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English Pages 855 [856] Year 1989
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
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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
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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.
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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
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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
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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
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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-
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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