modern Methods in Protein Chemistry, 1, J.J. LTtalien.ed., Plenum Press New York (1986) 5. Welling G.W., W . J . Weijer, R. van der Zee and S. Welling-Wester. 1985. FEBS Lett. l£g, 215-218. 6. Troger W., F. Gombert, G. Jung. 1986. unpublished results. 7. Karplus P.A., G.E. Schulz. 1985. Naturwiss. 72, 212-213. 8. Chou P.Y., G.D. Fasman. 1977. J.Mol.Biol. U 5 , 135-175. 9. Chou P.Y., G.D. Fasman. 1978. Ann.Rev.Biochem. 47, 251-276. 10. Gamier J., D.J. Osguthorpe, B. Robson. 1978. J.Mol.Biol. 120, 97-120. 11. Gnann J.W. J.B. McCormic, S. Mitchell, J.A. Nelson, M.B.A. Oldstone. 1987. Science 237, 1346. 12. Boltz T., R.-P. Hummel, W. Bessler, H. Riibsamen-Waigmann, L. Biesert, W. Troger, G. Jung. 1988. J.Virol.Methods in press.

FMDV INFECTION IS BLOCKED IN VITRO

BY RGD-CONTAINING

PEPTIDES

FROM THE IMMUNODOMINANT REGION OF VP,

A.Yu.

Surovoy

S h e m y a k i n Institute of Bioorganic C h e m i s t r y , USSR A c a d e m y of Sciences, 117871 M o s c o w , USSR

Introduction It has recently b e e n s u g g e s t e d that the A r g - G l y - A s p - s e q u e n c e (RGD) forms a part of a w i d e s p r e a d c e l l - e x t r a c e l l u l a r r e c o g n i t i o n system

matrix

(1). For e x a m p l e , the cell receptors

for

fibronectin, v i t r o n e c t i n and o s t e o p o n t i n were shown to interact w i t h R G D - s e q u e n c e s of these extracellular m a t r i x p r o teins. The RGD-sequence

is also a p a r t of the

r e g i o n of VP^ of f o o t - a n d - m o u t h disease virus

immunodominant (FMDV). We

report here that p e p t i d e s containing RGD-sequence

from the

immunodominant region of VP^ p r e v e n t viral i n f e c t i o n on cell m o n o l a y e r s . Specificity of the inhibitory a c t i o n suggests the RGD-sequence m a y also be u s e d by viruses to mediate attachment

that

cell-

phenomena.

Results a n d D i s c u s s i o n It follows from c o m p a r i s o n of the VP^ amino a c i d sequences different virus subtypes, shown below that the RGD-sequence absolutely conservative in spite of being p o s i t i o n e d in the extremely variable region of the protein.

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in G e r m a n y

of is

690

01K OJC

160 152 145 141 136 Y N R N A V P N L R G D L Q V L A Q K V A R T L P Y S R N A V P N V R G D L Q V L A Q K V A R T L P

C3B

Y T T G Y - -

-

R R G D L A H L A A A H A R H L P

C-jObb

Y T A S T - -

-

- R G D L A H L T A T R A G H L P

A ¡.WW

Y S T G G P -

-

R R G D M G S A A A

A

Y S A G G M G

-

22

R A A K Q L P R R G D L E P L A A R V A A Q L P

The question arises whether this sequence forms a part of FMDV cell attachment site. In order to check that hypothesis we studied in vitro

inhibition of the viral growth by a number of

earlier synthesized peptides

(2) belonging to the central im-

munodominant region of C^K VP^ sequence, both containing and not containing

the RGD site.

Table. Cell Protection Assay from FMDV Infection by Synthetic * Peptides

Synthetic peptides (the numbers refer to the amino acid sequence of VP.. VPNLRGDLQVLA

(141-152)

RGDLQVLA

(145-152)

VPNLRGDL

(141-143)

PNGAPEAAL

+ (30) + (60)

(control 90-99)

LLYRMKRAETYCPRP RGELQVLA

Cell protection (minimal peptide concentration, yg/ml)

(control 175-189)

(analog 145-152 with

D replaced for E) *

5 5 4*10 -6-10 of primary pig kidney cells per vial were incubated 3 days to generate confluent cell monolayers. Duplicate monolayers were pretreated with synthetic peptides for 1 hr at 37°C before infection with 100-200 tissue culture doses of specified virus. The cultures were incubated with virus for 2 hr at 37°C and washed with culture medium. After 2 days incubation monolayers were checked up for cytopathic effect.

691 It was found that RGD containing peptides 141-152 and 145-152 fully protect cells from infection at the concentrations 30 yg/ml and 60 ug/ml, respectively

(see the Table). Control

peptides lacking the RGD-sequence had no effect on the virus infection even at 1000 yg/ml. Peptide 141—148 also containing the RGD sequence did not affect the virus adsorbtion at 1000 ug/ml indicating that activity of RGD segment is incompatible with its C-terminal position. The protective activity of RGD-segment is quite sensitive even to minor structural changes. This follows from the inactivity of the specially

synthesized

analog of the peptide 145-152 in which aspartyl residue was reflaced for the glutamic residue. We also found that both peptides are active not only w i t h O^K but also with A 2 2 virus, the two strains showing no serological cross-reactivity. This results can serve as an additional evidence for a common receptor in different FMDV subtypes

(3).

Further studies of the molecular interaction between the cellular receptor and FMDV related peptides might lead to a better understanding of FMDV biology, and probably, to constructing novel antiviral drugs.

Acknowledgement I wish to thank Professor V.T. Ivanov for suggestions and helpful comments; A.V. Chepurkin and V.N. Ivanyushenkov for cell protection assay.

References 1. Ruoslahti, E. and M.D. Piershbacher. 1986. Cell 44, 517 2. Surovoy, A.Yu., O.M. Volpina, E.V. Snetkova, T.D. Volkova, V.T. Ivanov, A.V. Chepurkin, V.N. Ivanyushenkov, A.N. Burdov and N.N. Dryagalin. 1988 . Bioorg. Chem. (Russian) 14 (in press). 3. Colonno R.J. 1987. BioEssays 5, 270.

TOWARDS PEPTIDE VACCINE AGAINST THE FOOT-AND-MOUTH DISEASE

A.Yu. Surovoy, V.M. Gelfanov, L.A. Grechyaninova, A.V. Yarov, O.M. Volpina, V.T. Ivanov Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, 117871 Moscow, USSR

It is generally assumed that efficient peptide antiviral vaccine must contain segments adequately reproducing immunologically functional sites of the microorganism, viz. T-epitopes, B-epitopes and agretopes. We have found earlier (1,2) that free peptides, not conjugated with any protein carrier, 136152 (strain O^K) and 136-151 (strain

of foot-and-mouth

disease viral protein VP^ contain all the necessary information for inducing protective immune response. As expected this response proved a subject of Ir-gene control, i.e. reactivity of the animal is species dependent, or it varies from strain to strain in inbred populations (Table 1). In other words, this result provides an example of a common problem arising in the course of making a peptide vaccine: even if your preparation contains all the epitopes this does not guarantee the protective effect within the whole population of outbred animals, for which the vaccine is actually intended. In this work we found a novel way to broaden the species selectivity of peptide vaccines and possibly to overcome the Ir-gene control. Rabbits reactive to O^K peptide and non-reactive to &22 P e P~ tide were immunized twice with a mixture of the two peptides. As shown in Table 2 under these conditions antibodies are formed to both peptides. Analogous result was obtained with a non-responding BALB/c mice strain (Table 3). Moreover, the rabbit antibodies inhibited the growth of A ^ viral strain ¿n vitKo. It should be noted thereby that the two peptides do not crossreact

serologically (Table 2). In these experiments the

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin N e w York - P r i n t e d in G e r m a n y

693 Table 1. Reactivity of Animals to 0-^K and A 2 2 Peptides 0-^K

TyrAsnArgAsnAlaValProAsnLeuArgGlyAspLeuGlnValLeuAla

A_ _

TyrSerAlaGlyGlyMetGlyArgArgGlyAspLeuGluProLeuAla

Immunogen

Rabbit Guinea Catti Sheep plg

BALB/c CBA/J C3H C57BL/6

136-152 O-j^K

+

+

+

+

136-151 A„„

+

+

+

-

+

+ ±

136-152 0 1 K peptide served as an "inducer" of the immune response to the 136-151 A 2 2 peptide. Replacement of the 136-152 0-^K peptide with non-homologous VP^^ segments 10-25 or 197-213 abandoned the induction of anti-A 22 response. Data presented in Table 3 and obtained by successive immunization of mice with individual A 2 2 and 0-^K peptides allowed to infer that:

Table 2. Immunogenicity of C^K and A 2 2 Peptides in Rabbits3

Immunogen

Antipeptide titer, -log1L> ln Neutralizing titer (-log2) pig kidney cell culture, 200 tissue cytotoxic inanti-O^K anti-A 22 fection doses of A22 virus

°1 K + A 22 controls :

2.5-4.,0

2.5-4.0

3.0-5.0

136-152 0 1 K

2.5-4..0

< 13 - 29 >

< 1 -12 (18-29) > Fig. 1 : sequences of the selected

peptides

Circular dichroi.sm studies: the 17 amino-acids peptides ( 1-17 and 13-29) were significantly organized both in TFE and water.

In both cases, the

CD

spectra were characteristic of a partial alpha helical formation, with minima occuring at 202 and 216 nm

(peptide 1-17), or 197 and 220 nm (

peptide 13-29) . Helix content were calculated from the CD spectra, taking [0] 222 = - 35,700 deg.dmole - 1 .cm 2 1-17, and 6% for In

order

sequence

to was

carboxylic

destabilize also

group

for 100% helicity, and were 23% for

13-29 . the

organization

synthesized

instead

of

with

the

a

of peptide

C-terminal

carboxamide

13-29,

the

negatively

found

in

all

same

charged

the

other

peptides. CD studies of this analog ( 13-29-COOH) showed that although it remained partially helical in TFE, this organization disappeared totally in water. The shorter fragments 1-12 and 6-17 were unorganized in water. F.LISA inhibition experiments: antigenicity of the different peptides was determined towards human sera from individuals living in endemic area, selected for their ability to recognize the recombinant protein. All these sera recognized also the 41-peptide, which was thus used as the solid phase antigen.

The

different

peptides

were

tested

in

ELISA

inhibition

experiments. Among the human sera tested, three different behaviour were observed, that may be represented by the selected sera HS1 to HS3 (fig. 2). - HS 1 : both partially organized 17-peptides 1-17 and 13-29 were able to inhibit the binbing of the antibodies to the 41-peptide. - HS 2 : peptide 1-17 was a significantly better inhibitor. - HS 3 : only the 41-peptide was able to inhibit the binding. In all cases, the inhibiting capacity of the unorganized peptide 13-29-COOH appeared only at high concentrations, and was always significantly lower than

the

inhibiting

capacity

observed

with

the

partially

organized

carboxamide analog 13-29; peptides 6-17 and 1-12 were poor inhibitors(data not shown).

720

#

$

100

100

1-17 13-29 13-29-COOH 41-peptide

inhibiting peptide, flg/mi

inhibiting peptide, ng/ml

inhibiting peptides, pg/mi

fig. 2 : ELISA inhibition experiments, using human sera ( HS 1 to 3 )

Conclusion The fact that none of the unorganized overlapping sequences 1-12, 6-17, 13-2 9-COOH is endowed of any significant inhibiting capacity suggests that naturally developping human antibodies to the liver-stage of

Plasmodium

falciparum are mainly conformation-specific. When peptides 1-17 and 13-29 are tested, a significant inhibiting capacity is observed, that appears to be

correlated

with

their

helical

organization

in

water.

Moreover,

C-terminal alteration of 13-29 allows to modify the helicity of a unique aminoacid sequence by interfering with the helix dipole. In this case also, the

difference

observed

between

reactivities

is

clearly

dependent

on

helicity. Our results demonstrate the existence of at least one epitope present in the sequence 1-17 provided it adopts an helical organization. However, the existence of another epitope

inadequatly

folded in 13-2 9

cannot be ruled out, and could explain that only 41-peptide is endowed of inhibiting capacity towards the human sera represented by HS 3 . These observations strongly suggest that this repeated structure consits in long helical stretches at the parasitic surface. More generally, we believe that carefull screening according to both primary and secondary structure may help in the design of synthetic peptides to be used in diagnostic or serological studies. References 1 - C. Guerrin-Marchand, P. Druilhe, B. Galey, A. Londono, J. Patarapotikul, A. Tartar, 0. Mercereau-Puijalon, G. Langsley. 1987. Nature, 32 9, 164 - 167. 2 - K. R. Shoemaker, P. S. Kim, E. J. York, J. M. Stewart, R. L. Baldwin. 1987.Nature, 326, 563 - 567.

IDENTIFICATION OF A TOPOGRAPHIC EPITOPE AT THE SURFACE OF A CARDIOTOXIC PROTEIN.

G. Mourier, E. Gatineau , P. Fromageot, A. Ménez Service de Biochimie du Département de Biologie, CEN Saclay, 91191 Gifsur-Yvette Cédex, France P. Nicolas Institut Jacques Monod, Université Paris VII, 75005 Paris, France

Introduction Epitopes of native proteins are composed of residues which are separated from each other on the polypeptide chain, but are brought together in space by protein folding, as illustrated by X-ray crystallographic study of an antigen-antibody complex (1). Consequently, epitopes are difficult to delineate by a single chemical approach. In the present paper, we report the mapping of an epitope using two complementary procedures. Firstly, we determined the relative binding affinities for a monoclonal antibody of several protein variants and correlated these affinities with differences in protein amino acid sequences. Secondly, we synthesized a series of peptides and determined their binding affinities for the same antibody. Together, these approaches proved well suited to the identification of the residues forming a topographical antigenic determinant at the surface of toxin y, a cardiotoxin derived from the venom of the snake Naja nigricollis. The toxin is a polypeptide with a chain of sixty amino acids, cross-linked by four disulfide bridges (2). As shown in the figure, the chain is folded into three adjacent loops rich in P-pleated sheets (3). Myl, the monoclonal antibody used in the present study is capable of neutralizing the biological activity of toxin y both in vivo and in vitro (4). Results and discussion The relative binding affinities for Myj of fourteen natural variants of toxin y were determined by competition experiments (4,5). Some of the affinities were observed to be i) identical; ii) about six times weaker or; iii) 200 times

Peptides 1988 © 1989 Walter de Gruyter & Co., B e r l i n - N e w York-Printed in G e r m a n y

722

weaker, as compared to that of toxin y (K D = 4 10~10 M). The changes in binding affinities correlated with the substitutions that occurred in loop I (especially of residues 5, 9, 10 and 11) and/or in the C-terminal region (residue 58). We concluded that these positions may be involved in the epitope recognized by My^. We also monitored the binding affinities of three toxin derivatives modified either with nitrophenylsulfenyl chloride (6) at tryptophan 11 (loop I) ,or with tetranitromethane (7) at tyrosine 22 (loop II) or tyrosine 51 (loop III) (8). Only the modification at Trp-11 reduced the binding affinity (K D = 2 10 8 M), thus confirming the involvement of loop I in the epitope.

