r tl INTER- NATIONAL SYMPOSIUM NCHEMSTRY OF NATURAL PRODUCTS SYMPOSIUM PAPERS HALF-HOUR VOLUME PLENARY PART1 • tHnWB SANDS BULGARIA Q 17-23,9 X Golden Sands, Bulgaria September 17-23,1978 Bulgarian Aoademy of Sciences 1978 c/o Jusautor, Sofia PREFACE The present publication contains the proceedings of the llth IUPAC International Symposium on Chemistry of Natural Products, September 17-23v 1978, Golden Sands, Bulgaria, and Post-Symposium "Natural Products Research and Industry", September 24-26, 1978. The Symposium is to be held under the auspices of the International Union of Pure and Applied Chemistry/ Division of Organic Chemistry. The publication comprises four volumes. Volumes 1 to 3 include the original short communications, which have arrived at the Secretariate by July 10th 1978. Volume 4 includes the lectures of the invited Half-Hour Plenary Speakers of the Symposium and Post-Symposium, which sent their papers at the Secretariate by the same date. All papers are exact reproductions of the authors' original manuscripts. The Secretariate has hot made any corrections or changes in the texts. THE ORGANIZING COMMITTEE OF THE SYMPOSIUM CONTENTS OF THE VOLUMES Volume 1. Short- communications of Section "Bioorganic Chemistry" Volume 2, Short communications of Sections "Structural Elucidation and Chemical Transformation of Natural Products" and "Physical Methods for Investigation of Natural Products". Volume 3. Short communications of Section "Synthesis of Natural Products" Volume 4. Half-Hour Plenary lectures of the Symposium and Post-Symposium. EDITORIAL BOARD N. Marekov, I. Ognyanov, A. Orahovats, V. Tarpanov A. Trifonov, R. Vlahov ADDRESS OF THE SECRETARIATE t Dr. R. Vlahov, Secretary < 11th IUPAC Symposium on Chemistry of Natural Products c/o InsKtute of Organic Chemistry and Center of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria TABLE OF CONTENTS The invention of useful organic raacHcns Derek H.R. Bcrton . 1 The structure and reactivity of leghemogbbin, a monomeric hemoglobin C.A. Appleby 30 Polymers containing isonitrile functional groups as supports for the cova- lent fixation of biologically active molecules L.Goldstein, A.Freeman, R.Granot, M. Sokolovky 42 Biosynthesis of benzoisochrojfnane quinone antibiotics Heinz G. Flow, Ching-jer Chang 54 Secondary products and their possible function in plants V. Herout 69 Aspects of enzymic stereospecificity K. H. Overton 84 Detection and structure elucidation of leaf movement factors from Mimosaceae H. Schildknecht, B.Tauscher, H. Moeschler, J.Edelmann 97 Estrogen biosynthesis and its inhibition K. Schubert, K. Schade 112 Biotransformation of abscisic acid and related phytoeffectors H.R. Schutte 127 Some aspects of protein folding Jeannine Yon 139 Monoxidative cyclization of squalene by tetrohymena pyriformis. The incorporation of a 3beta-hydrogen /deuterium/ atom into tetrahymanol E. Caspi 166 Reactions of kaurene and its related compounds with thallium tri- nitraie Eiichi Fujita 182 Recent results in the chemistry of lichen substances Siegfried Huneck 197 The cyclization of acyclic terpenoids and some reactions of the products Sh6 Ito 2G7 Gama-glutamyl derivatives in fagaceae and other plants Peder Olesen Larsen 217 Recent chemistry of quassinoids Tatsushi Muro* 233 Studies in Hie chemistry of the oxazolidine ring-containing C2Q- diterpenoid alkaloids S.William Pelletier, Naresh V, Mody 248 Recent progress in the group of Veratrum and Buxus alkaloids J. Tomko, Z. VoMcky 260 The investigation of benzyl isoquinoline alkaloids from Papaveraceoe plants carried out during the past decade F. Santav? 272 Synthesis of biologically active 2'-deoxyuridine derivatives from thymidine D. BaYwolff 288 Synthetic peptides as molecular probes R, Geiger, J.. Sandow, H.Fruhbeis, R. Uhmann 301 The zip reaction: A new method for synthesis of polyaminolactams by ring enlargement A, Guggisberg, B. Dabrowski, Chr. Heidelberger, U. Kramer, E. Stephanou, M. Hesse 314 Synthesis of selected natural products Axel Nuerrenbach 325 Recent advances in Ergot chemistry P.A. Stadler 343 Synthesis of 8-azesteroids Csaba Szantay, Gdbor T6th 357 Antibiotic sugars by a totally synthetic approach Alexander Zamojski 369 A study by nitrogen-15 nuclear magnetic resonance spectroscopy of the state of histidine in the catalytic triad of aifa-lytic protease William W. Bachovchin, John 0. Roberts 379 Use of "in situ" reactions for structural elucidation of natural compounds by n.m.r. spectroscopy Z. Samek 390 New perspectives in natural products research in the pharmaceuticals industry George deStevens . 