Peptides: 17. Prague, Czechoslovakia, August 29–September 3, 1982 9783111694344, 9783110095746

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Peptides 1982

Peptides 1982 Proceedings of the 17th European Peptide Symposium Prague, Czechoslovakia August 29 - September 3,1982 Editors Karel Blaha • Petr Malon

W DE G Walter de Gruyter • Berlin • New York 1983

Editors Karel Bteha, Dr. Czechoslovak Academy of Sciences Institute of Organic Chemistry and Biochemistry CS-166 10 Prague 6, Czechoslovakia Petr Malofi, Dr. Czechoslovak Academy of Sciences Institute of Organic Chemistry and Biochemistry CS-166 10 Prague 6, Czechoslovakia

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Peptides nineteen hundred and eighty-two Peptides 1982: proceedings of the 17th Europ. Peptide Symposium, Prague, Czechoslovakia, August 29 - September 3,1982 / ed. Karel Bläha; Petr Malofi. Berlin; New York: de Gruyter, 1983 ISBN 3-11-009574-2 NE: Bläha, Karel [Hrsg. ]; European Peptide Symposium

Library of Congress Cataloging in Publication Data

European Peptide Symposium (17th : 1982 : Prague, Czechoslovakia) Peptides 1982 Bibliography: p. Includes indexes. 1. Peptides-Congresses. 2. Peptide synthesis-Congresses. I. Bläha, Karel. II. Malofi, Petr, 1947 . III.Title. [DNLM: 1. Peptides. W3 PE528] QP552.P4E9 1982 574.19'2456 83-5268 ISBN 3-11-009574-2

Copyright © 1983 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Preface

Nearly a quarter of a century has elapsed since the time when in Prague met a small group of chemists who decided to concentrate their scientific effort on a newly developing branch of chemistry - on the peptide synthesis. This meeting - in the original assumption rather of a private character - had an extraordinary successful course and became the base of present series of European Peptide Symposia. Professor Joseph Rudinger - spiritus agens of the first meeting - found many followers and the following meetings developed quickly in prestigious series of symposia of very high scientific reputation. The European Peptide Symposia are probably the oldest among specialized scientific meetings in chemistry and without doubt, the pioneering in peptide chemistry. We are proud of the fact that they also became a model for similar actions of our North American and Far East colleagues. The number of participants and the extent of the scientific programme of the first Prague Symposium reflected the situation in peptide chemistry of that time. These parameters of today's symposia are much greater and are not comparable with those of 1958. However, in one respect there is no change the European Peptide Symposia rely just on the initiative of peptide laboratories in the European region and are in a way their representative forum. The last, already the 17th European Peptide Symposium which was held again in Prague from 29th August to 3rd September 1982, retained these characteristics, and we trust that it had at least a part of the atmosphere and spirit which ruled during the first symposium 1958. The Proceedings volume you have in your hands contains all contributions really presented at the 17th EPS and the manuscripts of which reached the editors before the end of October, 1982. The arrangement of contributions in the volume is

VI

based on the four days scientific programme accepted by the Programme Committee. However, oral contributions and posters are not separate. The two main topics - synthesis of peptides and structure-activity relationships - have been privileged in ordering of contributions. Therefore, the contributions dealing with conformational aspects are only partly collected in the last section. We tried to unify as much as possible the presentation form and the lay-out of all manuscripts, unfortunately, with only limited success. We have to apologize for some inconsistencies due to our weakness. Cooperation with the publisher of these Proceedings, Walter de Gruyter, was excellent, and we would like to extend our thanks namely to Mrs. Evelyne Glowka for her enduring interest. The Organizing Committee found great understanding at all leading institutions and personalities representing the scientific life in Czechoslovakia. We appreciate very much that the 17th EPS was again sponsored by the Czechoslovak Academy of Sciences We acknowledge also with pleasure the permanent help of all members of the European Peptide Committee and, especially, of the Programme Committee. The symposium would not have been possible without great effort from all the members of the Organizing Committee and many other coworkers from the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences not listed under the heading. They performed efficiently and beyond the call of duty. Many thanks.

Karel Blaha and Petr Malon

January 1983

Contents

European Peptide Committee and Programme Committee .... XXVII Honorary Committee and Organizing Committee

XXVIII

Previous European Peptide Symposia

XXIX

List of Participants

XXXI

In Memoriam, Emil Taschner

XLIII

In Memoriam, Dieter Marquarding

XLVII

In Memoriam, Erhard Gross

XLIX

Plenary Lectures Recent Developments in the Chemistry and Biology of Cyclic Peptides Y. Ovchinnikov, G. Chipens, V. Ivanov

1

Conformational Considerations in the Design of Highly Potent and Long-Acting Peptide Hormone Agonists and Antagonists V.J. Hruby, H.J. Knittel, H.I. Mosberg, T.W. Rockway, B.C. Wilkes, M.E. Hadley

19

Semisynthesis of Proteins and Peptides R.E. Offord

31

Further Application of the Aqueous-Organic Two-Phase Approach to Enzyme-Catalyzed Peptide-Synthesis H.-D. Jakubke, P. Kuhl, A. Könnecke, G. Döring, J. Walpuski, A. Wilsdorf, N.P. Zapevalova

43

VIII Interactions of Lipid Bilayers with Corticotropin, Dynorphin, and Enkephalin Peptides R. Schwyzer, H.U. Gremiich, B. Gysin, U.-P. Fringeli

55

Gramicidin A: From Structure Towards the Molecular Mechanism of Action V. Ivanov

73

Structural Comparisons of Retroenantiomeric Cyclic Hexapeptides D. van der Helm, M.B. Hossain

91

Circular Dichroism and Conformation of Peptides Related to Oxytocin I. Fric, P. Malon, K. Jost, K. Bläha

103

Synthesis of Peptides Methods of Protection Adaptation of Side Reactions for Peptide Synthesis M. Bodanszky, M.A. Bednarek, Ai-xue Ji, A. Bodanszky

117

A New Type of Reagents for the Liberation of free Amino Components J. Izdebski, S. Drabarek

121

The Application of Recently Developed Amino Protecting Groups in Peptide Synthesis W. Voelter, J. Müller, C. Beni, W. Heinzel, H. Kalbacher, A. Morel, H. Wollman

125

Preparation of 4-Sulfobenzyl Esters and their Use in Peptide Synthesis R. Bindewald, E.E. Büllesbach, H. Zahn, M. Bodanszky, W. Danho

129

The 2-Chloroethy1 Group for Carboxyl Protection in Peptide Synthesis M.J.S.A. Amaral Trigo, M.I.M.R.E. Barbedo

133

IX

Some Properties of Amino Acid and Peptide Phenacyl Esters 0. Popova, R. Jung. Y. Mitin

137

Temporary Substitution of Asparagine by Aspartic Acid in the Synthesis of Peptide Segments of Vasoactive Intestinal Peptide (VIP) W.M.M. Schaaper, D. Voskamp

141

Methods of Coupling Recycling of Excess Peptides from Fragment Condensations with Carbonyldiimidazole/1-Hydroxybenzotriazole Activation C. Birr, I. Krück, R. Pipkorn, C. Voss

145

Fatty Acids as Additives Suppressing Racemization of Amino Acid Residues in Peptide Synthesis by the DCC Method J. Przybylski, H. Miecznikowska, G. Kupryszewski, H. Jeschkeit, M. Strube

149

HPLC as a Tool for the Investigation of Peptide Bond Formation M. Slebioda, A. Kolodziejczyk, T. Sokolowska

153

Application of Phosphinic Acids to Peptide Synthesis R. Ramage, C. Ashton, B. Atrash, D. Hopton, M.J. Parrott . 157 A Reinvestigation of the Mixed Anhydride Method V.K. Naithani, A. K. Naithani

163

Do Acylamino Acid and Peptide Anhydrides Exist? N.L. Benoiton, F.M.F. Chen

167

First Results with New Reagents for Peptide Coupling P. Henklein, K. Forner, H. Niedrich, P.Y. Romanovski

171

X Peptide Syntheses without Isolation of Intermediates Using Biphasic Solvent System R. Dolling, K.-D. Kaufmann

175

Some Applications of the Curtius Rearrangement P. Moutevelis-Minakakis, I. Photaki

179

Heterodetic Bonds A New Method for the Selective Synthesis of Unsymmetrical Cystine Peptides E. Wiinsch, S. Romani, L. Moroder

183

A Controlled Synthesis of Cyclic Unsymmetrical Cystine Peptides Bearing Two S-S Bridges in the Ring I. Photaki, M. Kolovos, A. Stathaki-Ferderigos, + L. Zervas

189

Synthesis of Cysteine and Cystine Containing Peptides through 3-0- jjSI-Benzyloxycarbony 1-S-AcetamidomethylCysteinyl] -Hydroxy-2-Phenylindenone S. Minchev, N. Sofroniev

195

Method of Cyclization of Carba Analogues of Oxytocin M. Krojidlo, M. Flegel, M. Lebl

199

Synthesis of Isopeptides of Lysine as Fundamental Structural Units of Clavicepamines G. Szokan, M. Gyenes, E. Tyihak, B. Szende

203

Synthesis of Leucine Enkephalin- and Aspartame Analogs Containing Thioamide Linkages at Specific Positions K. Clausen, B. Yde, S.-O. Lawesson

207

XI

Synthesis on Polymeric Supports Synthetic Studies on the Cecropins R.B. Merrif ie Id, L.D. Vizioli, H.G. Boman

211

Improved Syntheses of Substance P and Analogues on a Phenolic Resin Support E. Escher, J.-M. Lalonde, S. Caranikas

215

Peptide Ionophores. Synthesis and Characterization of Cyclic Octa- and Decapeptides Containing Leucine and Proline J. Halstern, S. Mozer, K. Brunfeldt

219

Histone Model Peptide Synthetized by Solid Phase Fragment Condensation V. Gut, H. Votavovä, K. Bläha, J. Sponar

223

The Biomimetic Gel Phase Synthesis of the RNA-Polymerase II Inhibitor Peptide 6'-Deshydroxy-Amanul1 in C. Birr, B. Schmitt

227

Methodological Studies on Gel Phase Synthesis of the Arginine-Rich Propeptide Extension of Proalbumin R. Pipkorn, C. Birr, M. Schmid, K. Weigand

233

Use of the Nbb-Resin and 4-HydroxymethylphenoxymethylResin in Solid Phase Peptide Synthesis E. Pedroso, A. Grandas, R. Eritja, E. Giralt

237

Comparative Syntheses of an Octapeptide Using Continuous Flow (Column) and Conventional Polyamide Solid Phase Methods E. Atherton, L.E. Cammish, R.C. Sheppard

241

The Solid Phase Synthesis of Neurotensin and Related Peptides on Polydimethylacrylamide Resins B.J. Williams

247

XII

Liquid-Phase Synthesis of Biologically Active Peptides on Easily Detachable Poly(Ethylene Glycol) Supports R. Colombo

2 5 1

Synthesis of Individual Peptides Synthesis of Tuftsin under HPLC Control 0.A. Kaurov, A.N. Prusakov, A.M. Pivovarov, V.A. Pasechnik

257

Synthesis of Tuftsin Analogs with Elongated Peptide Chain and a Possible Anticancer Activity D. Konopinska, V.A. Najjar, M. Luczak

261

Synthesis of an IgG Fragment Decapeptide and Preliminary Evaluation of its Role on Phagocytosis Stimulation of Human Polymorphonuclear Leukocytes J. Martinez, J. Laur, F. Winternitz

265

Synthesis and Pharmacologic Characterization of Glucagon (1-21)-Peptide H. Petersen, B.F. Lundt, N.L. Johansen, F.C. Gr^nvald .... 269 The Synthesis of Peptides Related to Prothrombin A. Hallett, A.P. Hope, M.S. Munns, R. Richardson, G.T. Young

273

Total Synthesis of Neurotoxin II from the Central Asian Cobra (Naja naja oxiana) venom V. Deigin, V. Ulyashin, I. Mikhaleva, V. Ivanov

277

Synthesis of Labelled Peptides Catalytic Exchange Labelling of Peptides D.E. Brundish, M.G. Combe, R. Wade

285

XIII

Solid Phase Synthesis of Neurotensin Analogues Susceptible to Radiolabelling C. Labbe, D. Blanot, E. Bricas, J.L. Morgat, P. Kitabgi, J.P. Vincent, C. Granier, J. Van Rietschoten

289

Tritium Incorporation into Histidine by Direct Catalytic Exchange: Labelling of Gonadotropin Releasing Hormone and Analogs E. Klauschenz, M. Bienert, H. Berger, B. Mehlis, H. Niedrich, U. Pleiss

293

3

The H-Labelling of Peptides by Reduction of Dehydroleucine P.M. Hardy, P.W. Sheppard, D.E. Brundish, R. Wade Analogues of [8-ArginineJ Receptor Research

297

Vasopressin for Hormone

F. Fahrenholz, P. Crause, J.-L. Morgat

301

Synthesis of Peptides Containing Non-Coded Amino Acids Synthetic Fragments of Bacterial Cell Walls. Physicochemical and Biological Properties M. Budesinsky, J. Jezek, V. Krchnak, M. Lebl, M. Zaoral, J. Rotta, R. Straka

305

Synthesis of Analogues of Precursors of Bacterial Peptidoglycan D. Blanot, A. Kretsovali, M. Abo-Galia, D. MenginLecreulx, J. van Heijenoort

311

Synthesis of Peptides with ot, B-Dehydroaminoacid Residue B. Rzeszotarska, M. Makowski, Z. Kubica

.315

Synthesis of Dehydro Amino Acids and Peptides R.H. Mazur, D.R. Pilipauskas

319

a-Hydroxymethylation of Peptides: A Synthetic Route to Peptides Containing a-Hydroxymethy1-Amino Acids Z.J. Kaminski, M.T. Leplawy, U. Siomczynska

323

XIV

L- a-Aminoadipic Acid S-N-Benzyloxycarbonylamide and its Use in Peptide Synthesis B. Liberek, R. Kasprzykowska

(Jablonska)

327

Synthesis of ß-Homo-L-Proline Analogues of Peptide Hormones K. Bankowski, A. Misicka

331

Optically Active Neopentylglycine and its Applications in Peptide Chemistry J. PospiSek, K. Bläha

333

Monodisperse Conjugates of Catecholamines K.A. Jacobson, R.P. Rosenkranz, M.S. Verlander, K.L. Melmon, M. Goodman

337

Total Synthesis of Althiomycin T. Shiba, K. Inami

341

Peptide Tricyclic Thia-Cyclols from Linear Precursors G. Zanotti, F. Pinnen, G. Lucente, S. Cerrini, W. Fedeli, F. Mazza

345

Semi-Synthesis Synthesis of Cytochrome C-(66-104) Nonatriacontapeptide and Analogues P.B.W. Ten Kortenaar, G.I. Tesser, R.J.F. Nivard

349

Cytochrome C Semisynthesis Using Enzymic Resynthesis Techniques A.E.I. Proudfoot, C.J.A. Wallace

353

Semisynthetic Analogs of Globomycin J.D. Glass, B. Hayes, M. Inouye

357

Semisynthetic Studies on the Bovine Trypsin-Kallikrein Inhibitor L. Biondi, B. Filippi, F. Filira, V. Giormani, R. Rocchi . 361

XV Synthesis of a Hybrid Chicken/Human Insulin H.-J. Wieneke, G. Wolf, W. Wolff, E.E. Büllesbach, H.-G. Gattner, D. Brandenburg

3 67

Semisyntheses of Insulin Analogues by Replacement of the Sequence Al-4 D. Saunders, K. Freude, V.K. Naithani, D. Brandenburg, S. Stoev

371

Synthesis of Peptides Using Enzymes Enzymatic Peptide Synthesis F. Widmer, M. Ohno, M. Smith, N. Nelson, C.B. Anfinsen ... 375 Amidation of Synthetic Peptides by Pituitary Enzyme: Specificity and Mechanism of the Reaction A. F. Bradbury, D.G. Smyth

381

Reaction Mechanism in Trypsin Catalyzed Synthesis of Human Insulin Studied by -^O-NMR Spectroscopy J. Markussen, K. Schaumburg

387

Trypsin-Catalyzed Oligomerization of the Ala-Ala-Arg Tripeptide V. Cerovsky, K. Jost

395

Thermitase-Catalyzed Hydrolysis of the C-Terminal Ester Group of Protected Peptide Ester Derivatives P. Hermann, L. Salewski

399

Reversibility of Tryptic Hydrolysis in a Model Nonapeptide Sequence V.S. Ganu, J.D. Glass

403

Synthesis and Characterization of a- and/or ^-Substituted Lysine Peptides P. Stehle, B. Kühne, A. Plessing, P. Fürst, P. Pfaender .. 407

XVI

Purification, Characterization and Analytics Isolation by Preparative HPLC and Characterization of Major Components after Cyclization of a Decapeptide P. Larsen, 0. Schou, T. Christensen

411

Separation of Basic, Hydrophilic Peptides by ReversedPhase Ion-Pair Chromatography. III. A Progress Report B. Fransson, U. Ragnarsson

415

Pre-Column Derivatization in HPLC of Peptides G. Szokän, G. Pinter

419

Fast Atom Bombardment Mass Spectrometry - a New Tool for Peptide Sequence Analysis W.A. König, M. Aydin, U. Schulze, M. Rinken

423

Progress Towards a Post-Synthetic Chiral Analysis of Peptides J.S. Davies, A.K. Mohammed, E. Hakeem

431

Direct Peptide Sequencing after TLC on Silica Gel without Substance Elution R. Kraft, A. Otto, G. Etzold

437

Structure-Activity Relations Neurohypophyseal Hormones Two Analogues of Oxytocin with Modified Proline Cyclic Structure Z. Prochâzka, M. Lebl, T. Barth, J. Hlavâcek, K. Jost .... 441 Synthesis of new Active and Highly Selective Analogues of Oxytocin and Arginine-Vasopressin Z. Grzonka, F. Kasprzykowski, B. Lammek, D. Gazis, I.L. Schwartz

445

XVII

CNS - Active Vasopressin Analogues F. Brtnik, T. Barth, J. Skopkova, K. Jost, I. Krejci, B. Kupkova

449

Investigation of Position 2 and 3 of Vasopressins I. Blaha, V. Krchnak, M. Zaoral, D. Konopinska

453

Analogues of Neurohypophysial Hormones Containing a D-Amino Acid in Position 2 M. Lebl, T. Barth, L. Servitovä, J. Slaninovä, K. Jost ... 457 Penicillamine-Containing Analogues of Neurohypophysial Hormones P. Simek, T. Barth, F. Brtnik, J. Slaninova, K. Jost

461

9-Substituted Vasopressin Analogs with Substantial Antidiuretic Activity D. Gazis, A. Buku, I.L. Schwartz

465

Interactions of Deamino-6-Carba-0xytocin Analogues in Rat Kidney and Liver Membrane Systems T. Barth, M. Lebl, K. Jo^t, B. Cantau, D. Butlan, G. Guillon, S. Jard

467

Opioid Peptides Synthesis and Biological Activity of New Enkephalin Analogues R. Paruszewski, R. Matusiak, E. Gmitrzuk, W. Gumuika, P. Janicki

471

Biochemical and Pharmacological Investigations of Peripheral and Brain 6-0piate Receptors Using Tyr-D-Thr-GlyPhe-Leu-Thr, a New Fully Specific 6-Ligand B.P. Roques, G. Gacel, P. Dodey, J.-M. Zajac, M.C. Fournie-Zaluski

475

Double Opiate Peptides. A Hypothesis of Two Different Mechanisms of Opiate Actions A.W. Lipkowski, M. Konopka, B. Osipiak, W.S. Gumuika

481

XVIII

Synthesis and Activity of Kyotorphin and its Analogs L. Balaspiri, K. Kovacs, A. Geese, G. Telegdy, K. Neubert

487

Opioid Activity of Synthetic "Small Demorphins" R. Tomatis, S. Salvadori, G. Sarto

495

Structure-Activity Relationship of Dermorphin K. Darlak, Z. Grzonka, P. Janicki, A. Czionkowski, S.W. Gumuika

501

Structural Modifications of B-Casomorphin-5: Synthesis and Pharmacology B. Hartrodt, K. Neubert, H. Matthies, H.-L. Riithrich, H. Stark, A. Barth

505

Substance P Substance P: The "Yin-Yang" of Behavior? J.M. Stewart, M.E. Hall

511

Hydrophilic Analogs of Substance P: Introduction of Sugar Acids and Sulfonium Groups M. Bienert, K. Forner, B. Mehlis, H. Niedrich, J. Bergmann, R. Kraft

517

Influence of the Methionine Sidechain on the Biological Activity of Substance P D. Theodoropoulos, C. Poulos, D. Gatos, P. Cordopatis, R. Couture, J. Mizrahi, D. Regoli, E. Escher

521

Substance P Analogues Containing Para-Substituted Phenylalanine: Synthesis and Pharmacological Properties E. Munekata, I. Kanazawa

527

Synthesis of Potent and Specific Antagonists of Substance P E. Escher, J. Mizrahi, S. Caranikas, P. D'OrleansJuste, D. Regoli

531

XIX

Substance P: Inactivation in CNS, Enzymatically Resistant Analogue and Development of "Brain Selective" Agonist B.E.B. Sandberg, M.R. Hanley, L.L. Iversen, J.E. Maggio, R.R.D. Pinnock, S.P. Watson

535

Other Biologically Active Peptides Some Pharmaceutical Aspects of Peptide Research L. Kisf aludy

543

The Synthesis and Gonadotropic Activity of [D-Tle 6 , Pro-NH-Et9]LRF M. Flegel, J. Posplsek, J. Picha, D. Pichovä

551

The Role of N-Acyl Groups in the Inhibitory Activity of LH-RH Analogues I. Mezö, J. Seprödi, J. Erchegyi, I. Teplän, M. Koväcs, B. Flerko

555

Gonadotropin Releasing Hormone and its Analogs Desensitize Pituitary GnRH Action G. Keri, K. Nikolics, I. Teplän DSIP: Novel Developments in Structure-Functional

559 Studies

I. Mikhaleva, A. Sargsyan, V. Ivanov

563

The Active Centres of Gastrin and Cholecystokynin: Syntheses, Conformational Problems, Correlation between Chemical Structure and Biological Activity B. Penke, M. Zarandi, G.K. Toth, K. Koväcs, M. Fekete, G. Telegdy, P. Pham

569

What is the Minimum Active Centre of Gastrin? M. Zarändi, B. Penke, J. Varga, K. Koväcs, G. Holczinger, T. Kadär

577

Structure-Activity Relationships of Highly Potent and Specific Gctapeptide Analogues of Somatostatin W. Bauer, U. Briner, W. Doepfner, R. Haller, R. Huguenin, P. Marbach, T.J. Petcher, J. Pless

583

XX Photoreactive Labelling

[Leu^l-a-MSH Derivatives for Receptor

A.N. Eberle, J. Girard, P.N.E. de Graan, F. F.C.G. van de Veerdonk

589

Degradation of a-Melanotropin by the Enzymes of the Soluble Fraction of Rat Brain Homogenate H. Medzihradszky-Schweiger, J. Szécsi, K. Medzihradszky .. 593 Solid Phase Synthesis of Amunine and Two Urotensin I

(CRF), Sauvagine

J. Rivier, J. Spiess, C. Rivier, R. Galyean, W. Vale, K. Lederis

597

Proctolin: Synthesis, Enzymatic Degradation and Effect on the Cyclic Nucleotide System H. Arold, C. Liebmann, S. Reissmann, J. Scheidt

603

Phosphonopeptides, Novel Inhibitors of Bacterial Cell Wall Biosynthesis C.H. Hassall, F.R. Atherton, M.J. Hall, R.W. Lambert, W.J. Lloyd, P.S. Ringrose

607

Urinary Excretion of Biologically Active Peptides and/or Peptide-Like Material in Various Disorders J.H. Johansen, J.B. BszSler, K.L. Reichelt, P.D. Edminson, N.K. Pape

613

Inhibitors and Binding Studies Synthesis and Properties of Peptide Ketones S. Fittkau, G. Jahreis

617

Enkephalin Diazomethyl and Chloromethyl Ketones: Synthesis and Biological Activity K. Medzihradszky, J. Szecsi, G. Csanady, J. Hepp

623

Use of Quantitative Lipophilicity Relationships for the Design of Renin Inhibitors J. Burton

629

XXI

Specific Inhibitors of Renin M. Szelke, A. Hallett, D.M. Jones, J. Sueiras, B. Atrash, B. Leckie, M. Tree, A.F. Lever

635

Anionic Inhibitors of Elastase E. Kasaflrek, P. Fric, J. Slaby

639

Inhibition of Thrombin with H- and Boc-D-Phe-Pro-Agm S. Bajusz, E. Barabäs, D. Bagdy

643

Analogues of Chymostatin I.J. Galpin, A. H. Wilby, R.J. Beynon

649

Binding Studies of Glucagon Antagonists V.J. Hruby, J.T. Pelton, R. McKee, D. Trivedi

653

Determination of the Toxic and Antigenic Sites of Peptide Snake Venom Short Neurotoxins A. Menez, J.-C. Boulain, P. Fromageot

657

Design and Synthesis of Two Peptides as Models for the Antigenic Sites of the Toxin II of the Scorpion Androctonus australis Hector C. Granier, E. Bahraoui, J. Van Rietschoten, H. Rochat, M. El Ayeb

663

Immunology Related Peptides Chemical Approaches to Improve Immunoassays of Peptide Hormones L. Moroder, M. Gemeiner, H. Kaibacher, R. Nyfeler, E. Wünsch

667

Antineoplastic and Immunogenic Effects of Tuftsin (Thr-Lys-Pro-Arg) and its Analogs V.A. Najjar, D. Konopinska, M.K. Chaudhuri, L. Linehan ... 673

XXII

Synthesis and Biological Assays of Peptides from a Tuberculin-Active Protein J. Savrda

679

Synthesis and Immunoreactivity of Escherichia Coli Heat Stable Enterotoxins Analogs A. Duflot, H. Gras-Masse, A. Tartar, E. Duflot, P. Boquet

683

Preparation and Antigenicity of the C-Terminal Fragments of Human Leukocyte Interferons a2 and c*i M. Ohno, F. Widmer, M.E. Smith, H. Arnheiter, K.C. Zoon .. 687 Immunological Properties of Hapten-Peptide Conjugates: The Effect of Size, Lipophilic Substituents and Haptenic Distance on Tolerogenicity and Anaphylactogenicity I. F. Luescher, C.H. Schneider

693

Conformational Studies Conformation: Model Peptides Conformational Features of Alternating L, D Peptides A. Bavoso, E. Benedetti, B. Di Biasio, V. Pavone, C. Pedone, V. Barone, G. Esposito, F. Lelj, G.P. Lorenzi . 699 Conformation of Cyclic Hexapeptides Studied by the Modified Factor Analysis of Circular Dichroism Spectra P. Panco^ka, K. Blaha, J. Vicar

705

Conformational Energy Calculations of Heterodetic Cyclic Hexapeptides Related to Oxytocin Ring Moiety P. Malon, G.V. Nikiforovich, K. Blaha

709

Conformational Energy Analysis of Oxytocin Molecule V. Krchnak

713

XXI 11

Conformational Analysis of Peptides Substrate of N-Glycosylation E. Giralt, M. Pons, J.M. Ricart, J.J. Perez, C. Granier, J. Van Rietschoten

717

Serine and ß-Folding M. Marraud, A. Aubry

721

X-Ray Structure and Conformation in Solution of Peptides Containing a-Aminoisobutyric Acid Residues G. Jung, R. Bosch, H. Brückner, E. Katz, H. Schaal, H. Schmitt, J. Strähle, K.-P. Voges, W. Winter

725

Preferred Conformations of Host-Guest Peptides M. Mutter, F. Maser, B. Klein, C. Toniolo, G.M. Bonora ... 729

Conformation: Biologically Active Peptides Conformation Calculations and Biologically Active Conformations of Cyclopeptides G. Nikiforovich, S. Rozenblit, I. Liepina, G. Chipens .... 735 Peptaibol Antibiotics: A Study on the Helical Structure of Emerimicins C. Toniolo, G.M. Bonora, E. Benedetti, A. Bavoso, B. di Biasio, V. Pavone, C. Pedone

741

Conformational and Ionophoric Properties of Prolyl Containing Analogs of Valinomycin T. Balashova, L. Fonina, G. Avotina, V. Xvanov

745

Synthesis of the Postulated Calcium Binding Site I of Calmodulin and Binding Studies F. Marchiori, G. Borin, G. Chessa, E. Peggion

751

X-Ray Structural Study of Ionophores of a Valinomycin Group V. Popovich, V. Pletnev

755

XXIV

Conformational Studies on Gaba-Enkephalinamides C. Di Bello, S. Andini, L. Ferrara, R. Napolitano, L. Paolillo

759

Conformational Studies of the "in vitro" Potent Opiate [D-Met2f Trp4, Pro5J Enkephalinamide by Fluorescence and Theoretical Calculations J.-M. Garcia Anton, F. Reig, G. Valencia, J.J. Garcia Dominquez, R. Guillard, J.-P. Demonte, A. Englert

763

Configuration of the Azo Bridge in Azo-Enkephalin I.Z. Siemion, A. Jankowski, M. Lisowski, Z. Szewczuk

767

Self-Association of Substance P and its C-Terminal Sequences K. Gast, J. Behlke, M. Rüger, M. Bienert, B. Mehlis

771

Spatial Structure of Bradykinin Potentiating Peptide BPPg a S. Reißmann, H. Arold, M.P. Filatova, N.A. Krit, I. Fric 775 Guest-Host Relationship in Bradykinin Peptides V. Dive, K. Lintner, S. Fermandjian, S.S. Pierre, D. Regoli

781

Importance of Residue 5 in Antiotensin IX C. Sakarellos, F. Piriou, M. Juy, F. Torna, H. Lam-Thanh, K. Lintner, S. Fermandjian, M.C. Khosla, R.R. Smeby, F.M. Bumpus

785

Circular Dichroism Aids Interpretation of StructureActivity Relationship of Somatostatin Analogs D.F. Veber, W.C. Randall, R.F. Nutt, R.M. Freidinger, S.F. Brady, D.S. Perlow, P. Curley, W.J. Paleveda, R.G. Strachan, F.W. Holly, R. Saperstein

789

The Conformational Equilibrium of the Peptide Hormone Somatostatin and some Analogues in Aqueous Solution K. Hallenga, F. Vlaeminck, G. Van Binst, M. Knappenberg, A. Michel, J. Zanen

793

XXV

Application of Homonuclear and Heteronuclear Two-Dimensional NMR Spectroscopy to Cyclolinopeptide A (1) H. Kess1er, R. Schuck

797

Role of the N-Terminal Fragment on the Stability of the Porcine Pancreatic Secretory Tryps in Inhibitors (KAZAL) A. Scatturin, V. Periotto, M. Guarneri, E. Menegatti, A. De Marco

801

Relationship between the Steric Structure of Insulin Derivatives and their Biological Activity G. Vlasov, N. Izvarina, N. Illarionova

805

Studies of Hydrogen Bonding in Relation to Oxytocin Activity D. Gazis, J. Roy, U. Roy, J.D. Glass, I.L. Schwartz

809

Synthesis, Conformation and Interaction with a Decanucleotide of the Fragment (26-39) of the "Cro" Protein from Bacteriophage X R. Mayer, G. Lancelot, C. Hélène

815

Abbreviations

819

Subject Index

829

Authors' Index

841

XXVII

EUROPEAN PEPTIDE COMMITTEE Aus tria: Belgium: Bulgary: Czechoslovakia: Denmark: Great Britain: France: PRG: GDR: Greece: Hungary: Israel: Italy: The Netherlands: Poland: Spain: Sweden: Switzerland: USSR:

H. Nesvadba A. Loffet B. V. Aleksiev K. Blàha It. Brurtfeldt R. C. Sheppard E. Bricas H. Hagenmaier H. Niedrich I. Photaki K. Medzihradszky A. Pa tchornik R. Rocchi H. C. Beyerman G. Kupry s z ewski E. Giralt U. Ragnarsson J.-L. Fauchere Y. Ovchinnikov

PROGRAMME COMMITTEE It. Blàha D. Brandenburg V.T. Ivanov K. Medzihradszky G.T. Young

H O N O R A R Y COMMITTEE B. Kvasil, J, Öermäk, Z. V. J. J.

Öeska, Holat, Chromik, Gabel

E. Hala V, Kubanek, L. KubiSek, J. Mostecky, K,

Sebesta,

F.

ätafa,

President of the Czechoslovak (chairman) Academy of Sciences Director, Institute of Chemical Process Fundamentals Rector of the Charles University 1st Deputy Minister, Czech Ministry of Health Director General, SPOFA 1st Deputy Minister, Federal Ministry for Technical Development and Investment Academician Head of the Science Department of the Central Committee Scientific Secretary of the Academy Rector of the Prague Institute of Chemical Technology Director, Institute of Organic Chemistry and Biochemistry Lord Mayor of Prague

ORGANIZING COMMITTEE K. T. I. F. M. I. V. J.

Blaha (chairman) Barth Blaha Brtnik Flegel Friö Gut Hlavaöek

K. M. P. J. Z. P. J. M.

Joät Lebl^ Ma Ion PospiSek Prochazka Simek Skopkova Zaoral

XXIX Previous E u r o p e a n 1.

Czechoslovakia,

1958;

Peptide

Symposia

Prague.

C o l l e c t . Czech. Chem. Conunun. 24, S p e c i a l I s s u e

(1959)

1-160. 2.

FRG, 1959;

Munich.

A n g e w . Chem. 71 3.

Switzerland,

(1959)

I960;

741-743.

Basel.

Chimia l4 (i960) 366-38O; 393-418. 4.

USSR, 1961;

Moscow.

C o l l e c t . Czech. Chem. C o m m u n . 27 also in Z h u r n a l M e n d e l e y e v s k o v o 5.

Great B r i t a i n , 1962;

6.

Greece, 1963;

7.

H u n g a r y , 1964;

Peptides Peptides Acta

(L. Zervas,

Peptides 9.

France,

(1962).

Oxford

(1963).

e d . ) P e r g a m o n Press, O x f o r d

(1966),

e d . ) P e r g a m o n Press,

Budapest.

Academiae Scientarium Hungaricae

The Netherlands,

1966;

eds) 44

Peptides 1968

1-239.

A. v a n de Linde, W . M a a s s e n

eds) N o r t h - H o l l a n d ,

1968;

(1965)

(V. B r u c k -

Noordwijk.

(H.C. B e y e r m a n ,

der Brink,

7

Athens.

n e r and K . M e d z i h r a d s z k y , 8.

2229-2262,

Oxford.

(G.T. Young,

Chimica

(1962)

Obshchestva

Amsterdam

van

(1967).

Orsay. (E. Bricas,

ed.) N o r t h - H o l l a n d ,

Amsterdam

(1968). 10.

Italy,

1969; A b a n o T e r m e .

Peptides 1969

(E. Scoffone,

ed.) N o r t h - H o l l a n d ,

Amsterdam

(1971).

11.

Austria,

1971;

Peptides 1971

Vienna. (H. Nesva.dba , ed,) N o r t h - H o l l a n d ,

Amsterdam

(1973). 12.

GDR, 1972; R e i n h a r d s b r u n n Peptides 1 9 7 2

Castle.

(H. H a n s o n a n d H.D. Jakubke,

Holland, A m s t e r d a m

(1973).

eds) N o r t h -

XXX 13.

Israel, 1972*; Kiryat Anavim. Peptides 197^ (Y. tfolman, ed.) Keter Press, Jerusalem (1975).

14.

Belgium, 1976; Wépion. Peptides 1976 (A. Loffet, ed.) Editions de 1 Université de Bruxelles.

15.

Poland, 1978; Gdansk. Peptides 1978 (i.Z. Siemion and G. Kupryszewski, eds) Wroclaw University Press (1979).

16.

Denmark, 1980; Helsing^r. Peptides 1980 (K, Bruni*eldt, ed.) Scriptor, (1981).

Copenhagen

XXXI

LIST OF PARTICIPANTS Albericio F., Dr., Laboratoire de Biochimie Faculte de Médicine, Secteur Nord, 13 326 Marseille Cedex 15, France Aleksiev B., Prof., Higher Institute of Technology, 1156 Sofia, Bulgary Allen M., Dr., Ciba Geigy Pharmaceuticals Division, Wimblehurst Rd, Horsham, Vest Sussex, HH 12 4AB, U.K. Amaral Trigo M. J.S.A. , Prof., Faculda.de de Ciencias do Porto, Departamento de Química, kOOO Porto, Portugal Andreatta R., Dr., Ciba Geigy AG, CH-4056 Basel, Switzerland Arielly S., Dr., Violgatan 3A, S-^34 00 Kungsbacka, Sweden Arold H., Prof., Sektion Biologie, Friedrich Schiller Universität, Humboldtstrasse 10, 6900 Jena, G.D.R. Bajusz S., Dr., Institute for Drug Research, P.O. Box 82, H-1325 Budapest, Hungary Balashova T.A., Dr., Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Vavilova. 32, 117 988 GSP-1, Moscow 33^, U.S.S.R. Baláspiri L., Dr., Institute of Medical Chemistry, Szeged University, Medical School, Dom ter 8, 6720 Szeged, Hungary Baiíkowski K., Dr., Faculty of Chemistry, University of Warszawa, Pa.steura 1, 02-093 Warszawa, Poland Barth T., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia Bauer W. , Dr., SANDOZ Ltd., Bldg. 507/204, Pharmaceutical Division, CH-4002 Basel, Switzerland Di Bello C., Prof., Istituto di Chimica Industriale, Via Marzolo 9, 35100 Padova, Italy Benoiton N.L., Prof., Department of Biochemistry, University of Ottawa, Ottawa, Ontario KIN 9A9, Canada Bienert M., Dr., Institut für Wirkstofforschung, Alfred Kowalke Strasse 4, 1136 Berlin, G.D.R. van Binst G., Prof., Vrije Universiteit Brüssel, Fakulteit Wetenschappen, Dienst Organische Chemie, Pleinlaan 2, B-1050 Brüssel, Belgium Birr C., Prof., Max Planck Institut für Medizinische Forschung, Jahnstrasse 29, D-69OO Heidelberg, F.R.G. Bláha I., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia

XXXII

Blàha K,, Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia Blanot D., Dr., Equipe de Recherche no 24-5 du CNRS, Institut de Biochimie, Université de Paris Sud, 9l405 Orsay Cedex, France Bodanszky M., Prof., Case Western Reserve University, Department of Chemistry, Cleveland, OH 44lO6, U.S.A. Borin G. , Dr., Istituto di Chimica Organica, Via Marzolo, 1, 1-35100 Padova, Italy Bradbury A.F., Dr., National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Brandenburg D., Dr., Deutsches Wollforschungsinstitut, Technische Hochschule, Veitmanplatz 8, D-5100 Aachen, F.R.G. Brenner M., Prof., Institut für Organische Chemie, Universität St. Johans Ring 19, CH-4056 Basel, Switzerland Bricas E., Dr., 11, Avenue de la Résidence, 92l60 Antony, France Brtnik F., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia Brunfeldt K., Prof., The Danish Institute of Protein Chemistry, 4, Venlighedsvej, DK-2970 H^rsholm, Denmark Burton J., Dr., Laboratory of Cellular and Molecular Research, Massachusetts General Hospital, Boston, MA 02114, U.S.A. Clausen K., Dr., Kemisk Institut, Laboratory for Organisk Kemi, DK 8000 Arhus C, Denmark Colombo R., Dr., Cattedra di Chimica Biologica, Ospedale 5. Raffaele, via Olgettina 60, 1-20132 Milano, Italy Cordopatis P., Dr., Laboratory of Organic Chemistry, University of Pa.tras, Patrai, Greece Davies J.S., Dr., University College of Swansea, Department of Chemistry, Singleton Park, Swansea SAI 8PP, U.K. Deigin V., Dr., Institute of Protein Chemistry, Poustchino, Moscow Region 142292, U.S.S.R. Durieux J. P. , Dr., UCB Bioproducts, 68 rue Berkendael, B-IO6O Bruxelles, Belgium Eberle A., Dr., Research Department, Kantonsspital, Hebelstrasse 20, CH-4031 Basel, Switzerland Escher E., Dr., Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec JIH 5N4, Canada

XXXIII

Etzold G., Dr., Academy of Sciences of the GDR, Central Institute of Molecular Biology, Berlin-Buch, G.D.R. Fahrenholz F., Dr., Max Planck Institut für Biophysik, Kennedy Allee 70, D-6000 Frankfurt 70, F. R.G. Fauchère J.L. , Dr., Institut für Molekularbiologie und Biophysik ETH,Hanggerberg, CH-8093 Zurich, Switzerland Fauszt I., Dr., Institute for Drug Research, P.O. Box 82, H-1325 Budapest, Hungary Fermandjian S., Dr., Service de Biochemie, C.E.N, Saclay B.P. no 2, F-911 91 Gif-sur-Yvette, France Filippi B., Dr., Istituto di Chimica Organica, via Marzolo, 1 1-35100 Padova, Italy Fischer R., Bachem AG, Hauptstrasse l44, CH-44l6 Bubendorf, Switzerland Fittkau S., Dr., Institut fur Physiologische Chemie, Martin Luther Universität, Hollystra.sse 1, 402 Halle/Salle, G.D.R. Flegel M., Dr., LÉÔIVA - Pharmaceuticals, Laboratory of Peptides, l43 10 Prague 4-Modrany, Czechoslovakia Fournié-Za.luski M. C. , Dr.. Department Chimie Organique, Faculté de Pharmacie, 4, avenue de 1 Observatoire, F-75006 Paris, France Friö I., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia. Friedrich A., Dr., Hoechst A.G., Pharma Synthese, Postfach 8OO32O, D-623O Frankfurt/Main 80, F.R.G. Fromageot P., Prof., Service de Biochimie, C.E.N. Saclay B.P. no 2, F-91191 Gif-sur-Yvette, France Galpin I.J., Dr., University of Liverpool, Department of Organic Chemistry, P.O. Box 147, Liverpool LÓ9 3BX, U.K. Garcxa-Anton J.M., Dr., Instituto de Techología, Química, Jorge Girona, Salgado s/n. , Barcelona 34, Spain Gazis D., Dr., Room 21-84. Annenberg Bldg., Mount Sinai School of Medicine, 100th St. and 5th Ave., New York, N Y 10029, U.S.A. Gillessen G., Dr., F. Hoffmann-La Roche & Co. Ltd., Bldg. 15/241, CH-4002 Basel, Switzerland Giralt E. , Prof,, Department de Química Orgánica, Fa.cultat de Química, Universität de Barcelona, Diagonal 645, Barcelona 28, Spain Glass J.D. , Dr., Mt. Sinai School of Medicine, Department of Physiology, 5th Avenue at 100th Street, New York, N Y IOO29, U.S.A.

