ENANTIOSELECTIVE CHEMOENZYMATIC SYNTHESIS OF OSELTAMIVIR (TAMIFLU)(R) INTERMEDIATES A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY HACI EŞĐYOK IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOTECHNOLOGY JANUARY 2008 Approval of the thesis: ENANTIOSELECTIVE CHEMOENZYMATIC SYNTHESIS OF OSELTAMIVIR (TAMIFLU)(R) INTERMEDIATES submitted by HACI EŞĐYOK in partial fulfillment of the requirements for the degree of Master of Science in Biotechnology Department, Middle East Technical University by, Prof. Dr. Canan Özgen _____________________ Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Gülay Özcengiz _____________________ Head of Department, Biotechnology Prof. Dr. Ayhan S. Demir Supervisor, Chemistry Dept. , METU _____________________ Prof. Dr. Ufuk Bakır Co-Supervisor, Chemical Engineering Dept., METU _____________________ Examining Committee Members: Assoc. Prof. Dr. Özdemir Doğan _____________________ Chemistry Dept., METU Prof. Dr. Ayhan S. Demir _____________________ Chemistry Dept., METU Assoc. Prof. Dr. Nezire Saygılı _____________________ Faculty of Pharmacy, HU Assoc. Prof. Dr. Emine Bayraktar _____________________ Chemical Engineering Dept., AU Assoc.Prof. Dr. Nursen Çoruh _____________________ Chemistry Dept., METU Date: 30.01.2008 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name : Hacı Eşiyok Signature : iii ABSTRACT ENANTIOSELECTIVE CHEMOENZYMATIC SYNTHESIS OF OSELTAMIVIR (TAMIFLU)(R) INTERMEDIATES Eşiyok, Hacı M.S., Department of Biotechnology Supervisor : Prof. Dr. Ayhan S. Demir Co-Supervisor: Prof. Dr. Ufuk Bakır January 2008, 72 pages The objective of this presented study was to synthesize optically active compounds considered to be key intermediates in the synthesis of Oseltamivir (Tamiflu) by performing chemical and biotechnological methods. Thereof, the carboethoxy cyclohexenone skeleton first was synthesized utilizing easily available substances. The synthesis of alpha-hydroxy ketones in enantiomerically pure form offers a great importance in the synthesis of biologically active compounds. Toward this fact, the enantioselective synthesis of alpha-hydroxy carboethoxy cyclohexenone scaffold has been accomplished by following the routes which were manganese(III) acetate- mediated chemical oxidation followed by enzyme-mediated hydrolysis and additionally microbial direct biooxidation by whole cells of fungi expressly A. oryzae and A. flavus. A very satisfying results have been obtained by both of the methods. Keywords: Alpha-Hydroxy Ketone, Manganese(III) Acetate, Enzymatic Kinetic Resolution, Microbial Hydroxylation, Fungi. iv ÖZ OSELTAMIVIR (TAMIFLU)(R) ANTĐVĐRAL ĐLAÇ HAMMADDELERĐNĐN KEMOENZĐMATĐK SENTEZLERĐ Eşiyok, Hacı Yüksek Lisans, Biyoteknoloji Bölümü Tez Yöneticisi : Prof. Dr. Ayhan S. Demir Ortak Tez Yöneticisi: Prof. Dr. Ufuk Bakır Ocak 2008, 72 sayfa Bu çalışmanın amacı antiviral bir ilaç olan Oseltamivir (Tamiflu) sentezinde kullanılabilecek optikçe aktif hammaddelerin kimyasal ve biyoteknolojik yöntemlerle sentezlenmesidir. Bu nedenle ilk olarak karboetoksi siklohekzenon yapısı kolayca bulunabilen basit maddeler kullanılarak sentezlendi. Alfa-hidroksi ketonların enansiyomerikçe saf hallerinin eldesi biyolojik açıdan aktif bileşiklerin sentezinde büyük bir önem teşkil eder. Bu gerçeğe yönelik olarak, alfa-hidroksi karboetoksi siklohekzenon yapısının enansiyoseçici sentezi, mangan(III) asetat vasıtasıyla kimyasal oksidasyon ve beraberinde enzim yardımıyla hidroliz ve ayrıca A. oryzae ve A. flavus türündeki mantarların tüm hücreleri kullanılarak yapılan mikrobiyel biyooksidasyonla gerçekleştirildi. Her iki metodla da tatmin edici sonuçlar elde edildi. Anahtar kelimeler: Alfa-Hidroksi Keton, Mangan(III) Asetat, Enzimatik Kinetik Resolüsyon, Mikrobiyel Hidroksilasyon, Mantar. v To My Family vi ACKNOWLEDGMENTS I would like to remark my feelings of appreciation and gratitude to my supervisor Prof. Dr. Ayhan S. Demir for his support, encouragement and guidance throughout the study. I would like to thank to my co-supervisor Prof. Dr. Ufuk Bakır for her helps, and understanding. My distinctive thanks extent to Peruze Ayhan for her endless support and suggestions during my study. I would like to thank also to my labmates Betül Sopacı, Umut Erkılıç, Umut Demirtaş and Đlke Şimşek for their friendship. I also express my sincere appreciation to all of the members of Demir’s Research Group. I am grateful to the Scientific and Technological Research Council of Turkey (TÜBĐTAK) for their financial support during my study. And I am indepted to my family… vii TABLE OF CONTE NTS ABSTRACT……………………………………………………………………… iv ÖZ………………………………………………………………………………… v ACKNOWLEDGMENT…………………………………………………………. vii TABLE OF CONTENTS………………………………………………………… viii LIST OF TABLES………………………………………………………………... xi LIST OF FIGURES………………………………………………………………. xii CHAPTER 1. INTRODUCTION 1.1 Bioconversions in Organic Chemistry……………………….. 