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Synthetic Applications of Homochiral Glycidic Esters Derived from Enzymatic Reductions

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SYNTHETIC APPLICATIONS OF HOMOCHIR AL GLYCIDIC ESTERS DERIVED FROM ENZYMATIC REDUCTIONS By BRENT DEREK FESKE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Brent Derek Feske

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This document is dedicated to my loving parents.

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ACKNOWLEDGMENTS First, I would like to thank my advisor, Dr. Jon D. Stewart, for his guidance and tutelage. He was always there to answer my questions and was willing to assist me with learning new techniques. In addition, I would like to thank him for taking me on my favorite bike ride. I would also like to thank Dr. Thomas Lyons for his friendship, helpful advice, and a listening ear. I thank the rest of my committee, Dr. Nigel Richards, Dr. Dolbier, and Dr. Madeline Rasche, for their variety of contributions to my education. I would like to thank all of the Stewart group members for their time, advice, scientific help and laughs. I thank Kavitha Vedha-Peters for taking me under her wing and teaching me the basics of organic chemistry. Many thanks go to Iwona Kaluzna for her research collaboration in the laboratory and also her friendship. I thank Brian Kyte for his help in molecular biology, proofreading, and being a good friend. I would also like to acknowledge Despina Bougioukou for giving me her advice EVERYDAY, whether I wanted it or not. In addition, I would like to thank Neil Stowe, Dimitri Daschier, Heather Hillebrenner, Magdalena Swiderska, and Parag Parekh for their combined assistance. I am also grateful for the help I have received from Luke Koroniak with his thorough knowledge of synthetic chemistry. I thank Ion Ghivirigia for his help in determining absolute structures by NMR and other related problems. I also iv

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acknowledge Lori Clark, Dr. Ben Smith, and Dr. Jim Deyrup for their continuous guidance, assistance, and friendship. I would like thank my loving fiance Valerie for her supportiveness and for her understanding during the periods that I lived in the laboratory. Lastly and most importantly, I want to thank my parents for their support during this process. They have continually made sacrifices for me throughout my life and this could never have been achieved without them. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .......................................................................................iv LIST OF FIGURES .............................................................................................viii LIST OF SCHEMES .............................................................................................x ABSTRACT ........................................................................................................xiv CHAPTER 1 HISTORICAL BACKGROUND OF TAXOL....................................................1 Discovery of Taxol..........................................................................................1 Synthetic Strategies for Taxol........................................................................3 Total Synthesis of Taxol..........................................................................4 Isolation of Taxol from Plant Tissue Cultures..........................................7 Taxol Producing Fungus..........................................................................7 Semi-synthesis of Taxol: Synthesis of the Taxol Side Chain................10 Asymmetric metal catalysis.............................................................11 Enzyme catalysis............................................................................15 Resolution of enantiomers...............................................................22 2 HISTORICAL BACKGROUND OF BESTATIN.............................................27 Discovery of Bestatin...................................................................................27 Synthetic Approaches to Bestatin................................................................28 3 STEREOSELECTIVE, BIOCATALYTIC REDUCTIONS OF -CHLORO--KETO ESTERS............................................................................................41 Introduction..................................................................................................41 Results and Discussion................................................................................42 4 SYNTHESIS OF THE C-13 TAXOL SIDE CHAIN........................................47 Racemic Synthesis.......................................................................................47 Biotransformation Strategy...........................................................................48 Optimization of Our Whole-cell System.................................................50 vi

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Whole-cell Assays.................................................................................52 Whole-cell Reduction of 126..................................................................54 Base Catalyzed Ring Closure......................................................................57 Ritter Reaction.............................................................................................59 Ring Hydrolysis to the Taxol Side Chain......................................................60 5 SYNTHESIS OF BESTATIN........................................................................62 Syntheis of -Keto Ester 213.......................................................................62 Chlorination of -Keto Ester 213..................................................................62 Enzymatic Reduction of 214........................................................................63 Whole-cell Reduction of 214........................................................................65 Base Catalyzed Ring Closure of 215...........................................................66 Ritter Reaction.............................................................................................66 Synthesis of Bestatin from 217: First Generation........................................67 Synthesis of Bestatin from 217: Second Generation...................................68 Synthesis of Bestatin from 217: Third Generation.......................................69 6 SYNTHETIC APPROACH TO CHUANGXINMYCIN....................................71 Introduction..................................................................................................71 The Akita labs Approach to Chuangxinmycin..............................................71 Enzymatic Reduction of 220........................................................................73 Whole-cell Reduction of 220........................................................................74 Base Catalyzed Ring Closure of 248...........................................................75 7 CONCLUSIONS AND FUTURE WORK.......................................................77 APPENDIX A EXPERIMENTAL.........................................................................................80 B ADDITIONAL INFORMATION......................................................................97 LIST OF REFERENCES..................................................................................108 BIOGRAPHICAL SKETCH...............................................................................116 vii

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LIST OF FIGURES Figure page 1-1 Natural products with anticancer activity: Vinblastine 1, Vincristine 2, Taxol 3, Camptothecin 4..............................................................................1 1-2 Taxol bound to a tubulin dimer...............................................................2 2-1 Structure of Bestatin 131 (ubenimex) 46,47 ...................................................27 3-1 Biocatalytic reductions of -chloro--keto esters.......................................45 4-1 Production of (S)-ethyl 3-hydroxybutyrate by engineered E. coli cells under non-growing conditions....................................................................50 4-2 SDS-Page of the overexpression of YGL039w over a 4-hour time period. The arrow marks the expected position of the YGL039w fusion protein....52 4-3 Specific activity of the E. coli cells overexpressing YGL039w, which have been grown under different induction temperatures (37 C, 30 C, and 24 C)..............................................................................................................53 4-4 Whole cell activity of an overexpressed YGL039w with a GST tag and YGL039w without a GST tag versus time..................................................54 4-5 Concentration of the product for the biotransformation using YDL124w and YGL039w............................................................................................56 4-6 Diagram of our gentle extraction technique...............................................56 4-7 Effect of Lewis acids on the Ritter reaction................................................60 6-1 Chuangxinmycin 236.................................................................................71 6-2 Final product concentrations for 249, 248, and 215 by the corresponding engineered E. coli......................................................................................74 7-1 Other pharmaceutical drugs that can be synthesized from homochiral glycidic ester intermediates: Diltiazem 252, KRI-1230 253, Amistatin ent-253, and Indolmycin 254............................................................................79 viii

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B-1 Long range coupling constants (Hz) for the major enzymatic reduction product of YDR368w, YGL157w, and YGL039w........................................98 B-2 The chemical shifts for the major reduction products of YDR368w, YGL157w, and YGL039w..........................................................................99 B-3 Coupling constants in the staggered rotamers of threo and erythreo diastereomers of reduction products........................................................100 B-4 Range of values for the large and small coupling constants (Hz)............100 B-5 Line equation for the Bradford assay.......................................................101 B-6 NMR of (+)-AHPA synthesized by our strategy........................................102 B-7 NMR spectra of authentic (+)-AHPA........................................................103 B-8 NMR spectra of the Taxol side chain 130................................................104 B-9 NMR spectra of the Taxol side chain enantiomer ent-130.......................105 B-10 1 H NMR for derivatized 130 and ent-130: Top spectra is the (S)-MPA ester of ent-130; bottom spectra is the (S)-MPA ester of the Taxol side chain 130.................................................................................................106 B-11 1 H NMR spectra of syn product from YDR368w (top) and anti product from TGL157w (bottom)...........................................................................107 ix