XXXIV Gras-Masse H., Dr., Faculté de Pharmacie, Université de Lille, rue du Prof. Laguesse, F-59045 Lille, France Grzonka Z. , Dr., Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80 952 Gdansk, Poland Guegan R,, Dr., Centre de Recherches Clin-Midy, rue du Pr. Blayac, F-34082 Montpellier, France Gustavsson S., Dr., AB Kabi, Peptide Research Molndal, Box 156, S-431 22 Moûnda1, Sweden Gut V., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia Halstr^in J. , Dr. , The Danish Institute of Protein Chemistry, 4, Venlighedsve j, DK-2970 Hj^rsholm, Denmark Hallet A., Dr., University of London, Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, Dueon Road, London ¥12 OHS, U.K. Hardy P.M., Dr., Department of Chemistry, University of Exeter, Stocker Road, Exeter EX4 4QD, U.K. Hartrodt B., Dr., Martin Luther Universität, Halle-Wittenberg, Sektion Biowissenschaften, WB Biochemie, Domplatz 1, 4020 Halle/Salle, G.D.R. Hassall C.H., Prof., Roche Products, Ltd., P.O. Box 8, Welwyn Garden City, Herts AL7 3AY, U.K. Hauzer K., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 1 6 6 10 Prague 6, Czechoslovakia van der Helm D., Prof., Department of Chemistry, of Oklahoma, 620 Parrington Oval, Room 211, Norman, OK 73019, U.S.A.

University

Hermann P., Dr., Institut für Physiologische Chemie, Martin Luther Universität, Hollystrasse 1, Postfach 184, 402 Halle/Salle, G.D.R. Hlavaöek J., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia Hollôsi M., Dr., Institute of Organic Chemistry, Eotvös University, Mûzeum krt. 4/B, H-1088 Budapest, Hungary

Lorând

Hruby V.J., Prof., Department of Chemistry, University of Arizona, Tucson, AZ 85721, U.S.A. Ivanov V.T., Prof., Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Vavilova 32, Moscow 117312, U.S.S.R.

XXXV Izdebskl J., Dr., Department of Chemistry, University of Warsaw, ul. L. Pasteura 1, 02-093 Varszawa, Poland Jacobson K., Dr., Department of Organic Chemistry, Institute of Science, Rehovot, Israel

Weizmann

Jakubke H.D., Prof. Karl Marx Universität, Sektion Biowissenschaften, Bereich Biochemie, Talstrasse 33, 7010 Leipzig, G.D.R. Jaquenoud P.A., Dr., SANDOZ Ltd., Lichtstrasse j6, CH-4056 Basel, Switzerland Jeschkeit H., Dr., Sektion Chemie Martin-Luther-Universitat, Weinbergweg 16, 402 Halle, G.D.R. Jezek J., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia Johansen J., Dr., CARLBIOTECH, l6, Tagensvej, DK 2200 Copenhagen N, Denmark Johansen J.H., Dr., The Peptide Group, Pediatric Research Institute, Children Clinic, University Hospital, Oslo, Norway Jost K., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia Jung G., Prof., Institut für Organische Chemie der Universität, Auf der Morgenstelle 18, D-7400 Tübingen, F.R.G. Kasafxrek E., Dr., Research Institute for Pharmacy and Biochemistry, Kourimska 17, 130 60 Prague 3> Czechoslovakia Kaufmann K.D., Dr., Institut für Wirkstofforschung, AW DDR, Alfred Kowalke Strasse 1136 Berlin, G.D.R. Kaurov 0., Dr., All-Union Institute of Extra Pure Biopreparations, Bolsoj pr. 71, 199026 Leningrad V.O., U.S.S.R. Kessler H., Prof., Institut für Organische Chemie, J.W, Goethe Universität, Niederurseier Hang, D-6000 Frankfurt 50, F.R.G. Kisfaludy L., Dr., Chemical Works G. Richter, P.O. Box 27, 1475 Budapest 10, Hungary Kohlbeck W.^ Dr., Diamalt A.G., Georg-Reismüller Strasse 34, D-8000 München 50, F.R.G. Kojfodzie jczyk A. , Dr., Faculty of Chemistry, Technical University of Gdansk, Majakowskiego 11/12, 80 952 Gdansk, Poland König W.A., Prof., Institut für Organische Chemie und Biochemie der Universität, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, F.R.G.

XXXVI Konopinska D., Dr., Institute of Chemistry, University of Wroclaw, ul. Joliot Curie l4, 50 3 8 3 Wroclaw, Poland Krchnak V., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak. Academy of Sciences, 166 10 Prague 6, Czechoslovakia Krojidlo M., Dr., LEClVA - Pharmaceuticals, Laboratory of Peptides, 143 10 Prague 4-Modrany, Czechoslovakia. Kubler U., Dr., VEB Berlin-Chemie, Glienicker Weg 182-184, 1199 Berlin-Adlershof, G.D.R. Kupryszewski G., Prof., Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80 952 Gdansk, Poland Lammek B., Dr., Institute of Chemistry, University of Gdansk, ul. Sobieskiego 18, 80-952 Gdansk, Poland Larsson L.E., Dr., Pharmacia AB, Box 181, S-75104 Uppsala, Sweden Lebl M., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia Lefrancier P., Dr., Institut Choay, 10, rue Morel, F-92 120 Montrouge, France Leplawy M., Prof,, Institute of Organic Chemistry, Technical University, ul. Zwirki 3 6 , 90 924 Lodz, Poland Leukart 0., Dr., BACHEM Feinchemikalien AG, Hauptstrasse 144, CH-44l6 Bubendorf, Switzerland Liberek B., Prof., Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80 952 Gdansk, Poland Lindeberg G., Dr., Pentapharm AG, Engelgasse 109, CH-4002 Basel, Switzerland Lipkowski A., Dr., Faculty of Chemistry, University of Warsaw, Pasteura 1, 02 093 Warszawa, Poland Loffet A., Dr., UCB Bioproducts, 68 rue Berkendael, D-1060 Bruxelles, Belgium Loozen H,J,, Dr., Scientific Development Group, Organon, Int. B. V., P.O. Box 20, 5340 BH Oss, Netherlands Low M., Prof., Chemical Works G. Richter, P.O. Box 27, H-l475 Budapest, Hungary Lowe L.A., Dr., The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K. Lundin R., Dr., Kabi Vitrum AB, Avd. FO, S—112 87 Stockholm, Sweden Makineni R., Dr., BACHEM Inc., 3132 Kashiwa Torrance, CA 90505, U.S.A.

Street,

XXXVII

Malori P. , Dr. , Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia Markussen J,, Dr., Novo Research Institute, Novo Allee, DK-2880 Bagsvaerd, Denmark Marraud M. , Prof., Laboratoire de Chimie-Physique Macroraoléculaire, Equipe de Recherche Associée au CNRS no 23, 1, rue Grandville, F-54042 Nancy Cedex, France Martinez J., Dr., Centre de Pharmacologie Endocrinologie, Rue de 20 Cardonille, F-34000 Montpellier, France Mayer R. , Dr., Centre de Biophysique Moléculaire, F-45045 Orléans Cedex, France Mazur R.H., Dr., Searle Research and Development, Division of G.D. Searle & Co, P.O. Box 5110, Chicago, IL 60680, U.S.A. Medzihradszky K., Prof., Central Research Institute of Chemistry, Hungarian Academy of Sciences, Pusztaszeri ut. 59, H-1025 Budapest, Hungary Mehlis B., Dr., Institut für Wirkstofforschung, Alfred Kowalke Strasse 4, 1136 Berlin, G.D.R. Merrifield R.B., Prof., The Rockefeller University, New York, NY 10021, U.S.A. Mezë I., Dr., 1st Institute of Biochemistry, Semmelweiss University Medical School, P.O. Box 260, H 1444 Budapest 8, Hungary Mikhaleva I.I., Dr., Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Vavilova 32, 117988 Moscow, U.S.S.R. Minchev S., Dr., Higher Pedagogical Institute, 97OO Shoumen, Bulgary Mitin Yu.V., Dr., Institute of Protein Research, USSR Academy of Sciences, 142292 Poustschino, Moscow Region, U.S.S.R. Morley J.S., Dr., 24 Cheadle Road, Cheadle Hulme, Cheshire SK8 5EW, U.K. Moroder L., Prof., Max Planck Institut für Biochemie, Abteilung fur Peptidchemie, am Klopferspitz, D-8033 Martinsried b. München, F.R.G. Munekata. E., Prof., University of Tsukuba, Institute of Applied Biochemistry, Ibarakiken, 305, Tsukuba., Japan Naithani V.K, , Dr. , Deutsches Wollforschungsinstitut an der Technischen Hochschule Aachen, Veltmanplatz 8, D-51OO Aachen, F.R.G. Najjar V.A., Prof., Tufts University School of Medicine, American Cancer Society, 136 Harrison Avenue, Boston, MA 02111, U.S.A.

XXXVIII

Nesvadba. Ii., Dr., SANDOZ Forschungsinstitut, Brunner Strasse 59, Postfach 80, A-1235 Wien, Austria Niedrich H., Prof., Institut für Wirkstofforschung, Alfred Kowalke Strasse 4, 1136 Berlin, G.D.R. Nikiforovich G.V., Dr., Institute of Organic Synthesis, Acad. Sci. of the Latvian SSR, Aizlcraukles 21, 226006 Riga 6, U.S.S.R. Nikolics K., Dr., First Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary van Nispen J.W.F.M., Dr., Scientific Development Group, Organon Int. B.V., P.O. Box 20, 5340 BH Oss, Netherlands Offord R., Prof., Dópartment de Biochimie medicale, 21 rue Lombard, 1211 Geneve, Switzerland Ohno M., Dr., Laboratory of Chemical Biology NIADDK, Bldg.10, 9N-307, National Institute of Health, Bethesda, MD 20205, U.S.A. Ovchinnikov Ya.A., Prof., Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Vavilova 32, Moscow II7312, U.S.S.R. Paruszewski R., Dr., Medical Academy, Department of Pharmaceutical Chemistry, Banacha. 1, 02 097 Warszawa, Poland Patchornik A., Prof., Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel Pedone C., Dr., Università di Napoli, Istituto chimico, via Mazocannone, 4, 80134 Napoli, Italy Pedroso E., Dr., Department of Química Orgànica., Facultat de Química., Universität de Barcelona, Diagonal 647, Barcelona 28, Spain Penke B. v Dr., Institute of Medicinal Chemistry, Dom ter 8, 6720 Szeged, Hungary Perseo A., Dr., Farmitalia Carlo Erba, via dei Crasci 3, Milano, 20l46-Italy Petersen H., Dr., Novo Research Institute, Novo Allee, DK-2880 Bagsvaerd, Denmark Pfaender P., Prof., Abteilung Biologische Chemie, Universität Hohenheim, Postfach 106, D-7000 Stuttgart 70, F.R.G. Photaki I., Prof., University of Athens, Laboratory of Organic Chemistry, Navarinou Street 13 a , Athens 144, Greece Pipkorn R., Dr., Ferring AB, Box 3 0 5 6 1 , S-200Ó2 Malmö, Sweden Popovich V.A., Dr., Shemyakin Institute of Bioorganic Chemistry, Vavilova 32, Moscow 117988, U.S.S.R.

XXXIX Pospiëek J., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia. Poulos C., Dr., Laboratory of Organic Chemistry, University of Patras, Patrai, Greece Prochâzka Z., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia Przyby^ski J., Dr., Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80 952 Gdansk, Poland Ragnarsson U., Dr., Institute of Biochemistry, University of Uppsala, Biomedical Center, Box 576, S-751 23 Uppsala, Sweden Ramage R., Prof., University of Manchester, Institute of Science and Technology, P.O. Box 88, Manchester M60 1QD, U.K. Reig F., Dr., Instituto de Tecnologia. Quimica y Textil C.S.I.C., C/Jorge Girona. Salgado s/n, Barcelona. 34, Spain van Rietschoten J., Dr., Inserm S.C. 10, Faculté de Médecine, Sec. Nord, Boulevard P. Dramard, F-13 326 Marseille Cedex 15, France Rivier J.E., Dr., Peptide Biology Laboratory, The Salk Institute, P.O. Box 858OO, San Diego, CA 92138, U.S.A. Rocchi R., Prof., Istituto di Chiraica via Marzolo 1, 351 Padova., Italy

Organica,

Roques B., Prof., Laboratoire de Chimie Organique, UER Sciences Pharmaceutiques et Biologiques, Université R. Descartes, 4, Av. de 1 Observatoire, F-75270 Paris Cedex 06, France Roy P.D., Dr., Océ-Andeno B.V., P.O. Box 81, 5900 AB Venlo, Netherlands Rzeszotarska, B., Prof., Institute of Chemistry, Higher School of Pedagogy, Oleska 48, 45-052 Opole, Poland Sakarellos C., Prof., Laboratory of Organic Chemistry, University of Ioannina, Ioannina., Greece Sandberg B., Dr., MRC Neurochemical Pharmacology Unit, Medical School, Hill's Road, Cambridge CB2 2QH, U.K. Sa.vrda J. , Dr. , Institut Pasteur, Service de la Tuberculose, 25, rue du Dr. Roux, F-75724 Paris, France Scatturin A., Prof., Istituto di Chimica. Farma.ceutica, via Scandiana, 21, 1-44100 Ferrara, Italy Schaaper W., Dr., Delft University of Technology, Laboratory of Organic Chemistry, Julianalaan 1 3 6 , 2628 B L Delft, Netherlands

XL Schneider C.H. , Prof., Institut fur Klinische Immunologie, Inselspital, CH-3010 Bern, Switzerland Schön I., Dr., Chemical Works of Gedeon Richter, Ltd., Budapest, P.O. Box 27, H-1^75 Budapest, Hungary Schou O., Dr., Danish Institute of Protein Chemistry, k, Venlighedsvej, DK-2970 H^rsholm, Denmark Schwartz I.L., Prof., Room 21-86, Annenberg Bldg., Mt. Sinai School of Medicine, 100th Street and 5th Avenue, New York, NY 10029, U.S.A. Schwyzer R., Prof., Institut für Molekularbiologie und Biophysik ETHZ, CH-8093 Zürich, Switzerland Seprodi J., Dr., First Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary Servitova L. , Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia. Sheppard R.C., Dr., Laboratory of Molecular Biology, MRC Centre, Hill's Road, Cambridge CB2 2QH, U.K. Shiba T., Dr., Department of Chemistry, Faculty of Science, Osaka University, Toyonaka 560, Osaka, Japan Siemion I.Z., Prof., Institute of Chemistry, University of Wroclaw, ul. Joliot Curie l4, 50 383 Wroclaw, Poland Skopkova J., Dr., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia. Stehle P., Dr., Institut fur Biologische Chemie und Ernährungswissenschaft, University of Hohenheim, D-7000 Stuttgart 70, F.R.G. Stewart J.M,, Prof., University of Colorado, Medical School, Biochemistry B-126, Denver, CO 80262, U.S.A. Stoev S., Dr., Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulga.ry Straka R,, Dr., Institute of Hygiene and Epidemiology, Srobarova 48, 100 kZ Prague 10-Vinohrady, Czechoslovakia. SÜli-Vargha H., Dr., Research Group for Peptide Chemistry, Hungarian Academy of Sciences, Muzeum krt. 4/B, H-1088 Budapest, Hungary Szelke M., Dr., Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 OHS, U.K. *

r

••

••

Szokan G., Dr., Institute of Organic Chemistry, Eotvos University, Muzeum krt. 4/B, H-1088 Budapest, Hungary Teetz V,, Dr., Hoechst A.G., Pharma Synthese G 8 3 8 , Postfach 800320, D-6230 Frankfurt/Main 80, F.R.G.

XLI Ten Kortenaar P.B.W., Dr., Laboratorium voor Organische Chemie, Toernooiveld, NL-6525 ED Nijmegen, Netherlands Teplân I., Dr., Hungarian Academy of Sciences, Department of Life Sciences, P.O. Box 6 , H - 1 3 6 1 Budapest, Hungary Theodoropoulos D. , Prof., Laboratory of Organic Chemistry, School of Natural Sciences, University of Patras, Patras, Greece Thoreil J., Dr., Ferring AB, Fack, S-200 60 Malmö,

Sweden

Thornber C., Dr., ICI Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K. Tomatis R., Prof., Istituto di Chimica Pharmaceutica, via Scandiana, 21, 1-44100 Ferrara, Italy Toniolo C., Prof., Istituto di Chimica Organica, University di Padova, via Marzolo, 1, 1-35100 Padova, Italy Vavrek R., Dr., Department of Biochemistry B12ó, University of Colorado, Medical School, 4200 East 9th Avenue, Denver, CO 802Ó2, U.S.A. Veber D. F. , Dr., Department of Medicinal Chemistry, Merck Sharp and Dohme, Research Laboratories, West Point, PA 19486, U.S.A. Vicar J., Dr., Department of Chemistry, Medical Faculty, Palacky University, S. Allende 3» 77515 Olomouc, Czechoslovakia Vlasov G.P., Dr., Institute of High Molecular Weight Compounds, USSR Academy of Sciences, Leningrad, U.S.S.R. Voelter W., Prof., Physiologisch-Chemisches Institut, Universität Tübingen, Hope-Seyler Strasse 1, D-7400 Tübingen, F.R.G. Vogel E., Dr., Fluka AG, CH-9^70 Buchs,

Switzerland

Voss C., Dr., Max Planck Institut fur Medizinische Abtlg. Naturstoff-Chemie, Jahnstrasse 29> D - 6 9 O O Heidelberg, F.R.G.

Forschung,

Wade J., Dr., Laboratory of Molecular Biology, MRC Centre, Hill's Road, Cambridge CB2 2QH, U.K. Wade R., Dr., CIBA GEIGY, Pharmaceuticals Division, Wimblehurst Road, Horsham, West Sussex RH12 4AB, U.K. Wallace C.J.A., Dr., Départment de Biochimie médicale, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Genève 4, Switzerland Walz H., Dr., VEB Berlin-Chemie, Glienicker Weg 182-184, 1199 Berlin-Adlershof, G.D.R, Widmer F., Dr., National Institutes of Health, Laboratory of Chemical Biology NIADDK, Bg.lO, 9 N - 3 0 7 , Bethesda, MD 20205, U.S.A.

XLII Wieland T., Prof., Max Planck Institut für Medizinische Forschung, Jahnstrasse 29, D-69OO Heidelberg, F.R.G. Williams B.J., Dr., Neurochemical Pharmacology Unit, Medical Research Council Centre, Hill s Road, Cambridge, U.K. Winter H., Dr., Institute fur Organische Chemie Universität Tübingen, auf der Morgenstelle 18, Tübingen, F.R.G. Wünsch E., Prof., Max Planck Institut für Biochemie, Abteilung für Peptidchemie, am Klopferspitz, D-8033 Martinsried b. München, F.R.G. Young G.T., Dr., The Dyson Perrins Laboratory, South Parks Road, 0X2 6UR Oxford, U.K. Zahn H., Prof., Deutsches Wollforschungsinstitut der Technischen Hochschule Aachen, Veitmanplatz 8, D-5100 Aachen, F.R.G. Zanotti G., Dr., Xstituto di Chimica Farmaceutica, Citta Universitaria, Pz. le Aldo Moro, 1-00100 Roma, Italy Zaora.l M. , Dr. , Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, l66 10 Prague 6, Czechoslovakia

XLIII Emil Taschner 1900 - 1982 Emil Taschner was born in Cracow in 1900. He entered the Jagiellonian University in 1920 to study chemistry and later moved to Vienna where he received in 1927 his PhD degree. After postgraduate studies in Paris (Pa steur Institute, Department of Biochemistry and Department of Internal Medicine of the University of Paris he returned to Cracow and worked in the Pharmacology Department of the Jagiellonian University (Professor J. Supniewski) and in the research laboratory of the pharmaceutical factory "Dr. A. Wander".

The years of World War II he spent in Lwow partly as a workman, partly in hiding. Vhentfie atrocities were over he moved to Wroclaw where he joined the General Chemistry Department of the University and Technical University. In 1953 he folior

wed a call to Gdansk and was appointed Head of the Department of General Chemistry at the Technical University. In 1964 he became Head of the Peptide Chemistry Department at the same University. He retired from that post in 1971« Taschner s scientific carreer began in Vienna with the isolation of compounds from Brionia dioica. (doctor thesis). In

XLIV Paris he widened his horizons into biochemistry (investigations on serum lipase). During the Cracow period his interest was focused on biochemistry and drug research. For his studies on tumor-inducing compounds he was awarded in 1938 the Pilsudski prize of the Polish Society for Fighting Cancer. His studies on derivatives of tyrosine hydantoin as potential antithyreotoxicosis agents, started in Cracow and continued in Wroclaw, are the first indica.tion of his future interest in amino acids and peptides. After World War II studies had begun in Wroclaw on acylation with diacetylamides, and this was a further indication of his future interest in peptide research. The move to Gdansk provided the opportunity for expansion of his research activities. He recognized amino acids and peptides as a field of expanding importance and was fortunate enough to gather a group of co-workers who laid the founda«tion for a school of peptide chemistry in Poland. He started by introducing newer methods of organic chemistry to peptide synthesis. In a series of papers various aspects of fision reactions of ester, urethane and amide groups were studied. These investigations culminated in the elaboration of new procedures for acidolytic removal of benzyloxycarbonyl amino protection. In this context, I should also mention the cleavage reaction of esters by lithium halogenides. This method, albeit not used widely in peptide chemistry, found its way into organic chemistry as a method of choice for solving specific problems connected with the carboxyl protection. The seniors among us will recall the first meeting of the European peptide community in Prague where Taschner reviewed the problem of carboxyl protection. It is a pity that the manuscript of the lecture was not published, but the printed discussion following his lecture gives evidence that he being aware of shortcomings of the methods available at that time and inspired from the field of penicillin chemistry -

XLV proposed to use the tert-butyl group for amino and carboxyl protection. To envisage protection with tertiary alkyl groups required great foresight and inspiration. It remained in his mind until in 1959 in Munich at the XVTIth Congress of Pure and Applied Chemistry and 2nd EPS he presented the first approaches to the synthesis of tert-butyl esters of amino acids. In 1961 Taschner and his group published the first paper on the synthesis of tert-butyl esters of amino acids by transesterification of tert-butyl acetate. This

transesterifica-

tion - or esterolysis, as Taschner called it - set a milestone not only in the history of carboxyl protection in peptide research but also as a method of choice for the generation of tert-butyl cations in organic chemistry. The approach was developed further and methods became available for the direct esterification of amino acids with unmasked amino group, selective esterification of the gamma carboxylic group of glutamic acid, etc. The studies of Taschner's group were contemporary to those of Robert Schwyzer's group. Both authors became aware of the synthetic potentialities of tertiary esters which were subsequently exploited in numerous syntheses. Another topic - epimerization

(ra.cemization) studies - conti-

nued to be Taschner's main theme for several years and culminated in the "two spots Taschner's ra.cemization test" as it was denoted in "Houben Weyl" by Wunsch et al. Using paper and/or thin-layer chromatography various activation procedures were tested for the degree of loss of stereochemical homogeneity in the a.cylating residue during coupling. A further point of interest was the evaluation of the danger of ra.cemization in the amino acid residue penultimate to the C-terminal one. Having established the degree of epimerization in various coupling procedures, including the azide method, and having defined the experimental conditions which

XLVI suppress the undesirable formation of epimers in peptide synthesis, Taschner considered even more ambitious targets. His last papers on peptide chemistry described new methods for peptide bond formation which, according to two spots test, were free or almost free from racemization. These routes included the use of active 3-Pyridyl, N-hydroxyquinolinimide, and benzhydroxamic esters. /

I have tried to offer an account of Taschner s more important contributions believing that this would be the best tribute to his innovative ability and foresight. Throughout his life, he was hard working and productive with many original ideas, and evinced the greatest enthusiasm for research devoting full vigour to his beloved peptide field. Taschner was a person who did not care for rank, honours or grades. The achievements which brought him international reputation gave him no splendour in his homeland. My portrait of a person and a scientist would not be complete without mentioning that he will always be remembered for his wit, and for anecdotes about him. I would like to quote from Theodor Wieland's recollections at the Gdansk meeting in 1978: "There was Etail Taschner who contributed at this place to the genius loci, and whose presence always brought a sort of pepper to the symposia". Einil Taschner died in Gdansk on Hay 4, 1982 in his 82nd year. He left over 100 scientific publications and patents. The Polish peptide community has lost its most prominent personality who will always be remembered.

(Bogdan Liberek)

XLVII Dieter Marquarding 193^ - 1982 Dr. Dieter Marquarding, Professor at the Technische Universität in Munich died untimely July 9, 1982 after a short, but severe disease. He was 47 years old. Born in Königsberg (presently Kaliningrad) on November 3°> 193^ Dieter Marquarding received the degree of Diplom-Cheraist from the University of Hamburg in 1961 and working with Professor G. Snatzke the Doctor degree in chemistry in 1965 from the University of Bonn. He then went to the research laboratories of Bayer AG, Leverkusen and finally in 1973 he joined the research group of Professor Ivar Ugi at the Technische Universität in Munich.

Dieter Marquarding made numerous research contributions to the stereochemistry of ferrocene derivatives in collaboration with Ivar Ugi. Among these as most significant results one has to mention the retentive nucleophilic substitution, the stereoselective 2-lithiation of a-ferrocenylamines, the use of OC-ferrocenyl alkylamines for asymmetric syntheses as well as a new method for the determination of the absolute configuration.

XLVIII

Besides his specific research contributions to the theoretical stereochemistry as pseudorotation and Tournstile rotation, hyperchira.lity or group theoretical analysis of conformational flexibility, since seven years in collaboration with Rosemarie Kopp, James Dugundji and Ivar Ugi he enthusiastically worked on a. new theory of chemical identity. The results of these extensive studies will be published by the Springer Verlag in the Lecture Note Series. Dieter Marquarding's main contribution to the peptide chemistry derived from his engagement in collaboration with Ivar Ugi in the chemistry of isonitriles. The development of new methodology based on the four component condensation procedure (Ugi's reaction) as well as the morpholinoethyl isocyanide as a. new coupling reagent enriched remarkably the possibilities of peptide chemistry. For these new developments Dieter Marquarding will be remembered by the peptide chemists and his active participation at Peptide Symposia, and other meetings he attended will be missed. He was a. constant contributer to scientific discussion, a distinguished scientist and a. dear colleague. Erich Wünsch Luis Moroder

XLIX

ERHARD GROSS, 1928-1981

The tragic death of Erhard Gross, Chief of the Section on Molecular Structure, Reproduction Research Branch, National Institutes of Health, Bethesda, Maryland, in a traffic accident in Germany on September 12, 1981, deeply shocked scientists in all parts of the world.

In an instant, Gross passed away,

in the most active period of his scientific career. Throughout the years Gross participated in almost every meeting on peptides and proteins in the United States or abroad. Thus, for many years he became an international ambassador in this field of research - a role served earlier by Josef Rudinger, the founder of Peptide Symposia.

The entire scien-

tific career of Gross has been characterized by the highest kind of motivation and accomplishment; because of this his presence was missed very much in this 17th European Peptide Symposium and will be missed for many years in our future scientific gatherings.

L

Erhard Gross was born in 1928 in Wenings, near Frankfurt/Main. After graduation from the Wolfgang Ernst Gymnasium at Büdingen, he studied chemistry at the Universities of Mainz (1949-1953) and Frankfurt/Main (1953-1958).

He received his doctorate

degree (Ph.D.) in 1958, working with Professor Theodor Wieland on the synthesis of a bicyclic model peptide related to the mushroom toxin phalloidin.

Upon completion of his studies

Gross went to the United States in 1958 to work with Professor Bernhard Witkop at the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health in Bethesda, Maryland.

He also spent some time with Professor Lyman C.

Craig at the Rockefeller University in New York City, further broadening his fields of expertise.

In 1968 Gross was ap-

pointed Chief of the Section on Molecular Structure, Laboratory of Biomedical Sciences, and in 1973 Chief of the Section on Molecular Structure, Reproduction Research Branch at the National Institute of Child Health and Human Development, National Institutes of Health. The scientific work of Erhard Gross was distinguished by excellence and innovation, and the foundations for these qualities were undoubtedly developed during his studies with Theodor Wieland in Frankfurt.

The style of Gross' work, the precision

and attention to detail as well as his extraordinary perseverance, were developed during these years.

The admirable com-

bination of basic organic chemistry, natural products chemistry and peptide synthesis in Wieland's laboratory determined the directions of Gross' research.

Of equally lasting influence

was the close friendship that existed between Wieland and Gross and which extended throughout his career.

The association

with Lyman C. Craig, an innovative genius and yet so kind and modest a person, was in Gross' own words "of the most profound

LI influence on my subsequent career"

(1).

Of, perhaps, similar

consequence was his affiliation for ten years with Bernhard Witkop, a superb analytic and philosophic mind at the National Institute of Arthritis and Metabolic Diseases. A series of publications started to appear in 1960 dealing with non-enzymatic cleavage of peptide bonds.

The paper by

Gross and Witkop on the "selective cleavage of methionyl peptide bonds in ribonuclease with cyanogen bromide"

(2) dis-

closed the most useful of all chemical cleavage procedures, which combined mild reaction conditions with a high level of selectivity.

It continues to be an indispensable method

for the structural elucidation of peptides and proteins that contain methionine

(3) and has recently gained additional

importance for rapid HPLC peptide mapping and for recombinant DNA synthesis. In 1967 the first publication on the peptide antibiotic nisin was published by Gross and Morell.

The report of the presence

of an unusual amino acid, i.e. dehydroalanine, in nisin was not to cause any special attention because unusual constituents had long become the hallmark of most antibiotics.

However, it

soon became apparent that nisin possessed a most fascinating complexity of unprecedented structural features.

The complete

structure was reported in 1971 (4), and of the 29 amino acids in nisin, eight are rarely found in nature.

Three of these are

dehydroamino acids, i.e. two dehydroalanine and one dehydrobutyrine residue.

There are five cyclic structures in the

peptide chain, formed by sulfide bridges that are contributed by one lanthionine and four 8-methyllanthionine units.

Penta-

cyclic heterodetic type of peptides were found for the first

Lll time, two of which were fused in a unique double-ring structure consisting of 13-membered rings each at the carboxyl terminal region of nisin. Surprisingly, other peptide antibiotics from different microorganisms, such as subtilin, cinnamycin, and duramycin were found to have similar backbone structures, although of slightly different amino acid sequence

(for review see 5).

The study of

these antibiotics developed into a lifelong project for Gross and his collaborators.

Plans and work toward the synthesis of

these complex molecules

(6) and on the further exploration of

their interesting biological effects remain uncompleted. The occurrence of so many unusual amino acids in nisin and subtilin became the origin of another fascinating trail of research for Gross, i.e. the chemistry and synthetic utility of a,0-unsaturated amino acids.

Dehydroalanine, isolated from

nisin in 1967 (7) was the first a,3-unsaturated amino acid discovered in nature.

It can be readily incorporated into peptide

chains via g-elimination of intermediate O-tosylserine residues, and its side chain reactivity then permits cleavage into peptide acyl amide and pyruvyl peptide by mild acid hydrolysis. Recently an elegant solid phase synthesis of oxytocin was described

(8) in which the carboxy terminal amide was generated

from a dehydroalanine anchor to the solid support and both the asparaginyl and glutaminyl amide groups from dehydroalanyl ethylamide, by acid hydrolysis at the end of the synthesis

(for

review see 9). Another family of antibiotic peptides with highly unusual albeit highly ordered structural features, the linear gramicidins, proved to be of continuous and lasting interest to Gross.

LIU Efficient solid phase procedures were developed for the synthesis of gramicidins A, B and C (10).

These formylpentadeca-

peptide ethanolamide molecules with an alternating pattern of L- and D-configurations were found by Dan Urry to assume the conformation of the ir -helix and to form channels for cation LD transport in lipid membranes. An intensive study of these systems was carried out in collaboration with Peter Läuger at the University in Konstanz using synthetic analogs of the gramicidins (for review see 11). The most recent interest of Gross was focused on the chemistry and function of chemotactic peptides, which he pursued in collaboration with E. Schiffmann (12). Erhard Gross will also be widely remembered for his outstanding literary contributions to the peptide and protein field. At a very critical time, in the early 1960's, when the methodology of peptide synthesis was rapidly expanding, Gross undertook the enormous task of translating the two volumes, "The Peptides", compiled by Eberhard Schröder and Klaus Lübke from German into English, published by Academic Press in (1965/66).

The impact of these books on the rapid development

of peptide research was invaluable.

Ten years later we dis-

cussed whether the monumental Houben-Weyl of Erich Wünsch should be translated into English, or whether an expanded second edition of Schröder/Ltlbke should be produced.

It was

Gross' dre am to collect all the information on the now exponentially growing peptide field in concise form.

Eventually, this

assumed the format of multi-authored, medium-sized reviews in the open-ended treatise, "The Peptides, Analysis, Synthesis, Biology .{Academic Press).

Due to the tireless efforts of Gross,

LIV four volumes have appeared (13).

Two other volumes under his

editorship are in preparation (14) . Gross was very active in the American Peptide Symposium Committee and made major contributions to the organization of several of the symposia.

One of the most outstanding meetings was one

held under his chairmanship in Georgetown University, Washington, D.C., in 1979.

The number of participants, as well as of

lectures and posters, and the size of the Proceedings Volume (Peptides: Structure and Biological Function) exceeded those of all previous symposia.

The Winter Gordon Conferences on

Peptides established in 1976 were regularly attended by Gross who was an outstanding contributor to the discussions and provided much stimulation. Gross always promoted international collaboration and exchange. Many of his collaborators were visitors from overseas, especially Japan and Poland.

In 1979 he was awarded the Alexander von

Humboldt prize to carry out collaborative studies in Germany a well deserved honor. Gross had a wide span of extracurricular interests including theater, literature and music. enjoyed a party.

He was very sociable and always

He did not hesitate to drive for hours to

obtain a selected bottle of wine.

At his home in Bethesda

Gross preferred to maintain a German style of life with his wife, Gertrud, and his two sons, Johannes and Christoph.

Every

year they spent their summer vacations at their German home town.

On each Christmas Eve the family followed him into the

nearby Virginia woods to personally cut a Christmas tree to be decorated in their home. Along with his family the large constituency of his friends throughout the world mourn his passing and will always remember him

-

(J. Meienhofer)

LV

References 1.

Gross, E.: In "Peptides: Chemistry, Structure and Biology (R. Walter, J. Meienhofer, eds.), p. 31. Ann Arbor Sei. Publ., Ann Arbor, Michigan 1975.

2.

Gross, E., Witkop, B.: J. Am. Chem. Soc. £3, 1510-1511 (1961).

3.

Gross, E.: In "Methods in Enzymology" (C.H.W. Hirs, ed.), pp. 238-255. Academic Press, New York 1966.

4.

Gross, E., Morell, J.L.: J. Am. Chem. Soc. 93., 4634-4635 (1971).

5.

Gross, E.: In "Protein Crosslinking-B" (M. Friedman, ed.) pp. 131-153. Plenum Publ., New York 1977.

6.

Pallai, P., Gross, E.: In "Peptides 1978" (I.Z. Siemion, G. Kupryszewski, eds.), pp. 357-363. Wroclaw University Press, Wroclaw, Poland 1979.

7.

Gross, E., Morell, J.L.: J. Am. Chem. Soc. 89^, 2791-2792 (1967) .

8.

Nöda, K., Gazis, D., Gross, E.: Int. J. Peptide Protein Res. 19 , 413-419 (1982) .

9.

Gross, E., Nöda, K., Matsuura, S.: In "Peptides 1974" (Y. Wolman, ed.), pp. 403-413. Wiley, New York 1975.

10.

Nöda, K., Gross, E.: In "Chemistry and Biology of Peptide (J. Meienhofer, ed.), pp. 241-250. Ann Arbor Sei. Publ., Ann Arbor, Michigan 1972.

11.

Bamberg, E., Apell, H.-J., Alpes, H., Gross, E., Morell, J.L., Harbaugh, J.F., Janko, K., Läuger, P.: Federation Proc. 37_, 2633-2638 (1978).

12.

Aswanikumar, S., Schiffmann, E., Corcoran, B.A., Pert, C.B., Morell, J.L., Gross, E.: Biochem. Biophys. Res. Commun. 80., 464-477 (1978) .

13.

The Peptides, Analysis, Synthesis, Biology (E. Gross, J. Meienhofer, eds.): Vol. 1, Major Methods of Peptide Bond Formation (1979); Vol. 2, Special Methods in Peptide Synthesis, Part A (1980); Vol. 3, Protection of Functional Groups in Peptide Synthesis (1981); Vol. 4, Modern Techniques of Conformational, Structural and Configurational Analysis (1981). Academic Press, New York.

14.

See (13): Vol. 5, Special Methods in Peptide Synthesis, Part B (1983); Vol. 6, Physical Methods in Peptide Confor mational Studies. (V. Hruby, Volume ed.) (1983).

RECENT DEVELOPMENTS IN THE CHEMISTRY AND BIOLOGY OF CYCLIC PEPTIDES

Yuri Ovchinnikov, Gunar Chipens* and Vadim Ivanov Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, 117988 Moscow, USSR *Institute of Organic Synthesis, Latvian Academy of Sciences, Riga, USSR

Cyclopeptides comprise a large group of compounds isolated from natural sources or obtained by chemical synthesis. There are many reasons due to which cyclic peptides over a number of decades remain a popular object of study. All cyclic peptides (except cyclic disulfides) are synthesized by specific

poly-

enzymic systems rather than by conventional ribosomal procedure. In addition to being cyclic they all have other peculiar features not present in the peptides obtained by processing of proteins. The most obvious is presence of various noncoded D,_ N u -methyl, A , etc. amino acids or a-hydroxy acids. Chemistry of cyclopeptides continuously raises interesting theoretical problems concerning their structure elucidation, synthesis or explanation of physicochemical properties. Reaction of cyclization appeared to be a complex process in which linear oligomerization competes with cyclization of both the original activated peptide and its oligomers, hence the term

R I H - (NHCHCO )n- X

I

R I ^-(NHCHCO)n^ Fig. 1.

R I H-(NHCHCOX

I

R ' ^(NHCHCO

R I *~H-(NHCHCO ), - X

I R

1

NHCHCO)^

Cyclooligomerization of a linear peptide (1).

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

, etc .