We determined then the binding affinities of a series of synthetic peptides gradually encompassing loop I up to residue 14. Peptides I (12-14) and II (1014) had no binding affinity for Myi although peptide II possessed one antigenic element (Trp-11) and possibly another (Phe-10). Peptide III (6-14) displayed a weak but clear affinity for Myj (K D = 5 10"4 M), suggesting that the P-turn-forming residues 6-9 are involved in the epitope. Circular dichroism analysis indicated that in water, peptides I and II had no detectable

723

secondary structure, whereas peptide III has some (3-turn conformation. Peptide IV (1-14) had a higher affinity than Peptide III for Myl (KD= 2.6 10 5 M). A disulfide bond between cysteines 3 and 14 was then formed and the peptide, called peptide V, had the highest affinity for Myl (K D = 6 10"6 M). No further increase of secondary structure content was observed in this peptide, despite the presence of a disulfide bond. Clearly, the folding of each peptide, including peptide V, was different from that of the homologous fragment in the native toxin. The absence of appropriate conformation may account, at least in part, for the weak antigenicity of die peptides. The present investigation indicates that numerous residues of loop I belong to the epitope recognized by My^ It also suggests that conformation plays a crucial part in antibody binding to synthetic peptides . However, the design of synthetic epitopes with an appropriate secondary structure is still a chemical challenge. References 1. Amit, A.G., R.A. Mariuzza, S.E.V. Philips and R.K. Poljak (1986), Science. 233. 747-753. 2. Karlsson, E., (1979), Handb. Exp. Pharmacol., 52, 159-212. 3. Rees, B.,J.P. Samama, J.C. Thierry, M. Gilibert, J. Fischer, H. Schweitz, M. Ladzunski, D. Moras. (1987), Proc. Natl. Acad. Sci. U.S.A., M , 31323136. 4. Grognet, J.-M.,E. Gatineau, P. Bougis, A.L. Harvey, J. Couderc, P. Fromageot, A. Menez. (1986), Mol. Immunol., 23, 1329-1337 5. Mourier, G., E. Gatineau, P. Nicolas, P. Fromageot, A. Menez. (in preparation). 6. Fontana, A . , E. Scoffone. (1972), Methods Enzymol., 15, 483-494. 7. Riordan, J.F. , B.L. Vallee.(1972), Methods Enzymol., 25, 515-521. 8. Gatineau, E., F. Toma, Th. Montenay-Garestier, M. Takechi, F. P. Fromageot, A. Menez. (1987), Biochemistry, 26, 8046-8055.

VARIABLE AND ACCESSIBLE RESIDUES OF SCORPION TOXINS: PREDICTION OF IMMUNODOMINANT RESIDUES OF (5-TOXINS

P. Fourquet *, J. Novotny C. Granier *

J.C. Fontecilla-Camps + , E. Bahraoui * , H. Rochat * and

* Laboratoire de Biochimie, Faculté de Médecine Secteur Nord, 13015 Marseille ° Molecular and Cellular Research Laboratory, Massachussetts General Hospital, Boston + Laboratoire de Cristallographie, Faculté de Médecine Secteur Nord, 13015 Marseille

Introduction

Scorpion toxins constitute a family of pharmacologically potent proteins. Despite some structural similarities, a-type and p-type toxins act in different ways on the sodium channel of excitable membranes and do not cross-react with antibodies raised against the other class of toxins. We have progressively deciphered the antigenic structure of an a-scorpion toxin (AaH II) (1,2, 3). Little is known, however, about the antigenic features of p-type toxins. We investigate here the possibility to forecast immunodominant residues of p-scorpion toxins. Starting from the two observations that antigenic sites are often located in those regions of proteins that are evolutionary unstable and that antigenic activity is a surface property , we propose to select as putative antigenic determinants of scorpion p-toxins the subset of variable residues that are particularly accessible at the surface of the protein.

Results

- To locate the most variable positions of the amino acid sequence of scorpion toxins, we applied the variability measure of Wu & Kabat (4) to the set of 25 known amino acid sequences of scorpion toxins. It appears that a great part of the structure is conserved and that only 25 of 64 residues are evolutionary unstable. - To pinpoint the residues of toxins that are particularly exposed at the molecular surface, we used the procedure of Novotny et al., (5). The atomic coordinates of the a-type toxin AaH II (6) and of the P-type toxin CsE v3 (7) were utilized in a Lee & Richards type algorithm (8). Those portions of the molecular surface contacting the surface of a spherical probe rolling over the protein are computed. Here we used a large probe of 10 A radius (5). A value of the surface contact of each residue of the two toxins was thus obtained.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w York- Printed in G e r m a n y

725 For AaH II, whose antigenic structure is known, we observed that 13 out of the 15 residues that possessed the joined properties of high accessibility and high variability are residues with experimentally assessed antigenicity. This fact encouraged us to consider that variable and accessible residues of p-scorpion toxins may indicate us where antigenic determinants are located. Twelve amino acids of CsE v3 were found to exhibit high surface exposure and high variability (Table 1).

Residue

Contact area (À2) (mean 2.64)

Lys Ser Leu Lys Gin Thr Lys Phe Ser Pro Lys Ser

5 .35 . 6.. 72 13..867 6,. 197 8.. 164 4 .. 69 13.. 146 8..463 5.. 837 5 .. 155 5 .222 . 4 .. 037

8 9 17 18 23 27 32 44 54 59 63 64

Variability index (mean 10.62) 21..8 55 29.. 17 32.. 14 29.. 17 25 17 .5 . 15 25 37 .5 . 11..36 11..54

Table 1: Residues of the fi-type tpxin CsE V3 showing above-the-mean variability and high surface-contact with the 10 A probe. Four of these residues appeared to be clustered in the vicinity of the disulfide bridge 1265 ( residues 8, 9, 63, 64). Thus, we attempted to mimic a putative conformational antigenic determinant encompassing these 4 residues. We prepared sequences 4-13 and 61-66 of the p-toxin Css II and linked the two fragments by a disulfide bond, as it occurs in the native toxin. This model peptide was shown to actually possess a significant antigenic activity: in solid-phase immunosorbants assays, the Sepharose bound peptide was found to be able to bind anti-Css II antibodies in a specific and saturable manner. This indicates that the region of the p-toxin modeled by the peptide is antigenic.

Conclusion

Only a few amino acid residues of scorpion toxins possess the joined properties of high variability and special surface exposure, two properties often associated with regions of antigenic reactivity. For a-type toxins, the subset of variable and accessible residues corresponds well to residues previously shown to be endowed with antigenic reactivity. For p-type toxins, our first attempt to mimic an antigenic region on the basis of this type

726

of predictions was successful. Thus, we have now an useful guide for the design of either chemically modified toxins or synthetic peptides aimed at mapping the epitopes of ß-toxin. Abbreviations used: AaH 11= Toxin II of Androctonus australis Hector CsE V3= Toxin 3 of Centruroides sculpturatus Ewing Css 11= Toxin II of Centruroides suffusus suffusus

References

1. El Ayeb, M., Bahraoui, E.M., Granier, C., Delori, P., Van Rietschoten, J. and Rochat, H. 1984. Molec. Immun. 21, 223. 2. Bahraoui, E.M., El Ayeb, M., Van Rietschoten, J., Rochat, H. and Granier, C. 1986. Molec. Immun. 22, 357. 3. Fourquet, P., Bahraoui, E.M., Fontecilla-Camps, J.C., Van Rietschoten, J., Rochat, H. and Granier, C. 1988 Int. J. Peptide Protein. Res, in press. 4. Wu, T.T. and Kabat. 1970. J. Exp. Med. 132, 211. 5. Novotny, J., Handschumacher, M., Haber, E., Bruccoleri, R., Carlsson, D., Fanning, D„ Smith, J. and Rose, G. 1986. Proc. Natl. Acad. Sei. USA £2, 226. 6. Fontecilla-Camps, J.C., Habersetzer-Rochat, C. and Rochat, H. 1988. Proc. Natl. Acad. Sei. USA., in press. 7. Fontecilla-Camps, J.C., Almassy, R., Suddath, F., Watt, D. and Bugg, C. 1980. Proc. Natl. Acad. Sei. USA 2, 6496. 8. Lee, B. and Richards, F. 1971. J. Biol. Chem. 55, 379.

EPITOPE MAPPING OF APAMIN: PARTIAL OVERLAPPING OF ANTIGENIC AND PHARMACOLOGICAL SITES

M.L. Defendini, E. Bahraoui, M. El Ayeb and C. Granier CNRS UA 1179, INSERM U 172. Laboratoire de Biochimie, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard, 13326 Marseille Cedex 15, France.

Introduction

The bee-venom neurotoxin apamin provides an interesting model peptide for structureantigenicity and structure-activity relationships studies. The latter have already given some insights into the structural basis for toxicity (1, 2, 3, 4, 5) ; little is known , however, on the antigenic properties of the toxin (6). As antibodies frequently recognize conformational epitopes, and as the 3D structure of apamin (7, 8) shows a central ahelical region (residues 6 to 13) surrounded by the turn-folded N- and C- terminal parts, we addressed the question of what part of the molecule is preferentially recognized by anti-apamin antibodies. Furthermore, we wanted to know to what extent residues involved in the binding of apamin to rat-brain membrane receptors are also antigenic.

Results

We prepared polyclonal anti-apamin antibodies by immunizing rabbits with a mixture of BSA-coupled apamin and free apamin ( 200 micrograms, including 30% of coupled peptide). After a few booster injections we obtained high-titer anti-apamin antisera (titer : 1/40000 in radioimmunoassay) from which we prepared specific IgGs by affinity chromatography on apamin-Sepharose. A sensitive radioimmunoassay was set up using monoiodo ^ j ^ p a n ^ n a s a tracer (0.6 nM ) . Nanomolar concentrations (0.5 to 2nM) of native or synthetic apamin displaced 50% of the labeled molecule from antibodies. A panel of 18 synthetic or chemically modified analogs of apamin was used in the radioimmunoassay in order to understand the structural requirements for antibody binding to the toxin. All analogs competed with the tracer but with efficiencies varying broadly. A representative set of the results thus obtained is given in Table 1 .

P e p t i d e s 1988 © 1989 Walter d e G r u y t e r & C o . , Berlin • N e w York - P r i n t e d in G e r m a n y

728

Analog

Apamin Tetra S-Acm Apamin

Antigenic activity relative to apamin 1 1964

4-biotinyl Apamin 1,4 diacetyl Apamin Ala 2 Apamin Ala 4 Apamin

0.9 5.5 2 2.6

(1-17) Apamin (1-16) Apamin (1-15) Apamin

2 2.5 3.3

Ala 7 Apamin Ala ^ Apamin Ala 1 0 Apamin Pap Apamin Pap 1 4 Apamin

11.3 14.3 755 168 16.8

Table 1: Relative antigenic activity of various synthetic or chemically modified derivatives of apamin. Each analog was tested in increasing concentrations for its ability to displace labeled apamin (0.6 nM) from antibodies ( 0.75 or 5 nM ). The concentration giving half-maximal effect was measured and compared to that obtained for activity. apamin in the same series of experiments to yield the relative (Acm=acetamidomethyl; Pap =p .aminoPheny¡alanine) It appears clearly that the conformational integrity of the molecule is required, that residues belonging either to the N-terminal ( a -and e-amino groups, Asn 2, Lys 4) or to the C- terminal end (Gin 16, Gin 17, His 18) are not antigenically relevant and, finally, that residues important for the antigenic activity of apamin (Leu 10, Arg 13, Arg 14, Glu 7, Thr 8) are clustered in the helical region of the molecule. The previous analysis indicated that the epitope recognized by rabbit antibodies included Arg 13 and Arg 14. These two amino acids have been previously shown to play a pivotal role in the toxic and receptor binding properties of apamin (1, 2, 3, 4 , 5). "Die possibility that the binding of antibodies via the Arg residues might preclude the fixation of the toxin to its rat-brain receptors was considered. Figure 1 shows that the preincubation of the toxin with its specific antibodies inhibited the binding of apamin to its pharmacological receptors: a concentration of 2.5 nM of IgGs inhibited 50% of the effect. This experiment confirms the overlapping of the antigenic and pharmacological sites . However, Arg 13 and Arg 14 seemed to be the only two residues that share antigenic and receptor binding properties inasmuch as Leu 10, Thr 7 and Glu 8 are not pharmacologically important (C. Labbe-Jullid, personnal communication)

729

Figure l: Inhibition of the binding of 125j. apamin to rat-brain membranes by anti-apamin antibodies. apamin (10~H M) was incubated with varying concentrations of antiapamin IgGs ( o )for 90 min. at 37 °C then 60 min. at 4 °C. Rat-brain synaptosomal membranes, expressing the apamin receptor (9), were then added for 60 min. at 1 °C. Bound ligand was separated from free by filtration on polyethylene imine-treated glassfiber filters. Bo = maximun binding in the absence of antibody; B = binding in the presence of the mentioned concentration of antibody . Shown in black symbols (m) is the binding curve obtained with non-immune IgG. Acknowlegedment We are grateful to C. Labbe-Jullie, A. Regnier-Vigouroux and F. Albericio for providing us with some of the synthetic analogs of apamin. We thank also B. Marqueze for help and advice in the receptor assay. References 1. Vincent, J.P., Schweitz, H. and Lazdunski.M. 1975. Biochemistry, 14, 2521. 2. Cosand.W.L.and Merrifield, R.B. 1977. Proc. Natl. Acad. USA, 24, 2771 3. Granier, C., Pedroso-Muller, E. and Van Rietschoten, J. 1978. Eur. J. Biochem., £2, 293. 4. Sandberg, B.E.B. 1979. Int. J. Peptide Protein Res. J I , 238 5. Albericio, F., Granier,C., Labbe-Jullie, C., Seagar, M., Couraud, F. and Van Rietschoten, J. 1984. Tetrahedron, 4Q, 4313 6. Komissarenko,S.V., Vasilenko,S.V., Elyakova, E.G., Surina,E.A and Miroshnikov, A.I. 1981. Molec. Immunol., i £ , 533. 7. Bystrov.V.F., Okhanov.V.V., Miroshnikov, A.I. and Ovchinnikov,Y.A. 1980. F.E.B.S. Lett. Ü 2 , 113. 8. Wemmer, D. and Kallenbach, N.R. 1983. Biochemistry, 22, 1901. 9. Hugues, M„ Duval, D„ Kitabgi, P., Lazdunski, M. and Vincent, J.P. 1982. J. Biol. Chem. 251, 2762.