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LIST OF SCHEMES Scheme page 1-1 Summary of Holtons approach....................................................................4 1-2 Summary of Nicolaous approach................................................................5 1-3 Summary of Danishefskys approach...........................................................5 1-4 Summary of Wenders approach..................................................................6 1-5 Summary of Mukaiyamas approach............................................................6 1-6 Summary of Kuwajimas approach...............................................................7 1-7 The biosynthetic pathway to Baccatin III......................................................9 1-8 The biosynthetic pathway of Taxol side chain and its coupling to Baccatin III..................................................................................................................9 1-9 Semi-synthesis of Taxol: Coupling of Baccatin III to the Taxol side chain11 1-10 Greenes Strategy to the Taxol side chain.................................................12 1-11 Jacobsens strategy to the Taxol side chain..............................................13 1-12 The Sharpless strategy to the Taxol side chain.........................................13 1-13 Hams strategy to the Taxol side chain......................................................14 1-14 Baruas strategy to the Taxol side chain....................................................15 1-15 Kims strategy to the Taxol side chain........................................................17 1-16 Kayser and Stewartss first strategy to the Taxol side chain using bakers yeast..........................................................................................................18 1-17 Kayser and Stewarts second strategy to the Taxol side chain..................18 1-18 Cardillos strategy to the Taxol side chain..................................................19 1-19 Hamamotos strategy to the Taxol side chain............................................20 x

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1-20 Mandais strategy to the Taxol side chain..................................................21 1-21 Bottas strategy to the Taxol side chain.....................................................22 1-22 McChesneys strategy to the Taxol side chain...........................................23 1-23 Zhous strategy to the Taxol side chain......................................................24 1-24 Our proposed synthesis of the Taxol side chain........................................25 1-25 Dynamic Kinetic Resolution.......................................................................26 2-1 Sudas strategy to Bestatin........................................................................29 2-2 Umezawas strategy to Bestatin.................................................................30 2-3 Pearson and Hines strategy to Bestatin....................................................31 2-4 Norman and Moriss strategy to Bestatin...................................................32 2-5 Palomos strategy to Bestatin.....................................................................32 2-6 Kosekis strategy to Bestatin......................................................................33 2-7 Bergmeier and Stanchinas strategy to Bestatin........................................34 2-8 Sekis strategy to Bestatin..........................................................................35 2-9 Semples strategy to Bestatin.....................................................................36 2-10 Parks strategy to Bestatin.........................................................................37 2-11 Jurczaks strategy to Bestatin....................................................................38 2-12 Wassermans strategy to Bestatin..............................................................39 2-13 Our proposed synthesis of AHPA and Bestatin..........................................40 3-1 Four possible reduction products of -chloro--keto esters: (2S-3S)-white, (2R-3S)-black, (2R-3R)-black/white lines, (2S-3R)-black dashes....42 3-2 Synthesis of all 4 diastereomers by sodium borohydride, which can be separated by chiral gas chromatography...................................................43 3-3 Derivatization technique used to separate all 4 diastereomers for chlorohydrin 218........................................................................................44 4-1 Racemic synthesis of the Taxol side chain................................................48 xi

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4-2 Two chlorination methods for -keto esters...............................................48 4-3 The Taxol side chain and its enantiomer can be synthesized by utilizing two different enzymes; YDL124w and YGL039w, respectively..................50 4-4 Final results for the whole-cell biotransformations after purification...........57 4-5 Results for the ring closure of chlorohydrins: Potassium carbonate results in the kinetic product, whereas sodium ethoxide affords the thermodynamic product.............................................................................58 4-6 Mechanism for the sodium ethoxide promoted epoxidation and a Newman projection describing syn versus anti configuration of chlorohydrins..............................................................................................58 4-7 Base promoted formation of cis-glycidic ester 128.....................................59 4-8 The Ritter reaction of glycidic ester 128 and benzonitrile...........................59 4-9 Hydrolysis of oxazoline 129 under mildly acidic conditions versus strongly acidic conditions...........................................................................61 5-1 Synthesis of -keto ester 213....................................................................62 5-2 Two different approaches to chlorinate 213 with sulfuryl chloride..............63 5-3 Three bakers yeast reductases that accept 214 as a substrate; YDR368w, YGL039w, and YGL157w........................................................64 5-4 The whole-cell biotransformation resulted in the chlorohydrin 215 and dechlorinated product 231.........................................................................65 5-5 Results for our optimized whole-cell reduction of 214................................66 5-6 Base promoted ring closure for glycidic ester 216.....................................66 5-7 The Ritter reaction of 216 and benzonitrile only afforded the trans-oxazoline 217.............................................................................................67 5-8 First attempt for the synthesis of Bestatin..................................................68 5-9 Second attempt for the synthesis of Bestatin.............................................69 5-10 Final Strategy to AHPA 137 and Bestatin 131...........................................69 6-1 Akitas synthesis of (+/-)-Chuangxinmycin 236..........................................73 6-2 Proposed scheme to the Chuangxinmycin intermediate (2R, 3S)-epoxy butanoate 243............................................................................................73 xi i

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6-4 Ring closure promoted by sodium ethoxide...............................................75 6-5 Ring closure of chlorohydrin 248 using potassium carbonate and water...76 6-6 Proposed synthesis to (2R, 3S)-epoxy butanoate 243..............................76 xiii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNTHETIC APPLICATIONS OF HOMOCHIRAL GLYCIDIC ESTERS DERIVED FROM ENZYMATIC REDUCTIONS By Brent Derek Feske December 2005 Chair: Jon D. Stewart Major Department: Chemistry A library of eighteen known bakers yeast reductases has been screened for their ability to reduce several -chloro--keto esters. By using these enzymes in whole-cell biotransformations an easily scaleable method to synthesize gram quantities of homochiral chlorohydrins can be achieved. A stereoselective, base catalyzed ring closure can be used to transform these compounds into enantiopure glycidic esters, which are useful intermediates to several biologically active molecules. Using a whole-cell reduction and a Ritter reaction as key steps, we were able to develop a new route to both antipodes of the C-13 Taxol side chain and a formal total synthesis of (-)-Bestatin. We synthesized the protected form of the Taxol side chain in four steps with an overall yield of 49%. (2S, 3R)-3-Amino-2-hydroxy-4-phenylbutyric acid was synthesized in six steps with an overall yield of 42%, thus completing a formal synthesis of (-)-Bestatin. xiv

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Depending on the substrate, we were able to consistently yield 1 to 4.5 g/L of product for our whole-cell biotransformations. Thus, the overexpression of bakers yeast reductases in whole-cells proved to be an adequate and scaleable method for the synthesis of homochiral chlorohydrins. xv

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CHAPTER 1 HISTORICAL BACKGROUND OF TAXOL Discovery of Taxol In 1960, the National Cancer Institute established a program that purified organic molecules from biological samples and screened the compounds for their pharmaceutical activities. This program was triggered by the early success of Beer et al., who found the antileukemic agents vinblastine 1 and vincristine 2 in periwinkle leaves from Madagascar. 1 Throughout this programs 22 year tenure, only two compounds were found to show medicinal potential, Taxol 3 (paclitaxel) and camptothecin 4 (Figure 1-1). NH3CONOHNHOHCOOCH3OCOCH3NH3CONNHOHCOOCH3OCOCH3COOCH3COOCH3NOHOHH3C12 OHOBzOHOAcOHOAcOABCDOOHONHO3NNOOOOH4 Figure 1-1. Natural products with anticancer activity: Vinblastine 1, Vincristine 2, Taxol 3, Camptothecin 4. Taxol was isolated from the bark of the Pacific Yew tree (Taxus brevifolia) and shown to have anti-tumor activity in 1962. Even though Taxol showed 1

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2 promising anti-cancer activity, further testing slowed because it was highly insoluble in water, which would make it almost impossible to deliver to patients. In addition, as the demand for Taxol increased, the supply of Pacific Yew trees and thus Taxol was diminishing. In 1979, there was a breakthrough in understanding Taxols anti-cancer activity. Susan B. Horwitz, a molecular pharmacologist, along with Schiff et al. found that Taxols mode of action was completely different from those of traditional cancer drugs. 2 Many of these drugs destabilize a cells ability to make microtubules, which are essential for cell replication. Taxols mode of action is actually the opposite: it stabilizes the microtubules during cell replication, preventing their separation (Figure 1-2). Due to the cells inability to divide they will eventually grow so large as to trigger their own death. Figure 1-2. Taxol bound to a tubulin dimer