2

"cyclooligomerization"

(Fig. 1). Relative yields of the pro-

ducts obtained are determined by an intricate combination of factors: dilution, conformation of linear peptides and the cycles to be formed, structures of the reaction complexes, stability of the reactive intermediates, and so on. The problem of cyclic disulfide formation has been met and solved in early 50-ies by Du

Vigneaud in his pioneering

studies of oxytocin and related molecules. Here, additional possibility arises that the cyclic dimer might be parallel and antiparallel, as shown in Fig. 2. The regularities found at the study of cyclic disulfides are directly related to such aspects of protein chemistry as recombination of A and B chains of insulin and renaturation of cystine-containing proteins after reduction (ribonuclease A, trypsin inhibitor, etc.). Cyclopeptides, especially with smaller rings sometimes possess unusual chemical properties due to transannular interactions resulting, for example, in formation of cyclols (5) (ergot alkaloids, antamanide, Fig. 3). Besides, it is the chemistry of cyclols from which an original method of cyclopeptides synthesis has evolved, the so called hydroxy (or amino) acyl incorporation. This method afforded facile preparation of a series of cyclic depsipeptides, including the antibiotic serratamolide (Fig. 4) .

H - C y s - G l y n ~ Cys - OH

> H - Cys-Glyn"Cys-OH

H-Cys-Glyn-Cys-OH

H - Cys - Gly n - Cys - OH

H - C y s - G l y n - Cys - O H Fig. 2. (2, 3).

HO - C y s - G l y n - C y s - H

Cyclization of Cys,Cys-containing peptides

3

H0

H QH

r C-C-NH

-NH-CJ 0

*

,0

r c-c'

-r—' -N-C n .0

NH,

* ,0

rc-c-

:R • HjN- •—COOMe I MeOH _J -N -N-C II _0

-H20/ /H20 H -C-C=Ni H» rC-C-NH ^ N-C-'

_H

* ^ N-C 1

^ N-C—'

n t g Fig. 3. Suggested formation of cyclols (b) during acid hydrolysis of antamanide, leading to "false" sequences (d) and racemization due to (b)i(f)£(g)*(h) equilibrium (4).

Absence of N- or C-terminal groups and availability of model cyclopeptides have made them favourable objects for physicochemical studies. Especially popular are the simplest cyclopeptides, the diketopiperazines. They easily form even on acid hydrolys is of proteins. This fact/ discovered in the last century at the reign of Emil Fischer led to a number of diketopiperazine theories of protein structure; some of them survived until the late 50-ies. At present diketopiperazines serve a

ACOCHJ

NH

0

OBZL CH 3 (CH ; )°CHCH 2 C0CI

O^nh-^ch2OAC

Bzl

CH,0Ac

OCAL^S

co^y10 o\-, Bzl

C 7 H ,5N

ri

1

1

CH2OH Serratamolide

Fig. 4. Total synthesis of serratamolide by hydroxyacyl incorporation (6, 7).

4 more prosaic but still a useful purpose in the studies of racemization kinetics (8-13), photolysis (14), radiolysis (15), mass-spectrometric fragmentation (16-19), solution thermodynamics (20) . Many naturally occuring cyclopeptides possess high biological activity. Extreme diversity of their structures, of mechanisms of action and of molecular targets is quite remarkable (see the review (21)). To this group belong the widely known cyclic disulfides - hormones oxytocin, vasopressin, calcitonin, somatostatin, toxins of the phalloidin and amanitin families, apamine, tertiapin, secapin, the antitoxin antamanide, cyclopeptides - carriers of alkali metal ions (valinomycin, enniatins) and Fe"*+ (ferrichromes), antibiotics disturbing the structure of biological membranes (gramicidin S, polymixins), blocking the ribosomal function (etamycin, virginiamycin S and related cyclodepsipeptides, containing a 3-hydroxypicolinic residue), the biosynthesis of bacterial cell wall (vancomycin and penicillins, though attribution of the latter to cyclopeptides might be rather far fetched) or binding to DNA (actinomycins, triostins and echinomycins); considerable interest is currently attracted by a powerful immunodepressor, cyclosporine A. Conformationally cyclic peptides are less flexible than their linear counterparts, a feature which has far-reaching consequences. Comparative simplicity of the conformational equilibrium greatly facilitates studies of three dimensional structures, both by X-ray analysis (conformationally homogeneous samples are easier to crystallize) and by spectral methods in solutions which are so difficult to apply to linear peptides because of averaging of the spectral parameters. Conformational studies provided a key to understanding the mechanisms of action of antibiotics-ionophores

(valinomycin,

enniatins), antamanide, gramicidin S, actinomycins, echinomycin, and some other cyclopeptides. Only a few examples from other classes of pharmacologically active compounds can be cited with a comparable insight into molecular mechanisms of action.

5

Understanding of regularities of cyclopeptide mitted goal-directed

folding per-

synthesis of new compounds of this class,

which are used either as enzyme substrates (22-30) or as models of enzyme active sites (31-40). Advances in stereochemistry of depsipeptides - ionophores stimulated preparation of novel syn2+ thetic ion-, particularly Ca -transporting cyclopeptides (41, 42). They also accelerated investigations into chemistry of other types of metal binding molecules - crown ethers, cryptands (43, 44) and even linear amides

(45-47).

Cyclopeptides are favourite objects for developing the methodology of the spectral analysis of peptide conformation. Assignment of CD bands and evaluation of their calculation methods, quantitative analysis of hydrogen bonding from IR and NMR spectra

(see the reviews (19, 48)), formulation of stereo-

chemical dependencies of vicinal coupling constants (49) these and many other studies became possible due to accessibility of cyclopeptides and their unique properties. It is not surprising that cyclopeptides became a sort of test field for formulating and verifying new stereochemical concepts subsequently extended to linear peptides and proteins. In 50-ies Schwyzer (50) having only sparse firm data at hand proposed "pleated sheet" structures for cyclic hexa- and decapeptides, a concept which we all accept

today. Conformational

parameters of g-turns stabilized with 4->-l hydrogen bonds were established on the example of cyclopeptides

(valinomycin,

gramicidin S, cyclic hexapeptides). Now B - turns are considered along with the a-helix and g-structure as the major, basic elements of the protein structure. In 1963 Dale (51) included cyclopeptides into his more general analysis of relative thermodynamic stabilities of various macrocycles. At the end of 60-ies Prelog et al. (52-54) developed on the example of cyclopeptides his original ideas of cycloenantiomerism and cyclodiasterebisomerism. The idea of total transformation of the peptide (retro-, enantio-, retroenantio isomers, substitutions COO

CONH, CONH

COO and so

on) with preservation of the overall molecular topography was

6

formulated by us on the example of cyclic peptides (55, 56). According to this approach, called topochemical, a number interesting analogs of valinomycin (57, 58), enniatins (59, 60), antamanide (61-64) and gramicidin S (65-67) was obtained. Similar transformations of linear peptides afforded various active preparations - inhibitors of enzymes, a-chymotrypsin (56, 68, 69), pepsin (56, 70), thrombin (71), analogs of angiotensin (72, 73) and enkephaline (74-76). Recent developments in experimental and theoretical approaches to conformational states of peptides afforded some progress in unraveling the spatial structures of biologically active linear peptides: bradykinin, angiotensin, corticotropin, enkephalins, neurotensin, 6-sleep inducing peptide, tuftsin and others. Despite the clearly expressed conformational mobility folded structures seemed to participate in the conformational equilibrium of all these peptides. Calculations showed that these structures may be stabilized by ionic interactions of oppositely charged carboxyl and amino (or guanidinium) moieties. These interactions are weak in the strongly solvating water but are expected to increase considerably in the lipophilic phase of bioreceptor as shown in Fig. 5. Thus, we arrive at the

Polar (aqueous) solution

L i p o p h i l i c receptor

biophase

Fig. 5. Receptor binding favours ionic interactions and folding (77, 78, 84).

7 suggestion that covalent crosslinking of these quasicyclic structures, i.e. cyclization of linear peptides might be a plausible way to novel biologically active preparations.

7 In fact as early as in 1966 Meienhofer synthesized cyclo (-Gly —

kallidin-) (78), in 1975 DeCoen et al. prepared (79), in 1977 Donzel et al.

cycloangiotensin

(80) and in 1978 Seprodi et al.

(81) have made several cycloluliberins. Since all these preparations proved inactive, work was not continued in that direction. However, a simple analysis shows that general topography of the cycloanalog, i.e. its complementarity to the receptor strongly depends upon the choice of cyclization site, this conclusion is schematically shown in Fig. 6. Therefore in the absence of concrete data on "bioactive" conformation of the peptide the search of active analogs should include variation of the cyclization site and the ring sizes. Such search was carried out for a number of peptides

and selected results

of this work are presented below. Quite interesting data were obtained in the bradykinin/kallidin series (Table 1). It appeared that e-cyclokallidin (IV) is a

full

agonist

(a=l) both in uterus contraction and arte-

ria pressure tests in rat -(although less active than bradykinin itself, pD2=7.6) . Noteworthy, in the in vivo test the cyclic analog expressed its action for more than 2 hr whereas bracfykinin

N

O D D u N C

Fig. 6.

"

r

L

C

N

c

Linear peptides can fold in a number of ways.

8

00

CN Q CM

IO

Ol

•P

00

1-H S 0

2

U

a)

o

rH (0 o •H 01 0 rH 0 •rH CQ

T3 g i rH O 0 M CM 0 M CM Oi M


1 •X) ta M XJ i

43 CM 0 U CM >i rH O 0) Sí >i CM i—i >i O rH 1 — O 0 0 rH h O CM 0 >1 M u 1 CM 01 a h

60

Val-HH, (70BM J Z-tlo-Ph«-OM«

lav-NH} ( 70 «M )

60 I-AI«-Pha-lav-HH;

V«I-HH, (70 *M ) I- l*u-Alo-OM*

Z-Ab-Tr-OMa

• sc-Lau-Fha-OMa

76 B

40 60

l«»-NH2 I 70 AMI

Z-lM-ila-lau-NH,

30

Val-HH, ( 70 mM)

Z-Lau-At«- Val-HH,

40

95"

J20

lay-HH, (105 mM)

I-Ala-T»

lohelix transition of ACTH N-terminal segments in trifluoroethanol (a solvent mimicking hydrophobic membrane surroundings [22])has been well documented by circular dichroism [23] and infrared spectroscopy [13]. Our results prove that the conformation of ACTH in membranes is similar to that in trifluoroethanol, but that, in membranes, the a-helical segments are oriented approximately parallel to one-another and to the lipid hydrocarbon chains. In thus appears that the lipid phase of target-cell membranes (that contain excess anionic lipid [24] could capture ACTH(l-24) molecules and facilitate receptor interaction by reduction of dimensionality [2],

The

membrane interaction would leave the address segment [25] of ACTH(1-24) on the hydrophilic surface and lead to an incorporation of the message segment [25] as an oriented a-helix into the hydrophobic layers.

This would

expose those elements of the message that are responsible for triggering different receptors for different responses on different surface elements of the helix [25].

Thus, the peptide/membrane interaction might well be

responsible for the correct orientation of the message within the membrane, so that it would then be in a position to interact properly with different receptors.

For such an interaction to take place, the active sites of the

receptors might be buried in the hydrophobic layers of the membranes and not necessarily be exposed on the hydrophilic surface.

This would explain

69 the extremely weak biological activity of the 'free message', ACTH(l-lO), that hardly interacted with membranes and prefered the p-pleated sheet over the 'active' a-helical structure of the highly potent ACTH(l-24). We might predict that a necessary condition for being a strong ACTH agonist is the presence in the molecule of a 'potentiator' or an 'address' segment [25] that will promote the tendency of the 'message' segment to be inserted into the membrane and assume a helical conformation.

If the

message is impaired, so that it cannot trigger the receptor (examples in [25], but can still meet the insertion and conformation condition, then we could expect such a molecule to be a potent antagonist. Preliminary experiments with our methods indicate that these assumptions might be correct, at least in the case of the native hormone ACTH(1-39), the partial agonist ACTH(5-24), and the strong antagonist ACTH(7-24).

(The peptide ACTH(ll-24)

is indeed only a very weak antagonist and could act by electrostatic competition on the membrane surface and not at the receptor itself.)

4.2 Opioid peptides

We have not yet performed ATR-IR experiments with these peptides, but our HL experiments strongly indicated differential ('specific') interactions with lipid bilayer vesicles. Dynorphin(1-13), which is a strong K-receptor agonist containing (like ACTH) a pharmacologically defined, N-terminal message segment (Tyr-Gly-Gly-Phe-) followed by an address segment (5-13) [26] , behaved very much like ACTH(l-24):

It inserted its message sequence into the membrane.

Thus a similar role of the membrane for the molecular mechanism of dynorphin action can be postulated.

Particularly, the recognition and active

sites of the •4

ap

d ^g

an

6

(D-Ala-D-Ala) and e ( I ) ( G l y - G l y ) bends:

d i angles d i f f e r by more than 100°, and t h i s i s i n

agreement w i t h the expectations f o r e ( I ) and p ( I ) ' II).

This i s

]

bends (Figure 5 ( I and

A least-squares f i t using the 18 atoms i n the r i n g and C„ atoms i s

shown i n Fig. 7.

P

o

The average d i f f e r e n c e i n l o c a t i o n o f the atoms i s 0.85 A.

From the drawing i t can be seen t h a t i t i s e s p e c i a l l y the peptide linkages i n the middle of the bends which do not f i t .

I f i t i s indeed p r i m a r i l y the

side groups which determine the topochemical equivalence, t h i s may not be too important because there i s compensation of the angles such t h a t the peptide linkages before and a f t e r the bends f i t q u i t e w e l l .

The d i f f e r e n c e

i n the l o c a t i o n o f the t o p o l o go i c a l equivalent C„P atoms i n the peptide and i t s retroenantiomer are 1.24 A. Prelog and coworkers (12) synthesized the peptides c ( L - A l a - L - A l a - G l y - G l y L-Ala-Gly) and c ( L - A l a - L - A l a - G l y - L - A l a - G l y - G l y ) merism or cycloisomensm i n c y c l i c peptides. mined the s t r u c t u r e s of both peptides (13).

to prove s t r u c t u r a l

iso-

Several years ago we deterF r e i d i n g e r and Veber (14)

96

5CLY 'fCO 6CLY

L-*U 3L-AL* GLY5^7T)2GLY GLY6 1GLY

X GLY1 gGLY GLY23(^(¡^ 5GLY 4 U-ALA L-4U Fig. 6. Retroenantiomer from enantiomer for c(2-D-Ala-4-C.ly).

0* «y 0 «MO

Fig. 7. Least-squares fit for c(2-D-Ala-4-Gly) and R.E.

L-Al» • Glv OA*« MU

Fig. 8. L.S. fit for c(L-Ala-L-AIa-Gly Gly-L-AlaGly ) and R.E.

Fig. 9. L.S. fit for (Mol. A c(Gly-D-Leu-L-Leu-Gly-D-LeuL-Leu) and R.E.

CkUu» L-Uu

97 TABLE 1

105 (

14 ß( I ) 1 1 3 1 -35

173

35 (

31

-53

-131 (

145

*,) -43 ( * . )

-14 (

145

-84 ( 4 )

>n) ß ( i ) - 6 6 (

31

0 UJ

*

3

)

*3) •4)

-105~ • 5 T ~ -168 -70

*

6

)

-16 ß ( I ) - 106

c(L Ala-L A l a Gly-Gly-L-AlaGly)

70

175 66

A

R.E

c(2 D Ala 4 Gly)

)

5

-175" (

70~

139

- 1 0 1 ( -e6)

A

-106

(-*3)

81

-14 (-k,2) 95 ( - * _ )

39 138 117

33

ß ( I )

62

62

(-*.)

28"

1P3) 84 U )

119 ( - < U 6 )

40 78

-17 (

53

)106 (

122

-113

CH2C12 washing

salt of the

out) and of m e t h y l a m i d e

of the products with MeNHg were

in

after

RCONHMe

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

168

CH R \ c=o

N

H

i R' - C

R

R-

! 1

R I C0-NHCH-C00H (4)

R1CO-NHCH-C R2C0-NHCH-C

/

1«

R

(3)

1

2 CO-NHCH-COOH R^

(2)

a :

R

R

= alkoxy;

R

= R

b:

R2

= al kyl /I ;

R3

=

c :

alkoxy;

obtained

rate

mole

one

drides. one m o l e of

salt

present

2)

in

R2

obtained

of a m i d e

and

in the

no

zolones acid

Down

were

mixed

(Id)

in C h ^ C ^

Z-Gly-Leu-OH.

in the

are

amount

f

R4

gene-

anhy-

and w h i c h

oxazolones.

of t h e to

and which

salt

iPr

iPr

= alkyl ; R 3

The of

generate amount

anhydride

1% of a n h y d r i d e

(9),

mixed

with

2-pheny1 - 5(4H) -

(l e ) , 4 - i s o p r o p y 1 - 5 ( 4 H— ) - o x a z o l o n e ,—•

and with

products

are

=

=

detected.

2-Benzyloxy-5(4H)-oxazolone oxazolone

of

in < 5 0 % y i e l d

salt

R

by w e i g h t

one mole

is a m e a s u r e

product. be

yield

and

Products

can

Ph;

>>50%

of a m i d e

generated

oxazolone

R4

,2 R = alkyl

Products

= P h C h 2 0 ; R3

R

solution

was

with The

1 equiv. nature

established

and

other

of t h e

of t h e using

oxa-

parent

neutral 60-MHz

NMR

spectroscopy.

Results 1)

Based

on a n a l y s i s

of E DC w i t h

of

pure

Z-Gly-Leu-OH,

and

crude

products,

Z-Leu-Gly-OH,

the

For-Val-OH

reaction and

169

For-Phe-OH gave

exclusively

the a n h y d r i d e

tides the

gave

with

proof

itely

did

in 88% y i e l d .

D C C are is

less

not

believed

give

(3d) / v

Z-Gly-Leu-OH, neutral

anhydride

ively (~2 b ) .

give

The

the

added

valine

was

component

when

2-alkyl-oxazolone

(2e).

Z-Gly-Leu-OH, derived

from

oxazolones reaction In the

However,

a mixture the

(£)

with

the

presence

into

a single

NMR

with

(le) w a s le w a s

(2d)

present

added

added

Bz-Leu-OH

substrate

was

present.

and

For-Phe-OH

6 = 5.7

complete

ppm

two

acids

did

the

chan-

exhibit

ring-protons.

for 4-iso p r o p y l - 5 ( 4 H J - o x a z o l o n e . (lb)

for

for

(6)

(7) w h i c h x

incomplete

resembles

no

acids.

but

Tfa-amino

or

Similarly, showed

was

oxazolone

the

exclus-

to

to

conversion

from

in

or

2-al k y l - o x a z o l o n e

4-a 1 k y l - 5 ( 2 H ) - o x a z o l o n e s —

kyl-5(4H)-oxazolones

give

f r o m the a c i d No r e a c t i o n

Et,N, 4 - a 1kyl-5(4H)-oxazolones

at

to

consisted

The

new

DCC.

the

For-Val-OH

signal

but

defin-

le a n d

parent

of

achiral

of

leucyl

from

when

acids

to B z - L e u - O H

of t h e 2 - a l k y l - o x a z o l o n e (5b) d e r i v e d Z - V a l - O H h a d b e e n p r o d u c e d f r o m Id. •—

Bz-Val-OH

pep-

products,

Z-Val-OH

Id was ^ f

of

same

reaction

with

neutral

o f the

formylamino

after

But w h e n

Z-Gly-Pro-OH

Reactions

The

reacts

side-chain

fraction.

occurred

ged

(lj)

(9). no

to

convincing.

2-Alkoxy-oxazolone ( Z - V a l ) oC 0

the o x a z o l o n e ;

4-sec-butyl-5(4H)-oxazolone

not

behave

2,4-Dial-

similarly.

"pseudo-oxazolones"

(8)

This derived

(10).

R

,'JA

N // H-C

C=0

(6)

Et3N C=0

(7)

C=0

(8)

170 Conclusion We

conclude

or

protected

that

H o w e v e r , we (3c) i—'

2b

into

of

formation

4d a n d

l_f a n d

exist

intermediate

is p o s t u l a t e d

mation

anhydrides

do n o t

postulate

as t r a n s i e n t

Id a n d (3f)

symmetrical

peptides

the

unsymmetrical

to a c c o u n t

intermediate

into

for

to a c c o u n t

an e q u i l i b r i u m

cyclizes

simultaneous ively. occurs

expulsion

In the for

to

the of

presence

(3) w h e n

R1

the

free

acid

of t e r t i a r y = R2

= OtBu

of

anhydride the

of

transfor-

l_f, 2 f ,

4f

w h e n a n h y d r i d e (3) a l k y l , the m o l e c u l e

2-al k y l - 5 ( - o x a z o l o n e

the

anhydride

conversion

for

mixture

acids

compounds.

Moreover,

a n d 5f. We p r o p o s e the f o l l o w i n g t h e s i s : '— 1 2 is f o r m e d a n d o n e or b o t h of R and R are immediately

of a c y l a m i n o

isolable

5b r e s p e c t i v e l y .

as

2f

of

(3b)

as

(4a) —

amines,

(5b)

with

or

(4b)

respect-

the

same

reaction

r~>

(11).

References

1.

Schüssler,

H.,

Zahn,

H.:

2.

Chen, F . M . F . , K u r o d a , K., B e n o i t o n , 9 2 9 (1 9 7 8 ) ; 232 (1 9 7 9 ) . I.Z., Nowak,

Chem.

Ber.

3.

Siemion,

4.

Chen, F.M.F., 232 ( 1 9 7 9 ) .

5.

Arendt, A., Kolodziejczyk , A.M.: 3868 (1978).

Kuroda,

E.: A n n .

K. : R o c z .

9J5, 1 076-1 080 N.L.:

Chem.

K., B e n o i t o n ,

Synthesis, 1 479-1 4 8 2

Tetrahedron

Schnabel,

7.

J o n e s , J . H . : in The P e p t i d e s : A n a l y s i s , S y n t h e s i s , V o l . 1. Gross, E., M e i e n h o f e r , J., eds. Academic N e w Y o r k , 1 979 , p. 71 .

8.

Meienhofer,

9.

Benoiton, (1981 ) .

10.

W e y g a n d , F., Prox, A., S c h m i d h a m m e r , C h e m . Int. Edn. 2, 1 8 3 - 1 8 8 ( 1 9 6 3 ) .

11.

Benoiton, 1225-1227

J.:

Ibid., Chen,

N.L., Chen, (1981).

p.

([88, 2 3 8 - 2 4 9

Lett.,

928(1 9 6 0 ) .

Synthesis,

6.

N.L.,

Chem.

34,

N.L.:

( 1 962).

2303867-

( 1 9 6 5 ). Biology. Press,

306.

F.M.F.:

F.M.F.:

Can.

J.

J.

Chem. L.,

Chem.

59^,

384-389

König, W.:

Soc.

Chem.

Angew.

Commun.,

FIRST RESULTS WITH NEW REAGENTS FOR PEPTIDE COUPLING

Peter Henklein, Klaus Forner, Hartmut Iliedrich Akademie der Wissenschaften der DDR, Institut für Wirkstoffforschung, DDR-1136 Berlin Peteris Ya. Romanovski Institute of Organic Synthesis, Academy of Sciences of Latvian SSR, Riga, USSR

Introduction In search of condensation reagents to be used alternatively to carbodiimides and without such drawbacks as allergenic properties and difficultly removable byproducts, we did some experiments with the Saccharin-derivative 3-chloro-1,2-benzoisothiazole 1,1-dioxide (BID-C1), a sulfimide acid chloride. Recently Inomata (1) reported the use of 3-(succinimidoxy)and 3-(5-nitro-2-pyridon-1-yl)-1,2-benzoisothiazole-1,1-dioxide for the synthesis of esters and peptides. The results of our studies on acylated derivatives of pentafluorophenol (2), the successful application of HOPfp (3) and HONB (4) in peptide chemistry and the limited stability of reagents like Cl3C-COOCgF5

(2,5) prompted us to prepare

3-(pentafluorophenyloxy)-1,2-benzoisothiazole

1,1-dioxide

(BID-OPfp) and 3-(5-norbornen-2,3-dicarboximidoxy)-BID

(BID-

ONB) and to use them for synthesis of peptides, see formula.

Results 1) Preparation of BID-OPfp and BID-ONB. Mixing of BID-C1 and HOPfp or HONB in acetonitrile and

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

172

dropwise addition of triethylamine at 0° C according to (1) yields BID-OPfp (87 %) and BID-ONB (89 %), respectively, as crystalline compounds.

|-0H S'N

•PC15 or S0CI2

rCl ^CX^S'

0

>F

^ 0

* %

0

BlO-OPtp

a) • HOPfp / NEt3 b) »HONB / N Eti

0

yliapld:,79 87% .,81.c

0 0

-S'N '/ W 0 0

II 0

Boc-NH-CHR-COOH + HjN-R =

>

BID - O N B NET 3

( Boc-NH-CHR-CO-ONB I

Boc-NH-CHR-CO-NH-R'

2) Peptide coupling. The protected amino acid and N-methylmorpholine, dissolved in C F ^ C ^ , DMF or CH^CN, are mixed at 0° C with BID-OPfp or BID-ONB. After 15 min. the amino component is added. The reaction is complete within 60 min. BID-OH, HONB and HOPfp, can easily be removed from the peptide by washing the organic phase with NaHCO^-solution. 3) Racemization. The Izumiya-test (6) was used to check racemization. Peptide coupling with BID-ONB or BID-OPfp alone gave racemization of 17 % and 15 %, respectively. No racemization was detectable when 2 moles of HONB or HOPfp were added before activation. Thus, these reagents can be applied for fragment condensation.

173

Table

1 : C o u p l i n g of p r o t e c t e d a m i n o

Peptide obtained

Method

Z-Val-Gly-OEt

acids Yield %

m.p.

°C

+

BID-OPfp

83

163 - 1 6 5

+

BID-ONB

85

163 - 1 6 5

i s o l • Z-Val'- O N B

83

163 - 1 6 5

isol. Z-Ala-Gly-OEt

+

Boc-Phe-Val-OMe

+

80

163 - 1 6 5

BID-ONB

81

9 4 .-96

Z-Val -OPfp

87

117 - 1 1 9

BID-OPfp

83

90'- 9 2

Z-Pro-Ala-OH

BID-ONB

81

154 - 1 5 6

83

159 -161

82

-141

Z-Asp(OtBu)-PheNH

2 Boc-Tyr-Met-Gly-OEt

dt

°dto.

a l l p e p t i d e s g a v e o n e s p o t in 4) F r a g m e n t c o n d e n s a t i o n .

CO

BID-OPfp

Boc-Trp-Gly-ONbz

t.l.c.

Using BID-ONB we synthesized

following protected peptides in good yields, graphically of fragments

(t.l.c.)

and optically pure by

the

chromato-

condensation

(2 + 2 a n d 4 + 4) .

Table 2 : Peptides o b t a i n e d by fragment

condensation

Boc-Trp-Met-Asp(OtBu)-PheNH2

77 %

m.p.

179-81

°C

Z-Val-His-Pro-AlaOH

86 %

m.p.

131-35

°C

Boc-Pro-Phe-His-LeuONbz

88 % [a]p°= - 3 8 , 1 ° c=1 , D M F

Z-Sar-Arg(N0„)-Val-Tyr-Val-His-Pro-AlaOH

65 %

174

Conclusions - BID-ONB and BID-OPfp have been shown to be useful condensing reagents for the preparation of both activated esters of protected amino acids in situ and peptides. - The reaction conditions are mild and yields are high. - The condensing reagents are stable and crystalline compounds. - Exce® of HONB and HOPfp is necessary to avoid racemization during fragment condensation. - BID-C1, the basic compound for this activation method, can be obtained from versatile Saccharin. - Side products formed in the coupling reaction as Saccharin, HONB, HOPfp, can easily be removed.

References 1. Inomata, K., Kimoshita, H., Fukuda, H., Miyano, 0., Yamashiro, Y, Kotake, H.: Chemistry Letters 1979 , 12651268 and Ahmed, A., Fukuda, H., Inomata, K., Kotake, H.: Chemistry Letters 1980, 1161-1164 2.

Romanovski, P.Ya., Azmanis, A.A., Romanovska, I.K., Juchnovich, A.D., Chipens, G.I.: 4.Soviet.Sympos.Chem. of Proteine; and Peptides, Kiew 1977

3.

Kisfaludy, L., Low, H., Nyeki, C., Szirtes, T., Schön, J.: Liebigs Ann.Chem. 1973, 1421-1428

4.

Fujino, M., Kobayashi, S., Obayashi, M., Fukuda, T., Shinagawe, S., Nishimura, 0.: Chem. Pharm.Bull. 2_2, 1 8571863 (1974)

5.

Gubkov, A.T. and Schechvatova, G.V.: Zh.obsc.Chim. 40, 2146 (1978) Izumiya, N. and Muraoka, M.: J.Amer.Chem.Soc. 9J_, 23912394 (1969) .

6.

PEPTIDE SYNTHESES WITHOUT ISOLATION OP INTERMEDIATES USING BIPHASIC SOLVENT SYSTEMS

Rudolf Dolling and Klaus-Dieter Kaufmann Humboldt-University, Section Chemistry, and Institute of Drug Research, Academy of Science of GDR, Berlin

Introduction Rapid peptide syntheses in solution without isolation of intermediates require complete coupling and deblocking without side reactions, a highly reactive carboxyl component and an excess of reagents. Application of mixed anhydrides (1), pentafluorophenyl- (2), o-nitrophenyl- (3), N-hydroxysuccinimide- (4) and N-hydroxybenzotriazole esters (5,6) or carbodiimides (7) differ in the excess of carboxyl component and in the way of its removal after the coupling, whereas the deprotection of the N ^ B o c group has been carried out mostly with trifluoro acetic acid. We found, that coupling of active Boc-amino acid esters of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) (8) proceeds rapidly and without racemisation, if a two layer system of dichloroethane or ethyl acetate with aqueous sodium hydrogencarbonate solution had been used. The catalysis in the intermediate layer and the simultaneous extraction of the acidic, water soluble HONB are obviously responsible for the acceleration of the coupling. In a synthesis scheme, developed on the basis of this coupling conditions, the growing peptide is consequently hold within the organic phase.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

176

Methods Sufficiently

hydrophobic amino acid or peptide ester (or ami-

de) and 1 . 2 - 2 equivalents of the Boc-amino acid ONB-ester are dissolved in dichloroethane, overlayed with 1 M HaHCO^ solution and intensively strirred. Coupling is finished mostly within some minutes up to one hour as detected by tic. The phases are separated and the active ester excess is transformed into an appropriate acid soluble amide by stirring the organic phase for 20 minutes with two equivalents of a prim./ tert. diamine, e.g. 3-dimethylamino-propylamine, which can be removed by washing (2). The Boc-group of the N-protected peptide is removed directly in the dichloroethane phase by treatment with a strong organic acid, e.g. methane-sulphonic acid, in 0.5 M concentration within 10 to 20 minutes (9). The excess of the acid is neutralized by 1 M NaHCO^ and separated. The two layer system than will be completed for the next coupling step. Results and Discussion Examples (see table 1) were synthesized in a 5 - 10 mmol scale with a time of 4 hours for one cycle. and 2 yielded the correct final products using an excess of 1.5 equivalents of the active esters without further purification. The example 2. shows, that tyrosin needn't any protection of the hydroxyl function. A partial tyrosin 0-acylation takes place forming depsipeptides, which however are cleaved together with the active ester excess in the acid soluble Boc-amino acid amides and the desired tyrosin peptides with free hydroxyl group by treatment with the prim./tert. diamine. The formation of the O-acyl-tyrosin derivative was observed for the coupling of Z-Gly-ONB and H-Tyr-OBzl already after some minutes before a complete N-acylation took place. The depsipeptide Z-GlyTyr(Z-Gly)-0Bzl, which was prepared with more excess of Z-Gly-ONB and by means of a tri-

177

ethylamine catalysis, could be cleaved quantitatively to the dipeptide with free hydroxyl group in dichloroethane by means of 4 equivalents 3-dimethylamino-propylamine within 30 min. Table 1 yield

m.p. OC

Boc-Tyr(Bzl)GlyLeu-OMe

83

55-•58

2

Z-GlyPhePheTyr(Bzl)-OMe

71

161-•164

-9.0°(DMF)

2

Z-GlyPhePheTyr-OMe

70

176-•178

-8.0°(DMP) x )

176 (10)

-13.5° (10) -17.0°(MeOH)

4

Z-Tyr(Bz1)GlyGlyPheLeu-OBzl 75

4 a H-TyrGlyGlyPheLeu-OH

95

151-•153 157-•159 160 (11)

578

-23.0°(DMP) -23.4°

(12)

x ) the rotation of the peptide differs from the literature value, but could be confirmed with a peptide prepared by a 2 + 2 fragment condensation For the synthesis of Leu-enkephaline ^a, a totally benzyltype protected precursor 4, w a s

chosen, to cleave all pro-

tection groups simultaneously. The leucin benzyl ester

and all

peptide intermediates were soluble in dichloroethane. N-(2-aminoethyl)-morpholine has been used to form the amides of the Boc-amino acids. Only the amide Z-Tyr(Bzl)HH(CH 2 ) 2 I^jD was difficult to extract with aqueous acids (13), so that traces of this compound had to be separated from protected Leu-enkephaline by gel filtration. This shows that amino acid active esters with an extreme hydrophobicity should be avoided. The final hydrogenation yielded the free Leu-enkephalin of high purity (70%).

The examples show

that this method permits to prepare pure

peptides in 70 - 80% yield. Prerequisites for a successful application are a sufficient hydrophobic character of the amino component to avoid its partition between the organic and aqueous phase, an appropriate solubility of the peptides

178

in the organic phase and a sufficiently hydrophilic behaviour of the Boc-amino acid amides for the extraction with aqueous acids. These properties can be attained mostly by selection of terminal and side chain protecting groups. After an appropriate adoption of this strategy it should be possible, to prepare peptides with a chain length up to about ten amino acids.

References 1.

Beyermann, H.C., DeLeer, E.W.B., Floor, J.: Rec.Trav. Chim.Pays-Bas ¿2, 481 (1973).

2.

Kisfaludy, L., Schön, I., Szirtes, T., Nyeki, 0., Löw.M.: Tetrahedron Letters 1785 (1974). Bodanszky, M., Punk, K.W., Pink, M.L.: J.Org.Chem. ¿8, 3565 (1973).

3. 4.

Schneider, C.H., Wirtz, W. : Helv.Chim.Acta

5.

Bratby, D.N., Coyle, S., Greggon, R.P., Hardy. C.W., Young, Q.T. : J.Chem.Soc.,Perkin I, 1901 (1979). Rolli, H., Blaser, K., Pfeuti, C., Schneider, C.H.: Int.J.Peptide Prot.Res. 399 (1980).

6. 7. 8.

1062 (1972).

Nozaki, S., Kimura, A., Muramatsu, I.: Chemistry Letters 1057 (1977). Fujino, M., Kobayashi, S., Fukuda, T., Shinagawa, S., Nishimura, 0.: Chem.Pharm.Bull. (Japan) 22, 1857 (1974).

9.

Yajima, H., Ogawa. H., Fujino, M., Funakoshi, S.: Chem. Pharm.Bull.(Japan) 2£, 740 (1977). 10. Katsoyannis, P.G., Ginos, G., Cosmatos, A., Schwartz, G.s J.Amer.Chem.Soc. 6427 (1973). 11. üeki, M., Inazu, T., Ikeda, S.: Bull.Chem.Soc.(Japan) 52, 2424 (1979). 12. Bower, J.D., Guest, K.P., Morgan, B.A.: J.Chem.Soc., Perkin I, 2488 (1976). 13. Schneider, C.H., Rolli, H., Blaser, K., Int.J.Peptide Prot. Res. 15,411 (1980).

SOME APPLICATIONS OF THE CURTIUS REARRANGEMENT Panayiota Moutevelis-Minakakis and Iphigenia Photaki University of Athens, Laboratory of Organic Chemistry 13A Navarinou str., Athens 144, Greece

As is known N-protected amino-acid azides are subject to the Curtius rearrangement and the resulting isocyanates can react with alcohols to give urethane derivatives. In 1936 Bergmann and Zervas (1) applied the following reactions in the so-called carbobenzoxy stepwise degradation method for the elucidation of the primary structure of peptides: BzNHCHCOOR1 I

• BzNHCHCONHNH,

R

I

R

• BzNHCHN=C=0

¿

R710H DZ,J W

H0N

^

- " . BzNHCHNHZ

¿

° > BzNHCHCON, I

J

R H

?/Pd

H=C1

—

• BzNHCHNH„ ,HC1

¿

•

• BzNH 2 + RCHO + NH 4 C1 Characterization of the liberated aldheyde gave information on the amino-acid originally present. Recently, the same series of reactions have been used for the synthesis of gem-diamino compounds incorporated into synthetical retro-isomers of biologically active peptides (2). Starting from the same L-amino-acid and applying the Curtius rearrangement followed by the reaction of the isocyanates with appropriate alcohols, we have been able to prepare optically pure enantiomers, R and S, of N,N'-substituted gem-diamines (assuming that no inversion or partial racemization occurs at the chiral centre which migrates from C to N).

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

180

CONHNH_ i.

1

ZNHCH t CH2CH (CH3) 2

1. Bu ONO,H 2

NHCOOEt

A

1

" „ .i. htUH

ZNHCH i CH2CH (CH3)

S-Enantianer (a)

i25 [cx]jr +12.4°(c 0.96,DMF) m.p. 123-125°C C0NHNHo

1.Bu ONO,H

'

2

i i . EtOCONHCH ch2ch(ch3)2

3

-

NHZ

A

'

EtOCONHCH

R-Enantianer (a 1 )

ch2ch(ch3)2

B z l 0 H

l25 [a]j: -12.4°(c 1 .01 ,DMF) m.p. 123-125°C I n an a n a l o g o u s way t h e p a i r s o f

enantiomers

have a l s o been s y n t h e s i z e d .

interchange of

This

tached at the asymmetrically atom o f

substituted

the parent amino-acid,

who has c o n v e r t e d

the p r i n c i p l e

tetrahedral

formation of

first

table

two g r o u p s

reac-

a racemate,

a p p l i e d by F i s c h e r

(+)-isopropylmalonamidic

at-

a-carbon

using analogous s e r i e s of

t i o n s and w i t h o u t t h e i n t e r m e d i a t e a n o t h e r example o f

shown i n t h e

acid to i t s

is (3)

(-)-en-

antiomer. Table Starting

Material

Alcohol

Used

Final

Product

¡2

[ a ] p 5 +17.1 ° (c 1 .05,DMF)m.p.143-145°C EtOCO-L-Ala-NHNH_

BzlOH

R-Enantiomer

(b')

[ a ] p 5 -16.0°(c 0.9,DMF)m.p. 143-146°C Z-L-Leu-NHNH„

Bzl(OMe)OH

Z(OMe)-L-Leu-NHNH0

BzlOH

R-Enantiomer

(c1)

- 1 . 8 ° ( c 0.9,DMF) m.p.138-140°C S-Enantiomer [a]n°

+1-75°(£

(c)

0.95,DMF) m.p. 138-140°C

181

Primary alcohols as used above give the best results when reacting with the isocyanates. However, starting from Boc-^-AlaNHNH2 and using benzyl alcohol we failed to obtain the N,N'~ -disubstituted S-enantiomer. This can be due to the known decomposition of the Boc group under heating (4). We have obtained the known R-enantiomer (2) from Z-L-Ala-NHNH2 only at the presence of anhydrous Et^N as catalyst (5). In the absence of Et^N, depending on the conditions, we have isolated two other products (III) or (VI). The formation of the former can be explained by the following reactions: ZNHCHN=C=0

(I)

+

Hn0

ZNHCHNH2 (II) + C0 2 ,

ch3

ZNHCHNHCONHCHNHZ (III)

(I) + (II)

CH-

CH_

Since after the formation of the azide the mixture was carefully dried, the necessary water for the above reaction could be derived from the known dehydration of t-BuOH in the presence of the isocyanate (6). The other compound (VI) was isolated in the presence of an agueous acid, i.e. either when the azide was insufficiently washed with aq.KHCO^, or when I,1-bis(trifluoroacetoxy)iodobenzene (PIT) (7a) was used in AcCN-H 2 0 for the degradation of Z-L-Ala-NH2 to a gem-diamine. This reaction is considered also to occur via an isocyanate with the simultaneous liberation of trifluoroacetic acid. We suggest the following mechanism for the formation of compound (VI): ZNHCHN=C=0

I

CH_,

(IV)

H

2°

+

H_0 £

f+) ZNHCHNH UN 3 (la) + (C02)

+

• ZNHCHOH + (H ) CH,

ZNHaf^ (IV) + (NH3)

CH, ZNH2 (V) + CH 3 CH=0

182

(IV) + (V)

Compound

H + + ZNHCHNHZ i CH^

• ZNHCliSli-Z i ^ CH^

(VI)*

(VI) was also prepared from Z-L-Ala-NHNH^ by the

Curtius rearrangement etc or from Z-L-Ala-NH2

and PIT in an-

hydrous DMF by the addition of benzyl alcohol.

H PIT „ „ . „ , „ 2° A na-n n • ZNHCHN=C=€> + (C,H I+2CF 7 C00H)—— (IV) etc ACUN H2U ^ b b H PIT

DMF

"

+

"

"

BzlOH

•(VI)

, , (VI)

References 1.