EPITOPE ANALYSIS: FROM HISTAMINE TO MELITTIN

C.H. Schneider, R. von Griinigen and H. Rolli

Institute of Clinical Immunology, Inselspital, CH-3010 Bern, Switzerland

Histamine as an Epitope It has been generally difficult to obtain an adequate immune response against histamine attached to protein carriers via conventional methods. This may not be surprising since in most of the work the histamine sidechain was used for conjugation, thus leaving as the haptenic moiety only the uncharacteristic aromatic ring (1,2,3). Our results are not better, but since epitopic histamine is presented with its free side-chain, the immunological failure becomes amenable to detailed discussion. 2-Carboxy-histamine was synthesized in four steps starting with a benzoyl chloride treatment of histamine which results in ring opening and benzoylation of all amino functions (4). Refluxing with trifluoroacetic anhydride and later methanol gives a 2-trifluoromethyl-histamine derivative (5) which, after prolonged NaOH treatment, affords 2-carboxy-histamine still carrying a benzoyl on the aliphatic amino group. The benzoyl group is removed in HC1 (6). For unambiguous conjugation to protein and peptide carriers, 2 - c a r boxy-histamine needs protection of its aliphatic amino function. Evaluation of Fmoc-, Nps- and Tcboc- groups led to Tcboc

(2,2,2-trichloro-tert.butyl-

oxycarbonyl) as the most satisfactory group. It seems of acceptable stability and its removal with NaBH^/ethanol/water with

cobalt-II-phthalocyanin

as catalyst (7) appears gentle and is expected to work with many protein conjugates. Control studies with human serum albumin (HSA) corroborate this. For smooth conjugation we introduced 6-aminohexanoic acid (SAH) as a spacer and a hydrophilic activated ester based on

4-hydroxy-3-nitrobenzenesulfonic

acid sodium salt (8) (Fig. 1). Using this reagent, we prepared histamine conjugates with HSA and a conjugate with the peptidic carrier PAL (9). The hexavalent 2-carboxy-histamine-PAL produced after injection into skin the same effects as free histamine. Immunization of rabbits with the HSA conjugates using conventional schedules involving Freund's complete adjuvant

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany

731

Fig. 1.

Tcboc-protected,

SAH-spaced 2-carboxy-histamine, activated for conjugation :

gave responses detectable by ELISA using solid-phase-bound 2-carboxy-histamine-PAL. Haptenic inhibition showed that the antibodies were directed mainly against the spacer and the 2-carboxy group but not against histamine or histamine metabolites. With a different immunization protocol, characterized by large doses of 2-carboxy-histamine-HSA, some responses also against the histamine moiety were obtained. - These results support the view that non-antigenic molecules of low molecular weight may interfere with the antigenic recognition step, provided they are able to constantly occupy antibody receptors on B-lymphocytes, a capacity we may indeed ascribe to histamine.

Epitopes on Melittin Melittin is able to induce a good antibody response in rabbits after injection as such, without prior covalent conjugation to a protein carrier. This provides a rather unique opportunity to do epitope analyses on a small unconjugated peptide. - For analysis, larger peptides were obtained via solidphase techniques whereas small peptides and derivatives were prepared by syntheses in solution. Conventional ELISA methodology was used for evaluation. Segments S(l-13), S(8-21) and S(21-26) but not S(8-13) (cf. Fig. 2) were shown to contain melittin-specific epitopes. S(21-26) represents a full epitope with virtually full binding capacity already shown by the shorter S(22-26). The second epitope included within S(8-21) seems centered around positions 14-17. The reactive segments showed their capacity in direct G I G A V L K V L T T G L P A L I S W I K R K R Q 20 15 1 10 S (1-13) S(8-13)

S(8-21) S(21-26) Fig. 2.

Amino acid sequence of melittin and segments.

Q-NH, '2

732 binding tests where they were attached to the solid phase and reacted with various rabbit anti-melittin antisera. They also were able to inhibit interactions between anti-melittin antibody and melittin-coated ELISA plates. The observation that S(l-13) was strongly active in direct binding tests, but not inhibitory at all, was interpreted as evidence for a helical epitope in the N-terminal region. This was substantiated by showing that two different segments 1-13 mimicking two faces of the N-terminal amphiphilic melittin helix were able to inhibit interactions between anti-S(l-13) antibody and ELISA plates coated with mimicking peptides. This inhibition required a homologous situation, i.e. the segment mimicking the left helical side inhibited left-side-segment but not right-side segment ELISA and vice versa (10). - It is of considerable interest that melittin exhibiting three epitopes seems able to induce an antibody response against virtually its entire potentially antigenic structure. It is further of methodological interest that with one antiserum, ELISA inhibition was significant with all peptides of epitope 21-26, including dipeptide 25-26 and even the C-terminal amino acid unit, provided these inhibitors carried biotinylated SAH at their N-termini (Fig. 3). If this can be substantiated, we may devise programs starting from the 400 possible dipeptides in order to find epitopes in virtually unknown protein chains. Fig. 3. Inhibition of melittin-specific ELISA by peptides of epitope 21-26.

Inhibiting peptide (mol/well) References 1. Mita, H., H. Yasueda, T. Shida. 1984. Agents and Actions 14, 574. 2. Guesdon, J.-L., D. Chevrier, J.-C. Mazie, B. David, S. Avrameas. 1986. J. Immunol. Methods 87_, 69. 3. Peyret, L.M., P. Moreau, J. Dulluc, M. Geffard. 1986. J. Immunol. Methods 90, 39. 4. Windaus, A., W. Vogt. 1907. Chem. Ber. 40, 3091. 5. Kimoto, H., K.L. Kirk, L.A. Cohen. 1978. J. Org. Chem. 43, 3403. 6. Kimoto, H., L.A. Cohen. 1980. J. Org. Chem. 45, 3831. 7. Eckert, H., Y. Kiesel. 1980. Synthesis, 947. 8. Bhatnagar, P.K., D.E. Nitecki, A. Raubitscheck. 1981. In: Peptides (D.H. Rich, E. Gross, eds.). Pierce, Rockford, Illinois, pp.97-100. 9. Rolli, H., C.H. Schneider. 1987. In: Peptides 1986 (D. Theodoropoulos, ed.). de Gruyter, Berlin, pp. 543-547. 10. von Grünigen, R., C.H. Schneider, to be published.

MODIFIED

C-TERMINAL

M.Casaretto,

C3A

PEPTIDE

D.Ambrosius,

ANALOGUES

M.Gier

Deutsches Wollforschungsinstitut Veltmanplatz 8 , D-5100 Aachen West

R.Gerardy-Schahn,

Germany

D.Bitter-Suermann

Universität Mainz, Medizinische Mikrobiologie Augustusplatz, D-6500 Mainz West Germany

D.Saunders Grünenthal

Human

C3a

GmbH,

is

D-5100

an a n a p h y l a t o x i c

inflammatory

processes.

parts

of

active

LGLAR

(J.). I n

with

Aachen

the

our

site

hands

of

activity

to

replace

enhancing

(2).

the

reside

in

NAP,

binding

sensitive

Therefore

the

and

short

NAP

group

by

of

that

pentapeptide

C3a

peptides

in a dramatic

activity

prepared

mediator

indicated

C-terminal of

resulted and

we

factor

investigations

photolabeling

For

light

residue.

Germany

complement

Previous

2-nitro-^-azidophenyl,

se

West

studies, a stabile

a series

of

increa-

we

wished

potencyN-acylated

hexapeptides. The

C-terminal

readily

Arg

cleaved

is

by

essential

serum

for

C-terminally

esterfied

or a m i d a t e d

of

analogues

resistant

producing

vivo All

model

to

activity,

N

We

peptides

but

is

prepared

with

carboxypeptidases

the for

aim in

studies. peptides

purified

by

were

and

by A T P

in

prepared

RP m e d i u m

characterized sis

biological

carboxypeptidase

by

HPLC,

special

release

introduction

was

performed

amino

cases

form

The

liquid

acid

pig

final

resin

The

platelets amino

using

the

on W a n g - r e s i n

chromatography.

analysis,

by F A B - M S .

guinea

of the

on t h e

by F m o c - s t r a t e g y

pressure

acyl

TLC,

activity

They

(4-,5) were

electrophorewas

measured

(6). group

to

corresponding

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

the

peptides

alanine

or

734 The rhodamine-residue was coupled via its carboxyl-group O-benzotriazoyl-tetramethyluronium

hexafluorophosphate,

(_7) • All substituted groups were stabile during

using HBTU,

detachment

form the resin by TFA. -amino hexanoic acid, Ahx, was the spacer giving the best potency. FmocAhxALGLAR was half as potent as NAPAhxALGLAR. The observed increase of activity after arylation might be due to hydrophobic interactions with the receptor or with the membrane.

Table 1 Biological activities of modified C3a peptides

ALGLAR Ac-Ahx-ALGLAR Ahx-ALGLAR Z-Ahx-ALGLAR Fmoc-Aun-ALGLAR Rho-Ahx-ALGLAR Dnp-Ahx-ALGLAR Fmoc-ALGLAR Fmoc-Abu-ALGALR Fmoc-Ahx-ALGLAR NAP-Ahx-ALGLAR ALGLAR-•NhT„ ALGLAR-• OMe Fmoc-ALGLAR-• OMe Fmoc-ALGLAR-•NH2

Concentration for 50% stimulation 6.0 uM 2. 3 uM uM 1 1.1 uM 80.0 nM 60.0 nM 52.0 nM 46,0 nM 37.0 nM 26.5 nM 15.5 nM 260.0 uM 36.0 uM 880.0 nM 600.0 nM

C-terminal modified analogues were prepared

Relative activity 1 2.6 3.5 5.4 75.0 100.0 115.5 130.4 162.1 226.4 392.2 0.02 0.16 6.81 10.0

in solution by

coupling Arg-amide or -methylester to ALGLA. The different strategies are shown in scheme 1, the third proved to be the best one. After amidation of the C-terminus, no

carboxypepti-

dase N digestion of the C-terminal Arg was observed. On the other hand a remarkable decrease of activity was measured. To compensate this effect the Fmoc-group was introduced. Not unexpectedly, the activity increased but never reached the level of Fmoc-Ahx-ALGLAR. These results in combination with our findings that changes in the C-terminal side chain in homo-Arg, citrulline and d-Arg analogues led to a complete lost of activity, prove the essential nature of unchanged Arg-residue at the C-terminus.

735 H-Leu-Gly-Leu-Ala-resin Fmoc-Ala Fmoc-A-L-G-L-A-resin

Fmoc-Ala

Z-Ala

Fmoc-A-L-C-L-A-resln

Z-A-L-G-L-A-resin

1. Piperidine 2 . TFA Fmoc-A-L-G-L-A-OH Arg-X / HBTU Fmoc-A-L-G-L-A-R-X Piperidine A-L-G-L-A-R-X

A-L-G-L-A-OH

Z-A-L-G-L-A-OH Arg-X / HBTU

Boc-O-Boc Boc-A-L-G-L-A-OH

Z-A-L-G-L-A-R-X

Arg-X / HBTU BOC-A-L-G-L-A-R-X

A-L-G-L-A-R-X

TFA A-L-G-L-A-R-X

Scheme 1 : Synthesis of C-terminal modified C3a analogues ( X = -OMe or -NH 2 ) References 1. Caporale, L., P.S. Tippet, B.W. Erickson, T.E. Hugli 1980. J. Biol. Chem. 255, 10758-63. 2. Gerardy-Schahn, R., M. Casaretto, D. Ambrosius, A. Grötzinger, D. Saunders, A. Wollmer, D. Brandenburg, D. BitterSuermann in press 3. Bokisch, V.A., H.J. Müller-Eberhard 1976. J. Clin Invest. _49, 2/f27-39 . A. Atherton, E., C.J. Logan, R.C. Sheppard 1981. J. Chem. Soc. Perk. 538-^6. 5. Wang, S.S. 1973. J. Amer. Chem. Soc. 95, 1328-33. 6. Becker, S., S. Meuer, U. Hadding, D. Bitter-Suermann 1984.. Scan. J. Immunol. _7> 173. 7. Dourtoglou, V., B. Gross 198-4. Synthesis 572-574.

CYCLIC

DISULPHIDE

ANALOGUES

OF

C3a

C-TERMINUS

D. A m b r o s i u s , M. C a s a r e t t o , M. G i e r , H. Zahn Deutsches Wollforschungsinstitut, Veitmanplatz

R. Gerardy-Schahn Med. Mikrobiologie,

J. G r ö t z i n g e r Physiologische

Uni.

Chemie,

Mainz,

RWTH

8,

Augustusp 1 atz,

Aachen,

5100

D. S a u n d e r s , W. S t r a ß b u r g e r Grünenthal GmbH, Forschungszentrum,

5100

5100

6500

Aachen

Mainz

Aachen

Aachen

Introduction

During a

complement

potent

from C3a

the is

mediator

is

then

specific

of

still

programme and

at

have

structure

region

of

N-terminus

located

approaches the

activation,

is to

to use

acute of

the

the

used

(CD,

the

C3a,

but

we

conformation of and

the

cyclic

to

human

based

expected

on

X-ray

strategies

and

solid-phase of

was

via

method.

to

the

by

We this

surface

of a

solution

peptide The

to

fit the

different

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany

with

design

modelling,

with

paper.

C3a,

the

in

bridges.

two

our

develop

seemed

of

Several

active

of

flexible

synthesized report

the

computer

octapeptides been

site

determine

of

investigate analogues

C3a,

generated

(1).

goal

binding

disulphide

supported

Two

in

To C3a

is

active

to

principal

stabilized

have

cyclization

X-ray)

information

C3a.

have

data.