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3 This newly discovered mode of action accelerated the clinical trials of Taxol. After many years of thorough testing, the Food and Drug Administration (FDA) approved Taxol for the treatment of ovarian and breast cancer in 1992. This newly approved and promising cancer drug quickly developed a strong commercial demand. This led to a problem, however, since the slow growing Pacific Yew tree was considered near the brink of extinction and this precluded an adequate supply of Taxol for the clinical use by isolating the natural product. To solve this problem, chemists were given the task of producing what would become the largest selling cancer drug ever placed on the pharmaceutical market, yielding sales in the billions of dollars. Synthetic Strategies for Taxol The isolation of Taxol from the Pacific Yew tree has low yields, so auxiliary strategies have been developed to obtain this drug. These include total synthesis, plant tissue cultures, engineering of a Taxol producing fungus, and the coupling of Baccatin III (which contains the ring structure of Taxol) with the Taxol side chain. Due to low yields and the high number of steps, the total synthesis of Taxol is unlikely to become an option for commercial production. Ongoing work on the engineering of plant tissue cultures continues, but many scientists believe that plant cells are too difficult to manipulate for high Taxol yields. As a result, engineering the Taxol producing fungus and semi-synthesis by coupling of Baccatin III to the side-chain are considered the primary strategies for commercial production.

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4 Total Synthesis of Taxol In 1971, the Wani et al. group published the structure of Taxol 3, which is composed of a complex poly-oxygenated diterpene and a phenylisoserine side-chain. 3 As a result, synthetic chemists were intrigued by the challenge of synthesizing such a complex organic molecule. It took nearly 20 years, but in 1994 the Holton group and Nicolaou group almost simultaneously reported the total synthesis of Taxol. Since that time, four more total syntheses have been completed on Taxol. The Holton lab approach 4,5 used (-)-camphor 5 as the starting material for a strategy in which the A and B rings of Taxol were created first, then this unit was fused to the C ring (Scheme 1-1). The oxetane (D) ring was subsequently formed through a tosylate intermediate. This synthesis was composed of 41 steps with an overall yield of 2%. HOOROPOPHOPOPOHOPOPO3765O Scheme 1-1. Summary of Holtons approach The Nicolaou group 6-9 first constructed the A and C ring systems from hydrazone 8 and bicyclic aldehyde 9, followed by a McMurry cyclization to form the ABC ring system intermediate 11 (Scheme 1-2). The D ring was added through the formation of a triflate silyl ether intermediate and treated under mildly acidic conditions to make the oxetane. This total synthesis of Taxol was completed in fifty-one steps with an unreported overall yield.

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5 NHSO2ArOTBSOBnOOHOTPSOOHCOBnOOHOOOOH38910HOBnOOHOOOOHHO11 Scheme 1-2. Summary of Nicolaous approach The Danishefsky group 10 used the Wieland Miescher ketone 12 and trimethylcyclohexane-1,3-dione 13 as starting materials for the synthesis of the C and D rings (Scheme 1-3). They then used an intramolecular Heck reaction to fuse the AB rings to the CD ring structure. This synthesis required 47 steps and resulted in an overall yield of less than 0.1%. OOOOHOOHOBzOHOAcOHOAcO3121314 Scheme 1-3. Summary of Danishefskys approach The Wender group 11 utilized -pinene 15 as a matrix to make the A and B rings through a fragmentation technique with an epoxy alcohol (Scheme 1-4). The C ring was added via an aldol condensation and then formation of the D ring following in several steps by direct closure of the diol. This synthesis of Taxol was accomplished in 37 steps with an unreported overall yield.

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6 GOHOOROHOBzOOAcOOROHO3151617 Scheme 1-4. Summary of Wenders approach In 1997, the Mukaiyama group 12,13 reported the total synthesis of Taxol utilizing L-serine 18 as the starting material (Scheme 1-5). In their strategy, the BC ring system was synthesized via a pinacol coupling cyclization; then addition of the A ring followed. In subsequent steps they added the D ring and the Taxol side-chain, thereby affording Taxol with an unreported yield. BnOTBSOPMBOOHOBnHOAcOOTESOHOTESOOO3181920HONH2OHO Scheme 1-5. Summary of Mukaiyamas approach The Kuwajima groups 14 synthesis started with a Peterson olefination to afford dienol silyl ether 21 to form the A ring (Scheme 1-6). Next, 2-bromobenzaldehyde dibenzylacetal 22 was used to synthesize the C-ring fragment. The coupling of the A and C rings and subsequent cyclization to form the B ring resulted in the tricarbocycle. This was converted to Taxol after several additional steps in an unreported overall yield.

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7 OTIPSHOHOSPhBrCH(OBn)2OOHOBzOHOAcOTrocOAcOTBS3212223 Scheme 1-6. Summary of Kuwajimas approach Isolation of Taxol from Plant Tissue Cultures Large-scale plant cell cultures have been shown to be useful sources of certain natural products. 15-17 This usually requires a cell selection with medium optimization, genetic engineering, elicitation of enzyme systems, precursor feeding, and overall process optimization. Research on the optimization of Taxol producing plant cells is still ongoing; however, due to the difficulty of engineering plant cells it is not expected to be a practical route for the production of Taxol. 18-20 As an example, Chang et al. recently collected tissues from Taxus mairei, a plant found in Taiwan at an altitude of 2000 m above see level, and discovered that the amount of Taxol and Taxol derivatives found in this plant were higher than the pacific yew and other Taxus species. 21 However, when using callus cells from Taxus mairei, an optimized cell line was only able to produce 200 mg/L of Taxol after a six week incubation period. Taxol Producing Fungus In 1993, Stierle et al. found that Taxol is produced by the fungus Taxomyces andreanae. 22 Unfortunately, Taxol is only produced at

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8 concentrations of 25-50 ng/L in this organism. On the other hand, fungal cells can be engineered more easily than plant cells, so the future of using engineered cells for the production of Taxol is promising. The Croteau lab has taken on the challenge of fully deciphering the Taxol biosynthetic pathway in Taxomyces andreanae. 23-28 His group has found that there are 19 enzymatic steps from the basic geranylgeranyl diphosphate used as the isoprenoid precursor. The biosynthesis of Taxol begins with the 2-C-methyl-D-erythritol phosphate pathway (MEP) (Scheme 1-7). Isopentenyl diphosphate 24 and dimethylallyl diphosphate 25 are then combined to form geranylgeranyl diphosphate 26 (GGPP) from geranylgeranyl diphosphate synthase. The next step is the cyclization of GGPP to taxadiene 27 by taxadiene synthase. Over the next three steps, taxadiene is decorated with an alcohol and acetate functionality. The order of additional oxygenations beyond this point has not been totally deciphered. MEPPathwayOPPOPPIPPIGGPPSIPPDMAPPtaxadienesynthaseHHHHOHcyctochrome P450taxadiene 5a-hydroxylase1) taxa-4(20), 11(12)-diene-5a-o-acetyl transferase2) cytochromeP450 taxane-10B-hydroxylase2425262728