Bergmann, M., Zervas, L.: J. Biol. Chem. 113, 341-357 (1 936) .

2.

Chorev, M., Willson, C.G., Goodman, M.: J. Amer.Chem. Soc. 99, 8075-8076 (1977).

3.

Fischer, E., Brauns, F.: Ber. _47, 31 81-31 93 (1 91 4).

4.

Felix, A.M., Heimer, E.P., Lambros, T.L., Tzougraki, C., Meienhofer, J.: J. Org. Chem. £3, 4194-4196 (1978).

5.

Baker, J.W., Holdsworth, J.B.: J. Chem. Soc. 713-726 (1947).

6.

Baumgarten, H.E., Smith, H.L., Staklis, A.: J. Org. Chem. 40, 3554-3561 (1975).

7.

(a) Radhakrishna, A.S., Parham, M.E., Riggs, R.M., Loudon, G.M.: J. Org. Chem. _44, 1 746-1 747 (1 979); (b) Loudon, G.M., Almond, M.R., Jacob, J.N.: J. Amer. Chem. Soc. 103, 45084515 (1981).

*After having developed the above mechanism, we noticed that the same scheme up to the formation of compound (V) was proposed, but rejected, by Loudon et al. (7b) for the hydrolysis of N(1-aminoalkyl)amides in acidic solutions.

A NEW METHOD FOR THE SELECTIVE SYNTHESIS OF UNSYMMETRICAL CYSTINE PEPTIDES

Erich Wünsch, Sigrun Romani and Luis Moroder Max-Planck-Institut für Biochemie, Abteilung Peptidchemie, 8033 Martinsried/München, FRG.

Introduction The tryptic digest of the cyanogen bromide fragment 10-118 of mouse nerve growth factor leads to an acid-insoluble fraction, from which an unsymmetrical cystine-peptide corresponding to the sequence portions 10-25 and 75-88 can be isolated; on molar basis this fragment exhibits a 100 fold activity if compared with the intact nerve growth factor (1). For a successfull synthesis of this unsymmetric cystine-peptide a selective disulfide pairing has to be achieved. For this purpose the procedures based on sulfenyl halides (2-4) cannot be applied because of the presence of tryptophan residues in both peptide chains with consequent well known side reaction to thio-indole derivatives.

Results The use of

azodicarboxylic acid derivatives for oxidation

of cysteine peptides (5) has been extended by us to the selective synthesis of unsymmetrical cystine peptides (6) as following: O O R1 - S -i N - CO,Bu' R,- SH » But O "- C - N "= N -t C - OBu »• | H - N - C02BU'

* ~ „2 -u

O O r' - S - S - R2 » Bu1 - O - C - NH - NH - C - OBu1

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

•

184

The sulfenohydrazide derivatives of cysteine peptides are readily isolated as stable and analytically well characterized sulphur-activated intermediates, which in turn react smoothly with the second cysteine-component to produce in optimal conditions exclusively the unsymmetrical cystine-peptide (6,7). A careful

screening of the reaction conditions has clearly

indicated that the best results are obtained by dropwise addition of the cysteine-component to a larger excess of the sulfenohydrazide-component, e.g. at a molar ratio of 1:3, in solvents such as dimethylformamide, dimethylacetamide or N-methylpyrrolidone. By this procedure concomitant oxidation of the cysteine-component to the symmetrical disulfide and/or disproportionation of the unsymmetrical disulfide induced by the thiol-component to a "statistical energy-dependent distribution", is efficiently suppressed. In fact, as shown by the preparative example reported in Fig. 1, the unsymmetrical cystine-peptide was insolated in over 80 % yield as homogenous material (Fig. 2).

BOC-Ala-Cly-Cys(NCOjBut-NHCOjBul) -OH • BOC-Thr( Bul)-Ala-Cys-Gly-CIn-Lys( BOC)-Ser( Bu')-Pro-OBu1 (3:1)

i

BOC-Ala-Gly-Cys-OH 1 t Bu )-P tro-OBu BOC-Thr(But)-Ala- Cys-Gly-Cln-Lys(BOC)-Ser( SH-content of the reaction mixture after 24 hrs hplc : 0% Ellmann's test : 0 % Yield of isolated unsymmetrical disulfide : 84 % mp. 1S3-155°C; (a) P0 = -67,7°, |a]^6^-80,8o (c = 0. 5,methanol); CggH11 ^JO^SJ- 1. 5 HjO ( 1533. 96). calcd. : C 51.68. H 7.75, N 11.87, S 4.18, found: C 51.87, H 7.87, N 11.24, S 4.23; amino acid analysis (6N HCI, 24h, 110°C) : Thr 1.04 (1), Ser 1. 02 (1), Clu t .04 (1), Pro 1. 05 {1), Cly 1. 99 ( 2), Ala 1.99 (2), Cys2 0. 94 (1), Lys 0.92 (1) ; peptide content (calcd. for Mr 1533.96) : 96 %. Fig. 1

185

Chromatographic

tests of purity of

Boc-Ala-Gly-Cys -OH

Boc-Thr(Bu*)-Ala-Cys-Gly-Gln-Lys(Boc)-Ser(But)-Pro-OBut

hpttc onprecoated silica gel-60 hplc :^Bondapak CI8 (0,4x30cm) lates (Merck AC) etuent:0,1 M ammonium acetateIpH 6,8)/ p solvent system: 1-butanol/ acetonitrit 52:48(v/v), a c en tic acid/water/pyridine i isocratic elution at a f low rate of l,5m(/ 6m0 : 6 24 :20 absorbance at 210nm

I

i a 12 16 20 2imin

Fig. 2

At molar ratios of the reaction components of 1:1 or higher a thiol induced disproportionation of the unsymmetrical disulfide was observed to occur as monitored by hplc and tic. To block in such cases the thiol-interchange at the maximum content of unsymmetrical cystine-peptide (for the examples discussed below after 24 h reaction time the concentration of symmetrical cystine-peptides was still negligible) and to concomitantly simplify the purification procedure thiol-trapping reagents on polymer matrix should be the most advisable. Among the various proposed SH-reagents N-alkylmaleimides are known to react rapidly and rather specifically with thiols. Thus, 3-maleimidopropionic acid was coupled via its N-hydroxysuccinimido ester (8) with amino-polyoxyethylene glycol (M

= 6000) to yield the water

soluble 3-maleimidopropionyl-amino-polyoxyethylene glycol (0.02 ^mol B-Ala/mg PEG). On the other side, the 3-maleimidopropionic acid active ester was also used to acylate L-leucyl - oxymethylpolystyrene [prepared according

186

to the method of Gisin (9) from Boc-Leu-OH cesium salt and chloromethylpolystyrene-co-1 % divinylbenzene resin (Merrifield polymer), followed by acidolytic removal of the Boc-group] to produce the insoluble 3-maleimido-propionyl-Lleucyl - oxymethylpolystyrene [0.51 mmol Leu/g resin; 6-Ala: Leu, 1:1 as determined by amino acid analysis of the acid hydrolysate; HCl/propionic acid; 1:1 (v/v), 130°C]. The usefulness of the cysteine-trapping procedure is illustrated in the two following examples: Example 1 S'-component:

BOC-Ala-Cly-Cys(NCC>2But-NHC02But)-OH

SH-component:

B O C - T h r ( But) - A 1 a - C y s - C l y - C l n - L y s ( BOC) -Ser( Bu*)-Pro-OBu1

S

component

•

SH

component

(1:1)

Mal-B-Ala-NH - ( PEC]

c o n j u g a t e of Mai B A l a - N H - [ P E C ) unsymmetrical

I SH-component

disulfide

S H - c o n t e n t of t h e r e a c t i o n m i x t u r e a f t e r 2« h r s hplc

: 37 °0

Ellmann's test

: 29 %

SH-content after treatment with M a i - A - A l a - N H - [ P E C ] hplc

:

0

Ellmann's test

:

0%

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

( 0 . 2 0 pniol m a l e i m i d e / m g

PEC}

: 53 »

A m i n o a c i d a n a l y s i s of S H - c o m p o n e n t / P E G c o n j u g a t e

:

37 ° S H

component

After treatment of the reaction mixture with an excess of 3-maleimidopropionyl-amino-PEG for 30 min

the unsymmetri-

cal cystine-peptide was isolated by precipitation with water followed by recrystallization from methanol-water in ca. 50 % yield [calculated taking into account the ca. 40 % unreacted cysteine-component;.the low yield is due to partial water solubility of the unsymmetrical cystine-peptide]. In the second example the reaction mixture was stirred with an excess of 3-maleimidopropionyl-L-leucyl-oxymethylpolystyrene. The capture of the thiol-component was monitored

187 Example 2

S* - component

:

5H- component

:

BOC-Ala-Cly-Cys(NCOJBU'-NHCOJBU1)-OH BOC-Thr( Bu^-Ala-Cys-Cly-Cln-Lyst BOC)-Ser{ Bul)-Pro-OBu1

S

- component

•

SH - c o m p o n e n t

(1:1)

Mal-ft-Ala-Leu-(g)

^

unsymmetrical

hplc

: 37 %

Ellmann's test

: 33

Ellmann's test

I ®

(0.51 ymol maleimide/mg

resin)

0 % 0 I disulfide:

95

I

A m i n o a c i d a n a l y s i s of S H - c o m p o n e n t / M a l - f t - A l a - L e u - ®

both by Ellman's

SH-component

2U h r s

SH-content after treatment with Mal-fi-Ala-Leu-

Yield of isolated unsymmetrical

/

disulfide

SH-content of the reaction m i x t u r e after

hplc

conjugate of Mal-G-Ala-

:

351 S H - c o m p o n e n t

test (10) and hplc [n-Bondapak C18

(0.4 x 30 cm); eluent: 0.1 M ammonium acetate (pH 6.8) / acetonitrile, 52 : 48 (v/v); isocratic elution at a flow rate of 1.5 ml/min, absorbance at 210 nm]. For the present example the reaction was found to proceed at a remarkable lower rate than with the PEG-bound reagent. A quantitative removal of the cysteine-peptide was achieved only after 12 h. The resin was then filtered off and the unsymmetrical cystine-peptide was isolated on addition of ether in nearly quantitative yield. In both examples the unsymmetrical disulfide compound exhibited analytical data identical to those reported above.

Conclusion The experimental models used to investigate the sulfenohydrazide method for the synthesis of unsymmetrical cystinepeptides clearly indicate its usefulness particularly if tryptophan is present in the peptide chain. No modification at the indole function was observed under the experimental conditions of this new procedure.

188

References 1. 2. 3. 4. 5.

Mercanti, D., Butler, R., Revoltella, R.: Biochim. Biophys. Acta 496^, 41 2 - 419 (1977). Kamber, B.: Helv. Chim. Acta 56^ 1 370 - 1381 (1974). Castell, J.V., Tun-Kyi, A.: Helv. Chim. Acta 62_, 2507 - 251 0 (1 979) . Matsueda, R., Kimura, T., Kaiser, E.T., Matsueda, G.R.: Chem. Lett. 737 - 740 (1981). Arold, H., Eule, M.: in Peptides 19 72, Proc. 12th Eur. Pept. Symp. Reinhardsbrunn Castle, GDR (H. Hanson and H.-D. Jakubke, eds) pp. 78 - 85, North Holland, Amsterdam.

6.

Wünsch, E., Romani, S.: Hoppe-Seyler's Z. Physiol. Chem. 363, 449 - 453 (1982).

7.

Romani, S., Göhring, W., Moroder, L., Wünsch, E.: in Proceedings of the 4th FRG-USSR Symposium on Chemistry of Peptides and Proteins, Tübingen, June 8 - 12, 1982, in press. Moroder, L., Nyfeler, R., Gemeiner, H., Kaibacher, H., Wünsch, E.: Biopolymers, in press. Gisin, B.F.: Helv. Chim. Acta 56, 1476 - 1482 (1973).

8. 9.

10. Ellman, G.L.: Arch. Biochem. Biophys. 82, 70 - 77 (1959).

A CONTROLLED SYNTHESIS OF CYCLIC UNSYMMETRICAL CYSTINE PEPTIEES BEARING TWO S-S BRIDGES IN THE RING.

Iphigenia Photaki, Miltiadis Kolovos, Ageliki StathakiFerderigos, and +Leonidas Zervas University of Athens, Laboratory of Organic Chemistry 13A Navarinou Str., Athens 144, Greece

Because of the importance in nature of cyclic peptides containing more than one S-S bridges, the problem of their synthesis has been intensively studied by several groups. Open-chain unsymmetrical cystine peptides are necessary intermediates for the synthesis of such cyclic multi-cystine peptides; they are known however to rearrange easily to the symmetrical ones (1) under conditions applied in peptide synthesis. Therefore, years ago (2) we expressed the opinion that an approach to a controlled synthesis of non-symmetrical "parallel" (3) cystine peptides with two or more S-S bridges would be facilitated if two peptide chains A and B containing cysteines bearing different and selectively removable S-protecting groups could be coupled through their amino-ends to a polyvalent N-protecting group G. I.

,NH-CHCO

/ \

-R

2

/ I \

CH2SR

1

NHCHCO

•

CH2SR

CH9SR1

| 2

NH-CHCO

2

A _R1

oxidation

CH0SR2

| 2

NHCHCO

and oxidation

A N D

B

/

/

V

\

.NHCHCO 1

CH2S

| 2

NHCHCO

NHCHCO

'

2

CH2SR

CH0SR2"

| 2

NHCHCO

etc

The establishment of the first S-S bridge would form a multimembered ring so that rearrangement of the cystine peptide chain could be prevented. An additional ring would be estab-

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

190

lished by the formation of the second S-S bridge. The next step would be the removal of the polyvalent N-protecting group G in such a way that neither the peptide bonds nor the S-S bridges would be affected. In the variation II of the above scheme the group G could be combined with two S-protected derivatives of cysteine which would form the first S-S bridge. The peptide chain could be propagated at both sides, and at a desirable length it could be supplied with new S-protected cysteine derivatives. II. NHCHCO / ç'

CH„SR 2

\

Cys(Trt)-Ala-0E)EM(4)

Cgl^S Na+

CH2OCO-Cys(Trt)Gly-OPac

^ ^

~

¿ ¿ ^ CH 2 OCO-Cys ( T r t ) -Ala-ODpn CH^O-Cys (Trt)-Gly

CH 2 OCO-Cys ( T r t ) -Ala-ODpm

O

(?)

CH 2 OCX>Cys-Gly-ODpm

2Cys(flan)-ODpn| m.a.

\

^

CH 2 OCO-Cys-Ala

Phenol"

CH 2 OCO-Cys-Gly

^ O C O - C y s - A l a - C y s (Aon) -ODpn k^®2C)CO-Cys-Gly-Cys(Aan)-ODpTi

CH 2 OCO-Cys-Gly-Cys-ODpm

-CH 2 OCO-Cys-Ala-Cys-QMe - CH 2 CXX)-Cys-Gly-Cys-OMe

Phenol

(12)

Scheme

(I)

(5)

MPOH

CH 2 OCO-Cys(Trt)-Gly-ODpn

CH 2 OCO-Cys-Ala-ODpn

'

C y s ( T r t ) -Ala-ODpn

^ ^ - C H 2 O C O - C y s (Trt)-Gly-OPac

(CgH5)2CN2

(2)

(g)

^ 1

'

DMF-AcOH

(^OCO-Cys-Gly-Cys

~

192 Compound

(7) is an unsymmetrically substituted cystine deriva-

tive of phthalyl alcohol containing the first S-S bridge in a ring. Elongation of the peptide chains from both of compound

(8) leads to compound

carboxy-ends

(10) bearing two S-S bridges.

All substances shown on the schemes having a number are moncmeric compounds and they have been characterized as optically and chemically pure. The removal of both the -ODpm groups with TFAphenol proceeded quantitatively at room temperature to give the monomeric peptide

(11). The Phthoc group being a protecting

group of the urethane type can be quantitatively removed by acidolysis. Treatment of the symmetrical cyclic compounds (13a-c) or

(14c) either with HBr/AcOH in the presence of anisol at rcon

temperature or with TFA-phenol at 70°C removed the phthoc group

0

X-Cys-Cys(Acm)-ODpm i X-Cys-Cys(Acm)-ODpm

CH 2 0C0-Cys-0R CH 2 0C0-Cys-0R

(13a_c)

a: R=Dpm b: R=Me c: R=H

a: X=Zb: X=Bocc: X , X = P h t h o c C Scheme

(II)

quantitatively, i.e. these reactions are comparable to the removal of the Z or the Boc group from open-chain cystine derivatives such as

(14a,b) or the cyclic peptide

(15) with one intra-

-chain S-S bridge. However, the removal of the same N-protectZ-Cys-Cys 1

1

(15)

X-Cys-Cys-ODpm i i X-Cys-Cys-ODpm a: X=Zb: X=Bocc: X, X = P h t h o c d Scheme

(III)

193 ing g r o u p s f r o m the c y c l i c s y m m e t r i c a l or n o n - s y m m e t r i c a l cystine peptides

(10),

(11),

(12) o r

(16a-c)

di-

r e s u l t e d in a m i x -

ture of p r o d u c t s . T h e s e f i n d i n g s s u g g e s t a n i n s t a b i l i t y of the r e s u l t i n g free s y m m e t r i c a l or n o n - s y m m e t r i c a l c y c l i c

peptides

b e a r i n g two S-S b r i d g e s . It h a s to be i n v e s t i g a t e d if this

in-

s t a b i l i t y is c o n n e c t e d w i t h the n u m b e r a n d the n a t u r e of the a m i n o - a c i d s b e t w e e n the two c y s t i n e m o i e t i e s ,

i.e. w i t h the

size of the r i n g a n d its p r i m a r y s t r u c t u r e , as it h a s s t a t e d for c y c l i c n o n - s y m m e t r i c a l p e p t i d e s w i t h o n e S-S bridge

been

intra-chain

(5).

References (a) Z e r v a s , L., B e n o i t o n , L., W e i s s , E., W i n i t z , M . , G r e e n s t e i n , J . P . : J. A m e r . C h e m . Soc. 81_, 1 729-1734 (1 959); (b) S a n g e r , F.: N a t u r e 171, 1 0 2 5 - 1 0 2 6 (1953); R y l e , A . P . , S a n g e r , F.: B i o c h e m . J. 60, 5 3 5 - 5 4 0 (1955); B e n e s h , R . E . , B e n e s c h , R.: J. A m e r . C h e m . Soc. 80, 1 6 6 6 - 1 6 6 9 (1958): K a m b e r , B.: H e l v . C h i m . A c t a 54, 3 9 8 - 4 2 2 (1971). Z e r v a s , L., P h o t a k i , I.: C h i m i a , 1_4, 375-376 C h e m . Soc. 84, 3 8 8 7 - 3 8 9 7 (1962).

(1 960); J.Amer.

W a d e , R., W i n i t z , M . , G r e e n s t e i n , J . P . : J. A m e r . C h e m . 78, 3 7 3 - 3 7 7 (1 956) .

Soc.

P h o t a k i , I., F e r d e r i g o s , A . , Z e r v a s , L.: in " P e p t i d e s : P r o c e e d i n g s of the T h i r t e e n t h E u r o p e a n S y m p o s i u m " , ed. Y. W o l m a n , W i l e y - I s r a e l U n i v e r s i t i e s P r e s s , N e w York-Jerusalem, 47-52 (1 975) . H a r d y , P . M . , R i d g e , B., R y d o n , H . N . , S e r r ä o , F . O . d o s S . P . : J. C h e m . Soc. (C) 1722-1731 (1971); L a r g e , D . G . , R y d o n , H. N., S c h o f i e l d , J . A . : J. C h e m . Soc. (C) 1 7 4 9 - 1 7 5 2 (1961); W e b e r , U., H a l t e r , P.: Z. p h y s i o l . C h e m . 355, 189-199 (1974) idem: i b i d . 2 0 0 - 2 0 4 .

SYNTHESIS 3-0-

OF

CYSTEINE

AND

CYST INCGNTA I N I N G

PEPTIDES

THROUGH

[n-benzyloxycarbonyl-s-acetahidomethyl-cysteinyl]-HYDROXY2-PHENYLINDENONE

Stoyan Higher

The

amide

Pedagogical

of

On

the

glycylcystine

cysteine of

the

uith

arise above

usual

at

such

isomerised (3).

of

2

ue

of

in

the

function

in

as

functions

uith

means

for

basic

reagents.

the

the

DCCI

difficulties

(2).

thiol And

the

is

the

for

the

reason the

the

quoted

respective

the

elimination

group

is

it

knoun

loosing

is

which

part

function

of

of

can

the

the

performed

can

their

difficulties

cysteine,

indandionyl

carboxylic

for

by

synthesis the

through because

of

respiratory

peptides

experimental

possible

of

(l).Uith

its

compounds

not

hydrobromide

be

that easily

biological ue

chose

removed

amides.

The

N-benzyloxy-

N-butyloxycarbonyl-S-acetamidomethyl-cysteine

uith

Y-Cys(Acm)-OH

on

of

mitochondria

well

eliminate

the

JL a n d

realized

2-amino-2-phenyl-

2-amino-2-ary1-1,3-indandiones

group

changes

activation

is

strong

To

diamide

considerable

which

Acm-protective

carbonyl-

as

mentioned

conditions

function

with

anticoagulating

effect

liver

protective

help

be

without

rat

amides

derivatives

the

an

the

a stimulatory

there the

has

Bulgaria

hand

cystine

Getting

Shoumeri,

2-amino-2-(4-methoxyphenyl)-1,3-indandione

isolated

method

9700

with

chain

in

Sofroniev

other

has

the

Institute,

(HjNPID)

(H2NAID)

of

Nedyalko

N-benzyloxycarbonyl-glycine

1,3-indandione blood.

Minchev,

+

3-hydroxy-2-phenylindenone

H0-

= 0

DCCI

Y= Z

according

Y - C y s ( A cm) - 0 -

(1);

Boc

(2)

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

(4,5):

=0

196 Esters

and

results ethyl

2 are

shou

ester

producing

that of

the

yellou-orange these

glycine

tuo

82.4 % the

peptides

f o r _5,

rest

of

aminolysis till

6a.

the

less

is

same

basic

more

constants

than

uith

is

by

are

and

7_a

X =

H

7b

X = DCHj

es t e r

deblocking

of

out of

by the

HgCl2

constants (7), the is

and

are

fact,

activated

ester

DCCI 7a

at

and

react

The and

runs

the

interaction

2-amiho-2-(4-methoxy-

uith

activated

X =

OCHj

o f _3 a n d g l u t am i c

follouing acid,

in

all

Acm-group as

from

(6).

In

the

from

the

the

first

accordance

uith

resulting of

hydrobromide

a m i d e s _8 a n d

good

thiol

compound

the

is

of

ue

Acm-

carried

oxidation

(T-glutathione,uhose published

compound is

12

case,after is

group

uith compound

groups.

the

result

same

e s t e r _1»

aminolysis resulting

functional

groups,the

deblocking

of

6b.Though

9

the

of

the

method -

(H2NAID)

the

5.

f o r _4 a n d

Successfully

H

uell

that

%

results,

X -

a m i d e _8 t h e

successful the

(j5) .

thiol

in

from

87.8

JB

according

released

of

obtained:

protected

g l u t a t h i o n e _ll,as

alanylglycine,

(H2NPID)

of

| p - g l u t a t h i o n e _10, The

9

decarboxylation

IT-activated

of

the

respectively

published

obtained

HjNPID 7_b a l s o

8

yields

the

coincide

and

After

uith

2-amino-2-phenyl-

amides

ester

by

Boc-Cys(Acm)-A1a-Gly-0Et

phenyl)-1,3-indandione the

ethyl

aminolysis

achieved

2-glycylamino-2-pheny1-1,3-indandione

compound

Z-Cys(Acm)-Gly-OH

undergo

the

obtained

o f _1 u i t h

The

A and

are

uhich

compounds.The

Z - C y s ( A c m ) - G l y - D E t _3,

Z-Cys(A cm)-A1a-Gly-0Et last

compounds a n d by

peptides

crystal

of

also

benzyloxycarbony1-cys tine

is

results

13a.

great

importance

obtained (5),

For from

after

the

its

197

Table Yield,

1*1. P . ,

Compound

tu

%

°C 1

Z - C y s ( A cm)-Gft"

64.2

159-160

M

D

141-143

B o c - C y s(A c m ) - O A

57 .8

3

142-143

Z-Cys(Acm)-Gly-0Et

74.4

6

Z-Cys(Acm)-Gly-NHPID

a 108-110

70.0

b

72.1

108-109

8

Z-Cys(Acm)-NHPID

79.8

amorph.

amo rph.

Z-Cys(Acm)-l\IHAID

85.7

-33.1

10

72.3

amorph.

Z-Glu-OM e

-26.3

Hbr.H-Cys(Acm)-NHAID

-18.4

13

(Z-Cys-NHPID)2

88.6

amorph.

a 213-215"" 77.6

215-216""

79.6

(Z-Gly-Cys-NHAID)2

148-15

0"**60.7

EtOAc)

+41.9

, 21

(Me) 2 ^ 0 )

+42.6 (1,

14

, 21

-

(1, b

, 19

EtOAc)

(1, 11

, 20

EtOAc)

(l,

C y s ( A c m ) - G l y - 0 Et

, 18

EtOAc)

(1, 9

, 24

EtOAc)

-14.2 (l,

, 18

EtOAc)

-13.6 (1,

, 20

EtOAc)

-26.8 (1,

, 27

EtOAc)

-19.8 (l,

, 21

(Ne)2C0)

-58.4

, 20

(1, DCIF) *A e

3-hydroxy-2-phenylindenone

""according """according

to (2) to (l)

m.p.

residue

215-216°C

m. p.- 1 5 0 - 1 5 1

C

^

solvent/

-27.9 (l,

2

.

/c,

198 aminolysis

uith

2 - a m i n o - 2 - p h eriy 1 - 1 , 3 - i n d an d i o n e 1 3 b .

compound

has

DCCI

(2).

method

been The

h y d r o b r o m i d e _12. groups

and

obtained

In

flcm-group this

interaction

characteristics

uith

already As

described

a result

suggested as

uell

of

for

as

is

in

for

very

1_4 i s

exactly

peptides

easily ester

The

uay

-

by

removed of

of

the

from

the

the

thiol

benzyloxy-

obtained,

uhich,

identical

uith

experiments of

another

oxidation

activated

synthesis

their

in

after

publications

these the

is

case,

c a r b o n y l - g l y c i n e, c o m p o u n d its

before

according

the

to

compound,

(l). a convenient

amides uith

of

method

cystine

and

is cysteine,

2-amino-2-ary1-1,3-indan-

diones. Some

of

the

compounds

most

are

characteristic

given

in

a

data

for

the

neu

obtained

table.

R e f e r en c es 1.

Minchev, G.B.:

S.,

Pol.

Sofroniev,

J.

Chem.

2.

Aleksiev,

B. , M i n c e v ,

3.

Aren,

Lencbergs,

kim.

A., ser.

1980

(6),

4.

I»!incev,

S. : Compt.

5.

Mincev,

S.,

54,

443

Aleksiev,

B.V.,

Kupryszeuski,

press).

S.: I.:

3.

pract.

Chem.

L atv,

PSR

Acad.

bulg.

316,

Zinat.

140

Acad.

(1974).

Vestis,

677. rend.

Derdouska,

Sci.

I.,

Kupryszeuski,

E.:

J.

32»

623

G. : P o l .

(1979). J.

Chem.

(1980).

6.

Hermann,

7.

Pastuszak, (1980).

N.V.,

(in

P.,

Schreier,

J.3.,

Chimiak,

A.:

pract. 3.

Chem.

pract.

316,

Chem.

719

322,

(1974). 495

METHOD OF CYCLIZATION OF CARBA ANALOGUES OF OXYTOCIN

Milan Krojidlo and Martin Flegel L6Siva - Pharmaceuticals, Laboratory of Peptides 143 10 Prague Czechoslovakia Michal Lebl Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia

Introduction Recently carba analogues of oxytocin found use in both veterinary and human medicine. ^2-0-Methyltyrosine^]deamino-1-carba-oxytocin (CARBETOCIN) has been introduced into veterinary therapy as oxytocicum with protracted activity and [j2-p- e thyl phenyl a lanine^j deamino-6-carba-oxytocin

(NACARTOCIN)

is being clinically tested as salureticum. The purpose of the present study is to find an easy method of cyclization without significant side reactions which would enable to prepare even large quantities of analogues of neurohypophyseal hormones.

Results The method is exemplified by the following procedure: The solution of -NHg

Nps-Tyr(Me)-Ile—Gln-Asn-Cys(C^H^COOH)—Pro-Leu—Gly-

(17.^ g; pure by chromatography) was treated with 50 ml

of 2M-HC1 in ether. After 15 min standing at room temperature the hydrochloride of the octapeptide amide was precipitated with ether, dried and dissolved in the mixture of DMFA (2^0 ml) and dioxane (150 ml). The cooled (0°C) solution was

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

200

stirred with DCCI (23.1 g) and 1-hydroxybenzotriazole (21 g) for 2 h at 20 C. The solution, was filtered and poured drop by drop into the stirred mixture of methanol (4 000 ml) and triethylamine (35 ml). After 3 h at room temperature the reaction mixture was evaporated to dryness; the residue was dissolved in 50$ aqueous acetic acid and purified on Sephadex G-15 column (4 000 ml) eluted with the same solvent. The crude product obtained by freeze drying (9.3 g) was further purified on Separon SI-C-18 column (500 g) using mobile phase composed of 55$ methanol and 45$ of 0.05M ammonium acetate (pH 7.0) by volume, see Fig. 1A. Evaporation and freeze drying yielded 3.38 g (22$) of

Q2-0-methyltyrosine]deamino-1-

-carba-oxytocin. The purity of the product was checked by HPLC, see Fig. IB.

O.D-220

—

60 A: B:

1 120

180

. ,

t(min)

0

± t(min)

Preparative HPLC Analytical HPLC: CGC column (0.3 x 15 cm) with SEPARON SI-C-18, mobile phase : 55$ methanol and 45$ 0.05M ammonium acetate (pH 7)

201

In the above way we prepared the following carba analogues; deamino-l-carba-oxytocin, [^2-0-methyl tyrosine] deamino-1-carba-oxytocin, deamino-6-carba-oxytocin, [^2-p-ethylphenylalanineJdeamino-6-carba.-oxy tocin, all in 20-25?o yield of HPLC pure products. Hie procedure is represented by the following scheme: Carba Is

Carba 6 s -S

CH,

I CH,

CH, CH2-COOH X

=

X-

CH„

CH, CH2-COOH

CH, X-

Nps, Boc, Z 1 - splitting off the protecting group 2 - protection by protonation

CH, CH, CH--CO -

3 - activation of carboxylic group k - cyclization S S CH,

CH,

CH, CH,

CH2-CO-

Discussion With the various types of peptides different cyclization methods were used for closing the carba bridge (see e.g. 1-3)• The most commonly used active ester method with the active ester prepared from properly substituted aryl sulphite suffers from difficulties connected with the preparation of the particular sulphite the stability of which is also limited. Hie synthesis of the active ester is time consuming. The most serious side reaction accompanying the cyclization with p-nitro- or 2,b,5-trichlorophenylesters in pyridine at eleva-

202 ted temperature is the one leading to products of higher molecular weight. Another disadvantage of the mentioned methods consists in the necessity to remove the protecting group after the synthesis of the active ester (often a discomforting feature). We found it possible to prepare the N-hydroxybenzotriazole active ester in situ with DCCI from a peptide protected only by protonation. Cyclization proceeds almost instantaneously after liberation of the amino group by pouring the solution of the active ester salt into alkaline medium where the intramolecularly cyclized product is predominantly formed. The reaction is fast enough even at room temperature. There was found no transesterifica.tion in methanol (on comparison by HPLC with an authentic sample of the corresponding linear peptide methyl ester). Trials were done also with other solvents (acetonitrile, tert-butanol, pyridine, isopropanol, DMFA). Sufficiently high reaction rate enables the reaction to be performed without risk in a. small volume compared with other methods of cycliza.tion. The described procedure was tested on cycliza.tion of both small (30 nig) and big

(26 g) batches.

References 1. 2.

3.

Jost, K.: Collect. Czech. Chem. Commun. 36, 218-233 (1971). Brady, S.F. , Vaxga., S.L. , Freidinger, R.M., Schwenk, D.A., Medlowski, M., Holly, F.W. , Veber, D.F.: J. Org. Chem. kk, 3101-3105 (1979). Sakakibara, S. , Ha.se, S. : Bull. Chem. Soc. Japan

2816 (1968).

SYNTHESIS OF ISOPEPTIDES OF LYSINE AS FUNDAMENTAL STRUCTURAL UNITS OF CLAVICEPAMINES Gyula Szókàn, Marian Gyenes Institute of Organic Chemistry, Eotvos University H-1088 Budapest, Hungary Erno Tyihàk Research Institute for Medicinal Plants Budakalàsz Béla Szende Institute of Pathology and Experimental Cancer Research, Semmelweis Medical University Budapest

Introduction The clavicepamines are lysine rich basic proteins isolated from saprophytic culture of ergot (Claviceps purpurea)(1). The lysine level in them changed between 30-95 mol %, their MW is between 2-17.000 dalton and they showed a cell proliferation retarding effect (2). The isopeptides of Lys occur rarely in nature: in some peptide antibiotics (3), in cell wall of bacteria (4) and in cross linkage of several proteins (5).

Results Dansylated, then hydrolyzed clavicepamines were analyzed exactly by HPLC and OPTLC methods elaborated for separating a, e and bis-Dns-lysines from each other. Chromatographic data: TLC -Merck silicagel G,EtOAc: i-propanol: NH^ 40:32:20 (v/v); HPLC - ODS Hypersil (125x4 mm), MeOH-phosphate buffer (pH 7.7) 50:50, 1.3 ml/min. On the base of finding only a-Dns-Lys it

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

204

was stated, that the lysine e-Nf^ groups are fully blocked, so isopeptides of Lys build up clavicepamines. To prove and to investigate the isopeptide structure e-oligo and poly-lysines were synthesized by our laboratory. Earlier e-lysine dipeptides were formed by classic peptide synthetic methods (1,6), via the NCA-method in aqueous media (7) and using lysine Cu-complex (8). We synthesized a few known and new Lys-peptides. The method of Theodoropoulos (8) was applied and improved. Lys-Cu complex was acylated by protected amino acids. Peptides were formed via MCA method, EEDQ and active ester in THF (or AcN)-aqueous system with 60-80% yield (Fig.l.). Decomposition of peptide complexes proceeded together with crystallization of half-protected Lyspeptides . X-Lys-Y f| Q

Fig.l

Q Gly, Phe, D Phe, Glu(OBil) Glu, Lys (OBzl)

Lys Lys—Gly

X H H

Y OH OH

Lys

OH

H

OH

ca-Polypeptides of basic amino acids were synthesized at first by Kushwaha (9) and Mathur (10) via polycondensation. We elaborated a new procedure using the^backing off-method (11) to obtain Lys-peptide active esters suitable for polycondensation. Lys and Lys peptide-ONp esters were acylated stepwise with protected Lys by rapid coupling method (for example MCA)(Fig. 2.). The method was systematically studied, optimalized and extended with Glu-peptide active esters (12). It can be used in the field of sequential polypeptides, branched peptides and multichain oligo and polyamino acids, too. The purity of active esters were controlled by HPLC. After deprotection and dialysis poly (s-L-lysine) was obtained with MW 20.000 dalton. The isopolypeptide with similar prqperties was prepared by polymerization of Z-Lys-OH with DPPA according to Nishi (13), too.

205

Z - L y s - O H

Fig.2 Polyeond«niatior

£

C)

{¿•protection: HBr / AcOH )

-tripeptide active ester

" baclcing-of f "proc*dur*(appt.) (Goodmon-Stutben) classic.method (Mathur)

Z - L y i - O H

I

-

BOC

I£AJ

Z-LyrOH^ I BOC Z - L y i - O N p

—

In DMSO with TEA 55-72 hrs. was needed to get solution. Some data concerning the enzymatic cleavage of the noncommon e-peptide bond were collected. In case of Lys(Gly) and Lys(Phe) the qualitative results were idential with those of Plessing (7). a*/ ,

/ml »1000

AlrHtmtnt

206 The hydrolysis of e-di and polylysines were compared to that of a-peptides. Trypsin, thermolysin, subtilisin did not split the poly(e-L-lysine), but aminopeptidases hydrolyzed it.The effect of synthetic isopeptides on K562 standard tumour strains was measured. The poly (e-L-lysine) showed the same effect as clavicepamines (Fig. 3.)

e-Dipeptides have no activity. On the.

base of structural and biological investigation it was demonstrated, that the e-lysine-peptides are the fundamental structural units of clavicepamines.

References 1.

Tyihäk, E.: Dissertation (1978)

2.

Tyihäk, E., Molnär, G., Patthy, A., Szende, B., Lapis, K.: Proc. 2 0 ^ Hungarian Annual Meeting Biochem,, SLofok (1980)

3.

Hausmann, W., Weisiger, J.R., Craig, L.C.: J.Am.Chem.Soc. 77, 723 (1955) Ghuysen, J.M., Shockman, G.D.: Bacterial Membranes and Walls, pp. 37-130, Marcel Dekker, New York (1973)

4. 5. 6.

Folk, J.E., Finlayson, J.S.: Adv.Prot.Chem. 31, 1 (1977) Chaturvedi, N.C., Khosla, M.C., Anand, N.: Indian J.Chem. 3, 554 (1965)

7.

Plessing, A., Siebert, G., Wissler, J.H., Puigserver, A.J., Pfaender, P.: Hoppe-Seyler's Z. Physiol.Chem.36 3,2 79 (1982) Theodoropoulos, D.: J.Org.Chem. 23, 140 (1958)

8. 9.

Kushwaha, D.R.S., Mathur, K.B., Balasubramanian, D.: Biopolymers 19, 219 (1980)

10. Mathur, K.B., Pandey, R.K., Jagannadham, M.W., Balasubramanian, D . : Int. J . Peptide Protein Res. ±1_, 189 (1981) 11. Goodman, M., Stueben, K.C.: J.Am.Chem.Soc.81, 3980 (1959) 12. Szokän,Gy., Kotai, A., Dobo, Gy., Kökösi, J., Schwartz, A.: Ann.Univ.Sei. Bp. R.Eötvös, 15, 169 (1979) 13. Nishi, N., Nakajima, B., Hasebe, N., Noguchi, J.: Int.J. Biol.Macromol, 2, 53 (1980)

SYNTHESIS OF LEUCINE ENKEPHALIN- AND ASPARTAME ANALOGS CONTAINING THIOAMIDE LINKAGES AT SPECIFIC POSITIONS

K. Clausen, B. Yde, S.-O. Lawesson Department of Organic Chemistry, Chemical Institute, University of Aarhus, DK-8000 Aarhus C, Denmark

Introduction Among peptide backbone modifications the replacement of an amide bond by a thioamide bond has hitherto attracted little attention (1-6). This may be explained by the lack of general synthetic methods for the preparation of endothiopeptides. Recently, we have reported a method leading to protected endothiodipeptide esters and endothiodipeptide ester salts (7, 8). We describe here a general and efficient method for preparing free endothiopeptides applied to the synthesis of the four possible monothioanalogs of leucine-enkephalin, and to the synthesis of the thioanalog of aspartame.

Results The [Leu5, Phet1* ]-enkephalin analog was prepared by the route shown in Scheme 1. The protected dipeptide Boc-Phe-Leu-OBzl was thionated by using 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Lawesson's Reagent), LR, as described previously (7, 8). Boc-group removal was effected by using 4M HC1 in dioxane. Next the fully protected monothiopentapeptide was obtained by a (3+2) segment condensation (DCC) . The Boc-, Bzl- and OBzl-protection groups were cleaved simultaneously by using HF. The free monothiopentapeptide was purified by Sephadex gel filtration.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

208 [ y r

G I y Boc -

G1 y

•OH

HCI-H-

-OEt

Boc-

- OEt

Boc -

DCC Boc Bz I -OH Bz l

BocBoc •

/

•OH T o s O H • H • DCC

-OEt

-OBzl

Boc

-OBzl

-OEt 1M

Bzl

¿M H C I / d i o x a n e S

N a OH OH

Boc •

-OBzl

LR

4M HCI / d i o x a n e HCI • H DCC/HOBt

L eu

Ph e

HCl-H DCC

Bzl

t

OBzl •OBzl

Boc • HF/ ar

¡soif -OH

H-

5

1

Scheme 1. Preparation of [Leu , Phet * ]-enkephalin.

Single thioamino acid residues were introduced in positions 1, 2 and 3, respectively, by using protected amino acid dithioesters, which were prepared in high yields by the route shown in Scheme 2. A new and promising MA method is involved (9) .