The

small

cyclization

analogues

structure,

polyamide

of

and

The

73-77)

conformation

receptor

structural

initially by

the

(residues

NMR

anaphy1atoxin

reactions,

component.

the

defined.

localize the

antagonist

2D- H-NMR,

third

been

poorly

77-residue

inflammatory

C-terminus

3-dimensiona 1 structure 1

the

the Fmoc-

737

R e s u l t s and

Discussion

T h e s o l i d - p h a s e s y n t h e s e s w e r e c a r r i e d out m a n u a l l y on p o l y a m i d e (2) r e s i n (1.0 m m o l / g ) , f u n c t i o n a l i z e d w i t h an acid labile linkage agent, using Fmoc p r o t e c t e d a m i n o a c i d s and t h e H B T U (3) c o u p l i n g m e t h o d . T h e s i d e - c h a i n of a r g i n i n e was p r o t e c t e d w i t h t h e M t r - r e s i d u e , and t h i o l s w e r e p r o t e c t e d w i t h e i t h e r A c m - or S - B u ^ - r e s i d u e s . T h r e e - f o l d e x c e s s of p r o t e c t e d a m i n o a c i d s , a c t i v a t e d in situ w i t h s t o i c h i o m e t r i c a m o u n t s of H B T U , w e r e used for all c o u p l i n g s . D M A P w a s used as c a t a l y s t d u r i n g t h e e s t e r i f i c a t i n s t e p . T h e s y n t h e s e s of t h e p e p t i d e s I: C ( A c m ) A A L G L C ( A c m ) R ( M t r ) , 11 a: C ( A c m ) A A L C ( A c m ) L A R ( M t r ) and lib: C ( S - B u t ) A A L C ( S - B u t ) L A R ( M t r ) w e r e u n p r o b 1 e m a t i c . All a c y l a t i o n s w e r e c o m p l e t e w i t h i n 30 m i n u t e s . C l e a v a g e and p a r t i a l d e p r o t e c t i o n w a s e f f e c t e d by t r e a t m e n t of t h e r e s i n s w i t h 9 5 % T F A / 5 % a n i s o l e for 5 h at RT. U n d e r t h e s e c o n d i t i o n s , a b o u t 8 0 % of t h e M t r - r e s i d u e s w e r e r e m o v e d , w h e r e a s both t h i o l p r o t e c t i n g g r o u p s (Acm and S - B u ^ ) w e r e s t a b l e . C y c l i z a t i o n of p e p t i d e s I and 11 a: T h e p e p t i d e i n t e r m e d i a t e s w e r e p u r i f i e d by R P - M P L C , d i l u t e d in •3 5 0 % a c e t i c acid to 4 x 10 M and s l o w l y a d d e d to a s t i r r e d s o l u t i o n of I 2 (50 x 1 0 ~ 3 M in a c e t i c a c i d ) . A f t e r 10 m i n . e x c e s s i o d i n e was q u e n c h e d w i t h t h i o s u l p h a t e and t h e p e p t i d e p u r i f i e d by R P - M P L C . T h e p r o d u c t s g a v e a n e g a t i v e E l l m a n a s s a y . T h e y w e r e c h a r a c t e r i z e d by a m i n o acid a n a l y s i s , T L C , HPLC and FAB-MS. C y c l i z a t i o n y i e l d : I: C A A L G L C R : 10% , 11 a: C A A L C L A R ; 16% C y c l i z a t i o n of p e p t i d e lib: T h e S - p r o t e c t i n g g r o u p s w e r e r e m o v e d n e a r l y q u a n t i t a t i v e l y by 100 e q u i v . m e r c a p t o e t h a n o l per S - B u ^ - r e s i d u e at pH 8 . 5 in 1 h at RT. T h e r e d u c e d p e p t i d e w a s p u r i f i e d by R P - M P L C . For c y c l i z a t i o n , d r o p s of 1.0 M s o l u t i o n w e r e a d d e d to t h e 4 p e p t i d e (4 x 1 0 ~ M in 50% a c e t i c a c i d ) u n t i l t h e m i x t u r e r e m a i n l i g h t y e l l o w o v e r 5 m i n u t e s . T h e c y c l i c p e p t i d e was p u r i f i e d by M P L C and a n a l y s e d as a b o v e . C y c l i z a t i o n y i e l d : lib: ¿ A A L C L A R : 31%

738 Using

both

analogues peptides also

were were

Peptide

successful,

the

preparations

although

yields

from

S-S-Bu^-peptides.

under milder

The

of

cyclic

S-Acmlatter

were

conditions.

potencies

I is

and d o e s

groups,

lower than

cyclized

Biological

poor

S-protecting

not

inactive inhibit

agonist,

but

in the p l a t e l e t ATP r e l e a s e a s s a y (4), 1 oc I - C 3 a b i n d i n g . P e p t i d e II is a v e r y

it has t h e

ability

to

desensitizate

p l a t e l e t s in a c o n c e n t r a1t OK i o n r a n g e f r o m 2 uM to 200 uM and to i n h i b i t t h e b i n d i n g of I - g p - C 3 a to g u i n e a pig p l a t e l e t s . The t e r t i a r y mined

structure

in d e t a i l

performed

ROESY

spectroscopy) data

has

not

from

D^O

with

of

antagonistic

NMR

to

CAALCLAR

studies

(rotating

and

analysis

activities

analogue

2D

experiments

been finished

unique

peptides

proton

in b o t h

The c o n f o r m a n t i o n a l of t h e

of t h e

h^O,

at 500

frame

but t h e

will

be

MHz.

Overhauser evaluation

deterWe effect

of

the

date. will

C3a

allow

a better

and will

enable

understanding the

design

activities.

Acknowledgement

This

research

was

supported

by B M F T

Grant

No.

01VM86048.

References 1. C a p o r a l e , L . H . , P . S . 1 9 8 0 . J. B i o l . C h e m .

Tippet, B.W. Erickson, 255, 10758-10763.

T.E.

Hugli.

2. A r s h a d y , R., E. A t h e r t o n , D . L . J . C l i n , R . C . S h e p p a r d . 1 9 8 1 . J. C h e m . S o c . P e r k i n T r a n s . J_, 5 2 9 - 5 3 7 . 3. D o u r t o g l o u ,

V. and

B. G r o s s .

1984.

Synthesis.

572-575.

4 . Z a n k e r , B . , H. R a s o k a , U. H a d d i n g , D. B i t t e r - S u e r m a n n . 1 982. Agent Actions Suppl. 147-1 5 7 .

of

GENERATION OF SPECIFIC ANTIBODIES AGAINST THYMOSIN 04-LIKE PEPTIDES BY IMMUNIZATION WITH N-TERMINAL FRAGMENTS

M. Mihelic, E. Hannappel, H. Kaibacher, W. Voelter Abteilung für Physikalische Biochemie des Physiologischchemischen Instituts der Universität Tübingen, Hoppe-SeylerStr.4, D-7400 Tübingen, F.R.G.

Introduction

Thymosin 04 (43 amino acids, 4982 d) was first isolated from calf

thymus.

species.

In

Its

sequence

mammals

is

tymosin

well 04

conserved

(T04)

second highly homologous peptide,

is

among

several

accompanied

i.e. thymosin 09

by

a

(T09) in

bovine and thymosin 01 o (T0io) in human, rat, murine, cat and rabbit tissues. For instance, thymosin 04 and thymosin 09 (41 amino

acids,

4717

d)

show

a homology

in their

amino

acid

sequence of 78.5% with maximal difference at the N-terminal region, residues 1-14 (64X homolgy). The aim of our study is to provide a model

for generating specific antibodies of one

of

04-like

the

thymosin

peptides

using

the

N-terminal

fragment for immunizaton, since this part of sequence is less conserved

in the

09[1-14] through and

were

fragment

able

to

thymosin tryptic show

crossreact

04-family. digestion

that

less

We

of

antibodies

than

specific antibodies are suitable

IX

generated

isolated

with

raised

thymosin

thymosin 09 against

thymosin

04 .

for immunohistochemical

other immunological studies.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

this Only and

740 Results Isolation minal

of

thymosin

fragment

09 and 04 and generation

thymosin

0a[1-14 J.

The

of

the

isolation

N-ter-

from

calf

thymus was performed according

to the method of Hannappel

2).

artificial

This

procedure

minimizes

isolation and

yielded

thymus.

digestion

After

50 pg T09 of

and

T09

proteolysis

150 ng of T04

with

trypsin

during

per

five

(1, g of

fragments

were obtained, which were separated by reversed-phase HPLC. T09: T04 :

acADKPDLGEINSFDK-AKLKKTETQEKNTLPTKETIEQEKQAK MA EK -S P S GES

S

Fig.l. Amino acid sequence of T09 and T04. T09 is cleaved by trypsin after lysine (K) at position 14 as indicated by the bond is resistant to trypsin. dash. The 3Lys-*Pro Generation

of

respectively,

specific was

limpet

hemocyanine

white

female

New

antisera.

conjugated (KLH)

T09

by

and

or

TPs[1-14]-fragment,

glutaraldehyde

administered

Zealand

rabbits.

to

keyhole

intradermally

After

two

to

booster

injections, the antisera obtained, were purified with DEAE by the batch method before their application. ELISA

procedure

petitive ELISA

and was

the

sensitivity

developed

the T09[1 — 14]-antiserum be

linear

from

2

of

for T09

the antiserum.

and

the

investigated. The

to

200

pmoles

per

ELISA was

assay

detection limit of about 2 pmoles (Fig.2). same

curve

amounts

was

of

the

the affinity

observed

when

T09

[1-14]thymosin

was

A com-

specificity with

of

found to a

lower

Interestingly, the

replaced

fte-fragment

by

which

equimolar shows

that

of antibodies against T09[l-14] is approximately

the same as that of T09 and T09[1-14]-fragment. In fact, this N-terminal

fragment

epitope

T09

of

[1-14]

showing

seems

most

to

represent

differences

in

an

important

comparison

with

TB4 . Cross-reactivity fragment

of

studies.

T09[l-14]

Using

generated

a

KLH

conjugated

antibodies

which

N-terminal crossreact

741

«MO 2o

Fig.2. Displacement curves of TfSg ( O ), T09[1-14] (X ) and T04 ( • ) in a competitive assay with T09 bound to microtiter plates, using anti-T09[114] antibodies.

10J

101

to4

10!

1

nMOlES IN ASSAY

less

than

against

IX

native

T09

show

T($4

is

with T09

TPi . On were

lower sensitivity

observed.

These

other

and

hand,

when

displacement

30-40%

antibodies

for further immunological Competing substance

the

produced,

are

antibodies

studies

cross-reactivity therefore

of no

with with value

studies. Cross- reactivity

Thymosin Rg [1-14] (bovine) Thymosin 139 (bovine) Thymosin Ra (bovine) Thymosin B4 (rat) Oxidized Thymosin Ra (bovine) Tyr1'-Thymosin R4[l~12] (synthetic) Thymosin ai (synthetic) Thymopoietin-pentapeptide (synthetic) Ubiquitin (bovine) Bovine serum albumin Insulin (pig)

100 100 1 1 0. 1 0.5 0 O 0 0 0

(*)

Tab.I. Crossreactivity of various peptides and proteins with anti-T09[1-14] antiserum.

References 1. Hannappel, E. 1986. Anal. Blochem. 156, 390. 2. Hannappel, E., Kaibacher, H. and Voelter, W. 1988. Arch. Biochem. Biophys. 260, 564. 3. Goodal1, G., Hempstead, J.L. and Morgan, J.T. 1983. J. Immunol. 131, 821. 4. Weiler, F.E., Mutchnick, M.G. Goldstein, A.L. and Naylor, P.H., 1988. J. Biol. Response Mod. 7 (1), 91. 5. Mihelic, M., Kaibacher, H., Hannappel, E. and Voelter, W.: J. Immunol. Met.h. (in press).

PROLINE RICH POLYPEPTIDE (PRP) FRAGMENTS AND THEIR IMMUNOREGULATORY PROPERTIES

Z.Szewczuk, I.Z.Siemion, A.Kubik Institute of Chemistry, University of Wroclaw 50-383 Wroclaw, POLAND Z.Wieczorek. K.Spiegel, M.Zimecki, M.Janusz, J.Lisowski Institute of Immunology and Experimental Therapy Polish Academy of Sciences, 53-114, Wroclaw, POLAND

al1'

A proline rich polypeptide (PRP) isolated by Lisowski et from ovine colostrum possesses

a

immunoregu1atory

activity.

The nonapeptide: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro,

obtained

by chymotrypsin digestion of PRP has the same immunoregu1atory properties as PRP

2>

.

The synthesis and immunotropic activity

of partial seguences of

PRP

have

been

previous European Peptide Symposium

3>

.

reported

All

by

known

us

on

synthetic

fragments of PRP were depicted in the Table. The evaluation of their immunotropic

activity

revealed

that

the

fragment of PRP (PRP-hexapeptide, compound 4)

hexapeptide

have

the

same

biological activity as natural PRP. The activity of C-terminal pentapeptide

(10)

is

unsignificantly

shorter fragments (16 and

17)

present now the new analogues PRP-pentapeptide

of

smaller,

not

active

PRP-hexapeptide

all

presented

solution

peptides

methods.

were

Compound

of

the

all. (5-9)

We and

a

linaer

hexapeptide

achieved 6

hexapeptide) was obtained by diazotization and coupling

while at

(11-15).

The synthesis of conventional

are

by

(azo-PRP-

intramolecular

(5)

containing

p-amino-pheny1 alanine in position 5 of the peptide chain.

The

influence

was

of

the

peptides

on

the

immune

response

established by two experimental assays. In the in the increase of the

humoral

immune

response

determined by the number of splenocytes

in

producing

vivo

assay

CBA

mice,

antibodies

against sheep red blood cells (SRBC) was determined. In the in

Peptides 1988 © 1989 Walter de Gruyter& Co., Berlin • N e w York - Printed in Germany

743 vitro assay, the peptides were added into

the

ceil

cultures

with antigen - SRBC together. As the rule, the similar

immune

response were obtained in both assays used. The details of the synthesis and biological tests will be published elsewhere.

Results and discussion

The biological activity of

synthetic

fragments

of

PRP

and

their 10 analogues are summarized in the Table 1 The

synthesized peptides

can be divided

into

3

series

of

analogues: 1.

To investigate the biologically active conformation of PRP

we

synthesized

a

cyclic,

PRP-hexapeptide with the aromatic side chains compound

(5):

semi-rigid

azo-bridge

(6).

The

initial

activity

for

of

of

of

azo-PRP-hexapeptide

the 5 Phe

and

this

synthesys

( [ (4 '-NI^) Phe] PRP-hexapeptide)

biological activity similar to that the

analogue 1 between Tyr

showed

PRP-hexapeptide (6),

(4);

however,

significantly higher then that of (4). This suggests

the was

that

in

the biologically active conformation of (6) both aromatic 5 1 rings (Tyr and Phe ) are situated close to each other. 2.