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9 HHOHOOHOBzOHOAcOHOAcOHOO3029 Scheme 1-7. The biosynthetic pathway to Baccatin III Once Baccatin III 30 has been formed, the C-13 Taxol side chain is added. The side chain begins from -phenylalanine 31, which is converted to -phenylalanine 32 by phenylalanine aminomutase PAM (Scheme 1-8). In the next step, 32 is activated as the corresponding CoA thioester followed by an aroyl transfer to Baccatin III resulting in 33. Lastly, the side chain is hydroxylated at the C-2 position and then N-benzoylated to afford the biosynthetic product, Taxol 3. OHONH2OHONH2ONH2PAM1) CoA thioesterligation2) aroyl transfer1) 2' hydroxylation2) N-benzoylation313233HOOHOBzOHOAcOHOAcOOOHOBzOHOAcOHOAcO30 Scheme 1-8. The biosynthetic pathway of Taxol side chain and its coupling to Baccatin III The strategy for engineering the Taxol-producing fungus is to locate the bottlenecks in the pathway and overexpress the genes responsible for the slow steps. This plan also includes knocking out competitive pathways that may lead to undesirable products; this can be done by adding elicitors or by gene

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10 knockout. If these two strategies are successful, it would increase the concentration of the final product and also allow for easier purification. Semi-synthesis of Taxol: Synthesis of the Taxol Side Chain Taxols path to becoming a commercial drug was an uphill battle, but in the late 1980s it became evident that Taxol would soon become FDA approved. Since a commercial source had not yet been found, Pierre Potier et al. began to study this problem. They found and extracted a compound from the Pacific Yew bush (Taxus baccata), which contains the terpene core of Taxol. 29 This compound, 10-deacetylbaccatin III 34, can be isolated from the leaves of the bush in high yield. In addition, only the leaves are removed from the bush, which can be regenerated by the plant, allowing it to be a renewable source. Upon isolation of 10-deacetylbaccatin III 34, it can be coupled to the protected form of the Taxol side chain 35 and/or 36 yielding the full structure of Taxol 3 (Scheme 1-9). Greene et al. developed a side chain synthesis and coupling procedure; however, it resulted in an enantiomeric excess (e.e.) of only 78%. 30 Due to the low enantiomeric excess, this synthesis did not meet the purity standards set by the FDA.

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11 HOOHOBzOHOAcOHOHONOORONOTESOONHOHOOOHOBzOHOAcOHOAcO3435363 Scheme 1-9. Semi-synthesis of Taxol: Coupling of Baccatin III to the Taxol side chain To answer the commercial demand for Taxol, Dr. Robert Holton developed a metal alkoxide process for the Taxol semi-synthesis in the early 1990s with a 74% overall yield. 31 This patent was licensed by Bristol-Myers Squibb and has been used for the commercial production of Taxol since its approval in 1993. Since then, a number of different approaches to the Taxol side chain have been reported. These approaches are listed within three catagories: asymmetric metal catalysis, enzymatic catalysis, and enantiomer separation. Asymmetric metal catalysis The main challenge for the synthesis of the Taxol side chain is to define the stereochemistries of the C-2 and C-3 positions with high selectivities. Popular solutions to this product involve asymmetric metal catalysts that afford the desired enantiomer in good to high enantiomeric excess. In 1986, The Greene lab was the first to publish the synthesis of the Taxol side chain using this approach (Scheme 1-10). 30 He applied the Sharpless epoxidation methodology to cis-cinnamyl alcohol 37 to afford chiral epoxy alcohol

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12 38 in 78 % e.e., which was then oxidized and esterified by diazomethane to yield glycidic ester 39. Ring opening of the 39 with azidotrimethylsilane yielded azide 40. Reaction with benzoyl chloride under Schotten-Baumann conditions followed by hydrogenation resulted in the Taxol side chain 41. HOHCH2OHt-BuOOH / Ti(OiPr)4(+)-DETHOH1) RuCl3 / NaIO42) CH2N270%90%84%OMeON3OH2) H2 / PdOMeONHOHO89%78% e.e.CH2OH1) C6H5COCl373839OMeO4041Me3SiN3 Scheme 1-10. Greenes Strategy to the Taxol side chain The Jacobsen group synthesis started by a Lindlar reduction of ethyl phenylpropiolate 42 (Scheme 1-11). 32 The key step used (salen) Mn (III) complex 48 as an inorganic asymmetric catalyst resulting in the glycidic ester 44. Ring opening with ammonia and hydrolysis of the amide resulted in -hydroxy--amino acid 46. Addition of benzoyl chloride and treatment with aqueous hydrochloric acid afforded the Taxol side chain 47. CO2EtH2Lindlar cat.CO2EtNaOCl(R,R)-1 (6 mol%)56%OCO2Et96% e.e.84%424344

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13 MnOONNHHt-But-But-But-BuNH3 / EtOH65%NH2ONH2OH1) Ba(OH)22) H2SO4OHONH2OH92%1) BzCl / NaOH2) HCl74%OHONHOHOCl45464748 Scheme 1-11. Jacobsens strategy to the Taxol side chain Sharpless et al. used an inorganic catalyst approach to carry out the asymmetric dihydroxylation of trans-methyl cinnamate 49 (Scheme 1-12). 33 The syn-diol 50 was converted to acetoxy bromo ester 51 by reaction with trimethyl orthoacetate in the presence of a catalytic amount of p-TsOH. After reacting 51 with sodium azide, the acetoxyazide ester was hydrogenated giving N-acetyl-3-phenylisoserine 52. The amide ester was then hydrolyzed to afford the acid and benzoylated to the Taxol side chain 47. OCH3O0.5 mol% (DHQ)2-PHAL0.2 mol% K2OsO2(OH)4NMO (60% in water)t-BuOHOCH3OOHOH72%99% e.e.1) CH3C(OCH3)3 p-TsOH2) AcBr / CH2Cl2OCH3OBrOAcOCH3ONHOHOMeOHONHOHO1) NaN3 / DMF2) H2 / 10% Pd/C1) 10% HCl (aq)2) BzCl / NaOH60%74%72%4950515247 Scheme 1-12. The Sharpless strategy to the Taxol side chain Ham et al. synthesis began with the protection of L-phenylglycine 53, followed by treatment with N,O-dimethylhydroxylamine hydrochloride to afford

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14 Weinreb amide 54 (Scheme 1-13). 34 Reduction of 54 by lithium aluminum hydride followed by a Wittig reaction resulted in ester 55. Treatment of 55 with DIBAL gave alcohol 56, which was acetylated over a three step process resulting in 57. The key step was a palladium catalyzed oxazoline formation yielding only trans-compound 58. This was then oxidized to acid 59 by sodium periodate. The resulting acid 59 was reacted with diazomethane resulting in the protected Taxol side chain 60. 1) (Boc)2O / NaOH2) HNCH3(OCH3)-HCl81%NONHOBoc1) LAH / ether2) (CH3O)2POCH2CO2CH380%NHOCH3BocODIBAL / BF3OEt289%NHOHBoc1) 3 N HCl2) BzCl / Et3N3) Ac2O / pyridine78%NHOAcBz5% Pd(PPh3)4 / K2CO378%NORuCl3 / NaIO478%NOCH2N2 / ether99%OHONOOMeOOHNH2O5354555657585960 Scheme 1-13. Hams strategy to the Taxol side chain The Barua lab began by reacting benzyl alcohol 61 and epichlorohydrin to afford epoxide 62 (Scheme 1-14). 35 Treatment of 62 with 30% HClO 4 gave the

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15 diol, which was oxidized to the aldehyde 63 with Pb(OAc) 4 The key step of this synthesis was a Shibasaki asymmetric Henry reaction with aldehyde 63 and phenylnitromethane 64 yielding alcohol 65 with a 90% e.e. Simple acetylation of the Henry adduct, followed by a hydrogenation resulted in 67. After oxidation of the alcohol using CrO 3 68 was benzoylated with benzoyl chloride and then the acetate was hydrolytically removed to the C-13 Taxol side chain 47. OHOONO2OBnNO2OHOBnNO2OAcOHNH2OAcOHNH2OAcOOHNHOHOOepichlorohyrdrin50% aq. NaOHTBAB80%1) 30% HClO42) Pb(OAc)468%La-(R)-BINOL80%Ac2O/Py93%10% H2 Pd/C80%CrO3 / AcOH / H2O82%1) BzCl / NaOH2) Et2NMe-H2O75%OOH616263646566676847 Scheme 1-14. Baruas strategy to the Taxol side chain Enzyme catalysis Another strategy to afford the proper stereochemistry at the C-2 and C-3 positions of the Taxol side chain involves the use of enzymes. Typically, acylases or lipases are the enzymes of choice for organic chemists. These enzymes can be an efficient source for chirality; however, the maximum yield possible from the racemate is fifty percent. Despite these problems, there have been several published syntheses using enzymes as a key step in their synthesis.