1 1.2 Chirality and Asymmetric Synthesis…………………………. 6 1.2.1 Chirality-Activity, Structure-Activity Relationships………………………………………... 7 1.2.2 Methods for the Synthesis of Chiral Compounds….. 10 1.2.2.1 Resolution………………………………… 10 1.2.2.1.1 Kinetic Resolution……………… 10 1.2.2.1.2 Other Methods Available For Resolution………………….. 12 1.2.2.2 Asymmetric Synthesis……………………. 12 1.2.2.2.1 Chiral Starting Material…………. 13 1.2.2.2.2 Chiral Reagents………………….. 13 1.2.2.2.3 Chiral Environments…………….. 14 viii 1.3 Synthesis of α-Hydroxy Ketones…………………………….. 14 1.3.1 Chemical Methods………………………………….. 14 1.3.1.1 Manganese(III) Acetate Mediated Oxidation of Enones……………………… 15 1.3.2 Biotechnological Methods………………………….. 16 1.4 Fungi Mediated Conversions: Fungi as Chemical Reagents… 18 1.5 Oseltamivir Phosphate (TAMIFLU)………………………… 21 1.6 Aim of the Work………………………………………………27 2. RESULTS AND DISCUSSION 2.1 Perspective of the Work……………………………………… 28 2.2 Retrosynthetic Analysis of Oseltamivir……………………… 30 2.3 Synthesis of Enone Structure………………………………… 31 2.4 Oxidation of Enone by Chemoenzymatic Method…………… 34 2.5 Microbial Direct Oxidation Reactions……………………….. 43 3. EXPERIMENTAL 3.1 Materials and Equipments……………………………………. 49 3.2 General Procedures 3.2.1 Synthesis of 1-hydroxycyclohexanecarbonitrile (65). 50 3.2.2 Synthesis of 1-hydroxycyclohexanecarboxylic acid (74)…………………………………………….. 51 3.2.3 Synthesis of ethyl 1-hydroxycyclohexanecarboxylate (66)……………. 51 3.2.4 Synthesis of ethyl cyclohex-1-enecarboxylate (67)... 52 3.2.5 Synthesis of ethyl 3-oxocyclohex- 1-enecarboxylate (68)………………………………. 53 ix 3.2.6 Synthesis of (±)-4-acetoxy-1- carboethoxy-cyclohexene-3-one (75)………………. 54 3.2.7 Synthesis of 4-hydroxy-1-carboethoxy- cyclohexene-3-one (69)……………………………. 56 3.2.7.1 Enzymatic Kinetic Resolution of (±)-4-acetoxy-1-carboethoxy- cyclohexene-3-one…………………….. 56 3.2.7.2 Direct Microbial Oxidation Of ethyl 3-oxocyclohex- 1-enecarboxylate (68)…………………. 56 4. CONCLUSION………………………………………………………… 67 REFERENCES…………………………………………………………………… 68 x LIST OF TABLES TABLES Table 1 Enzymatic hydrolysis of 4-acetoxy-1-carboethoxy- cyclohexene-3-one (75)…………………………………………………. 42 Table 2 Microbial hydroxylation of 1-carboethoxy-cyclohexene-3-one (68) by fungi A. oryzae OUT5048 and A. flavus 200120…………………….. 47 xi LIST OF FIGURES FIGURES Figure 1 Whole cell catalyzed biotransformation of nitriles…………………….. 2 Figure 2 Epoxide hydrolase mediated enantioselective hydrolysis……………… 3 Figure 3 Some reactions catalyzed by lipases in aqueous and organic media…… 4 Figure 4 Asymmetric reduction of enone with a reductase from baker’s yeast…. 5 Figure 5 Epoxidation of styrene catalyzed by peroxidase from the cultured cells of N.tabacum ……………………………………………. 6 Figure 6 Examples for the chiral biologically active compounds……………….. 8 Figure 7 The hypothetical interaction between the two enantiomers of a chiral drug and its binding site………………………………………………… 9 Figure 8 Catalytic kinetic resolution……………………………………………... 11 Figure 9 Kinetic resolution of allylic alcohol 16 as reported by Sharpless ……... 12 Figure 10 Cyclocarbonylation reaction of (1R, 4R)-isolimonene (19)…………... 13 Figure 11 α-Hydroxy ketones as building blocks for several active compounds... 15 Figure 12 Mn(OAc) 3 oxidation followed by enzymatic kinetic resolution yields α´-hydroxy and α´-acetoxy ketones……………………………. 16 Figure 13 Synthesis of (1R,2S)-ephedrine……………………………………….. 17 Figure 14 Thiamine pyrophosphate (TPP) structure…………………………….. 17 Figure 15 Fungi mediated conversion of benzil to benzoin and hydrobenzoin….. 20 Figure 16 Oseltamivir phosphate (34) and its active metabolite (35)…………… 21 Figure 17 Key intermediates to oseltamivir phosphate from shikimic acid……... 22 Figure 18 A new azide-free production of oseltamivir starting from shikimic acid.……………………………………………………. 24 xii Figure 19 Corey and co-worker’s approach……………………………………... 25 Figure 20 Shibasaki and co-worker’s approach………………………………….. 26 Figure 21 Aim of the work………………………………………………………. 27 Figure 22 Synthetic route to intermediate 69……………………………………. 30 Figure 23 Retrosynthesis of Oseltamivir………………………………………… 30 Figure 24 Synthesis of cyclohexanone cyanohydrin…………………………….. 31 Figure 25 Synthesis of 1-hydroxycyclohexanecarboxylic acid………………….. 32 Figure 26 Synthesis of cyclohexanol-1-ethylcarboxylate………………………... 33