In order to investigate configurational requirements of 5 Tyr and Phe residues for immunoregu1atory activity of (6) , 1

we synthesized a series of three analogues containing acid in positions 1 and/or 5 (7-9). The

immunological

showed that the configuration of the Phe

is

for

Tyr 1

biological

activity,

whereas

D-amino

for

not

assays

significant residue

the

L-configuration must be preserved. 3.

To determine the role of consecutive amino

acid

residues

in the immunoactivity of the shortest active fragment

of

PRP

(10), a series of analogues substituted by

in

the

successive positions of the

peptide

chain

Analogues substituted in positions 2, 3 active

was

and

deprived of the activity. The substitution positions 1 and 5 resulted in the

L-alanine 4 of

synthesized. (12-14) Ala

compounds

were

into

the

(11

and

744

Table 1. The immunoregulatory potencies of fragments of PRP and their analogues. The positions the peptide chain was modified are underlined. * - described previously.

synthetic in which

Sequence Va 1-G 1u-Ser-Tyr-Va1-Pro- Leu-Phe-Pro Gln-Ser-Tyr-Va 1-Pro- Leu-Phe-Pro Val-Glu-Ser-OMe Tyr-Val-Pro- Leu-Phe-Pro ,NHz Tyr-Val-Pro- Leu-Phe-Pro N • N — — « Tyr-Val-Pro- Leu-Phe-Pro D-Tyr-Va1-Pro- Leu-Phe-Pro Tyr-Val-Pro- Leu-D-Phe-Pro D-Tyr-Va1-Pro -Leu-D-Phe-Pro Val-Pro -Leu-Phe-Pro Ala-Pro -Leu-Phe-Pro Val-Ala -Leu-Phe-Pro Val-Pro -Ala-Phe-Pro Val-Pro -Leu-Ala-Pro Val-Pro -Leu-Phe-Ala Pro -Leu-Phe-Pro Val-Pro -Leu-Phe

15). [Ala5]PRP-pentapeptide (15) was PRP-pentapeptide (10). The loss of the

even

the case of compounds 12-14 points to the 2

residues Pro ; Leu

3

and

Phe*

of

more

activity

active

than

observed

importance

PRP-pentapeptide

of for

in the the

biological effect.

References 1. Janusz, M., J. Lisowski, F. Franek. 1974. FEBS Lett. 49, 267 . 2. Staroäcik, K., M. Janusz, M. Zimecki, Z. Wieczorek, J. Lisowski. 1983. Molec. Immun. 20, 1277. 3. Kubik A., W.A. Kliä, Z. Szewczuk, I.Z. Siemion, M. Janusz, K. Staro^cik, M. Zimecki, J. Lisowski, Z.Wieczorek. 1984. In: Peptides 1984. (U. Ragnarson ed.). Almqvist and Wikseil, Stockholm, p.457.

SPLENOPENTIN DETECTED BY

PEPTIDES: H-NMR

ITS A C Y L A T I O N AND E N Z Y M A T I C

K. F o r n e r , A. E h r l i c h , H.

Niedrich

Academy of S c i e n c e s of GDR, Institute Berlin, DDR-1136 O.L. Isakova, N.F. S e p e t o v , E.K. Academy of M e d i c a l C e n t e r , Moscow

of Drug

Cardiological

factors Thymopoietin

32-36, T h y m o p e n t i n

are able to trigger

Research,

Ruuge

S c i e n c e s USSR. A l l u n i o n s

From the i m m u n o r e g u l a t o r y pentapeptides

and S p l e n i n

(TP5) and S p l e n o p e n t i n

immunostimulation

in vivo attack by s u r r o u n d i n g

and a p p l i c a b i l i t y

is p r o t e c t e d

acetyl d e r i v a t i v e s ,

s t u d i e d to enhance e n z y m a t i c stability should also fullfill

use.

peptide chains. A lot of

thetic a n a l o g s of TP5, including

of

/1/.

use, SP5 in p r e p a r a t i o n for

In the native factors the signal s e q u e n c e

against syn-

has

been

/2/. A c e t y l a t i o n of

some d e m a n d s for better

the

(SP5),

by d i f f e r e n t i a t i o n

T - l y m p h o c y t e s . SP5 s t i m u l a t e s also B - l y m p h o c y t e s TP5 is in p h a r m a c e u t i c a l

DEGRADATION;

SP5

bioavailability

by increasing h y d r o p h o b i c i t y

and

proteolytic

stability. We s y n t h e s i z e d

SP5= A r g - L y s - G l u - V a l - T y r

via mixed a n h y d r i d e s using

s t e p w i s e from

Boc amino acids. After

TyrOBzl

deprotection

of side chains and/or N - t e r m i n a l Boc the a c e t y l a t i o n was successfully

done with AcONB

racemization

and a lot of b y p r o d u c t s ,

(see f o r m u l a ) . A c 2 0 gave see table

1. The

most

tyrosine acetyl-

d e r i v a t i v e s were p u r i f i e d on a m e t h a c r y l i c acid polymer Y 79 in a q u e o u s acetic acid.

Y 79 is a pilot p r o d u c t of

CKB/GDR.

The s t r u c t u r e of all d e r i v a t i v e s has been proved by H-NMR (Bruker W M - 5 0 0

in D 2 0 / K D 2 P 0 4 ,

internal s t a n d a r d

2,2-dimethy1-

2-silapentan-5-S0^Na . ) The acetyl N ? (Arg)

singuletts

are: N 2 , 0 1

2,06 ppm; O(Tyr)

2,31

ppm; N

c

1,95

ppm.

Peptides 1988 © 1989 Walter de G r u y t e r & C o . , Berlin - N e w York -Printed in G e r m a n y

ppm;

746 or

Z-

Boc-Arg(NO?)-Lys(Z)-Glu(OBzl)-Val-Tyr-OBzl

H2/Pd-C

H2/Pd-C

SP5

TF A

side chain p r o t e c t e d

BDC-SP 5

AcONB

Ac2-SP5

1. AcONB

1. AcONB

2. TF A

2. H 2 / P d - C

N£-Ac-SP5

(III)

R1-Arg-Lys-Glu-Val-Tyr I R

2

AcONB: C H , C 0 0 - N ^

(II)

N -Ac-SP5

(I)

I

R^ = CH-j-CG ; R 2 = H

II

R 1 = H, R 2 = C H 3 - C O

III

R ^ = CH3-CO;

R2 = CH3-CO

\

Table 1: B y p r o d u c t s of a c e t y l a t i o n of s p l e n o p e n t i n area percent of hplc peaks Compound

N*,

N £ '-Ac 2 -SP5 5

D-Tyr -Ac9-SP5

30 mol Ac„0 in HOAc 80 °C 20 °C 30 min 2 h

+ 4 mol AcONB 20 °C 2 h

29 , 2

62,0

93,7

14,4

12,0

•c 1

7,9

2-Z group has a high potential for protection schemes requiring more than two levels of protection. It can be safely used even under strong acidolytic conditions (HF) for the obtention of N-protected fragments or specifically cyclyzed peptides. Deprotection is easily achieved under mild conditions (I^-Pd/C or Zn-HOAc) even in presence of protected sulfhydryls. N-terminal tachykinin immunogens for SP, NKA and NKB have been obtained which all produce polyclonal sera free of any detectable cross-reactivity between these three tachykinins. Acknowledgements This work has been supported by grants from the Medical Research Council of Canada. E.E. is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Québec. We thank Mrs Couture for the preparation of this report. References 1. von Euler, U.S., J.H. Gaddum. 1931. J. Physiol. 72, 74. 2. Kimura, S., M. Okada, Y. Sugita, I. Kanazawa, E. Munekata. 1983. Proc. Jpn. Acad. Sci. B59, 101. 3. Kangawa, K., N. Minamimo, A. Fukuda, H. Matsuo. 1983. Biochem. Biophys. Res. Comm. 114, 533. 4. Nyberg, F., P. le Grevés, L. Terenius. 1985. Proc. Natl. Acad. Sci. (USA) 82, 3921. 5. Nakanishi, S. 1987. Physiol. Rev. 67, 1117. 6. Shields, J.E., W.M. McGregor, F.H. Carpenter. 1961. J. Org. Chem. 26, 1496. 7. Neugebauer, W., G. Champagne, M.-R. Lefebvre, E. Escher. 1988. In: Peptides 1987 (G.R. Marshall ed.) ESCOM, Leiden, Netherlands, p. 252. 8. Escher, E„ M. Bernier, P. Parent. 1983. Helv. Chim. Acta. 66, 1355. 9. Semenenko, F.M., S. Brainwell, E. Sidebottom, A.C. Cuello. 1985. Histochemistry 83, 405.

ANTIGENIC PROPERTIES OF PEPTIDES BOUND TO T-70 DEXTRAN AND ALBUMIN

P.G. Pietta, D. Agnellini, M. Pace, P.L. Mauri Dip. Scienze e Tecnologie Bicmediche, Via Celoria 2, 20133 Milano, I E. Manera 1st. Chimica degli ormoni, CNR, Via Bianco 9, 20131 Milano, I S. Cinquanta CNR, c/o 1st. Patologia vegetale, Via Celoria 2, 20133 Milano, I Introduction In recent years there has been considerable interest in the possibility of using synthetic peptides as vaccines against infectious agents and toxins (1). The necessary requisite for producing antibodies to peptides of low molecular weight is their coupling to protein carriers by means of watersoluble carbodiimides (2) or bifunctional agents (3). Nevertheless, this approach involves a random linkage as well as peptide modification. There fore, the development of new techniques for peptide coupling to polymeric carriers still remains a field of interest. This prompted us to immobilize a number of synthetic peptides to T-70 dextran, which is a polysaccharide used as a blood volume expander. The coupling was first carried out by means of CNBr-NaOH or CNBr-TEA (4) using the model peptides Gly-Phe, ValGly-Ser-Glu, Tyr-Lys and Tyr-Ser-Lys. This orocedure was abandoned due to the easy formation of dextran gels and concurrent reaction on tyrosine by the CNBr-TEA method. The sequences 24-37 of rat calcitonin gene related peptide (rCGRP) (5) and 92-101 of lectin-like protein (LLP) (6) were then synthesized by SPPS and coupled to T-70 dextran dialdehyde. The same peptides were bound to albumin by glutaraldehyde activation to yield conjugates, whose antigenic properties were compared with those of dextran-derivatives.

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

763 Results The pentadecapeptideamide (Tyr)-Lys-Asp-Asn-Phe-Val-Pro-Thr-Asn-Val-GlySer-Glu-Ala-PheNH2 (rCGRP) and the endecapeptide (Tyr)-Arg-Thr-His-Arg-Gln -Ala-Asn-Ser-Ala-ValOH (LLP) were synthesized by the step-wise solid phase nethod (7) . The side chain blocking groups on the Na-t-butyloxycarbonyl-a minoacids were benzyl based. The starting supports were the p-methylbenzh^ drylamine and the chloromethyl resins for rCGHP and LLP peptides, respecti vely. Coupling was DCC mediated except for BOC-Asn, which was attached via p-nitrophenylester. Each step was monitored by the ninhydrin test (8). Fi nal deprotection and cleavage frcm the resins was performed by HF treatment (9). Peptide purification was achieved by gel-filtration, ion-exchange and semi-preparative HPLC. Each peptide was bound to T-70 dextran activated by oxidation with sodium periodate (10) and the resulting derivatives were dialyzed before further characterization or use in immunization. The capacity ranged within 30-50 ymol of peptide/g of dextran. The peptide-albumin conjugates were prepared via glutaraldehyde (11). Antisera were obtained in rabbits by a series of 4 weekly injections emulsi^ fied with complete Freund's adjuvant and bleeded after a week from the last. Antibodies were purified according to the Rivanol method (12). Eventual an tibodies produced by bovine serum albumin were removed by affinity chromatography on a BSA-coated Sepharose 4B column and the antibodies against the peptide derivatives were linked to peroxidase by means of periodate (13). For an extended storage the derivatives were kept frozen in working aliquots and each sample was assayed by ELISA using o-phenylenediamine as substrate. The working dilution of each stock of enzyme-antibody conjugate in the ELI SA assay was 1:100 and the reaction lasted 30 minutes. The results obtained with rCGRP , LLP and their derivatives with dextran and BSA are shown in Fig.l. The data suggest that peptide derivatives produce more powerful antibodies than the peptide itself. In particular, a ccmparable trend among the dextran and the BSA derivatives can be observed.

764

Fig. 1. Comparison of the immunoreactivities of rabbit antisera raised against pure peptides and their derivatives. The data represent the mean of four determinations.

References 1. Steward, M.W., C.R. Hcward. 1987. Emtunol. Today 8, 51. 2. Staras, J.V., D.M. Swingle, R. Wright, P.S.R. Anjaneyulu. 1986. Proti des Biol. Fluids 34, 39. 3. Erlanger, B.F. 1980. In : Meth. Enzymol. (H. Van Kunakis and J.J. Lango ne, eds.). Vol. 70, p. 85, Academic Press, New York. 4. Kohn, J., M. Wilchek. 1982. Biochim. Biophys. Res. Commun. 1D7, 878. 5. Rossenfeld, M.G., J.J. Mermod, S.G. Amata, L.W. Swanson, P.E. Sawchenko, J. Rivier, W.W. Vale, R. Evans. 1983. Nature 304, 129. 6. Hoffman, L.M., Y. Ma, R.F. Barker. 1982. Nucleic Acid Res. 10, 7819. 7. Stewart, J.M., S.D. Young. 1984. In: Solid phase peptide synthesis, 2nd Edition, Pierce Chemical Co., Rockford, 111. 8. Sarin, V.K., S.B.H. Kent, R.B. Merrifield. 1981. Anal. Biochem. 1T7, 147. 9. Tarn, J.P., W.F. Heath, R.B. Merrifield. 1983. J. Amer. Chem. Soc. 105, 6442. 10. Horejsi, V. 1984. In: Meth. Enzymol. (W.B. Jakoby, ed.), Vol. 104, p. 275, Academic Press, New York. 11. Avrameas, S.,T. Ternynck, J.L. Guldson. 1978. Scand. J. Immun. 8_, 7. 12. Horejsi, J., R. Smeatna. 1956. Acta Med. Scand. 155, 65. 13. Wilson, M.B., P.K. Nakane. 1978. In: Immunofluoroscence and related techniques, Elsevier/North Holland Biomedical Press, Amsterdam, p. 215.