PAGE 31

16 The Kim labs Taxol side chain synthesis began with a kinetic resolution of racemic diol 69, using the lipase from Pseudomonas cepacia (Scheme 1-15). 36 Unlike other kinetic resolutions, in which there is at least a fifty percent loss in yield, Kims synthesis utilized both lipase products as precursors for the Taxol side chain. After the enzymatic reaction, 70 was tosylated, which allowed for ring closure by the addition of a weak base. Glycidic ester 39 was then reacted with sodium azide to form 40. The other enzymatic product, 72, was brominated by hydrobromic acid and acetic acid to afford 73. Reaction of 73 with sodium azide and then sodium acetate also gave compound 40. These compounds were combined and treated with benzoyl chloride, followed by a palladium catalyzed hydrogenation resulting in the Taxol side chain 41. OMeOHOHOOMeOHOOMeOHOOHOAcOMeOAcOOTsOMeOOAcBrLPS / vinylacetate44%54%87%89%TsCl /Et3NHBr-AcOH6970717273

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17 OOMeOOMeOOHN3OMeOOHNHO83%94%85%88%p-TsOH /K2CO3NaN31) NaN32) AcONa1) BzCl / Et3N2) H2 Pd-C394041 Scheme 1-15. Kims strategy to the Taxol side chain In 1999, Kayser and Stewart et al. also used an enzymatic approach to synthesize the Taxol side chain. 37 They developed two syntheses, both utilizing stereoselective ketone reductions by bakers yeast. In the first strategy, racemic glycidic ester 74 was opened with sodium azide, then the resulting alcohol was oxidized by Jones reagent to yield 75 (Scheme 1-16). Incubation of racemic 75 with bakers yeast gave the diastereomeric reduction products 76. syn-Azido alcohol 40 was purified by column chromatography and the Greene strategy (Scheme 1-10) was used to complete the Taxol side chain 41. OOMeON3OOMeON3OH27%1) NaN3bakers' yeast67%7 : 3 (syn : anti)OMeO7675742) Jones reagent

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18 OMeON3OH2) H2 / PdOMeONHOHO89%1) C6H5COCl4041chromatography Scheme 1-16. Kayser and Stewartss first strategy to the Taxol side chain using bakers yeast The second approach began with the LDA-mediated addition of 77 and 78, resulting in racemic -lactam 79 (Scheme 1-17). The ketal was hydrolyzed with concentrated sulfuric acid, then the ketone was reduced by incubation with bakers yeast resulting in three of the four possible diastereomers. syn-Alcohols 81 and 82 were separated from 83 by chromatography to yield a protected form of the Taxol side chain with an enantiomeric excess of 82%. EtOOEtOOEtNPMPNOEtEtOOPMPNOPMPOLDA91%H2SO485%NOPMPHONOPMPNOPMPHONOPMPHOHObakers' yeast41%4%8%0%7778798081828384 Scheme 1-17. Kayser and Stewarts second strategy to the Taxol side chain The Cardillo group synthesized racemic 88 by reaction of benzaldehyde 85 with malonic acid 86 and ammonium acetate 87, which was subsequently benzoylated with benzoyl chloride (Scheme 1-18). 38 Incubating 89 with penicillin G acylase resulted in formation of homochiral -amino acid 91. This was benzoylated and reacted with thionyl chloride and methanol, resulting in methyl

PAGE 34

19 ester 92. The addition of LiHMDS produced the lithium dianion, which after reacting with iodine, led to rearrangement product 60. Refluxing oxazoline 60 with weak aqueous hydrochloric acid afforded the Taxol side chain 41. HOHOOHOOOO-NH4+EtOH / reflux75%OHNH2OBzCl / Et3N75%OHNHOOPenicillin G acylase100%OHNHOOOHONH2OHONH21) Et3N / BzCl2) SOCl2 / MeOH70%OMeNHOO1) LiHMDS2) I295%NOOMeO1M HCl / MeOHOMeNHOOOH85%8586878889909191926041 Scheme 1-18. Cardillos strategy to the Taxol side chain The Hamamoto labs synthesis approach began with the Darzens condensation of 85 and 93 to produce -keto--chloro ester 94 (Scheme 1-19). 39 KS-Selectride reduced 94 to give predominately anti-chlorohydrin 95. Lipase then resolved 95 to afford homochiral chlorohydrin 96, which was reacted with sodium azide to afford 40. Reaction of 40 with benzoyl chloride followed by a palladium catalyzed hydrogenation afforded the Taxol side chain 41.

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20 HOOMeOClClOMeClOONaOMe91%KS-Selectride81%OMeClOHONaN382%LipaseOAcOMeClOHO46%99% e.e.OMeOHON31) BzCl / DMAP2) H2 Pd/C70%OMeONHOHO85939495964041 Scheme 1-19. Hamamotos strategy to the Taxol side chain The Mandai group began by reacting phenylacetic acid 97 with LDA to form the lithium dienolate, which was reacted with acrolein, then stirred with 3 N hydrochloric acid to afford acid 98 (Scheme 1-20). 40 In the next step, 98 was esterified with allyl alcohol to give 99, which was reacted with Chirazyme in 2-propenyl acetate and toluene to give 100 and 101. The latter was transformed into cyclic carbamate 102, via the Curtius rearrangement of the free acid produced by the palladium-catalyzed hydrogenolysis of the allyl ester. Carbamate 102 was then protected with (Boc) 2 O and oxidized with ruthenium oxide and sodium periodate to afford acid 103. Treatment with 2 M sodium hydroxide resulted in ring opening, and trifluoroacetic acid was used to remove the Boc protecting group. In the last step, benzoyl chloride was used to afford the Taxol side chain 47.

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21 CO2H1) LDA (2.1 eq)2) THF / acroleinHO2COHOHH2SO4 / CH2Cl2AllylO2COH70%85%AllylO2COHAllylO2COAcAllylO2COHChirazymeCH2=C(OAc)Me48% (98.5% e.e)49% (99% e.e)AllylO2COHOHNOOBocNOCO2HCO2HHNOHtoluene1) Pd(OAc)2 / PPh32) DPPA / Et3N80%88%1) (Boc)2O / Et3N / DMAP2) RuO2 / NaIO41) NaOH / MeOH2) TFA3) BzCl / NaHCO371%O9798999910010110110210347 Scheme 1-20. Mandais strategy to the Taxol side chain The Botta group began by adding the Grignard salt of acetylene to benzaldehyde 85 (Scheme 1-21). 41 Next, the racemic alcohol 104 was subjected to a Ritter reaction with acetonitrile and sulfuric acid. Alkyne 105 was then hydrogenated in the presence of Lindlar catalyst, and deacetylated by aqueous hydrochloric acid to yield 107. The enantioselective acetylation of 107 using Candida antartica lipase resulted in 108 and 109. Amide 109 was deacetylated with aqueous acid to afford 110, which was then benzoylated with benzoyl chloride. The addition of OsO 4 and NMO to 111 oxidized the alkene to a mixture of alcohol diastereomers. After oxidation with Jones reagent, L-Selectride yielded predominately 114. The Deoxo-Flour reagent was added which resulted in the ring closure product, oxazoline 115. Oxidation of 115 with PCC and then acid catalyzed hydrolysis resulted in the Taxol side chain 41.