X-NH-CH-C-OH I R

X a b c

1 ) 21}

NEt 3

t

LR

X-NH-CH-C-0-P-(O)-0Me S®NH®Etj

R

20°C

R H- N o°cN

H H --0-CH2

Z Boc Boc

S 11 X-NH-CH-C1 R

•o

LR D

80 C

\

0 II ^ X - N H - C H - C - N ) R

Mel THF

SMe X-NH-CH-C=N^ I ©

©

H, S 0°C

X-NH-CH-C-SMe I R

Scheme 2. Preparation of N-protected amino acid dithioesters.

209

By using Boc-protected amino acid dithioesters as thioacylating reagents we succeeded to prepare the three remaining fully protected monothio leucine-enkephalin derivatives 3

the [Glyt ]-analog by a (2+3) segment coupling

(Scheme 3),

(DCC)and the

[Glyt 2 ]- and [Tyrt 1 ]-analogs by stepwise procedures

(DCC and

DCC/HOBt). Final deblocking was effected by using HF. G I y

Tyr Bz I —OH B zI

Boc

V.

B 0c

HCI-HDCC/HOBt

II

G I y

-OEt

/

-OBzl

Bo c Boc -

-SMe

S

-H-

- OB z I

-OBzl 4M

H C I / d i o x a n e

-OBzl

HCI-H-

-OH

Bz I

HCI

L.

Boc Boc-

Leu

4M H C I / d i o x a n e - 0 Et

Bz I /

Ph e

DCC -OBzl H F / a n i s o l e -OH

G I y

Tyr

G I y Boc-

Phe

-0H

HC I • H -

Leu -OBz I

DCC -OBz I

Boc t Boc -

-SMe

M

HCI / d i o x a n e

HCI•H-

-OBz I -OBz I

Boc Bz I Boc-

4M

-OH

HCI

Bz I

DCC/HOBt

HCI / d

i o x a m

-OBzl

• H -

-OBzl

Boc • H F / a n i s o l e

• OH

G I y

Tyr

Bo c -

G I y

-OH

Ph e

HCI • H-

Leu • OBz I

DCC Boc Bz I B3 oocc ^-F - S M e

-OBzl ¿M

HCI

HCI / d i o x o n e OBzl

•H•

Bz I Boc

yt.

-OBzl H F / o n i s o l e

H-

L

Scheme 3. Preparation of [Glyt 3 ]-, [Glyt 2 ]-, and cine enkephalin.

OH

[TyrtM-leu-

210

Thioaspartame was prepared as outlined in Scheme 4. The fully protected aspartame was thionated by LR to give the corresponding protected thioaspartame, which was deblocked by using HF/ anisole.

?But Z-Asp-Phe-OMe

LR

?But Z-Aspt-Phe-OMe

HF

Aspt-Phe-OMe

Scheme 4. Preparation of thioaspartame.

Acknowledgements This work was supported by a grant from the Danish Natural Science Research Council

(K.C.).

References 1.

Ried, W., von der Emden, W.: Angew. Chem. 72^, 268 (1960).

2.

Ried, W., von der Emden, W-: Liebigs Ann. Chem. 642, 128 (1961).

3.

Ried, W., Schmidt, E.: Ibid. 695, 217 (1966).

4.

Jones, Jr., W.C., Nestor, Jr., J.J., du Vigneaud, V. : J. Am. Chem. Soc. 9ji, 5677 (1973) .

5.

Mock, W.L., Chen, J.-T., Tsang, J.W.: Biochem. Biophys. Res. Commun. 102, 389 (1981).

6.

Bartlett, P.A., Spear, K.L., Jacobsen, N.E.: Biochemistry 21, 1608 (1982) .

7.

Clausen, K., Thorsen, M., Lawesson, S.-O.: Tetrahedron 37, 1019 (1981) .

8.

Clausen, K., Thorsen, M., Lawesson, S.-O.: Chemica Scripta 20_, 14 (1982) .

9.

Pedersen, U., Thorsen, M., El-Khrisy, E.-E.A.M., Clausen, K., Lawesson, S.-O.: submitted.

SYNTHETIC STUDIES ON THE CECROPINS R.B. Merrifield and L.D. Vizioli The Rockefeller University New York City, New York, U.S.A. H.G. Boman University of Stockholm Stockholm, Sweden

The cecropins are a family of peptides that are induced in pupae of the giant silk moth Hylophora cecropia following injection of live bacteria

(1).

They are part of the humoral

immune response of the insect and are antibacterial against Gram negative and Gram positive bacteria.

Cecropins A and B

both contain 37 amino acid residues and a blocked C-terminus (2). To study its bactericidal activity, a solid phase synthesis of cecropin A(1-33) was undertaken.

The synthetic protected

peptide I was Boc-Lys(ClZ)-Trp(For)-Lys(CIZ)-Leu-Phe-Lys(ClZ)-Lys(C1Z)Ile-Glu(OBzl)-Lys(ClZ)-Val-Gly-Gln-Asn-Ile-Arg(Tos)-Asp(OcHex) Gly-Ile-Ile-Lys(C1Z)-Ala-Gly-Pro-Ala-Val-Ala-Val-Val-Gly-Gln-

The Pam-resin support (3) was used for increased acid stability. Double couplings with symmetrical anhydrides and hydroxybenzotriazole esters were monitored by a new quantitative ninhydrin procedure

(4) (Fig 1).

They proceeded to ^0.2% of completion in

the early part of the synthesis and within ^0.5% in the latter part.

There was a gradual loss of chains during the synthesis,

not due to acidolysis or to termination. resin was evaluated for deletions

The protected peptide-

(5) by solid phase sequencing.

Near the end of the synthesis 0.5 to 0.8% deletions were indicated for residues 4-7. significant deletion

The first 26 steps proceeded without

(ave

L e u

authentic

purification water;

hydrophobic

IR-45

l.

97 >

neuro-

on

5:1:3)

impurities

a

G10

or

on

and

to

homogeneity.

related

peptides

biological

acetylated

have

testing

o n the r e s i n

after

recently (table removal

been

1).

prepared All

in

a

acetylated

of the F m o c

group.

250

TABLE 1

hplcb

Kf3 nt

1-13

[Aha6]NT6_13 Ac-NT

0.25

9.4

0.19

8.6

Aha/Lys

1 05

Pro

Arg

Tyr

Leu

lie

1 .99 2 .04 0 .99 0 .93 1 .01

7-13

0.25

9.1

2 . 12 2 .16 1 .03 0 .90 1 .00

Ac-NT8_13

0.29

8.6

0 .90 2 .07 0 .98 0 .92 1 .00

[Ac-Lys8]NTg_13

0.26

8.4

0 97

0 .90 1 .01 0 .98 0 .90 1 .00

[Aha8]NTg_13

0.29

8.3

1 03

1 .00 1 .03 1 .00 0 .93 1 .02

Ac-NT9_13

0.41

8.7

1 .06 1 .09 1 .04 0 .87 1 .00

AC-NT10_13

0.71

9.2

0 .96

a. R f

(B:A:W

NH^OAc pH 4.5.

12:3:5).

b.

-

1 .01 0 .96 1 .06

CH3CN o v e r 20 m i n at 2 m l / m i n 0.01

5-100%

Aha=6-amino-n-hfixanoic

M

acid.

References

1.

Carraway, (1973) .

R. ,

and

2.

K i t a b g i , P., C a r r a w a y , 7 0 5 3 - 7 0 5 8 (1976).

3.

H a m m e r , R.A., L e e m a n , S.E., C a r r a w a y , Biol. Chem., 255, 2476-2480 (1980).

4.

Y a j i m a , H. , K i t a g a w a , K . , S e g a w a , C h e m . P h a r m . Bull . , _23_, 3 2 9 9 - 3 3 0 0

T. , N a k a n o , (1975).

5.

Carraway, (1975).

J.

6.

R i v i e r , J.E., L a z a r u s , L.H. , P e r r i n , M.H., and B r o w n , M . R . : C h e m . , 20, 1 4 0 9 - 1 4 1 2 ( 1 9 7 7 ) .

7.

Sheppard, R.C.,and in press ( 1 9 8 2 ) .

Williams,

B.J.:

8.

S h e p p a r d , R.C., and W i l l i a m s , 589 ( 1 9 8 2 ) .

B.J.:

9.

Atherton, E., Gait, M.J., Sheppard, B i o o r g . C h e m . , _8_, 3 5 1 - 3 7 0 ( 1 9 7 9 ) .

10.

S t e l a k a t o s , G.E., S o l o m o s - A r a v i d i s , C., K a r a y a n n a k i s , P., K o l o v o s , M.G., and Photaki, I.: Proc. 16th European Peptide symposium, H e l s i n g o r , B r u n f e l d t , D., Ed., pp. 133-138 ( 1 9 8 0 ) .

R., and

Leeman,

S.E.:

R., and

Leeman,

J.

Biol.

Leeman,

S.E.:

Chem.,

S.E.: R.

and

Biol.

Int.

J.

R.C.,

6854-6861,

Biol.

Chem.

Williams,

251 ,

R.H.:

M., and K a t a o k a ,

Chem.,

Pep.

J. C h e m .

248,

and

Soc. and

250,

Prot. Chem.

J. K. :

1912-1918 J.

Med.

Chem., Comm.,

Williams,

20, 587-

B.J.:

LIQUID-PHASE SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES ON EASILY DETACHABLE POLY(ETHYLENE GLYCOL) SUPPORTS Roberto Colombo Department of Biomedical Sciences and Technology , University of Milan, St. Raphael Hospital, 1-20132 Milan, Italy.

Polymer-supported peptide syntheses are based on the idea that a peptide chain can be conveniently assembled in a stepwise manner while it is covalently attached at one hand to a polymer.

In solid-phase synthesis (1) the growing peptide

chain is anchored to an entirely insoluble particle during all stages of the synthesis and all the reactions are carried out in a

heterogeneous system.

Therefore, after each of the

reactions has been completed, the solid phase allows

rapid

filtration and washing for the removal of excess reactants and by-products.

In place of the insoluble matrix, liquid-phase

synthesis (2) uses a linear polymeric support which is soluble in some solvents and insoluble in others, thus allowing a repetitive procedure for the removal of reagents and by-products while exerting a solubilizing effect on the peptide chain during the reactions required by the synthesis. The ever-increasing number of reports dealing with peptide syntheses which are carried out by the solid-phase technique shows the usefulness of this approach, discloses its limitations, and stimulates efforts to improve the present procedures.

On

the contrary, during the past years only a limited number of naturally occurring peptides was prepared on soluble supports; thus, it seemed necessary to examine more closely the feasibility and convenience of the liquid-phase technique as regards the synthesis of small- to medium-sized peptides.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

252

H 2 N-PEG-OCH 3

1. (BOC-Nle-) O 1. Et N/CH Cl 2 > H-Nie-NH-PEG-OCH^ 2. TFA/CH„C1„ • TFA 2. HO-C.H- (CH„) -COOSu 2

z

b

4

Z

Z

Bpoc-LTrp-OH , DCC , CH 2 C1 2

/=\ HO-A. V c H CH CO-Nle-NH-PEG-OCH YL_V

2. 1% TFA/CH 2 C1 2

'

» 3. Et 3 N/CH 2 Cl 2

1.

(Bpoc-DLeu-) 2 0

2.

and so on .

H-LVal-Gly-LAla-DLeu-LAla-DVal-LVal-DVal-LTrp-DLeu-LX(Ac)DLeu-LTrp-DLeu-LTrp-0-^^^-CH 2 CH 2 C0-Nle-NH-PEG-0CH 3 1. H 2 NCH 2 CH 2 OH , DMF , 36 h 2. HCOO-C^H -NO o

4

Z

3. chromatography 1

5

10

HCO-LVal-Gly-LAla-DLeu-LAla-DVal-LVal-DVal-LTrp-DLeu-LX15

DLeu-LTrp-DLeu-LTrp-NHCH 2 CH 2 OH Fig. 1. Outline of the synthesis of Val-gramici 345nm) yielding reactive carbenes. 2 [Phe(pNH2) ]deamino-dicarba-AVP was labelled with tritium by iodination of the 2-p-aminophenylalanine residue and catalytic dehalogenation in the presence of tritium; 3a specific radioactivity of 16 Ci/mMole was obtained. The H-labelled analogue 3 was converted into the H-azidophenylalanine derivative. 2 [pheipNf^) ]deamino-dicarba-AVP retained an antidiuretic activity in anaesthetized rats of 21 I.u./mg. From competition experiments with 3H-AVP (4) 60 the± apparent dissociation

R

NH2 JCl|| 3 H 2 ,Pd/Al 2 0 3 R

R

N=N

NH

NH

CO

I. NO*

2.NQN3

Br

CO [CH 2 ] 2

R

R=

I

NH-CH-CO-Phe-Gln-Asn-Asu-Pro-Arg-Gly-NH2 CH2

Fig. 1.

Introduction of (photo)reactive groups and of tritium label in [2-p-aminophenylalanine, 8-argininejdeamino-dicarba-vasopressin.

303 constants K for the binding of AVP-analogues to vasopressin receptors in plasma membranes were determined. [pheCp-N^) ]deamino-dicarba-AVP showed the highest binding affinities: For -9 -10 bovine kidney medulla K was 5.5-10 M (AVP: K = 9*10 M), —8 —1O for rat liver K Q was 4.1*10 M (AVP: K^ = 8-10 M). The high binding affinity was retained after labelling with tritium. For the introduction of reactive groups or reporter molecules g at the C-terminal end of vasopressin, 1-deamino-[Arg ]vasopressinoic-acid was prepared. Peptide synthesis (Fig. 2) was

Boc-ArglTosl-OH

H-Gly-NH-NH-Z | DCC I HOBt

Bx-Arg(Tos)-Gly-NH-NH-Z

jcFjCOOH I CHjCOOH Bx-Pro-OH

H-Arg{Tos)-Gly-NH-NH-Z

I

Mixed Anhydride Method Boc-Pro-ArglTosl-Gly-NH-NH-Z

jcFjCOOH I CHjCOOH Boc-Cys(Acm)-OH

H-Pro-Arg(Tosl-Gly-NH-NH-Z

1

r-1

I DCC / HOBt

Boc-Cys(Acm)-Pro-Arg(Tosl-Gly-NH-NH-Z

jcFjCOOH I CHjCOOH

Boc-Phe-Gln-Asn-OH

H-CyslAcml-Pro-ArglTosl-Gly-NH-NH-Z

1

'

'

I DCC I HOBt

Boc-Phe-Gln-Asn-Cys(Acml-Pro-Arg(7osl-Gly-NH-NH-Z

| CFjCOOH/CHjCOOH Bx-Tyr-OH

H-Phe-Gln-Asn-CyslAcml-Pro-ArglTosl-Gly-NH-NH-Z

I

Bx-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-Arg(Tos)-Gly-NH-NH-Z

^CFjCOOH I CHjCOOH p-MpalAcml-OH

H-Tyr-Phe-Gln-Asn-CyslAcml-Pro-ArglTosl-Gly-NH-NH-Z DCC I HOBt

p-Mpa(Acm)-Tyr-Phe-Gln-Asn-Cys(Acm}-Pro-Arg(Tos)-Gly-NH-NH-Z 1. CF3SO3H I m-Cresol, Dimethylsulfide

2. J,

p-Mpa-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-OH

I

Fig. 2

I

Synthesis of 1-deamino-[Arg ]vasopressinoic acid

304 started with the COOH-terminal glycine hydrazide in the N' protected form and gave yields between 60 and 90 % in all coupling steps. From the protected nonapeptide the N'-Z and N^-Tos groups were removed by trifluoromethane sulfonic acid. The resulting S-protected (Acm) nonapeptide was cyclized by treatment with iodine in acetic acid / water (4:1) and simultaneously the C-terminal hydrazido group was removed (yield:20%). Purification was accomplished by partition chromatography on Sephadex G-25 in n-butanol / ethanol / pyridine / 0.1 N acetic acid (4:1:1:7). [ot] D 2 0 = -68.3 (c = 1 , 1 N acetic acid). + Field desorption g mass spectroscopy: m/e 1071 = MH . 1-Deamino-[Arg ]vasopressinoic acid showed an antidiuretic activity of 19.6 ± 6 I.U./mg (n=4). It stimulated bovine renal adenylate cyclase and bound to vasopressin receptors _7 from bovine kidney inner medulla ( K = 4.5»10 M) and from r Li rat liver ( K = 1.1 • 10 M ).

References 1. 2. 3.

Fahrenholz, F., Thierauch, K.-H., Crause, P.: HoppeSeyler's Z. Physiol. Chem. 153-167 (1980). Fahrenholz, F., Husseini, H.S., Morgat, J.L., Thierauch, K.-H.: Hoppe-Seyler1s Z. Physiol. Chem. (1982) in press. Church, R.F.R., Weiss, M.J.: J. Org. Chem. 3j>/ 2465-2471 (1970).

4.

Crause, P., Fahrenholz, F.: Mol. Cell. Endocrinology (1982) in press.

SYNTHETIC FRAGMENTS O F BACTERIAL C E L L W A L L S . PHYSICOCHEMICAL AND BIOLOGICAL

PROPERTIES

MiloS BudgSinsky, 3an Oezek, Viktor Krchnak, Michal

Lebl,

M i l a n Zaoral Institute of Organic C h e m i s t r y and Biochemistry, Czechoslovak A c a d e m y of Sciences, 166 10 Praha 6, C z e c h o s l o v a k i a OiCi Rotta, R a d o v a n Straka Institute of Hygiene and Epidemiology, 100 42 Praha 10, C z e c h o s l o v a k i a

Introduction There are numerous compounds which show an immunoadjuvant and/or immunostimulating effect. Of their number, the

frag-

ments of bacterial cell wall, peptidoglycan, M D P , and other peptides and glycopeptides (1) are of key importance they permit us a direct examination of the

since

relationship

between the chemical structure and the biological

properties

of the components of bacterial cell walls. The character of the peptidoglycan is that of a sequential polymer. We have designed therefore the first part of our study as an examination of the relationship between biological effects and the complexity of synthetic glycopeptides,

fragments of the cell

wall peptidoglycan of Staphylococcus aureus, strain C o p e n h a gen and S t r e p t o c o c c u s pyogenes, group A. The peptides and glycopeptides needed for our study were prepared both by solid phase synthesis and by synthesis in solution.

Attention

deserves the simultaneous removal of the protecting

groups

from the glycopeptides by treatment with sodium in liquid ammonia (1,2) and the use of solid phase synthesis for the preparation of glycopeptides (1). In this communication we

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

306

have focused a special attention to the problem of the sugar component.

Results 1 1)

HPLC,

H - and

13 C-NMR studies. The high performance

chromatography (HPLC-reversed phase) of MDP,

liquid

galacto-,

alio-, and norallo-MDP showed the presence of three components at least, in each case (see Table I). The

position

of the individual peaks is characterized by the capacity factor k'. In the case of MDP the products represented by the individual peaks were isolated and equilibrated with an aqueous methanolic phosphate buffer. The

equilibrium

mixture contained pairs of the starting compounds. T h i s result shows that MDP represents a mixture of cr and /3 anomers and two impurities. The HPLC data were confirmed 1 13 by measurement of H - and C-NMR spectra. According to these data peak 1 corresponds to the /3-anomer and peak 2 to the oP-anomer. A n a l o g o u s results were obtained

recently

by Lederer et al. (3). Peaks 3 and 4 are impurities with the opposite configuration of the lactyl residue they yielded isomuramic acid after hydrolysis

since

(identifi-

cation by HPLC and in the amino acid analyzer). The densation of the protected glucosamine derivative

con-

with

the derivative of 2-halogenpropionic acid obviously does not proceed quite stereoselectively (cf., 4 ) . The HPLC of galacto-, alio-, and norallo-MDP indicated a mixture of three compounds (Table I). Each of these components afforded after equilibration, carried out as with MDP, the original equilibrium mixture. We account

for two of the

peaks by the presence of the of- and /3-anomer of the pyranoid form; the third peak corresponds to the

furanoid

form. T h i s assumption is supported by the results of e x p e r i m e n t s with the reduction of D - g a l a c t o - M D P by sodium

307

TABLE I C o m p o s i t i o n of glycopeptides in phosphate buffer pH 5.00 in equilibrium

compound MDP

No.of peak

k'

% (t

isomer3 ctf.j3;P,F

)

1

4.7

26.8

P

-

P

2

8.5

53.6

a

-

P

3

9.8

7.0

/3

-

4

15.8

12.6

Of

-

P (S) P (S)

1

4.2

34.1

|3

-

P

2

6.6

59.7

a

-

P and ¡3 - F

3

8.1

6.2

Of

-

F

product of NaBH^

1

7.7

88.4

reduction of D-galacto-MDP

2

10.4

11.6

D-galacto-MDP

D-norallo-MDP

D-allo-MDP

Q

1

4.9

56.0

/3

-

P

2

5.9

20.5

of

-

F

3

6.5

22.9

af

-

P

4

8.0

0.6

ug were

peptides

and i n v i t r o

are

pronounced

tidoglycan to g r o u p s

Multiple

in vivo or

1 of

1 3 . 1 (k

streptococcus

particularly the

k'

peptidoglycan

resemble

6

in

fever

dose of

20,

individual response

the h e x a p e p t i d e ,

no

309

Glycotetrapeptide

Glycohexapeptide

(a)

(b)

/

0 12

3 4 5 6

,

Glycononapeptide (c)

-

v

.

0 1 2 3 4 5 6

0 1 2 3 4 5 6

Hours

Glycotridecapeptide (d) 20 pq 100 pg — 500jjg

0 1 2

3

4

(a)

MurNAc-L-Ala-D-iGln-L-Lys|Ac)-D-Ala-NH2 |b): MurNAc-L-Ala-D- iGln-L-Lys-D-Ala-IL-Alal^OMe (c): MurNAc-L-Alo-O-iGln-L-Lys(Ac)-D-Ala-(Gly)5-NH2 (d); MurNAc-L-Ala-D-iGlnH_-L»sug. T h e p r o l o n g a t i o n

protein

antigens

reaction

potentiation

of was

310

recorded in humoral response as measured by the antibody answer to the antigen. Peptides were with a rare exception without effect. Thrombocytolysis of rabbit blood platelets could be provoked by glycopeptides whereas the peptides themselves had no effect or produced degranulation only. The lysis of blood platelets was dose dependent. In all three effects, in pyrogenicity,

immunoadju-

vancy and thrombocytolysis, an evident structure to function relationship could be demonstrated.

References 1. 2.

Zaoral, M., Oezek, Czech.Chem.Commun. Zaoral, M., Oezek, Czech.Chem.Commun.

0., 45, Ü., 43,

Krchnak, V., Straka, R.: Collect. 1424-1446 (1980). Straka, R., Maëek, K.: Collect. 1797-1802 (1978).

3.

Halls, T.D.O., Raju, M.S., Wenkert, E., Zuber, M., Lefrancier, P., Lederer, E.: Carbohydr.Res. 81, 173-176 (1980).

4.

Arendt, A., Kolodziejczyk, A., Sokolowska, T.: Rocz.Chem. 48, 1707-1711 (1974).

SYNTHESIS OF ANALOGUES OF PRECURSORS OF BACTERIAL PEPTIDOGLYCAN

Didier Blanot, Androniki Kretsovali, Mohamed Abo-Ghalia, Dominique Mengin-Lecreulx, Jean van Heijenoort E.R. n°245 du CNRS, Institut de Biochimie, Université de Paris-Sud, 91405 Orsay, France

Introduction

The cytoplasmic steps of the biosynthesis of bacterial peptidoglycan involve uridine diphospho-N-acetylmuramyi-peptide precursors, each step being catalyzed by a particular synthetase (1). In order to find specific inhibitors of these enzymes, which might act as antibacterial compounds, we have undertaken the synthesis of analogues of the glycopeptide part of the precursors. In this communication, we shall report the preparation of propionyl- and N-acetylmuramyl-di- and tripeptides, potential competitive inhibitors of some synthetases,as well as of Npropionyl-L-alanine chloromethyl ketone, potential irreversible inhibitor. The effect of these compounds on some of the peptidoglycan synthetases of E. coli will also be reported.

Preparation of the peptides

The propionyl-peptides Pr-L-Ala-D-Glu-OH

(I), Pr-L-Ala-D-Glu-OH (II) and L-L-Lys-OH

Pr-L-Ala-D-Glu-OH (III) were synthesized according to the scheme of Fig.l. H-D-Lys-OH Purification was carried out by preparative TLC on cellulose powder G 1805 (Schleicher and Schiill) in the solvent system n-propanol-water 2:1 (v/v) . The purity of the peptides was checked by TLC, HPLC and amino acid analysis . The N-acetylmuramyl-peptides MurNac-L-Ala-D-Glu-OH

(IV) and MurNac-L-Ala-

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

312 D-Glu-OH

(V) were prepared by mild hydrolysis (0.1 N HCl, 100°,

L(L)meso-DAP-OH 5 min) (2) of the corresponding UDP derivatives. They were purified by HPLC on y-Bondapak Cjg using 0.05 M ammonium formate pH 3.5, an eluent similar to the one recommended for the analysis of the nucleotide precursors (3). Each glycopeptide appeared as a mixture of two anomers. MurNac-L-Ala-D-Glu-NH 2

(MDP) was a gift from Dr. P. Lefrancier. H-L-Ala-

D-Glu-OH (VI) was synthesized according to Sachs and Brand (4). The chloromethyl ketone of N-propionyl-L-alanine

(Pr-L-Ala-CH^Cl) was

obtained by condensing the mixed anhydride (isobutyl carbonate) of propionic acid with H-L-Ala-C^Cl

(5). Purification was achieved by silica

gel column chromatography using CHCl^-MeOH 95:5 (v/v) as an eluent.

Pr

L-Ala

Nps .

D-Glu

, OSu

L-Lys

OBu

H

OH Nps , \

OBu OH

DCCI + HOSu -OBu

Nps ,

'OSu Nps _

H.

OH Boc

\

OH Boc

_0Bu C 5 H 5 N.HBr

OSu

\

+ indole

_0Bu .OH .Boc _0Bu OH Boc

cf 3 CO 2 H .OH

OH ^ H Fig.1

Scheme of synthesis of the propionyl-peptides

313 Enzymatic

results

The enzymatic p r e p a r a t i o n used was a crude E. c o l i e x t r a c t o b t a i n e d by the method of Mengin-Lecreulx e t a l .

( 6 ) . The enzymatic t e s t s were

out a c c o r d i n g to the published procedures 1)

UDP-MurNac-L-Ala-D-Glu s y n t h e t a s e .

carried

(6).

Contrary to the product of

the

reaction

(UDP-MurNac-dipeptide),

which had been shown to be an i n h i -

b i t o r of

t h i s enzymatic a c t i v i t y

(6),

n e a r l y no i n h i b i t o r y

the product analogue I

effect.

On the o t h e r hand, Pr-L-Ala-CH„C1, a f t e r p r e i n c u b a t i o n f o r the e x t r a c t ,

presented

t o t a l l y i n h i b i t e d the a c t i v i t y at 3.10 -3 : 14%) a t 10 M.

-2

15 min w i t h

M, and p a r t l y

(residual a c t i v i t y 2)

UDP-MurNac-L-Ala-y-D-Glu-meso-DAP Compound

synthetase.

Concentration

none

Enzymatic

I

10~3 M

88

I

10-2 M

59

IV

10~3

MDP VI Whereas the f r e e d i p e p t i d e by p r o p i o n y l b i t o r s of

(I),

activity

100

-

M

57

10" 2 M

79

10" 2

95

M

( V I ) had no i n h i b i t o r y e f f e c t , i t s

and mainly by N-acetylmuramyl

(IV),

acylation

gave good

the r e a c t i o n . On the o t h e r hand, the s u b s t i t u t i o n of

nal a-COOH by a primary amide (MDP) s t r o n g l y diminished the

inhitermi-

inhibitory

activity. 3)

UDP-MurNac-L-Ala-y-D-Glu-meso-DAP-D-Ala-D-Ala s u b s t r a t e analogues I I

(at

10~2 M), I I I

(at

presented a s i g n i f i c a n t i n h i b i t o r y a c t i v i t y

s y n t h e t a s e . None o f

10~2 M) and V ( a t towards t h i s

the

10~3 M)

synthetase.

Conclus ion

These p r e l i m i n a r y r e s u l t s a l l o w us to draw the f o l l o w i n g conclusions - The s t r u c t u r a l seem to be

requirements of

strict.

UDP-MurNac-pentapeptide

synthetase

:

314 - For the inhibition of UDP-MurNac-tripeptide synthetase, it seems necessary that the L-Ala-D-Glu moiety be acylated. The N-acetylmuramyl group gives a better result than the propionyl one. Experiments are being carried out in our laboratory in order to check the effect of

intermediary

groups such as lactyl. - The potential irreversible inhibitor P r - L - A l a - C ^ C l does inhibit UDPMurNac-dipeptide synthetase. Further experiments are necessary

to demons-

trate that this inhibition is irreversible, as it is the case for other enzymes with chloromethyl ketones

(7).

References

1. Rogers, H.J., Perkins, H.R., Ward, J.B. : in Microbial Cell Walls and Membranes, pp. 239-297, Chapman and Hall Ltd, London 1980. 2. Park, J.T.

: J. Biol. Chem. J 9 4 , 885-895

(1952).

3. Flouret, B., Mengin-Lecreulx, D., van Heijenoort, J. : Anal. 114, 59-63 (1981). 4. Sachs, H., Brand, E.: J. Amer. Chem. Soc. 75_, 4608-4610

Biochem.

(1953).

5. Thomson, A., Dennis, I.S. : Eur. J. Biochem. _38, 1-5 (1973) ; Thompson, R.C., Blout, E.R. : Biochemistry _1_2, 44-47 (1973) ; Powers, J.C., Tuhy, P.M. : Biochemistry 4767-4774 (1973). 6. Mengin-Lecreulx, D., Flouret, B., van Heijenoort, J. : J. Bacteriol., in press (1982). 7. Powers, J.C. : in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins (Weinstein, B., ed.), Vol. 4, pp. 65-178, Marcel Dekker, New York 1977 ; Walsh, C. : Horizons Biochem. Biophys. 3, 36-81 (1977).

S Y N T H E S I S O F P E P T I D E S W I T H oC,n-DEHYDROAMINOACID I.

RESIDUE

S y n t h e s i s of N - p r o t e c t e d D i p e p t i d e s w i t h C - t e r m i n a l Deh y d r o a l a n i n e and

Dehydrophenylalanine

B a r b a r a R z e s z o t a r s k a , M a c i e j M a k o w s k i and Z b i g n i e w

Kubica

I n s t i t u t e of C h e m i s t r y , P e d a g o g i c a l U n i v e r s i t y ul. O l e s k a 4 8 , 4 5 - 0 5 2 O p o l e , P o l a n d

Int r o d u c t i o n N-protected dehydroamino

acids obtained via condensation

of

a m i d e s w i t h oi-keto a c i d s are of l i m i t e d v a l u e in further tide chain b u i l d i n g . In the most c a s e s N - p r o t e c t i n g (Tos, Z, A l k y l - C O )

cannot be e a s i l y split off

from A A l a ) . M o r e o v e r , no g r o u p

reactivity

pep-

group

(especially

the of,n~double bond d i m i n i s h e s the

(especially

in A P h e ) (l-5). We have

d i f f i c u l t i e s can be c i r c u m v e n t e d by c o n d e n s a t i o n of

amifound

properly

N ^ - p r o t e c t e d amino a c i d a m i d e s w i t h oi-keto a c i d s , leading N - p r o t e c t e d d i p e p t i d e s w i t h C - t e r m i n a l oi,n-dehydroamino R I X-NHCHCO-NH2

CH2R I 0CC0-0H

+

to

acid.

R CHR' I II X-NHCHCO-NHCCO-OH

X = Boc , Z , TFA R = H , CH2C6H5 R

=

, CH(CH 3 ).

H , CgHg

C o n d e n s a t i o n s mere

c a r r i e d out in b e n z e n e w i t h a z e o t r o p i c

ter removing

in some c a s e s in the p r e s e n c e of

(2,5)

s u l p h o n i c acid T o s - O H as the c a t a l y s t semiquantitatively crystalline

wa-

p-toluene

(2). We e x a m i n e d

them

by T L C . T h e p r o d u c t s w e r e i s o l a t e d in a

form from e i t h e r warm or c o o l e d r e a c t i o n

T h e w e l l s o l u b l e but h a r d l y c r y s t a l l i z i n g c i p i t a t e d as d i c y c l o h e x y l a m i n e

mixture.

compounds were

salts.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

pre-

316

Table.

Prepared N - p r o t e c t e d riipeptides and their properties Solvent for crystallization

Mp °C

1 Boc-Gly-AAla

EtOH/pet.ether

157.5-150 157.5-159(8)

2 Boc-Phe-AAla.DCHA

EtOH/EtgO/pet.ether

170.5-173.5

3 Boc-Phe-AAla

crude

149-152

4 Boc-Val-AAla.DCHA

EtOH/Et^O/pet.other

155-158

5 Z-Gly-AAla

Me0H/Ho0

6 Z-Phe-AAla.DCHA

AcOEt/pet.ether

146-149

7 Z-Phe-AAla

crude

148-149 149-150(7)

8 Z-Val-AAla.DCHA

AcOEt

143-146

51

9 TFA-Gly-AAla

crude

193.5-195

76

AcOEt

168-170

42

Nr C o m p o u n d

10 T F A - P h e - A A l a . D C H A

(lit)

Yield £f /O 19a> 19 b ) 24 C >

186-188 55 187(5 1 188-189(7) 59

11 TFA-Phe-AAla

crude

193-195

12 T F A - V a l - A A l a

AcOEt/CHpCl?

162-165

42

13 Z - G l y - A P h e

AcOEt

168-170

32

14 Z-Phe-APhe

AcOEt

193-195

35

15 Z - V a l - A P h e

MeOH/H 0

207-211.5

39

\ / B 0 C

" \

0 Nr

H

//

were isolated, respectively

\

a

9/NH

^

CH3

'

6%, 183-185°C 37%

27%,

(AcOEt)

186

' 5 - 1 9 0 ° C (Ac0Et/Et 2 0) 176-179.5°C (Ac0Et/Et"0) 2

•'•H NMR s p e c t r a d " in ppm from HMDS and (jHz) Uret.proton Pept ide Olefinic Solven t oC-proton or C F o C 0 N H proton proton(s)

2

6.0s 6.6s

5.6b

4.8m

9.1s

CDC1?

4

6.0s 6.6s

5. 7d (8Hz)

4.4m

9.1s

CDC1, DM30-D

5

5.9s 6.5s

7. 8t

4 . O d (6Hz)

9.3s

11

5.9s 6.5s

9.9d(7Hz)

5.1m

9.8s

b DMSO-Dg

12

5.6s 6.2s

8.2d(7Hz)

4.3m

8.5s

Me^CO-D

13

Both protons o v e r -

4.Od(6Hz)

9.6s

14

lapped with aromatic

4.7m

9.9s

15

protons

7 .2 - 7.9m

4.2m

9.6s

DMSO-D. D DMSO—D_ 6 DMS0-D_ b

317 esults )

The model

compounds

of the

reaction

con-

prelimi

nary o b s e r v a t i o n s . Reactivity

amino

acid amides

decreased

was the m o s t Condensation for Z - N H

of the

protected

in the o r d e r B o c > Z > T F A .

reactive amino

acid was much more )

for i n v e s t i g a t i o n

d i t i o n s w e r e c h o s e n on the b a s i s of the f o l l o w i n g

acid among

reactive

tested and

than p h e n y l p y r u v i c

with pyruvic acid. The optimum

(2) o r C I C H ^ C O - N H ^

(5)

Glycine pyruvic

one.

conditions

condensations were

inade

quate for Nf*- p r o t e c t e d the b y - p r o d u c t

formation.The

sed by d i l u t i o n of c t i o n of the prolonged discussed,

the

reaction

heating

Tos-OH seems

amino acid amide ones because side

r e a c t i o n s can be

b e c a u s e of m i n o r a m i d e

to be an e f f e c t i v e

although

catalyst

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

d e n s a t i o n w i t h o t h e r of-keto acids(2) by-product

formations

e v e r , the t e n f o l d sufficient favours

the s i d e

appears

to

reaction

be and

amino

acid

the y i e l d s

are

on p y r u v i c a c i d a m o u n t . F o r m a t i o n be d e a l t w i t h i n

and the

paper.

Condensation

with phenylpyruvic

protected amino-acid

zed with Tos-OH. The dipeptides lyst oven 4 3 hrs h e a t i n g

isolated

in

Z-

catalymoderate

the

cata-

g i v e s o n l y m i n u t e a m o u n t s of

of s o m e of the p r o d u c t s

thesis was demonstrated

were

In the a b s e n c e of

p e p t i d e s and s e v e r a l b y - p r o d u c t s Utility

acid. Reaction with

amides occurs smoothly when

•'ields (30 - 4 0 % , s e o T a b l e ) .

)

How-

of T o s - O H by ZnCl,,

materials

the s t r u c t u r e of the b y - p r o d u c t s w i l l next

con-

many

reaction.

reactions. When Boc-protected

lower and dependent

reaction

u s e d in the

of the p r o p e r

a m i d e s are u s e d as the s t a r t i n g

)

concentration. of the

less T o s - O H c o n c e n t r a t i o n

results. Displacement

inspe

requires

it c a t a l y z e s

as. w e l l as the m a i n

for an a c c e l e r a t i o n

gives promising

decrea

r e a c t i o n m i x t u r e and c a r e f u l time. The dilute solution

of

as

are

for f u r t h e r

follows:

the

formed. peptide

syn-

318

Boc-Gly-AAla

+

Gly-OMe

TFA-Gly-AAla

CICOOiBu

Gly-OtBu

DCCI + BtOH

Boc-Gly-AAla-Gly-OMe

TFA-Gly-AAla-Gly-OtBu

1. H 2 0 / 0 H "

NH 3 -H 2 O

2. T F A - O H

Gly-AAla-Gly-OtBu

3. NET3 Gly-AAla-Gly

+

Boc-Gly-OSu (8)

Boc-Gly-Gly-AAla-Gly-OtBu

Acknowledgment The authors acknowledge financial support for this work by Grant MR.I.12.1.6.11 from the Polish Academy of Sciences .

References 1.

Schmidt, U., Häusler, 0., Ohler, E., Poisel, H . : in Progress in the Chemistry of Organic Natural Products 37. 252-327, ed. Grisenbach, H., Kirby, G. W., 3pringer-Ver lag, Wien . New York 1979.

2.

Yonezawa, Y., Shin, Ch., Ono, Y., Yoshimura, 3 . : Bull. Chen. Soc. 3pn 53, 2905-2909 (1980).

3.

Poisel, H . : Chen. Ber. 110, 942-947

4.

Shin, Ch., Yonezawa, Y., Takahashi, M., Yohsimura, 3.: Bull. Chem. Soc. Dpn 54, 1132-1136 (l98l).

5.

Edqe, A . B. S., Weber, P.: Int. 0. Peptide Protein Res. 18~ 1-5 (1981).

6.

Wieland, T., Ohnacker, G., Ziegler, W . : Chem. Ber. 90 194-201 (1957).

7.

Srinivasan, A., Stephenson, R. W., Olsen, R. K . : 0. Orq. Chem. 42, 2253-2256 (1977).

8.

Pawelczak, K., Krzyzanowski, L., Rzeszotarska, B. : Polish D. Chem. 53, 2002-2011

(1977).

(1979).

SYNTHESIS OF DEHYDRO AMINO ACIDS AND PEPTIDES Robert H. Mazur and Daniel R. Pilipauskas Department of Medicinal Chemistry G. D. Searle & Co., Skokie, IL 60077

Present methods do not permit placing a desired a,3-dehydro amino acid into a predetermined position in a peptide.

Fur-

thermore, E-isomers are not readily available so little is known about their biological properties.

The reactions

reported here have apparently solved these problems. We adapted a versatile synthesis of dehydro amino acids''" to allow the incorporation of the Boc N-protecting group. Condensation of an aldehyde or ketone with methyl isothiocyanoacetate, obtained from glycine methyl ester, with K0Bu t 2 in THF gave the oxazolidinethione as the potassium salt. The latter without isolation was treated with t-butyldicarbonate to yield the N-Boc derivative (structure A in Table I). Reaction of intermediate A with KOBufc in THF caused conversion to the Boc-dehydro amino ester (B in Table I).