IMMUNOCHEMISTRY OF POLARINS AND THEIR ANALOGUES

G. Chipens, R. Vegners, S. Skliarova, I. Perkone, L. Gnilomedova, Yu. Darinsky, I. Artemiev, D. Dorin Institute of Organic Synthesis, Latvian SSR Academy of Sciences, Riga, USSR, 226006

It has been assumed that, apart from the two known systems of bioregulation acting on the basis of peptides and proteins (viz. hormones and kinins), a third system exists in the organism, its main acting principles being represented by oligopeptides (cell hormones or tetines) formed from their precursors (growth factors, cytokines, hormones, etc.) in reactions of limited proteolysis in the vicinity of receptor systems (1-3). The present communication describes the structures and biological effects of oligopeptides composed of polar amino acids (polarins representing as tetine subgroup). The clusters of polar and hydrophilic amino acids present in peptides and proteins are highly informative (2). Polarins of various structure acting as natural bioregulators can be released from them in response to proteases. Oligopeptides were synthesized by the solid-phase method using Boc-amino acids and chloromethylated polystyrene resin (1% cross-linkage). The peptides were purified by HPLC. The effect of peptides on the survival of mice was evaluated as described in (4). Bioelectric activity in the hypothalamus was measured electrophysiologically with the aid of inserted gold electrodes. The activity of electrosensitive ionic channels was performed by potential fixation on neuronal somatic membrane. Polarins of the SKD (Ser-Lys-Asp) type often occurring in the molecules of neuro- and immunoactive peptides/proteins

were

chosen as model compounds. Given below are the structures of

Peptides 1988 © 1989 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany

766

SKD SKE TKE SKDK SKDT SKDE

V W VVVVVWWVvVVAWI ^ VVVVWM TTTI AVWWI S3 î

s KD SKE TKE SKDK SKDT SKDE

Control 3

Cbntrol AFC 60% 40 Fig.1. Survival (%) of mice Fig.2. Effect of oligopeptides infected with a lethal dose on the primary immune response of influenza virus (type A). of mice. Peptides were adminiAs single dose (0.1 mg/kg) of stered i.p. in the 0.1 jag/kg dose. The number of antibodypeptides were administered forming cells (AFC) was counted i.p. 30 min prior to infection. on the 7th day. 20

Current amplitude,% 140

Hrs after administration 24 24 3 3 JWWWW-A'AVAWVVs's'AVVA'J2

1

2 wuuuuuuuuwwt 1 1

100

200

3001

Fig.3. Increase in bioelectric potential (%) in subcortical structures of the rabbit brain in response to i.v. administration of SKD in the 0.1 mg/kg dose . I l- control; H - nucleus raphe centralis ; ES - hippocampus

Fig.4. Effect of SKD on electrosensitive ionic channels in neuronal somatic membrane of mollusc Lymnaea stagnalis. C^ - molar concentration of peptide ; 1 - sodium current; 2 - delayed potassium current.

767

synthesized peptides, their optical rotation at 20°C (c= 1 ; 1 M CH^COOH), electrophoretic mobility in paper electrophoresis (1 M CH3COOH) relative to His and TLC mobility in the chloroform : acetic acid : water (5 : 6 : 2 )system: SKD, -12.3, 0.91, 0.16} SKE, -1 7.4 , 0.83 , 0.23 ; TKE , -1 8 . 4 , 0 . 84 , 0 . 21 ; SKDE, -27 . 1 , 0 . 89 , 0.17. SKDK, -24.2 , 1 . 04 , 0. 1 9; SKDT, -23.3, 0 .87 , 0.1 5. The tripeptide SKD was found to exert a wide spectrum of biological effects. It prevents the death of mice infected with influenza virus (Fig. 1). This effect is structure-dependent. SKD and several its analogues are capable of decreasing the number of antibody-forming cells in mouse spleen (Fig. 2). Intravenous administration of SKD to rabbits brings about shifts (observation period - 24 hrs) of biological activity in several subcortical brain structures including nucleus raphe centralis and hippocampus (Fig. 3) . The effect reaches its maximum

in 2-3 hrs after peptide injection. SKD affects

sodium and potassium currents in nerve cells (Fig. 4), consequently, SKD can modulate the cell excitability. Presently, we are exploring models postulating that the activation of T-lymphocytes is effected by fragments of antigenic determinants represented by polarins and/or segmental amphiphilic oligopeptides that derive from immunogenic peptides in limited proteolysis reactions upon interaction of antigenpresenting cells with T-lymphocytes, i.e. after antigen processing.

References 1. Chipens, G. 1985. Surv.immunol.Res. A, 220. 2. Chipens, G. , R. Vegners, N. Ieviçia, G. Rosenthal. 1 986. Immunol. Res. 5^, 314. 3. Chipens, G.; Adv.Drug. Delivery Rev. (in press) 4. Sethi, K.K., Y. Omata, K.E. Schneweis. 1983. J.Gen.Virol. 64, 443.

Author Index

A

Aasmul-Olsen, S. 271,280 A b b a d i , A . 507 A d a m s , S.P. 580 Agnellini, D . 762 A i m o t o , S. 25 A l a k h o v , Y u . B . 286 A l b e r i c i o , F. 160 Aleksiev, B . V . 310 A l m d a l , K . 106 A l - O b e i d i , F. 598 A l o u f , J.E. 713 A m a r a i Trigo, M J . A . 82 Ambrosius, D . 733,736 Andersen, A J . 280 Andersson, L . 256 A n d r e u , D . 363 A n d r e w s , W . 483 Angliker, H . 405 A n w e r , M . K . 646 A p p e l , J . R . 217 A r a i , H . 58 Ariyoshi, Y . 652 A r n o l d , Z.S. 622 A r r o w s m i t h , R J . 393 A r s e n i e v , A . S . 471 A r t e m i e v , I . 765 A t h e r t o n , E. 619 A u b r y , A . 495,507

B

Bahraoui, E. 724,727 Bairaktari, E. 513 Baizman, E. 292 Baker, P . A . 163 Baiar am, P. 477 Balaspiri, L . 184 Balboni, G . 631 Bankowski, K . 552 Bannwarth, W . 37 Baranov, V . l . 286 Barbato, G . 450 Barbier, B . 423 Bardi, R . 477 Barsukov, I . L . 471 Bartfai, T . 223 Barth, A . 637 Barth, T . 546 Bartosz-Bechowski, H . 649

Batz, H . - G . 754 Baumann, J.B. 673 Bavoso, A . 465 Bayer, E. 61,109,199,316,390 Beck, A . 586 Becker, G . 103,157 Beckmann, J. 381 Bednarova, L . 516 Beißwenger, R . 462 Belté, I . 571 Beiton, P. 619 Benedetti, E. 447,453,465 Benkoulouche, M . 525 Bennett, C.D. 190 Bennich, H. 121 Benoiton, N . L . 43,46 Benovitz, D.E. 634 Berendsen, H J . C . 438 Berg, R . H . 196 Bernath, E. 676 Bessler, W . 686 Betins, J. 429 Beyermann, M . 28,205 Bienert, M . 28,205 Biesert, L. 686 B y l , W . A . A . J . 175 Bingcheng, L . 109 Biondi, F. 322 Birlirakis, N . 495,498,504,531 Birr, C. 103,157,235 Bitter-Suermann, D . 733 Bökönyi, G. 655 Bladon, C . M . 595 Bläha, K . 516 Blanchard, J.C. 319 Blaney, J. 438 Blanot, D. 348 Blomberg, J. 676 Blout, E.R. 453 B51tz, T . 686 B o d o , B . 351,354 Böldicke, T h . 220 Bornas, H . G . 363 Borea, P. 631 Born, I. 637 Bour, P. 516 Bousquet, Y . 577 Boussard, G . 507 Bovermann, G . 748 Bracci, L . 705 Brack, A . 423,718 Bradaczek, H . 19 Brady, S.F. 190,574

770 B r a n d e n b u r g , D . 307 B r a u n , R . 310 B r e i p o h l , G. 130 B r i e h e r , W . 549 Brocks, D. 402 B r ü c k n e r , H. 298 B r u g n o l o t t i , M . 589 B r u n g s , P. 79 B r u n n e , R . M . 459 B r y a n t , K J . 244 B u k u , A . 550 B u r k h a r d t , F J . 098 Burks, T.F. 010,034,043,040 Burton, J. 417,510 B u s q u e t s , M . A . 325 B y c r o f t , B . W . 340 Bystrov, V.F. 471

c

Carelli, C. 751 Carpino, L.A. 28 C a s a r e t t o , M . 733,730 Castro, B . 384,751 Castrucci, A . M . 598 Cerny, B . 540 Cerowsky, V . 205 C h a s s a i n g , G. 489 C h a t u r v e d i , D . 31 Chen, F.M.F. 43,40 C h e n , Z. 01 C h i n o , N . 100 C h i p e n s , G. 705 C h o r e v , M . 583 Ciccarone, T . M . 190,574 CiesSa, K. 414 Cinquanta, S. 702 C i o m e i , M . 589 C l a p e s , P. 208 Coffey, A . F . 202 C o h e n , K . A . 098 Collins, J . 220 C o m o g l i o , P. 589 C o n v e r t , O. 489 C o o k , R . M . 187 C o r d o p a t i s , P. 4 9 2 , 5 7 1 Corvol, P. 751 C o s t a , T. 028 C o t t o n , R. 019 Craig, A . G . 121 Crisma, M. 4 0 2 , 4 0 5 , 4 7 7 Cross, B . A . 007 C r o u c h , R . 483 Cuello, A . C . 759

C u n g , M . T . 513,528

D

D ' A l a g n i , M. 450 D ' A m b r o s i o , C.A. 4 4 1 D a n n , J . G . 393 Darby, N J . 372 Darinsky, Yu. 705 Darke, P. 190 Darlak, K. 034,040 D a r m a n , P. 598 D a u m a s , P. 474 D'Auria, G. 450 Davies, D . E . 393 Davies, J . S . 408 Davis, P. 010 D a v o u s t , D . 354 D e b e r , C . M . 300 de Castiglione, R . 88,589 D e Cock, E. 502 Defendini, M.L. 727 D e g e l a e n , J . 502 de Haas, G.H. 007 D e l m a s , A. 710 Derdowska, I. 707 Di Bello, C. 109 D i Biasio, B . 447,453,405 Diesis, E. 112 Dive, V . 399 D o m b o , B . 154,157 Dor, A. 319 Dorin, D . 705 D r o z d z , R. 073 Druilhe, P. 718 D u b e a u x , C. 718 D u c e p p e , J . S . 577 Duclohier, H. 357 Dufourcq, J . 713 Dukor, R . K . 519 D u n b a r , J . B . , Jr. 295,408 D u p l a a , H. 489 D u r i e u x , C. 319 D y l i o n Colton, C. 574

E

Eberle, A . N . 073 Eberle, I. 94 Echner, H. 181 Eckstein, H. 124 Eggena, P. 550 Ehrlich, A. 745 Eichler, J . 205,232,543,540

771 El A y e b , M . 727 El Hajji, M . 354 Elliott, P. 750 EngstrSm, E n t i a n , K . D . 309 E p p r e c h t , T . 420 E p t o n , R . 103,202 Erchegyi, J . 40 Erfle, V . 0 8 3 Escher, E. 750 E s n a - A s h a r i , A . 208 E t z o l d , G. 130 Ewenson, A. 411

F

Fahrenholz, F. 435,534,540 Fehrentz, J . A . 751 F e l d m a n n , A. 3 8 1 Felix, A . M . 001 Feliz, M . 522 Ferrandon, P. 5 0 2 Fischer, W . 04 Flerko, B . 004 F l o u r e t , G. 540 F o g d e n , Y . C . 303 Fok, K . F . 580 Folkers, G. 005 Fonteccila-Camps, J . C . 724 F o n t e h , A . N . 070 Forino, R . 88 Formaggio, F. 040 Forner, K. 745 F o u r q u e t , P. 724 Franke, P. 130 Franklin, K J . 571 Frank, R . 2 2 0 , 2 4 1 Freitas, A . M . 13 Freund, S. 005 Fridkin, M . 52 Friedrich, K. 220 F r o m a g e o t , P. 721 Fry, D . 0 0 1 Fiyii, N . 58 Fujita, H. 025 Funakoshi, S. 58

G

Gaida, W . 586 Galantino, M . 88 G a l e n , F . X . 751 Ganter, R.C. 571 G a r c i a - A n t o n , J . M . 208,325,480

Garsky, V . M . 100 G a s s m a n n , R. 103 Gatineau, E. 721 G a t t n e r , H.-G. 202 Gaul, H. 4 0 2 Gausepohl, H. 241 Gauthier, J . 577 Gazis, D . 537,550 Geiger, G. 130 Geiger, R. 4 0 2 Gelfanov, V . M . 002 Geoffre, S. 525 Geoffroy, C. 713 Gerardy-Schahn, R . 733,730 Gerothanassis, I. 408,504,531 Gesquiere, J . C . 112 Gibbons, W . A . 070 Gier, M . 733,730 Giles, M . B . 010 Gillessen, D . 37 Gilon, C. 4 1 1 , 5 8 3 Ginzel, K . - D . 70 Giralt, E. 100,522 Girard, J . 0 7 3 Glass, J . 537 Gnilomedova, L. 705 Gûnzler, V . 4 0 2 Goghari, M. 577 Golovinsky, E. 307 G o m e s , M J . R . 82 Gondol, D . 480 Go, N . 420 G o o d m a n , M. 432 G o o d p a s t u r e , J . C . 502 G o r d o n , T. 202 Gôrôg, S. 40 Gosteli, J. 133 Gozzini, L. 100,580 Graf, L. 040 Grandas, A. 100 Granier, C. 724,727 Granitza, D . 28 Gras-Masse, H. 718 Grechyaninova, L.A. 002 G r e e n , J. 172 Grellier, P. 378 Grogg, P. 133 G r o u t , R J . 340 Grötzinger, J . 730 Gruaz-Guyon, A. 710 Gruszecka, M. 10 Gruszecki, W . 10 Grzonka, Z. 435,540,034