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22 HOOHNHONHONH2NH2NHOMgBrMeCN / H2SO4H2 / LindlarsHClCandida antartica lipase /AcOEt99%38%43% / 98% e.e.46% / 98% e.e.NHOHCl32%NH2BzCl / Py / DMAPNHO1) OsO4 / NMONHO99%2) TBDPSCl ImidazoleOTBDPSOHJones reagent48%84%NHOOTBDPSOL-selectride99%NHOOTBDPSOHNOPhORNOPhOMeOOMeOOHNHODeoxo-Fluor94%1) PCC2) CH2N250%HCl85%105104851091081061071091101111121131141156041 Scheme 1-21. Bottas strategy to the Taxol side chain Resolution of enantiomers As seen above, popular published methods of the Taxol side chain use either an asymmetric metal catalyst or an enzymatic catalyst to afford the proper stereochemistry. However, some other strategies involve the racemic synthesis of the Taxol side chain, followed by the separation and purification of enantiomers.

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23 The McChesney labs approach utilized the Darzen reaction of methyl chloroacetate 116 and benzaldehyde 85 (Scheme 1-22). 42 Reaction of 117 with dry hydrochloric acid opened the epoxide with retention of configuration at C-3 affording chlorohydrin 118. Ring closure using basic Amberlite 400 resin gave cis-glycidic ester 119. Ring opening with ammonia followed by benzoylation afforded 120. Reaction of 120 with the acidic Amberlite resin 120 in methanol resulted in the racemic Taxol side chain. This was then resolved by entrainment to afford 41 with an enantiomeric excess of 95%. HOClOMeONaOMe / MeOHOCO2MeHCl / benzeneCO2MeClOHHHAmberlite 400 (-OH)OCO2Me73%70%60%NH3 / MeOHNH2ONH2OHBzCl / NaOHNH2ONHOHO1) Amberlite 120 (H+)2) ResolutionOMeONHOHO69%74%65%851161171181194512041 Scheme 1-22. McChesneys strategy to the Taxol side chain The Zhou group began with ammonolysis of the glycidic ester 121 to yield isoserineamide 122 (Scheme 1-23) 43 Benzoylation provided 123, and acid catalyzed methanolysis gave the methyl ester 124. The use of thionyl chloride and hydrochloric acid inverted the C-2 hydroxyl to produce 41, which was subsequently hydrolyzed to the racemic acid. This was resolved with R-(+)-

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24 methyl benzylamine to provide the Taxol side chain 47 with an unreported enantiomeric excess. OCO2EtNH2NH2OHONH2NHOHOOOMeNHOHOOOMeNHOOOHOHNHOOOHNH4OH81%Et3N / BzCl75%67%49%78%TsOH / MeOHSOCl2 / HCl1) K2CO3 / H2O2) Resolution1211221231244147 Scheme 1-23. Zhous strategy to the Taxol side chain As seen above, there are numerous approaches to the synthesis of the Taxol side chain. Many of these routes are often limited by the use of unsafe chemicals and/or conditions that are difficult for industrial scale-up. In addition, sometimes the synthetic routes do not yield a product with a high enantiomeric excess (e.e.) or diastereomeric excess (d.e.), which is essential for the commercial sale of pharmaceuticals. Many strategies to the Taxol side chain use reactions with lipase or the resolution of enantiomers, both of which can be very time consuming, thus inhibiting an industrial high-throughput process. With this said, it was our goal to develop an efficient and easily scaleable reaction for the homochiral C-13 Taxol side chain. We proposed a five step synthesis for the Taxol side chain, which begins with the reaction of ethyl benzoylacetate 125 with sulfuryl chloride to afford -chloro--keto ester 126 (Scheme 1-24). Ester 126 can be added to Escherichia coli (E. coli) that have overexpressed a single bakers yeast reductase, to yield

PAGE 40

25 homochiral chlorohydrin 127. Treatment of 127 with a weak base should yield the optically pure cis-glycidic ester 128. Subsequent treatment of the epoxide with benzonitrile and a catalytic Lewis acid can result in trans-oxazoline 129. Oxazoline 129 can then be treated under mildly acidic conditions to form the optically pure Taxol side chain 130. This reaction scheme utilizes mild conditions and reagents that can be used on a large scale. OOOEtOOOEtClOEtOHClOOCO2EtNOCO2EtONHOEtOOHSO2Cl2K2CO3BF3-OEt2 / PhCNH3O+YDL124w125126127128129130 Scheme 1-24. Our proposed synthesis of the Taxol side chain As seen in the proposed synthesis of the Taxol side chain we plan to utilize the reduction products of -chloro--keto esters using a single bakers yeast reductase. The enzymatic product has four possible diastereomers, given by a dynamic kinetic resolution (Scheme 1-25). This is made possible through the low pKa of the -proton in -keto esters. The average pKa for this functionality is 10, which allows the -carbon to quickly epimerize. Thus, if a reductase exhibits a preference for either substrate enantiomer, the rapid racemization re-establishes the equilibrium. This allows all of the starting material to be converted to a single diastereomer product. Once the substrate has entered the active site, the reductase will transfer a hydride from NADPH to the -carbon

PAGE 41

26 typically resulting in a highly stereoselective reduction. This characteristic is vital to our strategy, because it inserts two chiral centers in our scheme in a highly stereoselective manner. ROOOEtClHROOOEtHClROOEtClHROOEtHClOHOHHHFast[H][H] Scheme 1-25. Dynamic Kinetic Resolution

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CHAPTER 2 HISTORICAL BACKGROUND OF BESTATIN Discovery of Bestatin In 1975, The Umezawa group discovered and isolated an antitumor and antimicrobial agent from Streptomyces olivoreticuli named Bestatin 131 (ubenimex) (Figure 2-1). 44 Bestatin was found while screening cultures of actinomycetes for their ability to inhibit aminopeptidase B. This screening was ignited by the recent findings that exopeptidases have a strong effect on mammalian cell surfaces. 45 At that time, the research started as a hypothesis, but after 30 years, several aminopeptidase inhibitors are now used to treat a variety of cancers and antibiotic infections. NH2OHNHOOHO131 Figure 2-1. Structure of Bestatin 131 (ubenimex) 46,47 The function of Aminopeptidase B is to hydrolyze the N-terminal lysyl and arginyl residues from peptide substrates. 44,48 Aminopeptidase B is also thought to play a role in processing various peptide signals and precursor enzymes, by binding to membrane macrophages and lymphocytes through membrane aminopeptidases. 49,50 This binding induces a cascade of responses like increased cytokines, colony stimulating factors, and cell apoptosis. 50-52 27

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28 Presently, Bestatin is used as an oral medication for the treatment of cancer and bacterial infection in Japan. In addition, Bestatin is often used in conjunction with other antibiotics and anticancer drugs because it causes the proliferation of T cells thus enhances the immune response. 50 Bestatin also shows potential as an anti-inflammatory agent and for the treatment of HIV. 53-59 In 1976, shortly after its discovery, the Nakamura labs published the crystal structure of Bestatin, confirming that the compound was the dipeptide [(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine. 47 Upon verification of the correct configuration, Suda et al. began the first synthesis of this challenging molecule that contains three asymmetric centers. Since this publication, there have been several documented asymmetric syntheses of this popular anticancer and antimicrobial agent, which will be introduced in chronological order. Synthetic Approaches to Bestatin The Suda labs 60 1976 strategy began with Boc-protected D-phenylalanine 132, which was coupled with pyrazole to form pyrazolide 133 (Scheme 2-1). This was reacted with 2 equivalents of lithium aluminum hydride to afford aldehyde 134. The diastereomeric mixture of bisulfite adducts 135 was then reacted with sodium cyanide resulting in acyl cyanide 136. Hydrolysis of 136 with 6 M hydrochloric acid gave the diastereomeric acid mixture, which was separated by chromatography to afford the natural occurring amino acid (2S, 3R)-(3-amino-2-hydroxy-4-phenylbutanoic acid (AHPA) 137. This was reprotected with Boc-Cl before it was reacted with benzoyl protected L-leucine. A simple hydrogenation of 139 afforded Bestatin 131 in an overall yield of 14%.

