MIAMI UNIVERSITY-THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Lizhi Zhu Candidate for the Degree: Doctor of Philosophy ________________________________ Robert E. Minto, Director ________________________________ John R. Grunwell, Reader ________________________________ John F. Sebastian, Reader ________________________________ Ann E. Hagerman, Reader ________________________________ Richard E. Lee, Graduate School Representative ABSTRACT INVESTIGATING THE BIOSYNTHESIS OF POLYACETYLENES: SYNTHESIS OF DEUTERATED LINOLEIC ACIDS & MECHANISM STUDIES OF DMDS ADDITION TO 1,4-ENYNES By Lizhi Zhu A wide range of polyacetylenic natural products possess antimicrobial, antitumor, and insecticidal properties. The biosyntheses of these natural products are widely distributed among fungi, algae, marine sponges, and higher plants. As details of the biosyntheses of these intriguing compounds remains scarce, it remains important to develop molecular probes and analytical methods to study polyacetylene secondary metabolism. An effective pathway to prepare selectively deuterium-labeled linoleic acids was developed. By this Pd-catalyzed method, deuterium can be easily introduced into the vinyl position providing deuterolinoleates with very high isotopic purity. This method also provides a general route for the construction of 1,4-diene derivatives with different chain lengths and 1,4-diene locations. Linoleic acid derivatives (12-d, 13-d and 16,16,17,17,18,18,18-d7) were synthesized according to this method. A stereoselective synthesis of methyl (14Z)- and (14E)-dehydrocrepenynate was achieved in five to six steps that employed Pd-catalyzed cross-coupling reactions to construct the double bonds between C14 and C15. Compared with earlier methods, the improved syntheses are more convenient (no spinning band distillations or GLC separation of diastereomers were necessary) and higher Z/E ratios were obtained. The overall percent yield for (14E)-isomer was 21% and 29% for the (14Z)-isomer. The reaction between DMDS and 1,4-enynes in the presence of I2 was studied. 2,5-Disubstituted thiophene derivatives were produced as the main products under neutral and acidic conditions. The detailed mechanism of this reaction was studied. Current evidence is consistent with a mechanism that can be described as follows. Initially, electrophilic addition of a sulfenium ion to an alkene yields an episulfonium ion. The subsequent Wagner-Meerwein rearrangement leads to a cationic thietane intermediate through a ring expansion. This four-membered ring is opened by nucleophilic attack of iodide to give a MeSI adduct. Available protons activate the triple bond and promote the subsequent transformations to generate the final thiophene product and release MeI as a side product. The synthetic utility of this method was explored. The optimized reaction provides a mild synthetic route to 2,5-disubstituted thiophene derivatives. INVESTIGATING THE BIOSYNTHESIS OF POLYACETYLENES: SYNTHESIS OF DEUTERATEDLINOLEIC ACIDS & MECHANISM STUDIES OF DMDS ADDITION TO 1,4-ENYNES A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry & Biochemistry by Lizhi Zhu Miami University Oxford, Ohio 2003 Dissertation Director: Robert E. Minto TABLE OF CONTENTS CHAPTER PAGE List of figures iv List of tables vi List of abbreviations & acronyms vii Acknowledgement viii I Synthesis of regioselectively deuterated linoleic acids 1 1-1 Endiyne antibiotics 1 1-2 Naturally occurring polyacetylenes 5 1-3 Biosynthesis of polyacetylenic compounds 8 1-4 Synthesis of regioselectively labeled linoleic acids 13 1-5 Results and discussion 14 1-6 Conclusion 21 1-7 Experimental section 21 References 38 II Improved synthesis of methyl (14E)- and (14Z)-dehydrocrepenynate 47 2-1 Introduction 47 2-2 Results and discussion 49 2-3 Conclusion 51 2-4 Experimental section 51 References 59 III Mechanism of dimethyl disulfide addition to 1,4-enynes 61 3-1 Introduction 61 3-2 Results and discussion 63 3-2.1 Optimization of the reaction conditions 64 3-2.2 Effects of additives 67 ii 3-2.3 Proposed mechanisms for thiophene formation 70 3-3 Conclusion 73 3-4 Experimental section 74 3-4.1 General information 74 3-4.2 Materials 75 3-4.3 General procedure for making thiophene derivatives 75 3-4.4 Synthetic material 75 3-4.5 Experimental data 78 References 91 iii LIST OF FIGURES FIGURE PAGE 1 Members of enediyne antibiotics family 2 2 Cycloaromatization of enediynes and its utility in natural product synthesis 4 I 3 Mechanism of DNA cleavage by calicheamicin γ 1 6 4 Examples of linear biological active linear polyacetylenic natural products 7 5 Desaturase-like reaction catalyzed by an acetylenase 8 6 Expression of ELI12 in parsley induced