407 New industrial processes based on carene Sukh Oev 433 VI Chemical reactions on the alic/clic ring of daunomycin and its derivatives S.Penco, F. Angelucci, F. Gozzi, G. Franchi, B. Gioia, A. Vigevani, F. Arcamone 448 Research and development of 2alfa, 3alfa-epithioandrostan-17beta-ol and its derivatives anti-mammary cancer agents Ken'ichi Takeda 464 Total synthesis of natural and unnatural steroids U. Eder 478 Synthesis and properties of 6-thia-tetracyclines K. Irmscher 494 Antiulcer substances from a medical plant Y. Kishida, A. Ogiso 506 Industrial synthesis of prostaglandins and natural pyrethrins from common intermediates G. Kovacs 518 The aminoglycosides and the problems associated with their industrial synthesis Jean-Claude Gasc 533 Natural products research at Hoechst Pharmaceuticals Limited, India N.J. de Souza 544 Author index 559 VII 5c 100 15O FRACTION NUMBER 66*9 004X0 NONOXIDATIVE CYCLIZATION OF SQUALENE BY TETRAHYMENA PYRIFORMIS. THE INCORPORATION OF A 3&-HYDROGEN (DEUTERIUM) ATOM INTO TETRAHYMANOL Eliahu Caspi The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 The evidence accumulated in recent years indicates that the biosynthesis of C-3 oxygenated triterpenes and sterols involves the oxidative cyclization of squalene . In contrast to the initial views, it was shown that the oxidative cycli- zation of squalene is not a concerted process, but involves 2 3 at least one stable intermediate 2,3(S)-oxido squalene ' (1_). Certain microorganisms have the capacity to metabolize both the 4 2,3 (S)- and 2,3(R)-oxido squalenes . The liver squalene epoxidase was shown to be a microsomal monooxygenase which requires molecular oxygen, FAD, a superna- tant protein fraction and phbspholipids . The cyclization of the epoxide (1) to lanosterol (2_) is catalyzed by a liver micro- somal cyclase . Several years ago we have shown that the biosynthesis of the triterpene tetrahymanol (3_) by the protozoan Tetrahymena oyriformis involves a nonoxidative cyclization of squalene . This hypothesis was supported by the observations that in cora- 8 7,9 petitive ill vivo and iri vitro incubation experiments, 3 14 using mixtures of 2,3(RS)-oxido-[ H]-squalene and [ C]- squalene with T. pyriformis or homogenates of T. pyriformis, 14 the biosynthesized tetrahymanol (3_) contained only C and 166 167 14 was devoid of tritium. Accordingly, C-tetrahymanol (3_) was 14 obtained when C-squalene was incubated with a homogenate of 9 7 T. pyriformis under anaerobic conditions ' The nonoxidative biosynthesis of tetrahymanol was rationalised in terms of an enzyme-mediated proton attack on a terminal double bond of squalene. The proton attack on the 2,3-double bond could then generate a cation of type (4_) and the ensuing, indicated flow of electrons would cause cyclization, resulting in the C-21 cation {5). The C-21 cation could then be stabilized through the acquisition of a hydroxyl moiety from the water of the medium, as indicated in (5). It nay be assumed with certainty that the proton cyclase is in equilibrium with the medium and, therefore, both the "attacking" hydrogen atom and the oxygen atom will be derived from the water of the medium. We have, therefore, prepared an enzyme powder of T. pyriformis which was then suspended in 99.5% deuterium oxide ' . When squalene was incubated with this enzyme preparation, D^tetrahymanol (3) (37%-deuterium) was obtained. The mass spectrum of tetrahymanol (Figure 1) shows a molecular ion at ra/e 428 and, among other fragments, two characteristic ions at m/e 207 (7_) and m/e 191 (8). The m/e 207 (7) particle corresponds to fragmentation along pattern (6-a) with the loss of a hydrogen atom. Cleavage along pattern (6-b) and loss of a hydrogen atom results in the fragment m/e 191 {£). Hence, ions m/e 191 (£) and m/e 207 (7_) represent fragments corresponding to rings A-B and C-D of tetrahymanol, respectively. Mass spectrometric analysis of the D^-tetrahymanol indicated that all the deuterium was located 168 in the fragment (3) (m/e 193;37% D ) corresponding to rings A-B. When squalene was incubated with the enzyme powder suspen- 18 18 ded in OH (62.4% excess 0), the biosynthesized tetra- Ilr7 . 18 hymanol (3) contained 30.5% excess of O. The mass spec- 18 trum of the O-tetrahymanol showed fragments, at m/e 207;m/e 209 (207 + 2) (30%- 8O enrichments) (J) and m/e 191 (£). Clearly, all the excess of 180 is located in the fragment corresponding to rings D-E of tetrahymanol (:3). The fragment at 413 (M - CH-) (10) showed a 30% enrichment of the ion at m/e 415 (413 + 2). However, the peak at m/e 410 (9_) corresponding to the loss 16 18 of water from tetrahymanol 1(428- OH ); (430- OH,)] was un- changed and this confirmed that all the isotopic excess is asso- ciated with the oxygen atom located in ring E of tetrahymanol. The results indicate that, overall, the biosynthesis of tetra- hymanol from squalene by enzymes of T. pyriformis is equivalent to the acquisition by the squalene of a molecule of water. We now addressed ourselves to the question of the stereo- chemistry in tetrahymanol of the hydrogen atom introduced in the course of the enzymatic cyclization of squalene. The task of determining the stereochemistry of this hydrogen atom was •complicated because of the absence of a functional group which would facilitate access to ring A. Our initial attempts at mi- crobial hydroxylation of ring A, using sterane and triterpene 12 hydrocarbons as models, were not successful and were abandoned We then considered the use of deuterium n.m.r.