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29 OHNHOBocpyrazole / DCCNHOBocNNLiAlH4HNHOBoc83%2eq NaHSO374%SO3NaNHOHBocNaCN94%CNNHOHBocNH21) 6 N HCl 2) chromatographyOHOHONHOHOHOBocCl78%BocL-leu-OBz / DCC34%NHOHNHOBocOBzOH2 / Pd/C91%NH2OHNHOOHO132133134135136137138139131 Scheme 2-1. Sudas strategy to Bestatin The Umezawa labs 61 synthesis of Bestatin commenced by reacting N-acyl--aminoacetophenone 140 and glyoxylic acid 141 to yield 142 (Scheme 2-2). This was then subjected to a palladium catalyzed hydrogenation, which reduced the benzylic carbon to afford 143. The racemate of 143 was resolved with S-(-)--methylbenzylamine to give the optically pure salt 144. Refluxing 144 in aqueous hydrochloric acid gave the free amine 136 that was Boc-protected. The protected peptide 145 was formed by the DCC coupling of benzyl protected L-leucine. A simple hydrogenation resulted in Bestatin 131 in an overall yield of 10%.

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30 HNOOHOHOOOHOOHONHONaHCO362%H2 / Pd/C82%OHOHONHOS(-)MBAresolution43%OHOHONHO64%HClOHOHONH21) (Boc)2O2) L-leu-OBz / DCCHNOHONHOBnO1) H2 / Pd/C85%86%HNOHONH2OHOBoc140141142143144136145131 Scheme 2-2. Umezawas strategy to Bestatin Pearson and Hines 62 synthesis began with dioxalanone 146, which underwent an aldol condensation with phenyl acetaldehyde resulting in the mixture of diastereomers 147 (Scheme 2-3). The diastereomers were separated by column chromatography and homochiral 147 was reacted with diphenylphosphoryl azide to form azido compound 148. Refluxing 148 in aqueous hydrochloric acid resulted in ester 149. The ethyl ester was saponified, followed by a DCC coupling of the acid and the benzyl protected L-leucine. The peptide 150 was then deprotected by a palladium catalyzed hydrogenation resulting in Bestatin 131. OOPhOOOPhOOOPhON31) LiHMDS2) phenyl acetaldehyde56%Ph3P / DEAD / DPPA79%OH146147148

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31 EtOH / HCl59%OEtN3OHO1) LiOH / H2O2) L-leu-OBn60%NHNH2OHOOBnOH2 / Pd/C86%NHNH2OHOOHO149150131 Scheme 2-3. Pearson and Hines strategy to Bestatin Norman and Morriss 63 synthesis began with the diesterification of L-malic acid 151 resulting in 152 (Scheme 2-4). This was benzylated under basic conditions and the adduct was saponified to diacid 153. This was selectively esterified by forming the cyclic anhydride intermediate, which was opened regioselectively by ethanol to form 154. A base catalyzed Curtius rearrangement with diphenylphosphoryl azide resulted in the protected compound 155. Ester 155 was saponified with lithium hydroxide, which was coupled using EDC with L-leucine methyl ester affording 156. This was deprotected with 1 M sodium hydroxide to yield Bestatin 131. OHOEtEtOOOOHOHHOOO1) LHMDS / PhCH2Br2) 1 M NaOHOHOEtHOOO1) TFAA2) EtOH1) DPPA / Et3NONHOEtOO1) LiOH2) Leu-OCH3 / NMM / EDC / HOBt70%97%65%64%OHOHHOOOEtOH / H+151152153154155

PAGE 47

32 1M NaOHONHHNOOHOHONH2HNOOHOO100%156131 Scheme 2-4. Norman and Moriss strategy to Bestatin The Palomo group 64 started their approach by coupling 157 and 158 to form -lactam 159 (Scheme 2-5). This was followed by a two-step dehydroxylation of the benzyl carbon yielding 160. The -lactam 160 was protected with (Boc) 2 O and then reacted with L-leucine and sodium azide to form adduct 162. The addition of TFA followed by hydrogenation gave the deprotected compound 131. NOTBDMSPMPPySOBnO79%TiCl4 / TEANOBnOPMPOH1) NaH / MeI2) n-Bu3SnH / Et3BNOBnOPMPNOBnOBocHHHHHH1) (NH4)2Ce(NO3)62) (Boc)2O / DMAPL-Leu-OBn / NaN3NHNHCO2BnOBnOBoc1) TFA2) H2 / Pd/CH2NNHCO2HOHO73%60%88%98%157158159160161162131 Scheme 2-5. Palomos strategy to Bestatin The Koseki group 65 commenced their synthesis of Bestatin from 2,3-isopropylidene-D-ribose 163 (Scheme 2-6). The addition of phenyl magnesium bromide to the sugar gave diastereomeric mixture of Grignard adducts 164. The addition of sodium periodate gave the cyclic product 165, which was purified and

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33 reacted with Jones reagent to form cyclic ester 166. A hydrogenation followed by a DCC coupled addition of benzyl protected L-leucine afforded amide 167. Diol 168 underwent a two-step process mediated by 1-methyl-2-fluoropyridine to afford azide 169, which was hydrogenated to afford Bestatin 131. OHOOHOOPhMgBr / THFOOHOOHOHNaIO4 / etherOOOHO1) chromatography2) Jones reagentOOOO1) H2 Pd/C2) L-leu-OBn / DCC83%OOHNOOOBnTFA43%HNOHOOBnOOH1) 1-methyl-2-fluoropyridine2) NaN3 / HMPAHNOHOOBnON3HNOHOOHONH258%H2 / Pd/C163164165166167168169131 Scheme 2-6. Kosekis strategy to Bestatin Bergmeier and Stanchina 66 began with the protection of mannitol 170, which was oxidized to acid 172 (Scheme 2-7). This was reduced by sodium borohydride to form aldehyde 173, which was subjected to a Wittig reaction, and subsequent acid treatment to afford allylic alcohol 175. This was selectively monosilylated with tert-butyldiphenylsilyl chloride, followed by reaction with CDI and sodium azide to form 177, which was then heated in a sealed tube to yield cyclized product 178. Aziridine 178 was reacted sequentially with phenyllithium

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34 and TBAF to form 179, which was oxidized and coupled with protected L-leucine to provide dipeptide 180. The next three steps resulted in Bestatin 131 with an overall yield of 6% in 18 steps. HOOHOHOHOHOHOOOOOHOHOOOHOOOHO1) (COCl)22) NaBH4Ph3PCH2OOHOOHMe2C(OMe)2 /SnCl21) NaOI4 / NaHCO32) KMnO4 / KOH77%85%80%Dowex(H+)91%TBDPS-Cl /imidazole91%TBDPSOOH1) CDl2) NaN341%TBDPSOOON3109 oC / CH2Cl2sealed tubeTBDPSOONOH1) PhLi / CuCN2) nBu4NFHOONHO1) RuCl3 / NaIO42) L-Leu-OtBu / TBTU50%83%HNONHOOOt-BuO(BOC)2OHNONHOOOt-BuOHOHNOOOHNH21) LiOH2) TFA95%68%170171172173174175176177178179180181131 Scheme 2-7. Bergmeier and Stanchinas strategy to Bestatin The Seki labs 67 approach began with protected L-aspartic acid 182, which was converted in three steps to oxazolidinone 183 (Scheme 2-8). Treatment of 183 with benzyl bromide afforded 184. The lithium enolate of 184 generated by LiHMDS and subsequent treatment with 3-phenyl-2-(phenylsulfonyl)oxaziridine gave the chiral alcohol 185. Hydrogenation of 185 resulted in amino alcohol 186,