772 G u l y a s , T. 656 G u n n a r s s o n , K. 76 Gfinzler, V . 316 G u t t e , B . 420

H

H ä b i c h , R . 199 Hackenberg, M . 534,540 Hatjjidakis, I. 513 Hadley, M . E . 598 H a i b o t , N . 715 Halstrom, J. 601 H a m a d a , Y . 340 H a m i l t o n , E.A. 244 H a n a n i , M . 583 H a n n a p p e l , E. 730 H a n s e n , G. 608 H a n s e n , P. 202 Haro, I. 325,486 Harris, C J . 3 0 3 Harris, K. 608 H a r t r o d t , B. 6 3 7 H a s h i m o t o , C. 313 H ä u p k e , K. 106 H a u p t , A. 604 H a u p t , E . T . K . 450 H a u s s n e r , M . 28 H ü b n e r , C. 754 H e a r n , M . T . W . 007 H e h l g a n s , T. 420 H e i m e r , E.P. 001 H e i t z , A . 384,751 H e i t z , F. 474,751 H e l l s t e r n , H. 100 H e m m a s i , B . 01,310 H e n d r i x , B . M . M . 118 H e n k e , S. 4 0 2 H e p p , J . 010 H e r b o r n , C. 70 Herenyi, B. 40 H e r s p e r g e r , R . 103 Herz, A . 028 H e s s , G. 605 Höflacher, B . 6 7 0 , 6 8 3 H l a v ä c e k , J . 265 H . M . H a n a u s k e - A b e l , H . M . 310 H ö f l e , G. 100 H o j o , H. 25 H o l m , A . 100,208 H o n d a , M . 052 Hondrelis, J . 4 0 2 , 5 7 1 H o n g , A . 100 H o r i n o , H. 145

H o r m , M. 235 H o r t o n , J . 010 H o r v a t h , A. 655 H o r v a t h , J . 604 Horvat, J . 328 Horvat, S. 328 Hospital, M . 525 Ho, T.L. 502 H o u e n , G. 271 H o u g h t e n , R . A . 214,217 Hruby, V J . 508,616,043 H u d e c z , F. 701 H u d s o n , D . 187,211 H u m m e l , R.-P. 086

I

Iguchi, S. 64 I m m e r , H. 04 Isakova, O.L. 745 Ivanov, V . T . 602 Izdebski, J . 10

J

Jacquier, R . 22 Jakubke, H.-D. 247,250 J a n s , D . 534 J a n s s e n , W . P . A . 118 Janusz, M. 742 Jaramillo, J . 577 Jerobek-Sandow, G. 334 J o h n s o n , T. 103,202 J o h n s t o n , P.D. 178 J o n e s , D . M . 306 J o n e s , G.E. 408 J o n e s , H J . 280 J o n e s , R . M . L . 274 J ö r g e n s e n , K.H. 001 J u n g , G. 300,360,462,586,686,605 Jungfleisch, E. 73 Jurzak, M. 534

K

Kai, K. 145 Kaibacher, H. 73,730 K a l e t t a , C. 360 K a m b e r , B . 115 Kaminski, Z J . 208 K a n o u , K. 343 K a p t e i n , R. 438 K a p u r n i o t u , A. 07 Karagiannis, K. 01 Karayannis, T. 408,531

773 Kasprzykowska, R . 304 Kasprzykowski, F . 540 K a u f m a n n , K . - D . 106,260 Kazmierski, W . 643 Keiderling, T . A . 510 Keifer, D . 202 K e l e m e n , G . 127 Kellner, R . 366,360 Kelly, P J . 178 Kempny, M . 414 K e n t , S . B . H . 283 K e r i , G . 655 K e r s c h e r , L. 754 Kessler, H. 3 3 1 , 4 3 8 , 6 6 4 K i m u r a , T . 55,100 K i m u r a , Y . 343 Kirilov, M . 310 Kirstgen, R . 148 K i s a r a , K . 625 Kisfaludy, L. 40 Kiso, Y . 55 K i t a j i m a , H. 628 Kivirikko, K . I . 402 Klasse, P . - J . 676 Klauser, S. 420 K l e i n , C. 754 Klis, W . A . 552 Knolle, J . 136 K n o r r , R . 37 Kobayashi, M . 426 Kobayashi, Y . 426 K o d a m a , H. 628 Kojro, E. 435,534 K o l a r , C. 3 3 4 Kolodziejczyk, A . M . 43 K o n d o , M . 628 König, W . 334 Konopinska, D . 640 Kopina, N. 85 Koppenhoefer, B . 100 Kopple, K . D . 4 4 1 K o s c h , W . 154 K o t t e n h a h n , M . 331 Kovacs, K . , 1 8 4 , 6 6 1 Kovacs, M . 604 K o y a m a , S. 426 K o p p e n , H. 586 K r a f t , M . 241 K r a f t , R . 130 K r a u s e , E . 250 K r c h n ä k , V . 232 K r i e t e r , P. 580 K r u g , M . 605

Kruszynski, M . 552 Krzyzanowski, L. 4 1 4 Kubik, A. 742 K u b o , S. 100 Kucharski, A. 247 K ü h n e , S. 208 Kunz, H. 154,157,754 Kupryszewski, G . 707 Kurucz, I. 701 Kürz, L. 103 Kuwata, S , 301 Kyogoku, Y . 426

L

Lambert, P . F . 178 Lambros, T J . 6 0 1 Lammek, B . 552,707 Landavazo, A. 483 Langen, H. 420 Langer, M . 298 Langkjaer, L. 658 Lang, R . 586 Längs, D . A . 468 Lankhof, H. 226 Lankiewicz, L. 540 Lautz, J . 438 Lavallee, P. 577 Lavielle, S. 489 Lawton, P. 378 Lazaro, R . 474 Leban, J J . 483 Lebl, M . 205,232,543,546 Leckie, B J . 306 Lee, Y . 46 Leibfritz, D . 450 Lemieux, C. 613 Le-Nguyen, D. 384 Leplawy, M . T . 208,468 Leplawy, T . , J r . 253 Le R o u x , P. 348 Lesicki, A. 640 Lewall, B . 70 Liberek, B . 304 Liebmann, C. 568,637 Lifferth, A. 103 Lindenberg, G . 121 Linden, M . 420 Link, P. 07 Lisowski, J . 742 Liu, C . F . 751 Lockey, P . M . 346 Lombardi, A. 447 Londono, A. 718

774 Lonovics, J. 184 Lorenzi, G.P. 447 Lozzi, L . 705 Lucente, 6 . 460 L y n a m , N . 214

M

Mackiewicz, Z. 707 Madison, V . 601 M a e g a w a , C. 25 M a g y a r , A . 375,610 M a h a n , K . 548 M a i a , H.L.S. 13,456 M a j a m a a , K . 316 M a j e r , P. 265 Makara, G . B . 604 M a l o n , P. 516 M a n e r a , P.L. 762 M a n n i n g , M . 552 M a p e l l i , C . 646 Marastoni, M . 631 Markussen, J . 658 M a r r a u d , M . 495,498,504,507,513 Marseigne, I . 319 Marshall, G . R . 295,468 Marshall, K . W . 295 Mascagni, P. 283 M a t a - A l v a r e z , J. 268 Matsoukas, J . 492,571 M a t s u m o t o , T . 337 M a u r i , P.L. 762 M a y e r , R . 378 M a y w a l d , F. 220 M a z a l e y r a t , J.P. 387 M c B r i d e , K . 670 M c D e r m e d , J. 483 M c h a r f i , M . 507 M c K a y , F. 292 M c V i t t i e , L . T . 244 Medzihradszky, D . 375,610,640 Medzihradszky-Schweiger, H . 375 Mehlich, A . 381 M e l d a l , M . 208 M e l i n , P. 540 M e l o e n , R . H . 226 M e n a , R . 589 M é n e z , A . 721 Mengin-Lecreulx, D . 348 M e n t z , P. 637 M e n y h a r t , P.E. 184 M e r g l e r , M . 133 M e r r i f ì e l d , B . 196,363 M e z o , G . 701

M e z 5 , I. 604 Michel, A . 715 Michel, J.B. 751 Mierke, D . F . 432 Mihelic, M . 739 M i m o t o , T . 55 Minchev, St. 310 Minoshima, Y . 652 Mitchell, R . 595 M i t i n , Y u . V . 250 Miyazawa, T . 301 M o d r o w , S. 679,683 Molinari, I . 589 M o l l e , G . 357 Monsigny, M . 378 Montagne, J.-J. 387 M o o r e , G J . 571 M o o r e , G . 492 M o r g a n , B . 292 M o r g a t , J . L . 399 M o r o d e r , L . 748 M o r t o n , J . A . 393 M o r t o n , J - I . 396 Moser, E. 94 M o t t a , A . 501 Mourier, G . 721 Mowles, T . F . 601 Munekata, E. 652 Muschalek, V . 109 M u t t e r , M . 193 Muyshont, D . 715 M y l l y l a , R . 316,402

N

Naithani, V . K . 262 Nakamura, M . 652 Nakao, K . 340 Nakao, M . 301 Nalis, D . 384 N e d e v , H . N . 310 N e r i , P. 705 Nestor, J J . , Jr. 592 N e u b e r t , K . 637 Neugebauer, W . 759 Ng, F. 607 N g u y e n , O. 151 N g u y e n , T . M . - D . 613 Nguyen-Trong, H . 157 Nicklin, M J . H . 698 Nickolayev, A . 85 Nicolas, P. 721 Niedrich, H . 259,745 Nikiforovich, G . V . 429

775 N i o , N . 052 N o k i h a r a , K . 100 N o r r i s , K . 658 N o v a k , C . 235 N o v o t n y , J . 724 N o w o s l a w s k i , A . 707 N u t t , E . M . 100 N u t t , R . F . 190,574 N y é k i , O . 40 N y e r g e s , L. 142 Nyfeler, R . 133

o

O ' D o n o g h u e , M . F . 007 O f i o r d , R . E . 274 Ogden, H. 303 O g i e r , S.-A. 595 O g u n j o b i , O . M . 172 O h k u b o , T . 426 O k a d a , Y . 04 O k a m a c h i , A . 58 O l i n s , G . M . 580 O l m a , A . 552 Orchison, J . 408 Oren, D.A. 411 O r l o w s k a , A . 16 O r m b e r g , J . 31 O t a k a , A . 58 O t t o , A . 130 O v o d o v , S . Y u . 286 O y a m a d a , H . 145

P

P a c e , M . 702 P a l l a d i n o , D . E . H . 098 P a l l a i , P . V . 698 P a n c o s k a , P . 516 P a o l i l l o , L. 450 P a p a d o p o u l u s , A . 79 P a p a d o u l i , I. 513 P a p a i o a n n o u , D. 91 P a r o l a r o , D . 589 P a t t a r o n i , C . 432 P a u l , P . K . 477 Pauly, R. 313 P à v ó , I . 534 P a v o n e , V . 447,453,465 P a w e l c z a k , K . 414 P d o u s s a u t , S. 710 P e d e r s e n , W . B . 196 P e d o n e , C. 447,453,465 P e d r o s o , E . 160

P e d y c z a k , A . 417,510 P e e t e r s , J . M . 226 P e g g i o n , E . 322 Pelerin, J . P . 423 P e l z e r , H . 757 P e n k e , B . 67,142 P e n r o s e , A J . 340 P e p e r m a n s , H . 480 P e r k o n e , I . 765 P e t r e n i , S. 705 P é v e r e , V . 22 Piazzesi, A . M . 477 P i c a r d , I . 378 P i c h o n - P e s m e , V . 507 Picone, D. 501 P i e r r e , P . 710 P i e t t a , P . G . 762 P i n i l l a , C. 217 P i n n e n , F . 450 P i p k o r n , R . 676 P i r k o v â , J . 265 P l a n k e n h o r n , H . 402 P o n s , M . 522 P o t a m i a n o s , S. 5 1 3 P o t i e r , P. 313 P o u l o s , C . 34 P r a k a s h , O. 616 P r e c i g o u x , G . 525 P r o u d f o o t , A.E.I. 283 P r z y b y l s k i , J . 552 P u y k , W . C . 226

R

R a e , I . D . 607 R a g n a r s s o n , U . 76 R a k h i t , S. 577 R a m a g e , R . 34,172 R a n j & l a h y - R a s o i o a r i j a o , L. 474 R a p p , W . 199,390 R ü b s a m e n - W a i g m a n n , H . 686 R e b o u d - R e v a u x , M . 387 R e b u f i a t , S. 351,354 Redlinski, A.S. 468 R e i b a u d , M . 319 R e i c h l e , K . 01 R e i g , F . 208,325,486 R e i s , M . 369 R e i s s m a n n , S. 568 R e m â k , G . 184 R i c h a r d s , J . D . 619 R i e k e r , A. 462 R i n i k e r , B. 115 R i n k , H . 139

776 Rivalile, P. 710 R o c c h i , R . 322 R o c h a t , H . 724 R o d e , W . 414 R o d k e y , J . A . 100 R o d r i g u e s , L . M . 450 R o d r i g u e z , R . E . 325 R o l l i , H . 730 R o q u e s , B . P . 319 R o s e , K . 274 R o s e n , O. 52 R o s e r , K . - L . 444 R o s i n s k i , G . 649 R o t h e , M . 444 R o u m e s t a n d , C . 399 R u t e r j a n s , H. 4 3 5 R u b i n r a u t , S . 52 R u i z , P. 486 R u l e , W . K . 238 R u s t i c i , M . 706 R u u g e , E . K . 745 R y a b o v a , L A . 280 R z e s z o t a r s k a , B . 414

s

S a b a t i e r , J . M . 160 S a h l , H . G . 369 S a k a k i b a r a , S . 100 Sakarellos, C. 495,498,504,513,528,531 Sakarellos -Daitsiotis, M . 495,498,528,531 S a k u r a d a , S. 6 2 5 S a l v a d o r i , S. 631 S a n d o w , J . 334 Santini, A. 465 S a n t u c c i , A . 705 Sartore, L. 405 S a s a k i , N . A . 313 S a s a k i , Y . 625 S a t o , A . 426 S a u n d e r s , D . 733,736 S a w y e r , T . K . 598 S a w y e r , W . H . 552 S c a r s o , A . 562 S c h a a p e r , W . M . M . 226 S c h e e k , R . M . 438 S c h e l l e n b e r g e r U . 247 S c h e l l e n b e r g e r , V . 247,250 Schielen, W J . G . 70 Schiller, P . W . 613 Schmidt, J . 435 S c h n e i d e r , C . H . 730 S c h n i t t l e r , M . 568