These

reactions go rapidly at below room temperature. Table I S

S

II

BocNHCC0 2 Me CO 2 Me A

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

B

320

Table I (cont.) A(%)

B(%)

Me

82

77

Me

Et

67

84(E+Z)

Me

H

46

6 5 ( Z) , 8 (E )

Me

i-Pr

H

74

6 8(Z) ,10(E)

n-Pr

H

59

6 5 ( Z) ,16(E)

MeSCH,

H

56

63 (Z)

C

H

59

73(Z) ,9 (E)

H

33

6 9(Z) ,OH

6H5 ACOC,H . b 4

Conjugate addition of phenyl or benzyl selenol allowed selective protection of the double bond. The carbon-selenium bond is stable to acid so that deblocking with HC1 in anhydrous solvents can be carried out. Whereas enamines are very weak bases so that direct coupling to the amino group of a dehydro amino ester can be difficult,^ selenium addition restores normal basicity. The selenol reaction was run in refluxing THF with a 3-fold excess of selenol and a catalytic amount of NaOMe. R^-C-H

R^CHSeBz1 B0C-NHCHC02Me

BOC-NHCCO^Me R2

R-^CHSeBzl H 2 NCHC0 2 Me-HCl CHR,

R^CHSeBzl

B0C-NHCHC0NHCHC02Me

B0C-NHCHC0NHCC02Me E/Z

1-10

The double bond was regenerated by mild oxidation to the selenoxide and spontaneous elimination of a seleninic acid. Since addition is trans and elimination is cis, the consecu-

321

tive reactions should convert a Z dehydro amino acid to an E dehydro amino acid. This has been achieved and derivatives £ of A amino acids can be obtained pure in quantity. Also, E Z protected A and A amino acids and peptides are usually easy to separate by silica gel chromatography.

The reaction was

done in chloroform at -20° using one equivalent of m-chloroperbenzoic acid.

We found that addition of triethyl amine

improved the yield and prevented equilibration so that the E:Z ratio in the dehydro product agreed with the ratio of diastereoisomers in the starting selenium adduct.

The results

of one study are shown in Table II. Table II CHCHMe 2 B0C-NHCC0 2 Me Starting Material

^ ^

Yield (%) Diastereoisomer Ratio

BzlSeCHCHMe 2 B0C-NHCHC0 2 Me E t^N

Yield

(%) Ratio

AZ

80, 8.5

+

85E, 10Z

-

6 0E, 14Z

4.3

AE

77 , 2

+

65E, 27Z

2. 4

-

44E, 33Z

1. 3

Aliphatic A

Z

and A

E

8.5

protected amino acids and peptides

saponify normally and the resulting carboxyl group coupled Z E satisfactorily. This was also true of A -Phe. However, A Phe is susceptible to base catalyzed inversion to the Z 4 E isomer. E.g., Boc-Gly-A -Phe-OMe with hydroxide yielded 2 Boc-Gly-A -Phe-OH. But, saponification with aqueous methanolic potassium carbonate gave jrunrearranged E product. Coupling experiments with Boc-Gly-A -Phe-OH using either mixed anhydride or dicyclohexyl-carbodiimide gave mixtures of tripeptides having an E/Z ratio of about 1.5.

322 R,-C-H 1

II __> BOC-NHCCO^Me

R.—C—H 1

II B0C-NHCC0 H 2

R 1

>

-C-H

II I' B0C-NHCC0NHCHC0 Me 2

Z (or E)

References 1.

D. Hoppe: Angew. Chem. internat. Edit., 12^ 656 (1973)

2.

D. Hoppe; Angew. Chem. internat. Edit., .LI, 933 (1972)

3.

E. G. Breitholle and C. H. Stammer; J. Org. Chem., 41, 1344 (1976).

4.

T. J. Nitz, E. M. Holt, B. Rubin, and C. H. Stammer; J. Org. Chem., 46, 2667 (1981).

a- HY D R OXY MET HY LA T10N OF PEPTIDES: A SYNTHETIC ROUTE TO PEPTIDES CONTAINING a -IIYDR OXY MET HY LA MI NO ACIDS

Z.J.Kamihski, Li.T.Leplawy, Urszula Slomczynska Institute of Organic Chemistry, Technical University 90-924 Lodz, Poland

Due to steric hindrance, usual coupling methods are of limited value for incorporating a-hydroxymethy1-amino acids into peptide chain. Our work on a-hydroxyme thylation of amino A

.

.

.

acids led us to develop an indirect synthetic approach to peptides containing a-hydroxymethylamino acids, avoiding the use sterically hindered a-hydroxymethylamino acids as coupling components. This approach involves transformation of amino acid already present in the peptide chain into a-hydroxymethylated analogue by selective a-hydroxymethylation of 2

C-terminal moiety. In an earlier report a number of dipeptides have been transformed into corresponding analogues containing C-terminal a-hydroxymethylamino acids, however despite of good yield of conversion, its preparative value was diminished by concomitant racemization, and what more, by lack of the method for determining the enantiomeric purity of a-hydroxymethy lamino acids and their derivatives. These complications have now been mastered, and we are able to 5describe a prep2 arative method which in case of [Aib , Leu ]-enkephalin gives consistently a over-all conversion into well defined 2 5 - 2 5 epimeric [Aib , S-Hmlr]- and [Aib , R-HmL^]-enkephalins (HmL = hydroxymethylleucine). The extention of the method enabling the use hydroxymethylated peptides for fragment condensation is also described. The general synthetic sequence illustrating the transformation of C-terminal amino acid into a-hydroxymethylated analogue is shown on the Scheme.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

324

„1

r2

? » X-Pept-NHCHCGNHCHCOOH

CH3OCOCI

Et 3 N T H F Q O

LH

Rl

X-Pfept-NHCH-

COOCHn

1

CH2O/H2O/THF picoline, 5 h

*

X-Pept-NHCH R2

O

1) resolution by gel chromatography 2) IN NaOH, 25°,6h R^ 1 X-Pept-NHCHCO-NH-C—COOH Rl

4a

ch 2 oh

R1

ch 2 oh

X-Pept-NHCHCO-NH-Ç-COOH 4b

R2

The f o u r s t e p s i n v o l v e f o r m a t i o n of 1 , 3 - o x a z o l e d e r i v a t i v e 2 a s a r e s u l t of w e l l known r e a c t i o n with methyl c a r b o n o c h l o r i d a te i n the p r e s e n c e of t r i e t h y l a m i n e , subsequent r i n g enlargement to the 4-0X0-1,3-dioxane £ by i n s e r t i o n of 2 m o l e c u l e s of f o r maldehyde ( f o r m e c h a n i s t i c i n t e r p r e t a t i o n s e e r e f e r e n c e s 2 , 3» 4 ) , r e s o l u t i o n of d i a s t e r e o i s o m e r i c 5 - p e p t i d y l a m i n o - 4 - o x o - 1 , 3 dioxanes by g e l chromatography and f i n a l h y d r o l y t i c d e g r a d a t i o n of 4 - o x o - 1 , 3 - d i o x a n e r i n g r e s u l t i n g i n f o r m a t i o n of c o r r e s p o n d i n g C - t e r m i n a l a-hydroxymethylamino a c i d 4 . At t h i s time our most e f f i c i e n t example i s p r o v i d e d by t r a n s f o r m a t i o n 2 of p r o t e c t e d [Aib , Leu ] - e n k e p h a l i n . Hydroxymethylation of Z-Ty r(Bu^)-A ib-Gly -Phe—LeuOH gave a mixture of d i a s t e r e o i s o m e r i c 4-0X0-1,3-dioxane s Z-TyriBu^-Aib-Gly-Phel iBu*

325

i n 86>o y i e l d . They were s e p a r a t e d i n t o o p t i c a l l y pure 5S- and 5R-epimers by s h o r t column s i l i c a g e l (Merck, H-60) chromatog r a p h y . O p t i c a l p u r i t y proof (GLC a f t e r c o n v e r s i o n of the h y d r o l y s i s p r o d u c t s i n t o t h e i r N-L- a - c h l o r o i s o v a l e r y l - O - t r i methylsilyl methyl e s t e r s ) , and the f u l l a n a l y t i c a l and s p e c t r o s c o p i c d e t a i l s are a v a i l a b l e . A f t e r h y d r o l y t i c degrad a t i o n of 4—oxo-1,3-dioxane r i n g , b o t h 5 - e p i m e r s of Z-Tyr(Bu^)-Aib-Gly-Phe-HmL were o b t a i n e d i n over-all yield (equimolar a m o u n t s ) . Examination of the r e a c t i v i t y of 2 - p e p t i d y l - a m i n o - 4 - o x o - 1 , 3 - d i o x a n e s r e v e a l e d t h a t 4 - o x o - 1 , 3 - d i o x a n e system i s s t a b l e enough t o s u r v i v e amino group d e p r o t e c t i o n and p e p t i d e c o u p l i n g Scheme i l l u s t r a t i n g the use of p e p t i d y l a m i n o - 4 - o x o - 1 , 3 aioxane f o r [Aib2, HmL 5 ' J - e n k e p h a l i n s y n t h e s i s by f r a g m e n t c o n d e n s a t i o n Tyr

Aib

Gly

Phe

Leu •OH

I

r^i u.—hydroxymethylation % ;: m . N H | ] t^(• 7( 777% mixture of diastereoisomers)

(epimer after resolution) ¡Bug But -OH H-

NH [

' 100%

iBu Q Bu 1

z-

,Bu

l

.NH, i bu""

u

I I

82% ch2oh NH-C-COOH ¡bu

326

conditions; this permitted the use of peptidelamino-4-oxo-1,5-dioxanes as OH and COOH protected intermediates for fragment condensation and consequent extention of synthetic potential of a-hydroxymethylation (Scheme on the preceding page) . No method for determining the enantiomeric purity of a-hydroxyme thy 1-amino- acids is reported. iVe developed GI£ system which gave satisfactory results in separation of enantiomers of a-hydroxymethylated alanine, leucine, metionine, ethylglycine, phenylglycine. Separation of enantiomers of a-bydroxymethylphenylalanine and a-hydroxymethylvaline was incomplete. The method consists in converting the a-hydroxyme thy laiaino acid to its N-L-a-chloroisovaleryl-O-trimethylsilyl methyl ester. The resulting diastereomeric derivatives were analysed on 50m 0V-101 capillary column. The method is applicable to samples contaminated by the unhydroxymethylated amino acids and in effect permits the determining optical purity of a-hydroxymethyrlamino acids incorporated into peptide chain. Other approaches including the use of chiral stationary phase and nmr spectroscopy failed. Stereochemistry of cn.-hydroxymethy 1 leucine (HmL) is not known. To assign confi2 5 guration of HmL present in [Aib , Hmlj ]-enkephalin we made use of empirical rule based on results obtained in the separation of the known a-hydroxymethylamino acids. According to it, S-enantiomers have longer retention time than E-isomers. ACKNOWLEDGMENT The authors acknowledge financial support for this work by a grant MR-1.12.1.6.10 from the Polish Academy of Sciences REFERENCES 1. Kaminski Z.J., Leplawy M.T C , Zabrocki J., Synthesis 1973. 792; Leplawy M.T., Olma A., Polish J.Chem. 353 (W/3). 2. Kaminski Z.J., Leplawy M.T., Slomczyhska TJ., Synthesis m i , (593). 3. S teg lie h '.v., HOfle G., Chem.Ber. 102. 1129 (1969). Kaminski Z.J., Leplawy M.T., Olma A., Slomczynska U., Peptides 1978, Proc. 15th Eurp. Peptide Symposium, Wroclaw Press, Poland 1979, p. 113.

L-OC-AMINOADIPIC ACID Ö-N-BENZYLOXYCARBONYLAMIDE AND ITS USE IN PEPTIDE SYNTHESIS

Bogdan Ldberek, Regina Kasprzykowska

(Jablonska)

Institute of Chemistry, University of Gdansk PL-80-952 Gdansk, Poland

Introduc tion A route to LF-0(-aminoadipic acid

(Aad) from L-lysine was pre-

sented on the l6th EPS (l). The starting material was Lys(Z) and the key intermediate the E-oxidation product I. This compound is interesting for peptide syntheses involving L-oc-aminoadipic acid. ^H^CH-COOH (CH 2 ) -CO-NH-Z

&

L-Aad(NHZj

(i)

Amino protection Highest yields of Z-Aad(NHZ) and Boc-Aad(NHZ) are obtained w i t h mixed benzyl 8-hydroxyquinoline carbonate and di-tert-butyl dicarbonate. Other procedures gave lower yields and usually mixtures of products.

Esterification of the (X-carboxylic group Methyl ester with the intact 6-CONHZ grouping can be prepared by the action of diazomethane. Attempts to prepare Aad(NHZ)OMe by typical procedures of peptide chemistry (MeOH/HCl, MeOH/ /S0C1„) gave mixtures of products. Hydrochloride of

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

328

Aad(NHZ)OBu^ was obtained by Taschner s transesterification method with tert-butyl acetate. Procedures with isobutene gave mixtures of products. Benzyl monoester with the intact 6-CONHZ was prepared only from amino protected derivative by Williamson ester synthesis using benzyl bromide and dicyclohexylamine. Boc-AadiNHZjOBu* t

could be prepared by Taschner s transesterification method followed by acylation.

Peptide bond formation; reactivity of the 6-CONHZ grouping Model peptides with L-Aad(NHZ) residue incorporated to peptide chain were prepared. For activation and coupling of amino protected Aad(NHZ), EEDQ method, DCCX + HONSu procedure or mixed anhydrides are recommended. Monoesters of Aad(NHZ) can be acylated by N-protected amino acids affording peptides with the intact 6-CONHZ grouping. 6-Benzyloxycarbonylamide is susceptible to acid and base catalysed hydrolysis. This enables the easy removal of 6-carboxyl protection in amino acid and peptide derivatives. The unmasked

6 carboxylic group can be activated and 5-peptide bond

formed. In some cases the formation of cyano derivatives was observed in the presence of dehydration and activation reagents. The 6-C0-NH-C0-0-CH 2 C 6 H 5 derivative - containing an

"activated"

6-carboxylic group - is not suitable intermediate for controlled and repeated formation of the 6-peptide bond. The nucleophilic attack, may proceed either on the 6-carbonyl or on the urethan carbonyl. For instance, base-catalysed hydrolysis gave easily benzyl urethan on an alternative pathway to the normal route of hydrolysis.

329

R-CO-NH-CO-O-Bzl

RCOOH + NH 2 -Z RCONH2+ C0 2 + BzlOH

1

A m i n o l y s i s may p r o c e e d i n t r a m o l e c u l a r l y

or

intermolecularly.

On p r o l o n g e d h e a t i n g Aad(NHZ)Bu^ a f f o r d e d t h e tert-butyl lecular

e s t e r of

6-lactam d e r i v a t i v e

condensation

—

(Aad-OBut) * I 'n

Aad-OBu*

I

NH 2 -Z

+

6 CO-NH-Z g r o u p i n g

on CX .«

NH 2 -Z

6 acyl

ted products

of

(Z-Aad(NH2), »-6

esters

Characteristic Z-Aad(NHZ),

of

of

m.p.

derivatives

HCl.Aad(NHZ)OMe,

62-64° m.p.

m.p. m.p.

Boc-Aad(NHZ)0NSu, Z-Aad(NHZ)-GlyOMe,

l4l-l42° 102-104°

67-69°

m.p. m.p.

gave only

Boc-Aad(NH2),

147-149°

HCl.Aad(NHZ)OBut,

pep-

1 M HCl/

Z - and B o c - L - a d i p a m i c

150-152° 100-102°

the

expec-

Boc-Aad).

- w e l l known f r o m

- was f o u n d .

some Aad

Boc-Aad(NHZ) , m.p.

Z-Aad(NHZ)OBzl,

Z-Aad,

a.cyl s h i f t

and g l u t a m i c a c i d s f i e l d

of

shift

( Z - A a d ( N H 2 ) 0 M e and B o c - A a d ( N H 2 ) O M e )

e v i d e n c e f o r CX

treatments

30 m i n ) .

Base-catalysed hydrolysis acid

(n+l)

(AcOH/EtOH, N E t 3 / C H 2 C l 2 ,

k M HCl/dioxan f o r

Investigation

Aad(NHZ)OBu t

survived the usual

t i d e s o l i d phase s y n t h e s i s /AcOH,

intermo-

Aad-OBu t I

The

the

product.

Aad(NHZ) OBu*"

+

respective

besides

No

aspartic

330 Z-Aad-OBzl,

m.p.

Z-Aad(ONSu)OMe,

90-92° m.p.

Z-Gly-Aad(NHZ)OBut, Z-Gly-Aad(NHZ)OMe, Z-Aad-NH2,

m.p.

Z-Aad(NHZ)NH2, Z-Aad,

m.p.

m.p.

117-120°

151-153°

129-132°

Boc-Aad(NHZ)NH2,

m.p. m.p.

73-75° l47-l49°

c i t e d compounds g a v e c o r r e c t

Rp v a l u e s were

m.p.

96-97°

l40-l42°

Z-Aad(NH2)0Bzl,

All

45-47° m.p.

(TLC)

I n 12 s o l v e n t

C,H,N

systems,

analyses; and o p t i c a l

rotations

measured.

References 1.

Liberek, B., Jablonska, EPS, H e l s i n g ^ r Denmark, p . 242.

R . : P e p t i d e s 1980, P r o c . l 6 t h S c r i p t o r , Copenhagen 1981,

SYNTHESIS OF ß-HOMO-L-PROLINE ANALOGUES OF PEPTIDE HORMONES

Krzysztof Baiikowski and Aleksandra Misicka Laboratory of Peptides, Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland

Introduction Some of (i-homo-L-amino acids have already been introduced in peptide hormones in the hope of obtaining analogues that possess the desired biological activities and are not susceptible to enzymatic degradation. In some cases expected properties were found, for example,

|]3-homo-Pro] ''-bra.dykinin appea-

red to be equipotent with bradykinin and resistent to enzymatic cleavage

(l).

In the course of investigation on chemical structure - biological activity relationship we undertook the synthesis of fi-homo-L-proline analogues of thyreoliberin (TRH), MSH release-inhibiting factor (MIF) and oxytocin (OT).

Synthesis The protected [3-homo-L-proline intermediates were obtained by homologation of the corresponding L—Pro derivatives according to the Arndt-Eistert scheme. The preparation of carbobenzoxy(Z) derivatives followed the technique described by Balaspiri et al. (2); Boc derivatives were synthesized according to Ondetti et al. (l). The following compounds were obtained: Z-[3-homo-Pro-0Me, Z-(3-homo-Pro (m.p. 72-74°C, described as an oil (2)), Z-(J-homo-Pro-NH2, Boc-fi-homo-Pro-OMe, Boc-P-homo-Pro, Boc-|3-homo-Pro-NH2. In our hands Ondetti s procedure

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

332 was more convenient; we found that purification of Boc intermediates was not necessary if final Boc-fi—homo-Pro was purified by recrystallization of its dicyclohexylammonium salt. Splitting of protecting groups from substituted amides gave (i-homo-Pro-NH„ which was used for coupling with pGlu-His by DCC/HOBt method to give [(i-homo-Pro] -TRH. Z-(i-homo-Pro was coupled with Leu-Gly-OEt by mixed anhydride procedure to give protected tripeptide used in the synthesis of [G-homo-Pro] 1 -MIF. Boc-(i-homo-Pro was used in solid phase synthesis of [(i-homo-Pro^j ^-OT. Protected nonapeptide amide Z-Cys(Bzl)-Tyr(Bzl)-Xle-Gln-Asn-Cys(Bzl)-(i-homo-Pro-Leu-Gly-NH 2 was treated with Na/NH^ liq., oxidized with I^/MeOH and purified by column chromatography on silica gel column followed by gel filtration on Sephadex G-15. Biological activities of these analogues are under investigation.

Acknowledgement This work was supported in part by the Polish Academy of Sciences within the projects No MR 1-12 and MR 11-10.

References 1.

Bala spiri, L. , Perike, B. , Papp, Gy. , Dombi, Gy. , Kova.cs, K.: Helv. Chira. Acta 58, 969-973 (1975).

2.

Ondetti, M.A., Engel, S.L.: J. Med. Chem. 18, 761-763 (1975). ""

OPTICALLY ACTIVE NEOPENTYLGLYCINE AND ITS APPLICATIONS IN PEPTIDE CHEMISTRY

Jan Pospisek and Karel Blaha Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia.

Introduction Non-proteogenic amino acids (l-3) are useful in physicochemical (4,5) and biological (6) studies. Investigations of this kind require amino acids with pronounced manifestation of single structural features (bulkiness of the side-chain, hydrophobic! ty, etc.). Neopentylglycine (IV, 2-amino-4,4-dimethylpentanoic acid, Neo) represents one of such amino acids. In comparison with tert-leucine, the bulky tert-butyl group is in IV separated by one methylene group from the backbone. Two synthetic pathways (7,8) leading to IV have been described. We report now another synthesis of IV and also some experiments which show that the neopentylglycine (IV) can be used without difficulty in peptide syntheses.

Results 2-Cyano-4,4-dimethyl-2-pentenoic acid (i) was hydrogenated over Pd-bla.ck in acetic acid to give 2-cyano-4,4-dimethylpentanoic acid (il) in 98% yield. Treatment of II with alkaline hydrogen peroxide afforded 2-ca.rbamoyl-4,4-dimethylpentanoic acid (ill), m.p, 150-151°C, in 50% yield. Hypobromite degradation oX" III produced in quantitative yield D,L-neopentylglycine (iv) which was purified by elution with 55° amino-

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

334

nium h y d r o x i d e f r o m Dowex-50 (CH3)3C-CH=C-COOH

(Scheme »-

l).

(CH3)3C-CH2-CH-COOH

CN

CN

I

II

(CH3)3C-CH2-CH-COOH

(CH3)3C-CH2-CH-COOH

CONH 2

NH2

III

IV Scheme

Table

1

1

N-Substituted derivatives Deriva t i v e

Yield

of

L-neopentylglycine

[ext., s o l v e n t o„ m.p., C

$

Calcula ted/Found

za

55

-12.5°,methanol 15^-155

Boc

90

+13.9°,methanol IO2-IO3

NPSa

87

-49.7°,dimethylformamide 163-165

3

D,L

Dicyclohexylainmoniuin

lo C

till

r 0CH 3 1 I if Pyridine N • Trt-N'VV HN03-H2N-Sr Y^ 2) H2S 0 0 H 0 0 3

2

H NH 2-N ^oEt

H

0CH3

S 0

o c h 3J

]]

och3

0

HO'N^N-Sr^

8

o

S-n

H

0 -ONb: -0-N

1) HF-ani sole 2) NEt, '

0CH3

S Loh

0

althiomycin (1)

(ii) Photoreaction with diketene.

Fig. 2. Synthesis of Althiomycin.

0

343 A c c o r d i n g to this p a t h w a y ,

f i r s t the a c t i v e e s t e r

p r e p a r e d from N,S-ditritylcysteine endo-2,3-dicarboxyimide

and

ii-hydroxy-5-norbornene-

(HONb), w a s c o u p l e d w i t h

p y r r o l i n - 2 - o n e s o d i u m salt.

(2),

4-methoxy-3-

The c o m p o u n d thus o b t a i n e d w a s

d e t r i t y l a t e d i n the u s u a l w a y to g i v e the c y s t e i n e

derivative

(£) w i t h free a m i n o a n d m e r c a p t o g r o u p s w h i c h w a s t h e n c o u p l e d Q\

w i t h the imino e t h e r of b e n z y l o x y c a r b o n y l g l y c i n e the t h i a z o l i n e d e r i v a t i v e

.

to a f f o r d

The total y i e l d from

ditrityl-

c y s t e i n e to this c o m p o u n d t h r o u g h the r o u t e m e n t i o n e d a b o v e w a s m u c h h i g h e r t h a n t h a t via

the p h o t o r e a c t i o n .

T h e f o r m a t i o n s of

the imide b o n d a n d the t h i a z o l i n e ring in the c o u m p o u n d

(50

c o m f i r m e d by the d o w n f i e l d s h i f t of the m e t h y l e n e p r o t o n s p y r r o l i n e ring

(0.3 p p m ) , a n d the l o n g - r a n g e c o u p l i n g

exo m e t h y l e n e a n d C-4 of the t h i a z o l i n e r i n g ly in N M R s p e c t r u m . pyrroline compound

(2 Hz)

A l d o l c o n d e n s a t i o n of the (5) w i t h f o r m a l i n

(35%)

between

respective-

thiazoline-

in d i m e t h y l

sulfoxide

a t r o o m t e m p e r a t u r e a f f o r d e d the m i x t u r e of the d e s i r e d methylated derivative

were

in

hydroxy-

(£) a n d its a n h y d r o c o m p o u n d . H o w e v e r , by

u s e of p a r a f o r m a l d e h y d e i n s t e a d of f o r m a l i n , this r e a c t i o n g a v e only the h y d r o x y m e t h y l a t e d p r o d u c t

(£)

quantitatively.

In the final step of the s y n t h e s i s of a l t h i o m y c i n , hydroxymethyl derivative

(6) w a s d e b l o c k e d w i t h

the

anhydrous

h y d r o g e n f l u o r i d e i n the p r e s e n c e of a n i s o l e , a n d t h e n c o u p l e d w i t h the o x i m e of t h i a z o l e c a r b o x a z i d e f o r m a m i d e a t 5 °C for 5 d a y s .

(8) in

N,^-dimethyl-

Synthetic althiomycin which was

i s o l a t e d a n d p u r i f i e d by p r e p a r a t i v e t h i n - l a y e r (chloroform-methanol

9:1)

chromatography

was identical with natural

althio-

m y c i n i n all r e s p e c t s i n c l u d i n g T L C a n d H P L C . T h e m e a s u r e m e n t s of the b i o l o g i c a l a c t i v i t y of a l t h i o m y c i n a n d its f r a g m e n t as w e l l as the

synthetic

dehydroxymethyl

a n a l o g s are now i n p r o g r e s s .

References 1.

H. Y a m a g u c h i , Y. N a k a y a m a , K. T a k e d a , K. T a w a r a , K. M a e d a ,

344 T. Takeuchi, and H. Umezawa, J. Antibiot. A 10, 195 (1957). 2.

H. Fujimoto, T. Kinoshita, H. Suzuki, and H. Umezawa,

3.

H. Sakakibara, H. Naganawa, M. Ohno, K. Maeda, and H.

J. Antibiot. 22, 271 (1970). Umezawa, J. Antibiot. 27, 897 (1974); H. Nakamura, Y. Iitaka, H. Sakakibara, and H. Umezawa, ibid. 21_, 894 (1974). 4.

H. A. Kirst, E. F. Szymanski, D. E. Dormon, J. L. Occolowitz, N. D. Jones, M. 0. Chaney, R. L. Hamill, and M. M. Hoehin, J. Antibiot. 28, 286

5.

(1975).

T. Shiba, K. Inami, K. Sawada, and Y. Hirotsu, Heterocycles 13, 175 (1979).

6.

K. Inami, Y. Saito, and T. Shiba, Peptide Chemistry 1981,

7.

T. Shiba, K. Sawada, and Y. Hirotsu, Heterocycles 10_, 133

8.

Y. Hirotsu, T. Shiba, and T. Kaneko, Bull. Chem. Soc. Jpn.

137 (1982) . (1978). 40, 2945 (1967).

PEPTIDE TRICYCLIC THIA-CYCLOLS FROM LINEAR PRECURSORS

Giancarlo Z a n o t t i , Francesco Pinnen, Gino Lucente I s t i t u t o di Chimica Farmaceutica - Centro di Studio per la Chimica del Farmaco del C.N.R. - Città U n i v e r s i t a r i a 00100 - Roma - I t a l y

S i l v i o C e r r i n i , Walter F e d e l i , Fernando Mazza Laboratorio di S t r u t t u r i s t i c a Chimica " Giordano Giacomello " , C.N.R. c.P. 10 00016 - Monterotondo Stazione - Roma - I t a l y .

The chemistry of the oligo-cyclopeptides as valuable model systems for the elucidation of protein stucture, i s s t r i c t l y related to cyclol format i o n . C y c l o l i c tautomers are apparently stable forms of the nine-membered c y c l o - t r i p e p t i d e s in which a residue of a primary amino acid i s contained. In a preceedina paper^ we reported the synthesis of a peptidic t h i a - c y c l o l a compound which i s representative of a new c l a s s of cyclopeptides related to natural E r g o t - a l k a l o i d s . The f i r s t synthetic approach, in which an N-acyl-diketopiperazine was used as key intermediate, afforded t h i a - c y c l o l (1) whose stereochemistry i s d i f f e r e n t from that found in natural oxac y c l o l s . The synthesis of stable t h i a - c y c l o l s possessing the same stereochemistry zed

which i s found in natural E r g o t - a l k a l o i d s , has now been r e a l i -

through d i r e c t c y c l i z a t i o n of 2-mercaptopropionyl-dipeptides

p-nitro-

phenylesters. The synthetic strategy to achieve these l i n e a r precursors required the use of a thiol protecting group s e l e c t i v e l y removable under mild non-oxidative conditions so as to avoid any involvment of the a c t i v a ted carboxyl function and/or of the f i n a l c y c l i c product. Use of the t e r t butylmercapto group

2

was i n i t i a l l y discarded due to the d i f f i c u l t i e s

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

346 encountered during the synthesis of 2-(S-tert-butylmercapto)-mercaptopropionic acid . The acetamidomethyl group, because of its reported attitude 3 to be easily removed by mercury ions , was then considered and adopted in a sequence of steps which led eventually to 2(-S-acetamidomethyl)-mercaptopropionyl-Phe-Pro-ONp. Unfortunately, treatment of this active ester with mercury ions did not proceed satisfactorily. The problem was solved,as shown in the Scheme, by using the acetamidomethyl group during the early steps of the synthesis and by changing to the tert-butylmercapto group in the last steps. Thia-cyclols (4) and (5) were obtained in a single step by deprotecting S-tert-butylmercapto active esters (2) and (3) with tri-nbutyl-phosphine in a dilute solution of 70% aqueous propanol. The cyclolic structure assigned to (4) and (5) is based on spectroscopic data and for compound (5) is supported by X-ray crystallographic analysis. The i.r. spectrum (CHC1 ) of the thia-cyclols show a broad OH absorption centred at 3300 cm

-1

and two carbonyl bands at 1680 and 1650 cm

-1

; no absorptions

are observed in the ranges of amide second band and sulfur-hydrogen bond.

fHjPh CR®

CH-t-Acm

.

H^tCH

CO2H

f-^^l

CO-N

.

L e o

(R.S) C H - H l .

M.

CO-NH-CM-CO-«

COjMa CO^Me Ochromateflraphic aaparation lategra* ef diaateraoieomers itaraoiM A d t p r o t K t i a n with >2

Mi CM-»«.tBii [" CO-HH-CH-CO H CH-Ph CO

D OH~ aXM«)3C•SH/o2

""I I COOWp

3) p nrtrophewl/ DCCI '

- - S ^ H WandCS) CO-NH-ipH-CO

a c t i v e .«tar R . S . S

C3) a c t i v e aatar S . S . S

tri-n-butylphoephine

4—>L (4)

O'

X

'L,^ il ^ Scheme

o

C02M.

347 In lH nmr spectra the OH signal appears as a doublet at co

O) co I

rol Z5 a) i CD

-o

o_

CM

O)

"tul I

13 a> i

cu

i c

"5. JZ 1

I ,

x: 0 1

I

O

O

o

o

NI •O O

NI -O O

I

i CM

a)

a) •a 4-1 a) a) C¡ rH •tí o. m raß ra e rao »H O o • H P. O •O a)

—

a>

T

O tí ra

t—1

NI QQ O

o reí i i_ a> co i a>

01 • H ai 4J

•H

I

cu oo

O i

4-1 i H

cid

tí o

O. o

O a>

ai T-H 0 a) 4-1 W en 00 C 0 o

1 •O 0 •H

I

I

i O i o

i

360

treatment with a large molar excess of Cbz-glycine / carbonyldiimidazole in methylene chloride.

The product was isolated by HPLC in the same iso-

propanol / 0.1% TFA gradient described above. double absorbance at 210 nm and at 260 nm.

It was identified by its

Its identity was confirmed by

amino acid analysis of acid hydrolysates.

The benzyl ester of allothreonine was coupled with Boc-serine by treatment with one equivalent each of N-methylmorpholine and dicyclohexylcarbodiimide in methylene chloride.

S-(2,3-dihydroxypropyl)-cysteine was pre-

pared by treatment of L-cysteine with glycidol at pH 9.

The material

crystallized as a half hydrate from isopropanol / water, it comigrated with aspartic acid on the amino acid analyzer (5), and it was completely destroyed by periodate oxidation.

Repeated treatments with methanolic HC1

gave the methyl ester which was treated with methanolic ammonia to obtain the amide hydrochloride as a hydroscopic, but electrophoretically homogeneous solid.

Coupling of saponified globomycin with this product has

yielded a crude globomycin analog containing S-(2,3-dihydroxypropyl)cysteine, but the material must be further purified before biological evaluation.

ACKNOWLEDGMENT:

This work was supported by grants GM-19043 and AM 10080 from the National Institutes of Health.

References 1.

Inukai, M. , Takeuchi, M. , Shimizu, K. and Arai, M. : J. Antibiotics _31 1203-1205 (1978).

2.

Inukai, M.; Personal Communication.

3.

Inouye, S., Wang, S., Sekizawa, J., Halegoua, S., and Inouye, M.; Proc. Nat. Acad. Sci. US, 74 1004-1008 (1977).

4.

Maeda, T., Glass, J., and Inouye, M.; J. Biol. Chem. 256 4712-4714 (1981).

5.

Hantke, K., and Braun, V. ; Eur. J. Biochem. 34 284-296 (1973).

SEMISYNTHETIC STUDIES ON THE BOVINE TRYPSIN-KALLIKREIN INHIBITOR

Laura Biondi, Bruno Filippi, Fernando Filira, Virgilio Giormani, and Raniero Rocchi Centro di Studio sui Biopolimeri del C.N.R., Istituto di Chimica Organica dell'Università di Padova, 35100 Padova, Italia

Introduction

Bovine trypsin-kallikrein inhibitor (BTI) is a single chain protein of 58 amino acid residues containing three disulfide bridges (1, 2, 3, 4). The fully reduced inhibitor may be refo_l ded by air oxidation with nearly complete recovery of the inhi bition capacity (4). Chemical syntheses of the inhibitor have been achieved in different laboratories (for references see 5) and the partial synthesis of a fully active 52-homoserine-BTI has also been described (6, 7). Preparation of an active derivative of BTI in which the reactive site peptide bond Lys 15Ala 16 is cleaved (BTI*)(8, 9) opened the way to a series of elegant experiments through which reactive site modifications have been achieved (9). We decided to develop a semisynthesis of BTI which would allow the efficient preparation of analogs of the amino terminal region. The semisynthetic scheme used is shown in Figure 1. Since guanidination of the e-amino function of the four lysine residues and subsequent removal of the amino terminal tripeptide Arg-Pro-Asp do not affect the inhibitory capacity (10), the synthesis was designed to prepare a des (1-3) inhibitor derivative in which three out of four lysine residues (positions 26, 41, and 46) are replaced by homoarginine residues [des(l-3)-3-Guan-BTl] .

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

362

Figure 1. Semisynthesis of des(1-3)-3-Guan-BTI

363 Results Preliminary experiments showed that even the fully reduced, te traguanidinated native inhibitor (4-Guan-BTI) may be refolded by air oxidation with nearly complete recovery of its inhibiting capacity. To ascertain if truncated derivatives of 4-Guan-BTI were still able to undergo the correct refolding by air oxidation, the des(l)-, des(l,2)-, and des(1-3)-sequences have been prepared by successive Edman degradations (11) of 4-Guan-BTI. The degradation reactions were followed by determining, at each step, the amino acid composition and the inhibitory po wer of the isolated material. amino acid composition Arg

Pro

% BTI activity

Asp

A

B

4-Guan-BTI

6 .0(6)

4 .1(4) ,

5 .0(5)

100

100

Des(1)-4-Guan-BTI

5 .0(5)

4 .0(4) .

, 5 .0(5)

100

100

Des(1,2)-4-Guan-BTI

5 .0(5)

3..1(3)

4 .9 (5)

>95

> 95

Des(1-3)-4-Guan-BTI

5 .1(5)

3..1(3)

4 .1(4) .

>95

100

Table. Amino acid content and inhibitory capacity of the indicated 4-Guan-BTI derivatives before (A) and after (B) reduction and reoxidation. The reoxidized compounds have been purified by affinity chromatography on trypsinSepharose. The values denote number of residues per molecule. Only pertinent amino acids are included. All truncated sequences showed to posses nearly full inhibitory capacity against trypsin (12). Reduction followed by air oxidation yielded fully active

inhibitor derivatives. The mo-

dified inhibitor (BTI*) consists of two peptide chains linked through the disulfide bridges Cys 5-Cys 55 and Cys 14-Cys 38. The shorter segment corresponds to the sequence 1-15 of BTI and the larger one to sequence 16-58. By analogy with the nomenclature used for the ribonuclease S-peptide and S-protein (13) and considering that the selective cleavage of the

Lys

364 15-Ala 16 reactive site peptide bond has been achieved by try£ tic digestion (8), the 1-15 and 16-58 segments are respectively defined as T-peptide and T-protein. Reaction of BTI* with O-methylisourea converts essentially all lysines into homoarg^ nine residues yielding the tetraguanidinated modified inhibitor (4-Guan-BTI*). The a-amino functions of Arg 1 and Ala 16 are not significantly affected by the modification reaction,

as

shown by the amino acid analysis and determination of the amino terminal residues of 4-Guan-BTI* (14), and remain the only accessible sites of coupling with a carboxyl-activated, suitably protected peptide. The synthesis in solution of the protec ted 12-peptide hydrazide Msc-Phe-Cys(Acm)-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys (Acm)-Lys (Tf a) - ^ H ^ , corresponding to the sequence 4-15 of BTI [des(1-3)-T-peptide] , has been carried out through the fragment condensation procedure (Figure 2).

Msc-Phe-Cys(Acm)-Leu-N H -Boc 4 6 t Z-Glu(OBu )-Pro-Pro-Tyr-Thr-N29H29-Boc 7 11 Trt-Gly-Pro-Cys(Acm)-Lys(Tfa)-N„H -Boc 2 2 12 15

TFA

Acm Acm Tfa Msc-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-N7H 2 3 Figure 2. Synthesis of 4-15 sequence of BTI

Classical carboxyl activation, carboxyl and amino protection, and deblocking procedures were used during the synthesis. Coupling experiments have been carried out by reacting, through

365

the azide procedure, the synthetic des(1-3)-T-peptide hydrazide (6 eq.) with 4-Guan-BTI* in DMF/DMSO mixture (1:1, v/v). The reaction product was isolated, treated with 1 M piperidine, and finally dissolved in 5% aqueous acetic acid. Some insolu1ble material was removed by centrifugation and the supernatant was fractionated by gel-filtration on Sephadex G 50, and purified by ion-exchange chromatography on CM-Sephadex C 25 and gel-filtration on Sephadex G 25. Comparison of the amino acid composition of 4-Guan-BTI* and of the isolated material indica ted that the a-amino group of both Arg 1 and Ala 16 have been acylated. The diacylated derivative was treated with mercuric acetate in 50% acetic acid (15), to remove the acetamidomethyl groups, and fully reduced with 1,4-dithiothreitol (DTT) in 8 M urea, pH 8.6. Gel filtration on a Sephadex G 50 column, eluted with 5% acetic acid, allowed to isolate the reduced des (1-3)-3-Guan-BTI. The protein containing fractions were combined, the resulting aqueous solution was gradually brought to pH 8.0 by extensive dialysis against 0.02 M sodium phosphate buffer (pH 8.0), and the reduced product was renatured by air oxidation. Determination of the inhibitory capacity (12) indicated that the semisynthetic 3-Guan-BTI was about 10% effective in inhibiting the tryptic hydrolysis of the N^-benzoy1-L-arginine-4-nitroanilide. Further purification by affinity chromatography raised the inhibitory activity to 40-45% but full activity was not obtained. A possible explanation of this result could be found in side reactions occurring during the removal of methylsulphonylethyloxycarbonyl

(Msc) protecting group. Deblock-

ing experiments, carried out on both the synthetic des (1-3)-T-peptide and the model compound Msc-Phe-Cys (Acm)-Leu-^I^-Boc, showed that some unidentified products are formed, together with the desired o(-amino free compounds, by treatment either with 1 M piperidine or with the mixture dioxan-methanol-4 N NaOH being 0.1 N in base (16). The use of an alternative synthetic route for preparation of des(l-3)-T-peptide is under investigation.