by a fungal challenge 10 7 Proposed biosynthetic pathway of polyacetylenic compounds 11 8 Regioselectively labeled linoleic acids 13 9 Retrosynthesis analysis of vinyl deuterated linoleic acids 13 10 Synthesis of C1-C10 fragment 14 11 Synthesis of C11-C18 fragment 13a, 13b 15 12 Synthesis of C11-C18 fragment 13c 16 13 Proposed mechanism for Cu-catalyzed coupling reaction 18 14 Synthesis of regioselectively labeled linoleic acids 19 15 Crepenynate (30) and dehydrocrepenynate (31a, 31b) derivatives 47 16 Synthesis of methyl (14Z)-dehydrocrepenynate 31a 49 17 Synthesis of methyl (14E)-dehydrocrepenynate 31b 50 18 Dimethyl disulfide (DMDS) derivatization and its utilities 62 19 DMDS addition to 1,4-enyne derivatives 64 20 Effect of temperature on thiophene formation 65 21 Reaction of methyl 9-tridecynoate (49) with DMDS 69 22 Mechanism of DMDS addition to 1,4-eneyne compounds 70 23 GC-MS analysis of the reaction of cis-enynethiol ether 53 with HI at 60 °C 71 24 GC-MS analysis of the reaction of cis-enynethiol ether 54 with HI at 60 °C 72 25 Alternative mechanism through alkyne-allene rearrangement 73 26 Synthesis of methyl 9-tridecynoate 49 76 27 Synthesis of methyl (6Z)-tridecen-9-ynoate 45 76 iv 28 Synthesis of methyl cis-enyne thioether 53 77 29 Synthesis of methyl trans-enyne thioether 54 77 30 Synthesis of 1,4-enyne 46 77 v LIST OF TABLES TABLE PAGE 1 Results of copper catalyzed cross-coupling reaction 17 2 Optimization of the cross-coupling reaction for assembly of 1,4-diene unit 20 3 Solvent and concentration effects to the formation of thiophene derivatives 66 4 Effects of additives to DMDS derivatization of 45 67 vi LIST OF ABBREVIATIONS & ACRONYMS AcOH Acetic acid AcSH Thiolacetic acid DIAD Diisopropyl azodicarboxylate DIBAL-H Diisobutylaluminum hydride DMDS Dimethyl disulfide DMF N,N-Dimethylformamide DMA N,N-Dimethylacetamide DNA Deoxyribonucleic acid DPPF 1,1’-Bis(diphenylphosphino)ferrocene HMPA Hexamethylphosphoramide (hexamethylphosphoric triamide) KIE Kinetic isotope effect LAH Lithium aluminum hydride LDA Lithium diisopropylamide NAD(P)H Nicotinamide adenine dinucleotide phosphate, reduced NCS N-Chlorosuccinimide PC Phosphatidylcholine PE Phosphatidylethanolamine RBF Round bottom flask TBDMSCl tert-Butyldimethylsilyl chloride THF Tetrahydrofuran THP Tetrahydropyran (or tetrahydropyranyl) TLC Thin layer chromatography TMSCl Trimethylsilyl chloride vii ACKNOWLEDGEMENT I want to thank Dr. Robert E. Minto for his kindness, guidance, and giving me the opportunity to consult on all of the problems. I appreciate him for providing me the chance to become a member of his research group. From him, I learned not only the knowledge of chemistry, also his attitude and enthusiasm toward the academic work. I would like to thank all of my committee members for their generosity and giving me advice. I thank the faculty in the department of chemistry and biochemistry at Miami University for the instruction. I appreciate the Department of Chemistry and Biochemistry of Miami University for providing me the opportunity to utilize the scientific instruments and facilities. I also would like to thank my colleagues in chemistry department for their kind help and generous suggestion. Finally, I wish to thank my parents and all of my family members for their strong supporting and being behind me all the time. viii Chapter I. Synthesis of Regioselectively Deuterated Linoleic Acids Novel biologically active substances from nature often provide stimulation, challenges and opportunities for the scientific community. Naturally occurring acetylenes, as an example, have attracted wide interest during the past two decades. Even though acetylenic compounds have been studied by many research teams for more than a century, the most exciting results were obtained after 1980, through the discovery of a new antibiotic family, the so called enediyne antibiotics.1-6 Their phenomenal biological activities against selected cancer cells (calicheamicin I γ1 (4) is 1000 times more potent than adriamycin, a clinically useful antitumor antibiotic in murine tumor models2b) and their unusual molecular architecture elicited extensive research activities in chemical, biological and biomedical circles.7 As potential candidates for anticancer drugs, this family of compounds has been studied thoroughly. Several strategies exploiting modern synthetic technologies have led to the total synthesis of these compounds.8-15 Their fascinating modes of action have inspired the design of a number of novel analogs that mimic their chemical and biological functions. Continued studies will hopefully lead to the development of a second generation of designed enediyne antibiotics with improved