S c h n o r r e n b e r g , G . 586 S c h o b e r , P . A . 244 Schön, I. 40,49 S c h r ä d e r , U . 568,637 Schreiner, K . M . 698 Schrével, J . 378 Schudok, M . 664 S c h u m a n n , W . 61 Schwachula, G . 106 S c h w a r t z , I . L . 537 S c o l a r o , B . 322 Seiinger, Z. 583 S e p e t o v , N . F . 745 S e p r ö d i , J . 40 Shenderovich, M . D . 429 Shen, J . - H . 238 S h e p p a r d , R . C . 151 S h e r m a n , D . B . 646 S h i b a , T . 337,343 Shimohigashi, Y . 628 S h i m o k u r a , M . 55 Shioiri, T . 340 S h i r a t o r i , M . 625 Shoham, G. 411 S i e b e r , P. 139 S i e g r i s t , W . 673 S i e k m a n n , J . 381 Siemion, I.Z. 4 1 7 , 5 1 0 , 7 4 2 S i f f e r t , O. 713 Sigal, I. 190 S i n g h , J . 292 Skliarova, S. 765 Skylyarov, L . 85 Slaninova, J . 265,543,546 S l e b i o d a , M . 43 S ü l i - V a r g h a , H. 640 S l o m c z y n s k a , U . 253 Slonina, P. 106 S l o t b o o m , A J . 667 S m i t h , G . D . 295,468 S m y t h , D . G . 372 S o b ó t k a , W . 649 Sofroniev, N . V . 310 S o l d a n i , P. 705 S o m l a i , C s . 184 S o r e n s e n , A . R . 658 S p a c h , G . 357 S p a t o l a , A . F . 634,646 Spiegel, K . 742 Spiniello, O. 453 S t a v r o p o u l o s , G . 91,537 S t ü b e r , W . 757 S t e c k h a n , E . 79

S t e g l i c h , W . 148,229 S t e w a r t , J . M . 550,565 S t o e v , S. 3 0 7 S t o y a n o v , N . M . 310 S t r a f i b u r g e r , W . 736 S t ü b e r , W . 136 S u e i r a s - D i a z , J . 396 Sugiura, M . 301 S u j a k , P. 649 S u k u m a r , M . 477 Sundaram, G.274 S u r o v o y , A . Y u . 689,692 Suzuki, K . 625 S w i d e r s k a , H . 707 Szekerke, M . 701 Szelke, M . 396 S z e n d e , B . 127 S z e w c z u k , Z. 742 Szöke, B. 604,655 S z ô k â n , G . 127

T

T a h i l r a m a n i , R . 592 T a m a m u r a , H . 58 Tarn, J . P . 196,223 T a n a k a , E . 652 Tancredi, T. 501 T a n n e r , R . 133 T a r t a r , A . 112,718 Temussi, P.A. 501 T e n K o r t e n a a r , P . B . W . 118 Teplan, I. 40,604,655 T e p l o w , D . B . 187 Teshima, T . 337 T e s s e r , G . I . 70,226 T ê t e , F. 531 T h e o d o r o p o u l o s , D. 537 T h o b e k , P. 271 T h o m a s , A . 226 T j o e n g , F . S . 580 T o r n a , F . 399 Tomatis, R . 631 Toniolo, C. 462,465,477 Toome, V. 601 T o r r e s , J . L . 325,486 T 6 t h , G . K . 67 T o t h , G . 616 T o t h , M . 295 T o u r w é , D . 562 T r a p a n i , A J . 580 T r e g e a r , G . W . 178,238 T r e t t i n , U . 109 T r ö g e r , W . 686

T r o j n a r , J . 540 T r z e c i a k , A . 37 T s c h a n k , G . 316 Tschesche, H. 381 T s i g a , S. 528 Tsikaris, V. 513 T s o u , D . 187 T u c h s c h e r e r , G . 193 T y i h á k , E . 127 T z a r t o s , S. 513 T z o u g r a k i , C . 73

U

U c h i d a , H . 628 U e k i , M . 145 U n d e n , A. 223 U r b a n y i , Z. 375

V

V a d á s z , Zs. 40,604,655 V a e r m a n , J . P . 710 V á g n e r , J . 232 V a l e n c i a , G . 268,325,486 Valle, G . 462 v a n B i n s b e r g e n , J . 667 V a n B i n s t , G . 480,562 V a n D e r A u w e r a , L. 562 v a n d e R e e , E . C . A . C . 70 v a n G u n s t e r e n , W . F . 438 v a n H e i j e n o o r t , J . 348 V a n M a r s e n i l l e , M . 562 v a n N i s p e n , J . W . 118,175 V a n R i e t s c h o t e n , J . 160 v a n T i l b o r g , M . C . A . 175 V a r g a , L. 328 V a r r ò , V . 184 V a v r e k , R J . 559,565 V e b e r , D . F . 190,574 V e g n e r s , R . 765 V e r d u c c i , J . 22 V e s t e r m a n , B . G . 429 Vickery, B . H . 592 V i d e n o v , G . 307 V i t a , C . 169 Vitoux, B. 498,531 Viville, R . 562 V o e l t e r , W . 73,97,181,739 V o l p i n a , O . M . 692 v o n G r f l n i g e n , R . 730

w

W a d e , J . D . 178,238

778 Wahren, B. 683 Wakamiya, T. 337,343 Wakselman, M. 387 Waldmann, H. 277 Wallace, C J . A . 283 Wallace, E.C.H. 396 Wang, C.-T. 601 Wang, D.-X. 223 Wang, Y.-S. 441 Watanabe, T. 58 Waxman, L. 190 Wenzel, H.R. 381 Wernic, D. 577 Whittaker, G.G. 244 Wickstrom, E. 187 Widmer, F. 271,280 Wieczorek, P. 414 Wieczorek, Z. 742 Wiesmüller, K.-H. 605 Willer, A. 683 Williams, P. 346 Williams, T.M. 574 Willisch, H. 109,316 Wilson, L. 549 Wimmer, E. 698 Wingender, E. 220 Winquist, R J . 574 Wire, W.S. 634,646 Wisniewski, K. 304 Wünsch, E. 1,748 Wolfe, H. 31 Wolf, H. 679,683 Wolley, G.A. 360 Wo, N.C. 552 Wong, H. 187 Wood, S.G. 417,510

Y

Yajima, H. 58 Yamada, T. 301 Yamamura, H.I. 643 Yamanoi, K. 343 Yamin, N. 556 Yanagi, T. 301 Yarov, A.V. 692 Yiotakis, A. 399 Yoshida, M. 55 Yoshimura, S. 25

z

Zabrocki, J. 295,468 Zahn, H. 736

Zakhariev, S. 307 Zanotti, G. 450,453 Zhang, L. 199,390 Zimecki, M. 742 Zivny, S. 298 Zou, A.Q. 187 Zsigö, J. 534

Subject Index A

A c e t i c acid-labile resin 130 N-Acetyl amino acid - Oxygen-17 NMR 531 Acetylation - of splenopentin 745 N - A c e t y l i m i d a z o l e 214 A c i d - l a b i l e anchor - for peptide amides 136,252 ACTH - receptor binding assays for 673 A c t i o n p o t e n t i a l 070 Activating reagents - BOP 37,166,181,187,238,241 - DCC/HOSU 1 - HBTU 37 - HOBT 31 - phosphinyl chlorides 34 Activation - of Fmoc-amino acid, kinetics 31 - kinetics of active ester 235 A c y l a m i d i n e s 363 N - A c y l a m i n o a c i d s 46 A c y l a t i o n 28 - via diacylamines 19 - via Fmoc-amino acid chlorides 28 A c y l carrier p r o t e i n (65-74),151,211 N - A c y l u r e a 16 0-1 a n d / 3 - 2 - A d a m a n t y l a s p a r t a t e s 64 2-Adamantyl-aspartyl ester - in solid-phase peptide synthesis 166 A d a m a n t y l derivative 562 Affinity c h r o m a t o g r a p h y 124 - anti-prothrombin antibody 757 - of opioid receptors 574 - of thymidylate synthase 414 - of vasopressin receptor 534 Affinity p u r i f i c a t i o n - of rabies virus glycoprotein 704 A g g r e g a t e s of a-helices 357 A g g r e g a t i o n 250 Agonist (s) - of Arg-vasopressin 540 - of neuropeptide Y 586 - of substance P 486 (Aib-Ala)n peptides 465 AIDS - ELISA development 686 A l a m e t h i c i n 360 - analogue 357 - model helices 465

D,L- a-Alanine - substituted by 1,3-indandiones 310 ( A l a n y l ) „ -valine 151 Alkoxybenzyl alcohol s u p p o r t 133,220 Alkylation - of tryptophan 115 S- Alkylation of opioid r e c e p t o r s 628 /3-Alkyl a - a m i n o a c i d s 313 S-Alkyl t h i o e s t e r p e p t i d e 25 Allyl anchor H Y C R A M 154 - in solid phase synthesis 157 A m i d e b o n d s u r r o g a t e 205,646 A m i n o acid a - a m i d e s - dansyl derivatives 130 Amino acid(s) - synthesis of 310 Aminoacyl-4-hydroxycrotonyl -aminomethyl - (HYCRAM)-resin 157 { - [ I - a - A m i n o a d i p y l ] - p e p t i d e s 304 A m i n o a l k y l a m i d e s 136 a - A m i n o b o r o n i c acid 3 - ( S ) - A m i n o - d e o x y s t a t i n e 306 a - A m i n o i s o b u t y r i c a c i d 610 - in Ala-Aib(Dj )-Ala 459 - deuterated chiral Aib 459 - in CCK-8 analogues 265 - in peptaibols 468,360 2-Aminopimelic a c i d 348 Amino protecting groups - N,N-bis-Boc-amino acids 76 - N-Boc-N-Z-amino acids 76 Aminosuccinyl derivative 88 Amphiphilic o l i g o p e p t i d e s 357,423 A m p h i p h i l i c s e c o n d a r y s t r u c t u r e 103 Ampicillin - peptide conjugates 346 A n a e s t h e s i a 718 Analgesic p e p t i d e s 625 Anaphylatoxin - C3a cyclic disulfide analogues 736 - Porcine C5a 100 Anchor g r o u p s - allyl anchor (HYCRAM) 154 - for peptide amide synthesis 136,142 - multidetachable amide anchor 223 Angiotensin(s) 402 - conformation 429 A n g i o t e n s i n I I 571 A n g i o t e n s i n o g e n 306 Anodic decarboxylation - of N-protected amino acids 79

780 Antagonist(s) - of angiotensin II 571 - of bombesin 589 - of GN-RH 334 - of LHRH 592 - of a-melanotropin ( a-MSH) 598 - of the opioid ¿-receptor 619 - of oxytocin 549 - of vasopressin 435,552 Antamanide - conformation by MDS 438 Antibacterial peptides 340,348,303 Antibiotic peptides - bacitracin 522 - epidermin 366 - gallidermin 366 - gramicidin A 471,474 - /3-lactam 250,343 - Pep 5, a lantibiotic 369 - peptaibols 468,354,360 - tricholongino 351 - trichorziamines 354,357 A n t i b o d i e s against - apamin 727 - choleratoxin 710 - FMDV 689,695 - HIV-1 and HIV-2 676,679,686 - human renin 751 - influenza virus 217 - vasopressin receptor 534 - thymosin 0 9 739 A n t i c o a g u l a t i n g a c t i v i t y 310 Antidiuretic 552 Antigenic determinants - of FMDV VP1 689,695 - of HIVenv proteins 676,686 - of influenza virus 217 - of gD-1 701 Antigenicity 217 - prediction of scorpion toxin 724 - of synthetic peptides 718 A n t i g e n search 109,676,079,680,680 A n t i h y p e r t e n s i v e agents 5 7 7 Anti-idiotypic antibodies 534 Antinociception 325 Antipeptide antibodies - FMDV VP1 689,695 - prothrombin F2 757 - rabies virus 704 Anti-sense peptide 673 Antitumor activity 127 Apamin

- epitope mapping of 727 Apocytochrome 1-66 169 A p r o t i n i n 381 Arg-Gly-Asp-Ser 408 A r g - G l y - A s p site 6 8 9 Arginine analogue Arginine containing peptides - immobilization of 124 Arnstein's tripeptide 304 Asparagine coupling 2 4 1 A s p a r t i c acid - /3-cyclohexyl ester 67 - protection of 64 A s p a r t i c p r o t e a s e o f H I V 190 Aspartimide formation - suppression of 64 Asx-turn 507 Atrial natriuretic factor ( A N F ) - analogues 574,577 - synthesis on HYCRAM resin 157 - blood pressure 580 - cleavage sites 580 - conformation 426 - metabolism 580 - Met(O) 12-analogue 426 - specific endoprotease 580 - synthesis of ANP 11-27 148 A t t a c h m e n t to Sepharose 4 0 8 Autoantibodies 513 A u t o m a t e d synthesis 151 see Solid-phase synthesis Avellanins 340 Azide synthesis - of insulin derivatives 262 p-Azidophenylalanine 5 3 4 Azo r u b i n e 169

B

Baboon myometrium 549 B a c i t r a c i n 522 B a c k b o n e modifications - in enkephalin analogues 646 B a c k i n g off couplings 127 B a c t e r i o l y s i s 348 /3-Barrel 1 9 3 B a s e labile a m i n o p r o t e c t i o n - Mpc group 70 B-cell activator 695 /3-Bend c o n f o r m a t i o n - of Pip peptides 477 u>-Benzyloxycarbonylcarbamoyl substituent 3 0 4 o-Benzyloxyphenyl esters 289

781 a - B e n z y l p h e n y l a l a n i n e 13 Bicyclic p e p t i d e s - ANF analogue 574 - conformation 450 - ion binding 450 - synthesis 450 B i n d i n g affinities (tee alto receptor binding) - of neuropeptide Y analogues 586 - of toxin-antibody complex 721 Binding sites - of bradykinin 568 B i o e l e c t r i c activity - of Ser-Lys-Asp 765 Biological a c t i v i t i e s 577 - of bombesin analogues 589 - of C5a anaphylatoxin 100 - of CCK-8 analogues 265 - of enkephalin analogues 628 - of NPY analogues 586 Biologically active conformation 429 Biosynthesis - of lantibiotics 369, 366 Biotin - biotinylated insulin 262 - biotinylated LHRH 595 - in receptor purification 595 Bivalent ligands 031 B o c - a m i n o acids - dichlorobenzoyl derivatives 19 B o m b e s i n analogues - molecular dynamics 483 - nuclear magnetic resonance 483 - synthesis 483 B O P reagent 37,166,181,187,238,241 Boroarginine B o r o n i c acid Bradykinin - analogue(s) 295,562 - antagonists of 565 - potentiating peptide 5a 55 - structure-activity relationship 540 - receptor binding studies 568 B r a i n e n z y m e 375 B r o m o p h e n o l b l u e m o n i t o r i n g 232 B u i l t - i n a