366 Acknowledgements Financial support by the Italian C.N.R. Target-Directed Project "Chimica Fine e Secondaria" is gratefully acknowledged. References 1. Kassell, B., Laskowski, M. Sr.: Biochem. Biophys. Res. Commun. 20, 463-468 (1965). 2. Chauvet, J., Nouvel, G., Acher, R.: Biochim. Biophys. Acta 115, 121-129 (1966). 3. Dlouha, V., Pospisilova, D., Meloun, B., Sorm, F.: Coll. Czech. Chem. Commun. 31/ 346-352 (1966). 4. Anderer, F. A., Hörnle, S.: J. Biol. Chem. 241, 1568-1572 (1966) 5. Rocchi, R.: in Perspectives in Peptide Chemistry (Eberle, A., Geiger, R., Wieland, T. eds.), Karger, Basel, pp. 318328 (1981). 6. Dyckes, D.F., Creighton, T., Sheppard, R.C.: Nature 247, 202-204 (1974) 7. Dyckes, D.F., Creighton, T., Sheppard, R.C.: Int. J. Peptide Protein Res. 11, 258-268 (1978) 8. Jering, H., Tschesche, H.: Angew. Chem. Int. Engl. Ed. 13, 660-661 (1974). 9. Jering, H., Tschesche, H.: Angew. Chem. Int. Engl. Ed. 13, 662-663 (1974). 10. Kassell, B., Chow, R.B.: Biochemistry 5, 3449-3453 (1966) 11. Edman, P.: Acta. Chem. Scand. 10, 761-768 (1956) 12. Kassell, B.: in Methods in Enzymology (Perlmann, G., Lorand, L. eds.), Academic Press, New York, 19^, 844-852 (1970). 13. Richards, F.M., Vithayathil, P.J.: J. Biol. Chem. 234, 1459-1465 (1959). 14. Gray, W.R.: in Methods in Enzymology (Hirs, C.H.W., Timasheff, S.N. eds.), Academic Press, New York, 2_5, 121-138 (1972) 15. Veber, D. F., Milkowski, J.D., Varga, S.L., Denkewalter, R.G., Hirshmann, R.: J. Am. Chem. Soc. 94, 5456-5461 (1972) 16. Tesser, G. I., Balvert-Geers, I.C.: Int. J. Peptide Protein Res. 7, 295-305 (1975).

SYNTHESIS OF A HYBRID CHICKEN/HUMAN INSULIN

H.-J. Wieneke, G. Wolf, W. Wolff, E. E. Büllesbach, H.-G. Gattner, D. Brandenburg Deutsches Wollforschungsinstitut D 51oo Aachen, Federal Republik of Germany

Introduction Chicken insulin (turkey ins.) is the only known native analogue with enhanced receptor binding and in vitro activity (1, 2). The sequence differs from human insulin in positions A8-1o (HisAsn-Thr), B1, B2, B3o (Ala) and B27 (Ser). In order to detect the molecular basis for high affinity, we have set out to synthesize a series of analogues (3). The first is a hybrid insulin containing a chicken A-chain and human B-chain. In this communication we wish to describe the synthesis of the protected A-chain, its deprotection, conversion to the tetraS-sulphonate form, and its combination with semisynthetic human B1 32 327 B3oi B-chain to yield [Phe , Val , Thr , Thr J chicken insulin.

Results The synthesis was performed by segment condensation in solution. We used trityl groups for protection of the cysteines A6/A7, tert. butyl thio groups for A11- and A2o-Cys, tert. butyl etherand ester groups for the blocking of all hydroxy and carboxyl functions. For temporary protection of a-amino groups the Z- and the Bpoc groups were selected. Removal of these protecting groups was carried out respectively by catalytical hydrogenolysis, and with 80 % acetic acid in the presence of pyridine hydrochloride (4) (method A) or with HC1 in trifluoroethanol (5) (method B). Pep-

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

368

tides were purified by gel chromatography (g. c.) on Sephadex LH-2o in DMF. Plan of synthesis is outlined in the following figure: 1

2

3

U

5

6

7

8

Gly

lie

Vol

Glu

Gin

Cys

Cys

His

9

10

Asn Thr

11

12

13

Cys Ser

H

15

Leu Tyr

Gin

18

19

Leu Glu Asn

16

17

Tyr

20

21

Cys Asn

z->0Np ,But

z-

L »

z-k 2 h 3 Trt Trt l Trt c rTr.

Bpoc«

IV

fTrt rTrt

) 0) û£

4.5 5.5 6.5 7.5 8.5

pH

Fig.2 Effect of pH on the activity of the amidating enzyme in porcine pituitary The pH optimum of the enzyme was found to be 7.0 (Fig.2).

It

was observed that additi on of 0. linM cupric ion to the enzyme

384 increased its activity but higher concentrations abolished the effect.

Dialysis against ethylene diamine tetra-acetate

(lOmM, pH7.0) resulted in complete loss of activity;

however

the activity could be restored by the addition of cupric ion. To investigate the mechanism of amidation, the reaction was carried out with isotopically labelled tripeptides. D-Tyro14 sylvalylglycine was synthesized using both N-glycine and 15 N-glycine and the corresponding D-tyrosylvaline amides were isolated after incubation with the pituitary enzyme.

The

molecular structures of the products were confirmed by highland low-resolution mass spectrometric analysis of the trifluoracetyl derivatives (3) (Fig.3). Three of the ions obtained showed the single mass unit increments expected from the incorporation of the Nisotope. High-resolution mass measurements of the molecular ions at 549 and 550 AMU (atomic mass units) and the base peaks of the spectra at 346 and 347 AMU gave unequivocal evidence for the empirical formulae of these compounds. These results demonstrate that the N isotope from the glycine residue is The data support a mechanism retained in the peptide amide. involving removal of hydrogen from the C-terminal glycine and spontaneous hydrolysis of the resulting imino linkage: NH CO I R-CH I NH

CH20 I C00H

CO I R-CH I NH

X

NH, CH I C00H

CO I R-CH I NH

CHO I C0 2 H

The substrate specificity of the amidating enzyme was examined with a range of synthetic tripeptides.

It was found that

glycine was a mandatory amino acid in the C-terminal position of the tripeptide D-Tyr-Val-Gly;

substrates containing

385 leucine, alanine, sarcosine, lysine or glutamic acid in place of the glycine did not give rise to a detectable amount of dipeptide amide.

In addition amidation of the tripeptide

00

r._«5

1

standard

Oi

so

1 300

330

340

360

HA

430

450

»00

11»,

unknown troni

r-

>>ofc»'

»30

M0

560 T

N tripeptide^

1 «M -P

•H m fl 0)

170 J 300 330

i 10 340

360

430

4S0

500

530

540

560 y

•H

0 >) •H -P ai r-i «0)

standard

300

330

340

360

430

450

500

unknown from

300

330

340

360

430

450

500

530 540 560 15N tripeptide

530

540

560 j

Fig.3 Normalized partial mass spectra of the trifluoracetyl derivative of Tyr-Val amide, (a) Synthetic D-Tyr-Val-1 ''N-amide. (b) D-Tyr-Val amide formed enzymatically from Tyr-Val-1"N-Gly. (c) Synthetic D-Tyr-Val-'^N-amide. (d) D-Tyr-Val amide formed enzymatically from Tyr-Val- 15 -Gly

386 D-Tyr-Val-Gly was found to take place when the valine in position 2 was replaced by phenylalanine or glycine.

It

seems likely that the amidating enzyme in porcine pituitary will prove to be common to all tissues that biosynthesize the C-terminal carboxamide group.

References 1. 2. 3.

Bradbury, A.F., Finnie, M.D.A., Smyth, D.G.: Nature 298, 686 (1982). Jacobs, I.S., McKeel, D.W., Jarett, I., Daughaday, W.H.: Endocrinology 92, 477-486 (1973). Engelfried, C., Koenig, W.A., Voelter, W.: Spectrom. 3, 241-244 (1976).

Biomed. Mass

REACTION MECHANISM

IN TRYPSIN

INSULIN STUDIED BY

CATALYZED

SYNTHESIS OF

HUMAN

0-NMR SPECTROSCOPY.

and Kjeld Schaumburg 2

Jan MarkusserJ ^Novo Research

17

Institute,

Novo

Allé, DK-2880

Bagsvaerd,

Denmark 2

H.C. 0rsted Institute, Universitetsparken 5, DK-2100 Copenhagen 0, Denmark

Introduction Two methods

of

replacing

the

alanine

residue

B30

of

porcine

insulin with threonine by trypsin and thus achieving

a conver-

sion to

(1,2)

human

insulin

alanine residue is A (3)

resulting

are

known.

selectively

in

In

one

method

hydrolyzed with

des(Ala B 30)

porcine

the

carboxypeptidase

insulin

(DAI).

In

a

second step, DAI is coupled with a threonine ester in a mixture of water and organic solvents by trypsin, resulting in an ester of human

insulin

(HI-OR).

In the

other method

(4,5) porcine

insulin is directly transpeptidized with a threonine ester in a mixture of water and organic solvents by trypsin, resulting in an ester of human insulin under concomitant release of alanine. The pathways of the two reactions are shown in Fig. 1 in accordance with the acyl-enzyme concept (6) in which an intermediate ester (DAI-trypsin) is formed from the carboxyl group of Lys B 2 9 of DAI and the hydroxyl

group of Ser^&3

trypsin, state III and IV. transpeptidation method _i.e. transverses ponent R

and

the

returns

the active site of

In order to investigate whether the

is truly

states via

Qf

a transpeptidation

I-»-III, exchanges III+I, or

only

the

reaction, amino

apparently

comso

by

having free DAI as an intermediate state, the transpeptidation (experiment A) was carried out in a mixture of organic solvents and H 2 ^ 0 .

If

incorporated in of DAI-Trypsin

free the at

DAI

occurs

carboxyl step

as

group

IV+V, and

an of

intermediate Lys

B2

9

by

hydrolysis

transpeptidation

regarded as hydrolysis (I+VI) followed by coupling

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

is

could

(VI+I).

be

388

Trypsin + Porcine Insulin or Trypsin + Human Insulin Ester O o I. ii c •ex. / N\ _N 183 C- H

Trypsin + DAI o

VI.

/

H N'

/

cr" ,H

O'

H n N «

// B29

o

(CH ) -NH B30 N \ / 24 3 H' i ' ^Ch HN. ^ B 28 \ k

.N

0 w

O a II c/ -•Sk A _ N 183 C- H

1

II

^

C 183 C _N H I I I I H I 0'C /H 0'' //B29 ^ c > II / A183 C- N H I C \ o \/B29 H ; N _ ... yC (CH2). -NHj H' HN ^B28

V.

\ \

\/B29 H. X N---H-- -N \ / (CH2)4-NH; Ch

HN \B28 -Ala (B30) (irreversible)

\

Acyl-Enzyme Intermediates

\

46

,

H

-Ala Fig. 1

n \>/B29 C

y

\

-H,0.

\

(CH2)4-NH3

HN "^628 +Thr-OMe

S t e r e o c h e m i c a l v i e w of the s t e p s in (I+III and III+I) and coupling human insulin ester.

k-5 +H20

O V0 cs\\c / n\ A C 183 C-__N H I 1 / \ •H' C \ \ ) o-' H c \ffB29 H N" IIN\ C (CH2)4-NH3 / 46 V' HN^ ^28 IV.

183"C- ,NH H c

N

Nk-2 + Thr-OMe (B30)

o II

transpeptidation

( V I + I ) , leading to

D r a w i n g a d a p t e d from

(6).

389 The r e v e r s i b i l i t y

of

step

c o u p l i n g of DAI w i t h s o l v e n t and

H2O

the h y d r o l y s i s in L y s B 2 9 control

step

futher

in a m i x t u r e of

If

V + I V is

step

IV+V leads

C)

in

investigated

ester

B) .

consequently

(experiment

the

to

organic

reversible,

incorporation

product

by

HI-OR.

of

A

positive

17

0 - l a b e l l e d H I - O R w a s p r e p a r e d by R9Q 17 17 insulin in H 2 0 yielding DAI(Lys - 0)

hydrolyzing porcine f o l l o w e d by

a threonine

(experiment in

and

V+IV was

coupling

of

to

Thr-OMe

by

trypsin

in

a

mixture

in

H^70.

of

w a t e r and o r g a n i c s o l v e n t s , s t e p s V I + I . M a t e r i a l s and M e t h o d s A.

P r e p a r a t i o n of H I - O M e by t r a n s p e p t i d a t i o n

100 mg

monocomponent

0.5 ml

10 M a c e t i c acid.

porcine

12°C.

10

added to

mg

porcine

the

nr. 8 0 0 1 0 4 ,

proteins were

The

solutions

21.72

atom

precipitated

and d r i e d

_in v a c u o . HI-OMe.

B.

10

17

%

the

(Novo, M

were

i s o l a t e d by c e n t r i f u g a t i o n , s i o n into

p r o c e e d e d as which was

a c e t i c acid H2170.

and

the

acid with

addition

of

by

HPLC

M

and

hours 20

A,

showed

for

the

aqueous

solutions,

calcium

acetate

H P L C a n a l y s i s s h o w e d 96%

HI-OMe.

100 The

acetone,

of

mg

acetone conver-

of

DAI

preparation

reaction i.e.

were

M

12°C, the

of

92%

0.05

(Ventron

20 ml of

in H ? 1 7 0 .

except

the

at

ml

was

in ice and

H2^0

10 M a c e t i c acid.

under The

0.05

in

cooled

crystallized)

acetic

24

was

chilled

w a s h e d twice w i t h

in 0.5 ml

hours.

x

A^ter

Analysis

described

two

2

prepared

0).

by

mixture

acetate,

P r e p a r a t i o n of H I - O M e by c o u p l i n g

(3) w a s d i s s o l v e d

dissolved

to

and

trypsin

mixture.

was

added

in 0.2 ml 0.05 M c a l c i u m

calcium acetate

(Novo)

in

N,N-dimethylacetamide was dissolved

insulin

1.3 ml of 1.64 M T h r - O M e d i s s o l v e d

made

the up

time 10

M

with

390 P r e p a r a t i o n of D A I ( L y s 8 2 9 - 1 7 0 ) . s i m i l a r to

DAI 17

21.72 atom %

(3),

except

DAI(Lys829-170)

that

(Ventron

100 mg

of D A I ( L y s

acid.

The

829

-

17

0)

preparation

reaction

was

no.

800104,

b u f f e r and

Positive

was dissolved

proceeded

time

prepared for

A.

P r e p a r a t i o n of H I - O M e ( L y s 8 2 9 - 1 7 0 ) .

that the

0

was

0 ) w a s used for the 0.1 M N H 4 H C 0 3

d i s s o l u t i o n of c a r b o x y p e p t i d a s e C.

H2

17

in 0.5 ml

as d e s c r i b e d

2 hours,

control.

and

the

10 M

under

acetic

A,

aqueous

except

solutions

w e r e m a d e up in c o m m o n , d i s t i l l e d w a t e r .

A n a l y s i s by H P L C s h o w -

ed that

to the e x t e n t of

coupling

to H I - O M e

had

occurred

1 7 0 - N M R of H I - O M e p r e p a r a t i o n s . p r e p a r a t i o n s of HC1.

The

HI-OMe

solutions

were

Approximately

dissolved

the

ml

0.06

N

1.7-1.9,

The

in

2.5

3

prepara-

activity.

values

to

the

range

its

pH

2

of

w e l l b e l o w pH 3 w h e r e the t r y p s i n , still p r e s e n t in the t i o n s , loses

acquired

in

50 mg

96%.

concentrations

of

insulin

in

the s o l u t i o n s w e r e d e t e r m i n e d by amino acid a n a l y s i s , using

the

d a t a for p h e n y l a l a n i n e , of w h i c h there are 3 in b o t h insulin

and

trypsin.

are

T h e c o r r e c t i o n s due to the c o n t e n t of 10% t r y p s i n

then -(molecular 10%= - 6 0 0 0 / 2 4 0 0 0 in the

weight x

insulin/molecular

10%=

experiments

-2.5%.

A,

B

The

and

C

weight

trypsin)

concentrations

were

3.7,

3.5

of

and

x

insulin 2.6

mM,

respectively. T h e 1 7 0 s p e c t r a w e r e o b t a i n e d using a B r u k e r H X 2 7 0 with a

resonance

frequency

for

w i d t h of 36 kHz c o r r e s p o n d i n g of 30

usee c o r r e s p o n d i n g

points were

collected,

instrument was

^70

of

36.6

to 2 5 ° p u l s e s w e r e

the use

of

the s p e c t r a

natural

quadrature marked

"ghost s i g n a l "

is

abundance detection,

with 1.2%

water

x of

in

a

Fig.

that

of

used

and 16 K d a t a sample.

checked

The

on a

water refe-

"ghost the

width

The s p e c t r a are

signal

2.

spectral

Pulse

for e a c h

and the p e r f o r m a n c e

a c e t o n e s a m p l e p r i o r to the e x p e r i m e n t s . r e n c e d to the

A

to 975 p p m w a s used.

in ~ 1 0 0 , 0 0 0 scans

adjusted

spectrometer

MHz.

present.

signal"

T h e peak parent

Due

appears

height water

of

to in the

signal.

391 Results and Discussion The

17

0-NMR

in Fig.

spectra of the 3 preparations A, B and C are

2.

Only

signal arising

in the positive

from HI-OMe

200-500 ppm downfield In experiment would lead

incorporation

dance during

steps

increase of

^ ^O

The absence

of

the dominance III + I.

of

I+VI.

the B

is

one

an ^ range,

the

in

24

test

h

in 20%

period

HI-OMe

could

of

in

for

a

further place.

HI-OMe

mechanism,

the

abun-

take

ratio

support I+III and

of

IV+VI as compared to that of reaction

Provided

C

intermediate

i n Lys 29

amounts a

as an B

transpeptidation

belled DAI is dissolved in ^ 0 4.5.

experiment

in the expected

free DAI

During

detectable

Experiment

of reaction

of

incorporation

of

seen

from the water signal.

A, the existence

to

control

can be

shown

the

IV+I.

Unla-

enriched water at a "pH" of about

that the reaction VI + I proceed

fast

in compari-

son to exchange of oxygen between water and the carboxyl B

of Lys 29 f

the test

(7) that oxygen detectable

^0

is valid.

exchange

negligible under

the

a rate

orders

of

It appears

between

present

signal

oxygen in experiment

in B

from

carboxylic

the

expected

magnitude

The

range

reactions

larger

the

than

literature

for

absence

and

III+I.

Difference

related to

the

water

bond with

of

reactions

mechanism.

in

is substantiated. insulin

overall

difference

in

The

between

synthesis

of

and DAI both pass through

reaction rates

a

peptide

Furthermore, the assumption of lack of oxygen exchange from porcine

of

IV+I proceed that

IV+VI, thus supporting the transpeptidation carboxyl groups

group

acids and water is

conditions.

shows that

HI-OMe starting

rate

of

rates

is

acyl-enzyme

therefore formation,

i_.e. I+III for transpeptidation and VI+III for coupling. The reaction

rates

markedly, TN

(transpeptidation)=0.12 , TN

In Fig. states.

1, II and

expressed V

represent

the

kcal/mole.

number TN

differ

(coupling)=8, at 12°C

rate determining

Experiments aimed at determining

tion for the transpeptidation E a =12±2

by the turnover

transition

the energy of activa-

and coupling

reactions both

gave

392

H

600 Fig.

2

^O-NMR C.

500

400

300

200

s p e c t r a of the H I - O M e

100

0

ppm

of e x p e r i m e n t s A , B and

O n l y the p o s i t i v e c o n t r o l e x p e r i m e n t C r e s u l t s in a

band attributable

to

d o w n f i e l d from the H2

HI-OMe(LysB29-1 70) , 17

0 signal.

200-500

ppm

393 H e n c e the

observed

difference t e r m s of

in

Eyrings

by d i f f e r e n t _i.e.

of

the

transmission where

in

TN

values

transition

transition

~ 65'Kji,

k y

difference

energy

state

theory,

coefficients

not II

it m a y

(3

by IR, NMR

— C(CHj)j

by IR, NMR

O

-NH-CHCONHCHCO-CH,CH,OH 1 1 R' R"

analogy

422

The method elaborated can be applied to the study of the metabolism and enzymatic degradation of peptide hormones. The samples need pre-purification on SEP-PAK cartridges (Waters) with 40-80% recovery. Recently OPA-peptides separated have been used for MS peptide sequencing, too (10). The application of pre-column derivatization is growing in peptide-analysis.

Chromotagram «f »-phthalalrfohydo dorivativoi of a-MSH fumanti Column ODS HYPIRSIL-Sum Ili I 4mm Flow rata 1.4 ml/min Prouuro : 70 boi Salvonl : AtH-HjO-lFA i : IS »S : 0.1 B ' 90 : 10 : 0.1 Gradient : 1.2 *0 B/min UV profit Dotoction : W ot to .r,. H 20fluori, mo Pool«»: 1. 0 1 pi H-aOH -io-frosoni k' 6 5 2. 0.2fig H-1-NH, 1-13 »2

References

i (min)

Wilkinson, J.M.: J.Chrom.Sei. 16, 547 (1978) Hsu, K.T., Currie, B.L.: J.Chromatogr. 166, 555 (1978) Szokän, Gy., Gyenes, M., Tyihäk, E., Szende, B.: Proc. of 17th European Peptide Symposium (1982) McHugh, W., et al.: J.Chromatogr. 124, 376 (1976) Gruber, K., Stein, S., Brink, L., Radhakrishnan, A. Udenfriend, S. Proc.Nat.Acad.Sci. USA 73, 1314 (1976) Martin, W.R.: J.Chromatogr. 202, Wu, K.M., Sloan, J.W 500 (1980) Lindroth, P., Mopper, K.: Anal.Chem. 51,(11) 16 6 7 (19 79) Hodgin, J.C.: J.Liquid Chrom. 2,

1047 (1979)

Simmons, S.S. , Johnson, D.F.: J.Chem.Soc.Chem.Comm..11, 374 (1977) 10.

Larsen, B.R.: Life Sci. 30, 1007 (1982)

F A S T ATOM BOMBARDMENT MASS S P E C T R O M E T R Y A NEW T O O L FOR P E P T I D E

SEQUENCE

-

ANALYSIS

Wilfried A. König, Mitat Aydin, Uwe Schulze, Marian Rinken Institut für Organische Chemie und Biochemie der Universität Hamburg D-2000 Hamburg (F.R.G.)

Introduction Mass spectrometry has been used in peptide analysis for many years, particularly for structure elucidation of biologically active peptides. Conventional electron impact or chemical ionization techniques afford evaporation of the sample. For peptides this is only possible after conversion to volatile derivatives in a series of microchemical reactions. The use of diborane for reduction of acetylated peptides to polyamino alcohols has proved to be a valuable approach (1, 2, 3). Complete sequence and molecular weight information is available from these peptide derivatives upto a chain length of 10 to 12 amino acids (fig. 1). In 1981 a very promising new ionization technique, {¡ait

atom

bomban.drmnt

(FAB)

(4, 5), has been

sug-

gested for the analysis of high molecular, polar natural compounds. Sample molecules are dissolved in a polar, low volatile solvent, for example glycerol, and bombarded with highenergy argon or xenon atoms on a metal target in the ion source of a mass spectrometer. The molecules are ionized and evaporate spontaneously at ambient temperature.

Results Since peptides as polyfunctional compounds possess acidic and basic functions, they may form cations by proton addition or anions by proton abstraction. Therefore the spectra of positive

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

424

as well as negative ions may be recorded. rrn

pro

pro

,976

phe

, 893

\Z'

j 12

HjC-CHj^nn-^n-l,^'

phs

,610

\ **

pro

6 77

5ti

Zi

ZS

293

a

ch2

319 I

CHj

636 A7

AJ

6«« »5

S3S ?36,

i

ch3

/\ i

¿02,

A2

, CH«—I i2-HN-CHtCH2-HN-CH7CH2-otms

CH |

HjC OTMS

1236 \z9

! Oi C H , - H N - C H +t CCHH,, - N — H - C H j - N —

ij-K—vCH,-HN-CH' I CH

CHj

PRO

?H2

919. 9761 A9

7S3,

1109', aio

H

A, A2 319

a,.2, 236

*3 (02

I I * *' l u T . U u T [ „ h I V 5 , , , , i .,

-

«7 :929

(z7>

l»6> »6 :773

"5> »5 :674 758

:I0!9

t»3>

«4» (Zs>

(zt>

659

>96

h2n '• CH - C

«3 ;396

(Z3)

CH \ CHj CH3

C-0

: CH ch2 ch3 ch3

OH

366

(C,)

-p-aminophenyla.la-

|ji-p—nitrophenylalani-

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

468 neQdeamino-6-carba-oxytocin (VXIl) were prepared at the Prague Institute (2). ¡j2-[^H]Tyrosine-8-lysine] vasopressin (•'h-LVP) was prepared and purified according to (^,5). The binding of analogues to liver membranes (6) was followed by determining the ability of the individual analogues to compete for binding sites with o H-LVP (7)« The method for measuring the binding of analogues to the membrane fraction of the rat kidney medulla, and the activation of adenylate cyclase was the same as described in papers (8) and (•?)•

Results In a number of oxytocin analogues, deamina.tion and carba-substitution of the bridge leads to an increase of the natriuretic effect. The modification of the p-position of tyrosine resulted not only in an absolute change of the natriuretic effect but also affected other biological, responses. As can be seen in Table I, the modifications performed in the p-position of a. series of analogues of deamino-6-carba.-oxytocin resulted in a decrease of the affinity to the liver receptor system. Although the decrease in affinity was not dramatic at the most by 1.5 order as compared with oxytocin - the com3 pounds were found to be only weak antagonists of H-LVP binding (pKQ = 8.33) in. this system. In accordance with these data., the pressor action of the analogues was also lower than that of oxytocin ( 2 ) . The character of the dependence of the binding and the adenylate cyclase activation on the molecular structure is somewhat different in the renal system. The substitution of the hydroxyl group by methyl or ethyl groups increased the affinity and activation ability of both compounds (ill, IV), whereas the other modification decreased the affinity ten times and the activation ability in some cases even more (VI and VIII). The anti-

469 Table X Characteristics of binding and activation constants of oxytocin analogues Compound

Liver membranes

PKD I II III IV V VI VII VIII a

7.11 7.71 6.48 6.25 5.37 5.86 5.36 5.86

_

_

Renal system Binding

Adenylate cyclase

Na triuresis

D

PKD

1"

7.47 7.^9 7.80 7.60 6.28 6.39 6.62 6.57

7.21 7.^3 7.54 7.48 6.72 5.70 6.72 5.96

iooa 298 326 255 131 31 87 66

PK

The natriuretic potency of oxytocin was taken as a. basis

for calculations.

diuretic potency is in agreement

with the tendency of chan-

ges of the activation constants for the adenylate cyclase system; compounds III and IV have potencies in the range of tens of antidiuretic units per nig, the other compounds have a lower activity than that of oxytocin. As can be seen from the results presented in Table I, compounds II-IV have 2.5-3 times higher natriuretic potency than oxytocin (i), the others have 30-130% of the activity of oxytocin. In most of the structural modifications performed the lipophilic properties of the analogue molecule were enhanced; this effect was most pronounced in the case of compounds II—IV. Although these changes result in higher affinity to renal receptors and more pronounced activation of adenylate cyclase (compare the p K n and pK. values for oxytocin and compounds II and IV and

470

their natriuretic uretic

potency

(2)

action), of

the higher n a t r i u r e t i c

a b o u t by a l t e r e d d i s t r i b u t i o n and e l i m i n a t i o n p r o l o n g a t i o n of

the i n d i v i d u a l

responses,

c r e a s e d a f f i n i t y and by t h e a c t i v a t i o n in

and

t h e s e compounds i s a p p a r e n t l y

of

leading

rather

antidi-

brought to

the

than by

in-

adenylate

cycla.se

kidneys.

References 1.

ä k o p k o v a , J . , H r b a s , P . , B a r t h , T . f L e b l , M., J o s t , K . j Hormonal R e g u l a t i o n o f Sodium E x c r e t i o n ( L i c h a r d u s , B. , S c h r i e r , R.W., Ponec, J . , E d s ) , p. 103-168. E l s e v i e r , Amsterdam 1980.

2.

L e b l , M. , H r b a s , P . , A . , Barth, T . , Jost, in press.

3.

J o s t , IC., äorm, F . : 234-245 ( 1 9 7 1 ) .

4.

Pradelles, Cohen. P . , (1972).

5.

C a m i e r , M. , A l a z a r d , J . L . , Fromageot, P.s

6.

Neville,

7.

Cantau, B . , K e p p e n s , S . , de W u l f , t o r R e s . 1, 1 3 7 - 1 6 8 ( 1 9 8 0 ) .

8.

R a j e r i s o n , R.M., M a r c h e t t i , J . , Roy, C . , B o c k a e r t , J a r d , S . : J . B i o l . Chem. 249, 6390-6400 ( 1 9 7 4 ) .

9.

Butlen, D., G u i l l o n , G., R a j e r i s o n , R.M., Jard, S . , S a w y e r , W . H . , Manning, M . : M o l . P h a r m a c o l . 14, 1006-1017 (1978).

ä k o p k o v a , J . , S l a n i n o v a , J . , Ma.ch.ova, K. : C o l l e c t , C z e c h . Chem. Commun., Collect.

Czech.

Chem. Commun.

36,

P . , Morga.t, J . L . , F r o m a g e o t , P . , Bonne, D. , B o c k a e r t , J . , J a r d , S . : FEBS L e t t . 26, 189-192 ~~ R. , Cohen, P . , P r a d e l l e s , P . , Morga.t, E u r . J . B i o c h e m . ¿ 2 , 207-214 ( 1 9 7 3 ) .

D.M.: Biochim.

Biophys.

Acta. 154, H.,

Jard,

540-552 S.:

J.

(1968). RecepJ.,

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW ENKEPHALIN ANALOGUES

Ryszard Paruszewski, Roza Matusiak, Elwira Gmitrzuk, Witold Gumulka and Piotr Janicki Dep. of Pharmaceutical Chemistry and Dep. of Pharmacodynamic Medical Academy, 02-097 Warsaw, Poland

Introduction The analogues of enkephalin containing 6-aminohexanoic acid, which molecule is flexible and its length in extended conformation is about 5-1 S, were synthesized to study influence of substitution by this amino acid on the biological activity of peptides. Activities in vitro in the guinea-pig ileum /GPI//1/, mouse vas deferens /MVD//2/, rat vas deferens /RVD//3/ and in vivo analgesic activities /4,5/ were examined. Also resistance of the obtained analogues to proteolytic degradation in vitro by rat brain supernatant was investigated. The analogues synthesized were: H-Tyr-t-Ahx-Phe-Leu-OH /l/, its amide /il/ and ester /ill/, H-Tyr-Gly-Gly-Phe-£-Ahx-OH /iv/, its amide /V/ and ester /VI/, H-Tyr-D-Met-Gly-Phe-£-Ahx-OH All/, its amide /VIII/ and ester /IX/, H-Tyr-£-Ahx-Phe-£-Ahx-OH /X/, its amide A l / and ester /XII/. Synthesis Peptides esters were synthesized by DCC/HBT solution method /6/. Peptides amides were obtained by ammonolysis and free

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

472

peptides by alkaline hydrolysis of peptides esters. All peptides were purified by ion exchange and by partition column chromatography /7/.

Table 1. Analytical data of the analogues 5T5 Compd A/jj DMF

Amino acid ratio ¿-Ahx

Gly D-Met

Leu

TLC, R f

PB:400 V 2h

Tyr BAW BPW * E Phe 4:1 : 65: ' 5 35s pH= pH=

Phe

65

1 . 8 8.6

I

-23.3

0,87

-

-

1.06 1.11 0.95 0.60 0,70 211

II

-25.0

1.13

-

-

1.05 1.05 0.78 0.41 0.72 222 1933

III

-20.8

1.03

-

-

1.06 1.08 0.82 0.54 0.75 178 2200

IV

-26.7

0.97 1.92

-

-

1,07 1,05 0.55 0,59 311

V

-21,4

1.05 1.87

-

-

1.03 1,05 0,42 0.57 198 2133

VI

-26,0

1.19 1,69

-

-

1.07 0,94 0.56 0,67 188 1933

VII

-30.9

0.95 0.98

0.97

-

1,10 0.99 0,49 0.64

71

133

VIII -27,6

1.08 1.05

0.92

-

1.11 0,95 0.50 0.63

81

125

IX

-28.7

0.95 1,09

0,98

-

1-08 0.92 0.60 0.72

92

100

X

-12.5

1.73

-

-

-

1.21 1.06 0.53 0,52

95 -163

XI

- 7.9

2.01

-

-

-

1,07 0.92 0.54 0.54

111 -200

XII

-10.5

1,90

_

_

—

1.16 1.05 0.56 0.66

92 -150

0

0

Table 2. Degradation of the analogues by rat brain supernatant Compd

Percent, of amino acid rel. after incubation /20',37°C/ ¿-Ahx

Gly

D-Met

Leu,Gly-Gly

Phe

Tyr

I

0

-

-

0

0

8

IV

0

5

-

0

0

20

VII

0

0

0

-

0

0

X

0

-

-

-

0

2

_

7

_

30

32

Leu-E

46

473

Table 3. Biological activity of the analogues Compound

Agonist potency

Analg.activ., hot pi.test

IC 50 /M/

IC5 0 / V

IC 50 /M/

GPI

MVD

RVD

n=3-5

n=3-5

n=3-5

ED50/L'lAg/ Rel.potency i.e.v.,mice i.p.,rats n=6

2x10~ 6 MAg n=6

Morphine

x10 Dalamid

4.7^.3

9*2-1.8 3

phin I II III

10"

8

V

x10"

9

7

10"

10-

-4 >10 *

>10~3

4

3

>10~3 -4 >10 4

>10"

>10~

6

>10-

x10~

>10~3

0,35

3

0.40

5

0.58

>10" 4x10~

x10~

>10~3

1.3-0.5

8

x10"

6,8il.8

>10-

-

5

-8

4.5io.6

6

-

7

-

4.2il,1

8.2-1.2

6.7io.8 _7 x10 '

x10"

x10~

7

1.00

7

0.39i0.15

5

x10~

7

x10 IV

-8

11,8i2-8 1.oio,2

9.Oil.2 x1 0"

^-Endor-

x10

0.4i0.1

-

>10

-3

0.65

4

>10"3

0.57

1.6io.4

1.13

x10 ' VI

x10" VII

IX X XI XII

8

x10

5

3

x10~

4.oio.8

12.4^3•2 1.5^0.3

9.oio,4 x10"

VIII

>10~3

5•45—1,2 6.8io.9

8

x10

3

6

x10 '

x10~

8

7

5

ND

ND

-8

7

5

ND

ND

>10~3

ND

ND

3

ND

ND

3

ND

ND

1.1x10~ 2.3x10~ 3.0x10" 2,2x10

0.73

>10~3

5.6x10~ 3.0x10~ >10"3 6

3

8.5x10~

>10~

6

1,9x10"

-5

3.0x10

>10" >10~

474

Conelusions

1.

A l l of the synthesized analogues of enkephalin w i t h 6 - a m i nohexanoic acid / c - A h x / in positions 2 - 3 , show morphine-like

2.

and/or 5

activities.

The highest activity on theJH. receptors

is shown by

peptides VI-IX , on the S and £ receptors by the peptide

VII.

The highest analgesic activity is shown by peptides VI,VII. 3.

Activity

of peptide XI on the £ receptors is at least

sand times higher t h a n activity on they«- and £ 4.

thou-

receptors.

Substitution of £ - A h x in position 2-3 or 5 protects the binding of Phe and substitution of £ - A h x in position 2-3 p r o the

binding of T y r against in vitro proteolytic

tion by rat brain

degrada-

supernatant.

References

1. 2. 3.

Kosterlitz, H.'.V., V/aterfield, A . A . : B r i t . J . P h a r m a c o l . 53, 1 31-138 / 1 9 7 5 / . Hutchinson, IJ., Kosterlitz, H.'J., Leslie, P.M., '.'/aterfield, A . A . : Brit. J . Pharmacol. 55., 541-546 IT Schulz, R., Paase, E., V/uster, Ivl., Herz, A . : Life 24, 8 4 3 - 8 5 0 / 1 9 7 9 / .

/1975/. Sci.

4.

Ueda, H., A m a n o , H., Shiomi, H., Takagi, H.: E u r . J . P h a r m a c o l . 56, 265-268 /1979/.

5.

Tyers, LI.B.j Brit. J . Pharmacol. 69, 503-512 / 1 9 8 0 / . ti Konig, ,/., Geiger, R . : Chem. Ber. KV5, 788-798 / 1 9 7 0 / .

6. 7.

Paruszewski,R., i.iatusiak, R., Gumulka, ',/., Janicki, P . : P o l i s h Patent Applications P-233416, P-233417, P - 2 3 3 4 1 8 /1981/.

BIOCHEMICAL AND BRAIN

AND PHARMACOLOGICAL

¿"OPIATE

A NEW FULLY

RECEPTORS

SPECIFIC

INVESTIGATIONS

USING

ON PERIPHERAL

TYR-D-THR-GLY"PHE"LEU-THR,

6-LIGAND.

Bernard P. Roques, G i l l e s Gacel, P i e r r e Dodey, Jean-Marie Zajac, MarieClaude Fournié-Zaluski Département Biologiques,

de Chimie 4 avenue

Organique, VER des de l'Observatoire,

Sciences Pharmaceutiques 75270 Paris cedex 06,

et France.

Jean-Louis Morgat Service

Biochimie,

CEN Saclay,

91190

Gif

Sur

Yvette,

France.

Jean Costentin, Hélène Marçais-Collado and P i e r r e Chai 1 l e t . Laboratoire du Rouvray,

de Pharmacodynamie, France.

Université

de Rouen,

76800

Saint

Etienne

Introduction

The m u l t i p l e pharmacological

responses e l i c i t e d by morphine administration

could be r e l a t e d to heterogenous receptors s t i m u l a t i o n .

Indeed, from phar-

macological and binding experiments three kinds of binding s i t e s were well characterized i n the CNS (1) : i ) y-receptors i n t e r a c t i n g with morphine and surrogates, i i )

preferentially

6-receptors e x h i b i t i n g a highest

f o r enkephalins and peptide s t r u c t u r e s , i i i )

affinity

K-receptors corresponding to

ethylketocyclazocine and dynorphin b i n d i n g - s i t e s . These r e s u l t s are supported by a large number of d i f f e r e n t studies (rev i n 2). However i t

remains

to demonstrate : i ) the s p e c i f i c pharmacological responses r e l a t e d to a s i n g l e receptor a c t i v a t i o n , d i s t i n c t proteins,

iii)

ii)

the l o c a l i z a t i o n of each binding s i t e on

the p o s s i b l e occurrence of a l l o s t e r i c a l l y

coupled

u and 6 receptors accounting f o r p o t e n t i a t i o n of morphine analgesia by Leu-E (3). Such i n v e s t i g a t i o n s e n t a i l i n g ligands of high a f f i n i t y and complete s p e c i f i c i t y , were done using a new f u l l y s e l e c t i v e

6-agonist.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

476 Materials and Methods Tyr-D-Thr-Gly-Phe-Leu-Thr (DTTLE) was prepared by liquid phase method as described for DSTLE (4). Binding and pharmacological assays were performed as described (4, 5). Analgesic experiments (hot plate test) were done on mice (9). Bestatin is a gift of Roger-Bellon laboratories.

Results and discussion As proposed (6), changes in Leu-E sequence allowing enhanced 6-receptor recognition are : i) an hydrophilic D-aminoacid in 2 decreasing p interaction, ii) a peptide-lengthening with hydrophilic residues leading to a more extended conformation . Using these features, Tyr-D-Ser-Gly-Phe-LeuThr (DSTLE) was recently proposed (5) as a much better 6-ligand than the 2 5 commonly used D-Ala -D-Leu -E. In order to still decrease the cross-reac2 2 tivity of DSTLE (Table I) we replaced D-Ser by D-Thr in order to enhance receptor-affinity without change in 6-selectivity. Tyr-D-Thr-Gly-Phe-LeuThr (DTTLE) was synthesized by liquid phase method as schematized below (11),

TYR

D-THR

GLY

PHE B o c _ L OH Boc_

Boc_

OH

0CH 3 . 0CH 3 OH

OH

H_

LEU H J OCHOCH. OCH-

Boc.

OCH,

BOC_

OH

BOC.

THR

H

OBzI OBzI OBzI OBzI OH

477 y or & selectivity on peripheral

and brain receptors. As shown in table 1,

the proposed changes in Leu-E sequence lead to a large decrease in IC^q on MVD (rich in ô-receptors) with a concomittant increase in IC^q on GPI (yreceptors). So as evidenced by the ratio of IC 5 Q on both organs, DTTLE is 3000 more potent on peripheral 6 than on y-receptors. Table I. Inhibitory effects of morphine and opioid peptides on the electrically induced contractions of guinea-pig ileum and mouse vas defe3 rens and on the binding in rat brain homogenates of H DAGO (1 nM) and 3 H DSTLE (2 nM).

I C , . (GPI) nH

TYR-GLY-GLY-PHE-LEU (LEU-E)

I C . . (MVD) nK

I C , (GPI) —-I C , - (MVD)

393.

6.2

48.

«8.

0.55

88.bo

,

K. (nB) , "

H Df-"

2.40

0.31

TYR-D-SER-GLY-PHE-LEU-THR

(DSTLE)

406.

0.40

1015.

31.

4.80

0.15

TYR-D-THR-GLY-PHE-LEU-THR

(DITLE)

460.

0.15

3067.

25.30

1.35

0.0s

TYR-D-ALA-GLY-PHE-D-LEU

(DADLE)

lTYR-D-ALA-GLY-PKE-NH-(CH2)6-]2 TYR-D-ALA-GLY-KEPHE-GLY-OL

(DAGO)

" 11.5

. 76.1

.

43.

0.15

The IC^Q values are the mean of 3-5 observations.

7.70

3.9

Methionine

11.50 1300.

0.23 333.

enkephalin

was

used in each assay as internal standard. The Kj values are the mean of three determinations.

Each Kj values was calculated from Hill plots with 9 concen-

trations of unlabelled

ligand.

As expected the same pharmacological profile occurs on rat brain membranes, 3 since from displacement experiments of H DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) (7) and 3 H DSTLE, the cross-reactivity falls from 30% for DADLE to 5% for DTTLE. Therefore, DTTLE behaves at this time as the more specific 6-probe, largely better than the recently claimed (8) "extraordinarily 6-selective dimeric-enkephalins" which were not studied on a homogenous preparation as -5 required. Moreover DTTLE is poorly recognized by K-receptors (KT ~ 3 x 10 M 3 from H ethylketocyclazocine displacement).

Binding experiments

of

3 H DTTLE on brain tissue. According to the high selec-

tivity of DTTLE, this peptide was tritiated by exchange from its dibromo-Tyr precursor, synthesized through the same scheme as DTTLE itself. As expected,

478

Figure 1. 3 on crude rat brain membranes,

H DTTLE (35 Ci/mmole) binds to a single

classe of sites (6-receptors) with a high affinity, K^ = 1.45 nM and B

max

=

° ' 1 0 5 P m o l e / m 9 (Fi9-

Analgesic

activities

of y and 6 selective

ligands.

The very high difference

in y-recognition of DAGO (Kj = 3.9 nM) and DSTLE or DTTLE (Kj = 31 and 25 nM) allowed for the first time to evidence a significant correlation between y-affinity and analgesia since,measured on hot plate test in mice after i.c.v. administration, the ED 5 Q of DAGO = 0.5 nM is more than 50 times lower than those of DSTLE, 100 nM, and DTTLE, 25 nM. Obviously, these effects are naloxone-reversible evidencing for all compounds a y-receptor activation.

Studies

on y and 6 receptors

coupling and enkephalins

autoreceptors.

As

shown in table 2, subanalgesic doses of DTTLE do not potentiate the analgesic effect elicited by DAGO or morphine. These results seem to exclude an allosteric coupling of y and 6 receptors at least for antinociceptive regulation. Finally, as previously shown ( 9 ) , the analgesic effects induced by endogenous enkephalins can be studied following the protection of these natural peptides from enzyme degradation by Thiorphan

(enkephalinase

inhibitor (10) and Bestatin (aminopeptidase inhibitor). So, using these two

inhibitors,a strong analgesia is obtained (Table 2). Moreover, i.c.v.

479 Table 2. Analgesic effects in mice (hot-plate test) of simultaneous i.e.v. administration of DTTLE, DAGO, Bestatin (B) and Thiorphan (T).

Treatment (n=10)

Jump latency time (s)

Sai ine

59 ± 6

DTTLE (1 ng)*

63 ± 4

DAGO (1 ng)

151 ± 19

DTTLE (1 ng) + DAGO (1 ng)

140

DAGO (0.01 ng)*

*

14

66 ± 7

B (50 ng) + T (10 ng)

157 i 15

B (50 ng) + T (10 ng) + DTTLE (1 ng)

154 ± 18

B (50 ng) + T (10 ng) + DAGO (0.01 ng)

150 ± 18

p < O.OOl ;

A

*

subanalgesia

*

dose.

injection of subanalgesic doses of DAGO or DTTLE does not change the previous analgesic response indicating that enkephalins level is not significant modified in the synaptic cleft suggesting lack of autoreceptors of y or 6 types.

Acknowledgments. ille are indebted to Annick Bouju for typing

the manuscript. This work was

supported by grants from Rhône-Poulenc S.A. (Doctor J.M. Zajac is a research fellow from R.P.), the Université René Descartes, The Ministère de le Recherche et de la Technologie and the Fondation pour la Recherche Médicale Française.

480

References 1.

Kosterl i t z , H.W., Lord, J . A . H . , Paterson, S . J . , Waterfield, A.A. : Br. J . Pharmacol., 68, 333-342 (1980). 2. Roques, B.P., Fournie-Zaluski, M.C., Gacel, G., David, M., Meunier, J . C . , Maigret, B., Morgat, J . L . : in Regulatory Peptides from Molecular Biology to Function, E. Costa and M. Trabucchi, Eds, Raven Press New York, pp 321-331 (1982). 3. Vaught, J . L . , Rothman, R.B., Westfall, T.C. : Life Sei. 30, 1443-1455 (1982). 4. Gacel, G., Fournie-Zaluski, M.C., Roques, B.P. : F.E.B.S. Lett. 118, 245-247 (1980). 5. David, M., Moisand, C., Meunier, J . C . , Morgat, J . L . , Gacel, G., Roques, B.P. : Eur. J . Pharmacol. 78, 385-387 (1982). 6. Fournie-Zaluski, M.C., Gacel, G., Maigret, B., Premilat, S . , Roques, B.P. : Mol. Pharmacol. 20, 484-491 (1981). 7. Handa, B.K., Lane, A.C., Lord, J . A . H . , Morgan, B.A., Ranee, M.J., Smith, C.F.C. : Eur. J . Pharmacol. 70, 531-540 (1981). 8. Shimohigashi, Y . , Costa, T . , Chen, H., Rodbard, D. : Nature (London) 297, 33-335 (1982). 9. Fournie-Zaluski, M.C., Chaillet, P., Soroca-Lucas, E . , Margais-Collado, H., Costentin, J . , Roques, B.P. : J . Med. Chem. in press. 10. Roques, B.P., Fournie-Zaluski, M.C., Soroca, E . , Lecomte, J.M., Malfroy, B., Llorens, C., Schwartz, J . C . : Nature 288, 286-288 (1980). 11. Patent 80.17,523, August 8, 1980.

DOUBLE OPIATE PEPTIDES. A HYPOTHESIS OF TWO DIFFERENT MECHANISMS OF OPIATE ACTIONS

Andrzej W. Lipkowski and Miroslawa Konopica Laboratory of Peptides, Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland Beata Osipiak and Witold S. Gumulka Department of Pharmacodynamics, Warsaw Medical Academy 00-927 Warsaw, Poland

Introduction Since the isolation from the brain of endogenous opiate like peptides, Met- and Leu-enkephalin (l), numerous analogs of these substances have been synthesized. It is now apparent that 2-substituted analogs retain their analgesic activity with a variety of substitution or even deletion (2) at position 5 suggesting that this position might not be obligatory for opiate receptors affinity. The conclusion that a necessary element for opiate receptors interactions are elements from Tyr to Phe was a basic assumption for synthesis of an analog in which two of these elements were connected by hydrazide bridge, giving analog with high analgesic activity (3)» In 1979 Henschen et al. (k) isolated heptapeptide fragment of /3-casein with some opiate-like effect in G.P.I.

This hepta-

peptide, /3-casomorphin , shows no effect in the M.V.D. assay. In 1980 Erspamer group ( 5 ) isolated peptide with opiate-like activities from the amphibian skin. Both these peptides and enkephalins have almost completely different sequences. N-Terrainal Tyr and closely located Phe are the only two common residues. The consequent conclusion which could be drawn is that necessary elements interacting with opiate receptors

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

482 in the case of /3-casomorphin and dermorphin are N-terminal tripeptides and a D-amino acid or proline residues in those peptide replace dipeptide -Gly-Gly- which is present in enkephalins» This hypothesis is confirmed also by: (i) The synthesis of morphinoceptin with high specifity

toyu-receptors

which is reduced to tetra.peptide amide fragment of /3-casomorphin (6). (ii) The synthesis of tetrapeptide analogs of enkephalin (7) in which the removal of -Gly- in position 3 from enkephalin analogs, giving general formula Tyr-aa-Phe-Met-NHg (where aa - D-amino acid) preserves affinity to ^x-receptors. A fall of activity in the case of /3-casomorphin where sequence have been reduced to N-terminal

tripeptide

could be a. result of the peptide reducing into a, molecule too small for interacting with receptors or/and with transport biosystems. To check this suggestion we have synthesized analogs in which two necessary elements of dermorphine, /3-casomorphin , enkephalin and their analogs are connected by a hydrazide bridge, giving general formula

where —I

Tyr—{

I— Phe-NH I

Tyr-f

I—Phe-NH

h- means amino acid or dipeptide.

Materials and Methods Double opiate peptides were synthesized in solution by step by step elongation starting from (Phe-NH-) 2 (3) using DCC+ +HOBt as a coupling reagent. The final peptides were purified by absorption chromatography on silica gel followed by molecular sieve chromatography on Sephadex LH-20. Two of the assays were based on pharmacological responses: The depresión of the electrically - evoked contractions of the guinea-pig ileum (G.P.I.) and of the mouse vas deferens (M.V.D.). Antagonist potencies were characterized by ability

483

of

analogs

to reduce

pharmacologically

effect

stable

on M . V . D .

opiate

a n d on G . P . X .

agonist

of

the

(Tyr-D-Ala-Gly-Phe-

-NH-)2.

Results The p h a r m a c o l o g i c a l are

present

in Table

with glycine residue activity

in

1.

All

ly

as a g o n i s t

in peptide

of

residue

Table

agoof

synthesized double

on G . P . I .

amino a c i d s

I n M.V.D.

test

of

agonistic

antagonistic

but

significant-

all

activities.

activities

peptides

enkephalins

high

tests without low

in position

to

express

-Gly- are

and a n t a g o n i s t

to antagonist

related

chains

and M . V . D .

compounds w i t h o u t

pounds e x p r e s s a g o n i s t tions

of

Double p e p t i d e s

both G . P . I ,

activity. active

activities

analogs

these The

com-

rela-

depend

on

2.

1

Pharmacological

activities

of

Agonist Compound

M-

Tyr-D-Ala-Gly-Phe-Met-NH2

22.8

(Tyr-D-Ala-Phe-NH-)2

600

(Tyr-D-Thr-Phe-NH-)2 (Tyr-D-Thr-Gly-Phe-NH-)2 (Tyr-Pro-Phe-NH-)2

opiate

potencies

G-

IC5Q(nM)

(Tyr-D-Ala-Gly-Phe-NH-)2

double

1.94 1^30 6.40 1^50

Rel.x act."

763 29 8969 12 2718 ,0 12

in

IC„0(nM) V

17.1 6500 11.7 10.2 -

peptides

D

Inhibitory '

e

Rel. act.x

f , f i V ^ xj 0

n

191 0.26

weak

278 weak*1

+

319 , xxx weak

++

P o t e n c y r e l a t i v e t o M e t - e n k e p h a l i n = 100, n I8?b o f t w i t c h i n h i b i t i o n f o r c o n c . 30 . u M ; * * * 10$ o f t w i t c h i n h i b i t i o n f o r c o n c . 3 ° A»M. '

x

484

Discuss ion Ones of the well characterized isolated tissues are M.V.D, containing mainly 6 receptors and G.P.I, containing generally jw receptors. Both morphine like drugs and enkephalin analogs have affinity to both types of receptors. Nevertheless, morphine like drugs are considerably more potent than the enkephalin analogs in the G.P.I, but the opposite is true in the M.V.D. Additionally, opiate peptides generally better displace the bound enkephalin than narcotics but the opposite is true for morphine like thmgs. To elucidate these results the hypothesis have been proposed that narcotics and opiate peptides are interacting -with overlapping, but slightly different portions of the opiate receptors (8). We present a different point of view. The morphine like compounds are not flexible but opiate peptides have very flexible peptide chain. The preservation of a high agonistic activity on "typical" peptide receptors 3 requires of saving a flexible center -Gly -. The replacement of glycine residue in position 3 by D- or L-amlno acid residues (9), or abolishing this residue, and in consequence "freezing" molecules resulted in a dramatic reducing of agonistic activity on ¿receptors, but the agonistic activity on yu-receptors in most of the cases is maintained. On the other hand we found that double opiate peptide analogs composed from "freeze" elements express antagonistic activity on 6 receptors without antagonistic activity onyu receptors. Basing on these results we propose that general difference between 6 and yu receptors are concerning two different mechanisms of interactions with morphine like drugs and peptides. The classical "lock-and-key" mechanism (lO) is a way of interaction of molecules withyu receptors. In this mechanism the active site of the receptor by itself is complementary in shape to that of the morphine structure or some from the packed opiate peptide conformations. The interaction of peptides with 6 receptors is realized rather by "zipper" mechanism in which receptors

485

m a c r o m o l e c u l e s and p e p t i d e m o l e c u l e s action.

T h i s mechanism i s

s t a t e s of

receptor-peptide

les with a b i l i t y for

of

change shapes upon

characterized

binding with a lower

changing c o n f o r m a t i o n s i n p e p t i d e - r e c e p t o r activity.

The r i g i d

phine drugs a r e p r o b a b l y i n t e r a c t i n g by m o d i f i c a t e d

"induced-fit"

c h a n g e s shape upon b i n d i n g shape c o m p l e m e n t a r y substrate i s

to

that of

molecu-

possibility

complex

could

c o n f o r m a t i o n s of

w i t h t h i s kind of

mechanism i n w h i c h

substrate.

inter-

transient

complex. T h e r e f o r e p e p t i d e

receptor

express antagonistic tors

by s e v e r a l

The a c t i v e

the s u b s t r a t e

mor-

recep-

receptor

s i t e has a

only a f t e r

the

bound.

Acknowledgement T h i s w o r k was s u p p o r t e d i n p a r t

by t h e P o l i s h Academy

of

S c i e n c e s w i t h i n t h e P r o j e c t n2 1 0 . 4 .

References 1.

Hughes, J . , A . , Morgan,

Smith, T . t f . , K o s t e r l i t z , H . V . , F o t h e r g i l l , L . B . A . , M o r r i s , H . R . s N a t u r e 258, 577-579 ( 1 9 7 5 ) .

2.

McGregor, V . H . , S t e i n , 1371-1378 ( 1 9 7 8 ) .

3.

Lipkowski, A . V . , Konecka, 3 (1982), i n press.

L. , B e l l u z z i , A.M.,

J.D.:

Sroczynska,

Life

Sei.

I.:

23,

Peptides

Henschen, A . , L o t t s p e i c h , F . , B r a n t l , V . , T e s c h e m a c h e r , H.s Z . P h y s i o l . Chem. 360, 1211-1216 ( 1 9 7 9 ) . 5.

Erspamer, V . , M e l c h i o r r i , P . : i n " G r o w t h Hormone and Other B i o l o g i c a l l y A c t i v e P e p t i d e s " , P e r c i l e , A . , M u l l e r , E . E . , e d s . Exepta M e d i c a , Amsterdam 1980, p p . 1 8 5 - 2 0 0 .

6.

Chang, K . J . , K i l l i a n , A . , Hazum, E . , S c i e n c e 212, 75-77 ( l 9 8 l ) .

7.

C h i p k i n , R . E . , M o r r i s , D . H . , E n g l i s h , M . G . , Rosamond, J . D . , Stammer, C . H . , Y o r k , E . J . , S t e w a r d , J . M . : L i f e S e i . 28, 1517-1522 ( 1 9 8 1 ) .

8.

Lee, N.M.,

Smith,

A.P.s

Life

Sei.

26,

Cuatrecasas,

1459-1464

P.:

(1980).

486 9.

10.

Miller, R.J., Chang, K.J., Cuatrecasas, P., Wilkinson, S., Lowe, L., Beddell, C., Follenfant, R.: in "Centrally Acting Peptides", Hughes, J., ed. University Park Press 1978, pp. 195-213. Burgen, A.S.V., Roberts, G.C.K., Feeney, I.s Nature 253 753-75^ (1976).

SYNTHESIS AND ACTIVITY OP KYOTORPHIN AND ITS ANALOGS Lagos Balaspiri, Kaiman Koväcs, Institute of Medical Chemistry, Medical School of Szeged, Hungary Arpad Geese, Gyula Telegdy, Institute of Pathophysiology, Medical School of Szeged, Hungary Klaus Neubert, Section of Biosciences, Martin Luther University, Halle-Wittenberg, German Democratic Republic

Introduction In 1979, Takagi et al. [ l ] isolated and identified a morphine-like dipeptide from bovine brain by means of an in vivo assay utilized for evaluating analgesic activity The new analgesic dipeptide Tyr-Arg was named kyotorphin meaning an endorphin-like substance which was discovered in Kyoto. Using the sensitive analgesic bioassay method this novel opioid dipeptide administered intracisternally to mice has the analgesic effect /ED^Q 34.5 nmol per animal/ which is about 60 times less than that of morphine /ED^Q 0.61 nmol per animal/ but is about 4.2 times higher than that of Met-enkephalin /ED,-q 146 nmol per animal/ and is completely abolished by pretreatment with the narcotic antagonist naloxone f1, 3j. Kyotorphin did not bind to the specific opiate receptors, but did have a Met-enkephalin releasing effect from slices of guinea-pig striatum [ 3 ] and this effect is considered to be related to the analgesic action of the dipeptide. These findings suggest that the naloxone-reversible analgesic effect of Tyr-Arg is mediated by enkephalins or /3-endorphin. Therefore, it acts either as an enkephalin- or /S-endorphin-rele^sing factor or as an inhibitor of the degradation enzymes of enkephalins /aminopeptidase and enkephalinase/. The relatively long-lasting analgesia induced by Tyr-Arg may be attributed to a stabilising

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

488

effect on the released Met-enkephalin because it weakly inhibits enkephalin degradation enzymes. Thus, Tyr-Arg seems to be a Met-enkephalin-releasing factor, and its presence suggests the existence of a regulating mechanism for the release of Met-enkephalin. The regional distribution of kyotorphin in the rat brain and spinal cord [4] and effects on single neurons in the spinal dorsal horn of rabbits and the nucleus reticularis paragigantocellularis /NRPG/ of rats [5} suggest that kyotorphin has qualitatively similar actions as enkephalins and may play a physiological role in pain control in the CNS. According to Kastin et al. the kyotorphin as some other peptides can also affect behavior in chicks after peripherial administration. In a short time after isolation and identification of kyotorphin, this dipeptide, its long-lasting D-Arg analog and their retro isomers were synthesized by Yajima and his coworkers [6] . The same group synthesized eight Met- and Leu-enkephalin analogs [7] substituted with L- and D-Arg at position 2 containing the dipeptide unit kyotorphin or its analogs. The long-acting D-Arg analog and the two retro isomers, further four Met- and Leu-enkephalin analogs had higher analgesic activity than that of Tyr-Arg. The same data of synthetic Tyr-D-Ala and Tyr-D-Phe J8J have not been published.

Results Studying the structure-activity relationships in analgesia and/or Met-enkephalin releasing activity we have synthesized the kyotorphin and sixty-seven protected and unprotected structure analogs of it with a conventional and the solid phase peptidesynthesis methods. Optically active mainly natural but also unnatural amino acids were substituted mainly instead of L-tyrosine. With ^ C - and ^H-labelled derivatives of L-Tyr-L-Arg and L-Tyr-D-Arg were also prepared. Protected or partly protected, especially by carbobenzoxy-group blocked analogs were

489

synthesized on conventional way, in solution phase, starting from at the guanidine function by protonation blocked Arg or D-Arg, using the C-terminal amino acid a3 HONSu-esters 10 /Figure I./. Purification of the protected intermediates or analogs was effected by silicagel column chromatography. After deprotection by 10 % Pd-C/H2 the free peptides were treated with Amberlite CG-4B /acetate fom/ and purified by column chromatography on CM-cellulose.

1 Classical w a y Arg

Tyr OBu* OSu rOBut

H-|-0H TEA pH 8.5 AcOH/HCI fcKVH2 1.amberlite CG AB 2. CMC

-OH -OHxHCl OHxHCl OH

2. Solid phase w a y

-TOS Bil, BOC- A r g - O - C H2 - ( R e s i n ^ I

Ì

A c OH/MCI

®V rTOS T ' TtBfC^ r'"a ^ ^ »CCI BOC—Tyr- A r g - 0 - CH? - ( R e s i n

i

* Ma»«*

'

cnc

H-Tyr-Arg-OH

Figure I. Synthesis schemes of Kyotorphin and its analogs

490

At C-terminal amino acid by Boc-group blocked dipeptidea were prepared on original Merrifield-resin. After removing the protec ting-groups and the peptide from resin by HF/anisol the crude peptides were also treated with Amberlite CG-4B and purified by column chromatography on CM-cellulose. The homogeneity of the synthetic kyotorphin analogs and derivatives was assessed by TLC, HPLC and was characterized by amino acid analysis, NMR-data, melting points or optical rotation-data. Determination of analgesic activity was carried out on male CFLP-mice /25-35 g/. The peptides were administered intracerebroventricularly in 5 Jul saline under light ether anaesthesia. Two minutes later the animals were tested for analgesic activity on hot-plate [llj described by Ankier and the ED^Q values were compared /Table I./. We also examined naloxone antagonism with some of our peptides pretreating the animals with naloxone /I mg per kg/. Twenty minutes after i.p. injection analgesic effects of the peptides were tested on the same way.

Table I.: The Analgesic Effects of Synthetic Kyotorphin and its Analogs /intracisternally administered to Mice/ Hot-plate Test Compound Kyotorphin Me t-enkephalin Leu-enkephalin C b z-Tyr/Bu^/-Arg Cbz-Tyr-Arg.HCl Cbz-Tyr/BuV -D-Arg C b z-Tyr-D-Arg.HC1 L-Tyr-D-Arg Cbz-Gly-Arg Cbz-Gly-D-Arg Gly-L-Arg Gly-D-Arg

ED^Q ¿)g/animal 5.3 84.0 123.0 >20 >20 >20 >20 2.2 7.70 4.85 >20 11.6

Naloxone antagonism + + + n.i. n.i. n.i. n.i. + + + n.i. +

491

Cbz-Phe-Arg Cbz-Phe-D-Arg

17.0

.

+

+

Cbz-D-Phe-D-Arg L-Phe-L-Arg

1.95 >20

n.i.

>20

n. i.

L-Phe-D-Arg

>20

n. i.

D-Phe-L-Arg

> 20

n.i. +

D-Phe-D-Arg Cbz-Pro-Arg Cbz-Pro-D-Arg

4.9 2.65

L-Pro-L-Arg

2.65 >20

?

L-Pro-D-Arg

> 20

9

D-Pro-L-Arg

> 20

n.i.

Boc-Ser/Bzl/-Arg

11. i.

n.i.

S er/Bzl/-Arg.HC1

> 20

n.i.

Bo c-S er/Bz1/-D-Arg

n.i.

Ser/Bzl/-D-Arg.HC1

11. i. > 20

n.i.

L-Ser-L-Arg

> 20

n.i.

L-Ser-D-Arg

> 20

n.i.

Cbz-Tlir-Arg Cbz-Thx-D-Arg

4-97

L-Thr-L-Arg

6.4 > 20

L-Thr-D-Arg

> 20

+ + n.i. n.i.

Cbz-Pip-Arg

1.2

n. i.

Cbz-Pip-D-Arg Cbz-D-Pip-Arg

1.8

n.i.

8.25

n.i.

Cbz-D-Pip-D-Arg

4.45 7.6

n.i.

L-Pip-L-Arg

n.i.

L-Pip-D-Arg

>20

D-Pip-L-Arg D-Pip-D-Arg

> 20

n.i.

> 20

n.i.

Cbz-Pab-Arg

n.i.

n.i.

Gbz-Pab-D-Arg

inactive inactive

Pab-L-Arg

inactive

n.i.

Pab-D-Arg Boc-D-Tyr/Bzl/-Arg Boc-D-Tyr/Bzl/-D-Arg

inactive > 20 > 20

n. i. n.i.

Boc-Tyr/Br2/-Arg

> 20

n.i.

n.i. n.i.

492

Bo c-Tyr/Br 2 /-D-Arg L-Tyr/Br 2 /-L-Arg L-Tyr/Br 2 /-D-Arg 14, •^C-Tyr-L-Arg 3

H-L-Tyr-L-Arg

3

H-L-Tyr-D-Arg

>20

n.i

>20

n.i

>20

n.i + +

5.3 5.4 2.4

L-Tyr/B^/ L-Pip, D-Pip Pab

+

3,5-dibromo-tyrosine optically active pipecolic acid

p-amino-benzoic acid

Up to now,fifty-two of our synthetic peptides have been tested for analgesic activity and fifteen analogs have similar and/or better activity; some of them with longer-lasting effect. Amo;ig the peptides examined for naloxone antagonism some can be antagonised, some of them probably not. Nine of the eleven new active analogs are partly protected peptides. Further biological examinations are necessary to know more about chemical structure —biological activity relationships and biological role of kyotorphin and its analogs. Fourteen new synthetic analogs which have not been tested up to now are under

analgesia test. A number of analogs are under releasing and behavior effect studies. The radioactive derivatives of kyotorphin and Tyr-D-Arg analog help us to learn more about this new group of opioid peptides.

Acknowledgements We thank P. Eva Henyhart for chemical technical assistance, and Dr. Anna Ottlecz for tremendous help in biological tests.

493

References 1. Takagi, H., Shiomi, H., Ueda, H. and H. Amano: Eur. J. Pharmacol.

109 /1979/.

2. Satoh, M., Kawajiri, M., Yamamoto, M., Akaire, A. and H. Takagi: Ueurosci. Lett. 16, 319 /1980/. 3. Takagi, H., Shiomi, H., Ueda, H. and H. Amano: Nature 282. 410 /1979/. 4. Ueda, H., Shiomi, H. and H. Takagi: Brain Res. 198. 460 /1980/. 5. Ueda, H., Amano, H., Shiomi, H. and H. Takagi: Eur. J. Pharmacol. ¿6, 265 /1979/. 6. Yajima, H., Ogawa, H., Ueda, H. and H. Takagi: Chem. Pharm. Bull. 28, 1935 /1980/. 7. Kubota, M., Uagase, 0., Amano, H., Takagi, H. and H. Yajima: Chem. Phaxra. Bull. 28, 2580 /1980/. 8. Kastin, A. J., Honour, C. H. and D.H. Coy. Phys. Behavior 27, 1073 /1981/. 9. Balasiri, L., P. Menyhart, É., Geese, A., Ottlecz, A., Kovaca K. and Gy. Telegdy: Hungarian Patent announcement 1374/82, 5.30.1982 Budapest. 10. Voskuyl-Holtkaop, I., C. and Schattenkerk; Int. J. Peptide Protein Rea. 1^, 185 /1979/. 11. S. I. Ankier; Bur. J. Phannacol, 2J, 1 /1974/.

OPIOID ACTIVITY OF SYNTHETIC "SMALL DERMORPHINS»

Roberto Tomatis, Severo Salvador! and Gianpietro Sarto Institute of Pharmaceutical Chemistry and Pharmacology, University of Ferrara, Ferrara, Italy

Introduction We previously reported part of our investigation on dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH^),

leading to oligo-

peptides which are potent analgesics in vivo following subcutaneous administration (l). Sixty "small dermorphins" have now been prepared and tested for opiate activity in vitro and in vivo. Synthesis.

All peptides were prepared by solution methods

following procedures previously reported for H-Tyr-D-Ala-Phe-Gly-NH 2 and H-Tyr-D-Ala-Phe-Gly-Tyr-NH,, (l). Guanidino derivatives and aminoxy analogues were obtained according to reported procedures (2,3)« Th© purity of each sample was established as

> 95$.

Biological evaluation.

In vitro methods. The peptides were

examined for their ability to inhibit the electrically induced contractions of guinea-pig ileum (h) (GPI) and mouse vas deferens

(5) (MVD).

In vivo method.

The mouse tail flick assay

was essentially that of Janssen et al. (6) and analgesia was determined as previously described (l). All effects were completely blocked by naloxone. The activities of H-Tyr-D-Ala-Phe-Gly-NHg

(l) (Table I) and H-Tyr-D-Ala-Phe-Gly-Tyr-NH 2

(37) (Table II) were considered equal to 100, those of the analogues being expressed in percent, on a molar basis. The potency of reference compounds (Table I and II) in the tests were as follows (l): GPI (IC 5 Q a

5.2 + 3.19 nM and 5.38 +

0.06 nM); MVD (IC 5 Q = 510 + 31.82 nM and 86.28 + 6.16 nM); tail-flick test (ED _ = 68 pM/mouse and 22 pM/mouse).

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

496

Table X N° 1

Biological Da ta, of Dermorphin Tetrapeptid.es (On a molar basis) Structure

GPI

MVD

Tail-Fl:

H-Tyr-D-Ala-Phe-Gly-NH2

100

100

100

2

H-D-Tyr-D-Ala-Phe-Gly-NH^

0.03

0.03

0.03

3

H-Phe-D-Ala-Phc-Gly-NH^

0.8

1

0.7

4

H-Cly(Ph-4-OH)-D-Ala-Phe-Gly-NH^

0.5

0.4

0.2

5

H-Tyi—Gly-Phe-GlyNH^

0.7

1

0.6

6

H-Tyr-OAla-Phe-Gly-NH^

0.1

0.1

7

H-Tyr-DOAla-Phe-Gly-NH^

0.1

0.1

8

H-Tyr-D-Met-Phe-Gly-NH^

3

6

0.1

9

-

H-Tyi—D-Ala-Phe-Sar-NH^

130

8 6.7 • — O-Pro • OH i 4.03 150 (doc.) + 71.6° «2.4 * |o-*ro | OH 1.2 7.10 167-170 • 16.4° + 1 3»] D-Pro D-Pro OH 1 NO 169-172 «131.7° «2.4 OH — IfrPhQl 1 6.64 210 (dec.) - 55.5° • 1 200 1 O-Tyr - OH 1 3.60 212 (doc.) -122.3° «2.4 • D-Tyr O-Phe OH 1 5.03 214-218 -143.4° no activc — — Phe(Cl) — — OH 2 5.0 3.95 202 (dec.) - 53.5° Phe — - • — — OH 2 7.71 202-209 - 14.4° no active -

;;II XIII ;:iv

Tyr—Pro —Phe —Pro-OH • 1 5.52 • • • • one » 1 11.06 — N H 2 8 l" 3.91

No. coap. I II III

Structure

173-17G 160-173 17G-1C3

- 56.9° -

00.0°

- 55.5°

3.C 14.0 2.4 / 55*#)

«

•

'All compounds were characterized as hydrochlorides, except IV - free base. Homogeneity of the peptides wa.s verified by HPLC and by TLC on silica, gel plates (SilufolR, Kavalier, Czechoslovakia) in four different solvent systems. Amino acid analysis after acidic hydrolysis showed the expected composition. )For III and XIV the Boc-tripeptide and Boc-dipeptide methyl ester, respectively, was converted into the amide. ^Reversed phase HPLC (HPLC-Hewlett Packard 1084D) was performed on a RP 8 column and eluted with 0.025 M KHoPO^, pH 3.0 / methanol (65:35) at a flow rate of 1.2 ml/inin, UV detector at 210 nm. ND - not determined. 3) 'For the determination of the analgesic activity the tail flick test on male Wistar rats was used (electrical stimulation) measuring the effective dose for doubling the pain threshold 30 min/**)10 min after icv. administration of the peptide. For morphine a dose of 6.0 nmol/rat, icv. was determined. The relative activity of morphine is assigned as 100 and peptides are compared to it on the molar basis. k) 'For enzymatio degradation in vitro peptides were incubated at 30°C (pH 7.6) with the peptidase and the hydrolysis of the peptides was followed by TLC as a function of time: (+) = degradation, (-) = no degradation. - These pharmacological results demonstrate, that the introduction of D-Pro and D-Phe , respectively, in the p-CM-5 sequence leads to analogues with remarka.bly high analgesic

508

potency and enhanced s t a b i l i t y tion.

towards enzymatic

2

- Replacement of

Pro

g i v e an i n c r e a s e i n - The change o f

by i t s D-enantiomer, however, does n o t potency.

the C - t e r m i n a l carboacyl group by amida.tion

causes a marked i n c r e a s e i n a n a l g e s i c The

degrada-

activity.

h i g h e s t a n a l g e s i c a c t i v i t y wa.s f o u n d f o r compound V,

deprolorphin

(7).

A l r e a d y 0 . 5 nmol i c v .

significantly

sed the p a i n t h r e s h o l d . The e f f e c t i s dosedependent, maximal v a l u e s a t about 10 nmol i c v .

increareaching

1 . 6 8 nmol d e p r o l o r p h i n

proved t o be e q u i p o t e n t t o 6 . 0 nmol morphine and 1 7 . 0 nmol morphiceptin ( 6 ) ,

d o u b l i n g the pa.in t h r e s h o l d . A f t e r i c v .

m i n i s t r a t i o n of 5 . 0 nmol d e p r o l o r p h i n a. s i g n i f i c a n t

ad-

analgesic

potency c o u l d be o b t a i n e d up t o 90 min. T h i s a.ction was comp l e t e l y b l o c k e d by i c v .

pretrea.tment w i t h 2k,0

15 min b e f o r e p e p t i d e a d m i n i s t r a t i o n .

nmol n a l t r e x o n

Furthermore

deprolor-

phin r e v e a l e d an a n a l g e s i c a.ction even a f t e r i n t r a v e n o u s

ad-

m i n i s t r a t i o n - 7«0yumol/kg w e r e e q u i p o t e n t t o 4.0yumol/^cg morphine. Consequently,

[3-casomorphin analogues a r e a b l e t o produce

a n a l g e s i c a c t i o n s w i t h a p o t e n c y a t l e a s t as h i g h as by p o t e n t e n k e p h a l i n s , action with^u-receptors

obtained

but proba.bly due t o a. s e l e c t i v e

inter-

(2,6,8).

Further s t r u c t u r a l m o d i f i c a t i o n s of

the (3-oasomorphin m o l e -

cule are i n preparation to g e t a d d i t i o n a l information i n f i e l d of s t r u c t u r e - a c t i v i t y

the

relationship.

References 1.

Henschen, A . , L o t t s p e i c h , F . , B r a n t l , V . , H . : H o p p e - S e y l e r ' s Z. P h y s i o l . Chem. 360, (1979).

Teschemacher, 1217-1224

2.

B r a n t l , V. , Teschemacher, H, , B l a . s i g , J . , Henschen, L o t t s p e i c h , F . : L i f e S c i . 28, 1903-1909 (1981).

A.,

509 3.

G r e c k s c h , G. , S c h w e i g e r t , C h r . , L e t t e r s 27, 325-328 ( l 9 8 l ) .

k.

H a r t r o d t , B . , N e u b e r t , K . , F i s c h e r , G. . S c h u l z , B a r t h , A . : P h a r m a z i e 37, 165-1Ö9 ( 1 9 8 2 ) .

5.

H a r t r o d t , B . , N e u b e r t , K . , F i s c h e r , G . , Derauth, U . , Y o s h i m o t o , T . , B a r t h , A . : P h a r m a z i e 37, 7 2 - 7 3 ( 1 9 8 2 ) .

6.

Chang, K . - J . , Chang, J . - K . :

K i l l i a n , A , , Ha.zum, A . , Cua.treca.sas, S c i e n c e 212, 75-77 ( l 9 8 l ) .

P.,

7.

Matthies, Hartrodt,

Rüthrich, H . - L . , Barth, A., Neubert, Europ. J . P h a r m a c o l , ( i n p r e s s ) .

K. ,

8.

Zhang,

H., B,:

A.-Z.,

Chang,

28, 2829-2836 ( 1 9 8 1 ) .

J.-K.,

Matthies,

Pasternak,

H.:

G.W.:

Neurosci, H.,

Life

Sei.

SUBSTANCE P:

THE "YIN-YANG" OF BEHAVIOR?

John M. Stewart and Michael E. Hall

Department of Biochemistry, university of Colorado School of Medicine Denver, Colorado 80262 USA

It is generally accepted that substance P (SP) may play a variety of roles as a neurotransmitter or neuromodulator in addition to its longstudied actions in causing smooth muscle contraction, vasodilatation and salivary secretion (1). Its widespread occurrence in both the central (CNS) and peripheral (PNS) nervous systems has been esiablished in recent years, long since the original observation by Lembeck (2) of a high concentration of SP in dorsal roots of spinal cord. On the basis of that observation, he proposed in 1953 that SP is a neurotransmitter of sensory impulses in the spinal cord.

This prediction has been

confirmed, at least as far as thermal sensory neurons are concerned.

It

appears that red pepper (Capsicum app.) is "hot" (piquant) because its active principle, capsaicin, causes release of SP from these neurons (3). the past work with SP has established that the C-terminal pentapeptide, -Phe-Phe-Gly-Leu-Met-NH2, is the "active core" for all the biological activities of SP described so far; the N-terminal hexapeptide, Arg-Pro-Lys-Pro-Gln-Gln-, has been considered to have no inherent activity, but to increase potency by protecting against degradation or increasing receptor affinity. number of behavioral responses.

SP is known to produce a

SP-induced tranquilization was

described in 1957 (4), and its antinociceptive action in 1976 (5). In this latter work the suggestion was first made that SP might require metabolism to an active fragment before it exerted its antinociceptive effect. We wish to describe our experiments which further support this idea and show that antinociception (analgesia) is produced by peptides related to the N-terminus of SP. Such N-terminal peptides also shew other kinds of biological activity, in certain cases producing effects on behavior opposite to those produced by C-terminal SP fragments.

Peptides 1982 © 1983 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

512

IVo brain enzymes have been described which cleave SP: the "post-proline cleaving enzyme" (6) which cleaves SP between residues 4 and 5 and the enzyme described by Benuck and Marks (7), which cleaves SP between the two phenylalanine residues. Considering that the latter enzyme would completely inactivate SP for the previously described activities, we thought it might prove to be in fact a processing enzyme which would selectively activate SP for antinociceptive action. Thus we tested SP(17), a product of the action of this enzyme of SP, and found this fragment to possess the full antinociceptive potency of SP (8). Consistent with the processing hypothesis, SP(l-7) antinociception was rapid in onset and short in duration of action, whereas intact SP produced an antinociception of long duration, following a long lag period (even after intracerebroventricular injection). Remarkably, the SP(l-7) antinociception was also seen following peripheral administration (as was that produced by intact SP), and the ratio of intracerebroventricular to subcutaneous dose for production of an equivalent effect suggested that the peptide was acting centrally in each case. The dose-response curve, like that of SP, was bell-shaped; antinociceptive action was seen only within a relatively narrow dose range and disappeared at higher doses. The SP(l-7) analgesia, like that elicited by SP, was blocked by the opiate antagonist naloxone. In studying SP-induced antinociception we observed a marked interaction with stress reactions. When mice treated with SP rested quietly following peptide administration they showed antinociception, but when they were excited by agitation conccmitant with group housing, no antinociceptive action of SP was seen. It is known that restraintinduced stress produces a naloxone-reversible ("cpioid") analgesia in mice, while the acute stress of brief electrical foot-shock produces a very strong analgesia not reversible by naloxone ("non-opioid"). We found that SP blocked the opioid type of stress-induced analgesia but had no effect on the non-opioid type. It has been suggested that SP produces analgesia by stimulating release of Met-enkephalin in the CNS(9); it now appears that during restraint-induced stress SP can inhibit release of endogenous opioids.

513

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Substance P Post-Pro / enzyme /

\ Marks' \ enzyme

Arg-Pro-Lys-Pro SP(l-4) + Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 SP(5-11)

Arg-Pro-Lys-Pro-Gln-Gln-Phe SP(l-7) + Phe-Gly-Leu-Met-NH2 SP(8-11)

Having discovered this biological activity of SP(l-7), we then examined other behavioral models for effects of SP, SP(l-7) and the potent Cterminal fragment analog, pyroglutamyl-Phe-Phe-Gly-Leu-Met-NH2 ( to X u C O C O

( • L O

S

oo i O rH L O r~t

1 O rH

00 •

o rH

I

§

« N r. 1 X « I I X o——o — o —y 1

H C O co o co a UJ h H O u H H Q O s

P U E 1 < D C O

00

to S C U O

o C T .

e 3 0) T — ( • rH M •H O. cfl < D C •H 3 M C o

(1) fi E I _ I I Ì y—O—O—O—O

C i-H

+

\D 1 O i-H O

1 — 1 C M O

A o\° C •H

H CJ O 1 o

c¿ Et/3 I X I X X o —o — o — o — o

< D

T

ru

Z

V D L O

tu j

X p •r-t G •H 4H 4-1 al a)

>

co < H

O < X I I X y — < r > — o — o — o

P a >

S

O o ^

O ^

•H P ai rH (U tu tn •rH


•rH

Ul P O tu < D ai -o -(H X O p u P •H •H Pu t/l P 1) O Q J C P. 3 •H i—i H P U ai p p O C c 1 •H o o 0) X O i—l co ai rH ai Ci 3 4h e •H U •o O "O X c