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35 which was then treated with benzyl chloroformate to protect the free amine. Alcohol 187 was reacted with DEAD and formic acid resulting in formate 188. This 189 was reacted with L-leucine, then deprotected with hydrogenolysis to yield Bestatin 131. Cbz-ClHOOEtONHMeO2COONHOMeOONOBnMeOONOBnMeOOHOMeOOHNH2OMeOOH1) PCl5 / PhH / AlCl32) PhSiHMe2 / TFA3) NaOMe / THF34%BnBr / Ag2CO383%1) LiHMDS2) phenylsulonyl oxazaridineH2 / Pd/C / HCl86%62%85%HOOEtONHMeO2COONHOMeOONOBnMeOONOBnMeOOHOMeOOHNH2OMeOOHNHCbzOMeOOMeONHCbzOCHODEAD / HCO2H60%1) NH32) NaOH70%NHONHCbzOHOBnOOMeOOMeONHCbzOHL-leu-OBn / DCC99%182183184185186187188189190 H2 / Pd/C90%NHONH2OHOHO131 Scheme 2-8. Sekis strategy to Bestatin The Semple group 68 approached their synthesis by utilizing the coupling of 192 and 194 (Scheme 2-9). The synthesis began with N--Cbz-D-Phe-H that was reduced over two steps to form aldehyde 192. Next, benzyl protected L

PAGE 51

36 leucine 193 was reacted over two steps to form isonitrile 194. After the addition of trifluoroacetic acid, 192 and 194 were reacted to form the diastereomeric mixture of dipeptides 195. After a simple hydrogenation, the diastereomers of 131 were separated by liquid chromatography, resulting in pure Bestatin 131 in a 13% overall yield. CbzHNOOHCbzHNOHHCl-H2NOOCNOO1) BF3-THF2) Pyr-SO3 / Et3N83%1) CH3CO2CHO / Et3N2) Cl3CO2CCl / NMM84%TFA / pyridine65%H2NNHOOOBnOH1) H2 / Pd/C2) HPLC separationH2NNHOOOHOH29%191192193194195131 Scheme 2-9. Semples strategy to Bestatin The Park group 69 synthesis began with an alkyne Grignard reaction on (R)-phenylalaninal resulting in predominately syn-197 (Scheme 2-10). O-Benzylation of 197 afforded 199, and the alkyne was subsequently oxidized to acid 200. This underwent a DCC coupling to produce 201, which was subjected to a two-step deprotection process that resulted in Bestatin 131.

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37 HONHPfOHNHPfMgBrOHNHPf96%9.5:1 Major9.5:1 MinorOHNHPfOBnNHPfOBnNHPfOHOBnBr / NaHKMnO4 / HOAcOBnNHPfLeu-OCH3OOHNH2HNOCOOHL-Leu-OCH3 / HOBtDCC / TsOH1) LiOH 2) H2 / Pd/C97%87%91%93%196197198197199200201131 Scheme 2-10. Parks strategy to Bestatin In 2003, the Jurczak labs 70 began their synthesis with aldehyde 202. They utilized a nitro aldol reaction with 1-nitro-2-phenylethane 203 (Scheme 2-11). The aldol product was purified by chromatography to yield homochiral 204. A Nitro group reduction by Raney-Ni hydrogenation, followed by the addition of a Boc protecting group resulted in 205. This was cyclized in the presence of DMP to the protected form of -amino -hydroxy acid 206. Addition of L-leucine to 206 resulted in protected dipeptide 207. This was followed by two deprotection steps that yielded optically pure Bestatin 131.

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38 OHOONO21) Al2O32) ChromatographyOOOHNO21) H2 / Raney-Ni2) (Boc)2OOOOHHNBoc1) DMP / TsOH2) NaOMeBocNOL-Leu-OMeBocNOOMeOHNOOOMeH2NOHHNOOOH1) TsOH2) 1 N HCl81%93%78%95%70%202203204205206207131PhPhPh Scheme 2-11. Jurczaks strategy to Bestatin The Wasserman groups 71 synthesis began with N-Boc-D-phenylalanine 132 which was coupled with (cyanomethylene) triphenylphosphorane 208 to afford 209 (Scheme 2-12). This was reacted with ozone and L-Leu-OBn to form the doubly protected dipeptide 210. A stereoselective reduction with zinc borohydride resulted in 145 with a diastereomeric excess of 86%. Refluxing with aqueous hydrochloric acid followed by a palladium catalyzed hydrogenation resulted in Bestatin 131. OHNHOPPh3CNEDCl / DMAP88%NHOBocBocCNPPh31) O3 / CH2Cl22) L-Val-OBn62%NHOBocNHOOBnO1) Zn(BH4)2NHBocNHOOBnO85%OH132208209210145

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39 1) 2M HCl2) H2 / Pd/C92%NH2NHOOHOOH131 Scheme 2-12. Wassermans strategy to Bestatin Over the past 30 years, there have been several documented strategies for the synthesis of Bestatin. Most of these utilize amino acids or other naturally occurring chiral compounds for their starting material. Other strategies require enantiomer separation by chiral resolution; however, this process can be very difficult and often inefficient. It was our goal to develop a novel synthesis for AHPA and Bestatin through our simple glycidic ester approach. Our proposed synthesis begins with a literature procedure using meldrums acid 212 and phenylacetyl chloride 211 to yield 213 (Scheme 2-13). Reaction of -keto ester 213 with sulfuryl chloride affords -chloro--keto ester 214. Compound 214 can be added to E. coli that have overexpressed a bakers yeast reductase, affording (2R-3S)-chlorohydrin 215 as the homochiral reduction product. Treatment of chlorohydrin 215 with potassium carbonate will result in cis-glycidic ester 216. Epoxide 216 can be treated with borontrifluoride diethyl etherate and benzonitrile to afford the rearrangement product 217. Reflux with 6 M HCl should result in hydrolysis product (2S, 3R)-(3-amino-2-hydroxy-4-phenylbutanoic acid (AHPA) 137. Following the Suda labs synthetic strategy, AHPA 137 can be coupled with L-leucine over three steps to yield Bestatin 131.

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40 OClOOOO1) pyridine (2.5 eq)2) EtOHOOOEtSO2Cl2OOOEtClYDR368wOHOOEtClK2CO3OOEtOBenzonitrile / BF3OEt2NOOEtO6 M HClH2NOHOHOH2NOHHNOOOH1) DCC / L-Leu-OBz2) H2 / Pd/CNHOHOHOBocClBoc138211212213214215216217137131 Scheme 2-13. Our proposed synthesis of AHPA and Bestatin

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CHAPTER 3 STEREOSELECTIVE, BIOCATALYTIC REDUCTIONS OF -CHLORO--KETO ESTERS Introduction Homochiral glycidic esters are versatile intermediates that can be converted into a variety of high-value products. Optically active glycidates can be prepared by a number of routes including asymmetric Darzens reactions, chiral alkene oxidation methodologies and by ring closure of homochiral -halo--hydroxy esters. 72-86 We were particularly interested in the last strategy because asymmetric reductions of -chloro--keto esters might afford each of the four possible glycidate precursors via dynamic kinetic resolution processes from single, inexpensive starting materials (Scheme 3-1). Here, we explore the potential of individual reductase enzymes from bakers yeast (Saccharomyces cerevisiae) as solutions to the problem of obtaining homochiral glycidate precursors. Reductions of -chloro--keto esters by whole cells of commercial bakers yeast generally produce disappointing mixtures of alcohol diastereomers. 87-90 Recent work has revealed that the yeast genome encodes a large number of reductases and it seemed likely that their simultaneous participation was mainly responsible for the modest stereoselectivities commonly observed in yeast-mediated ketone reductions. 91-93 In response, we have adapted a fusion protein strategy 94 that allows the properties of yeast reductases to be assessed 41

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42 ROEtOClO[H]R = MeR = EtR = n-PrR = PhR = BnRCO2EtRCO2EtOHOHClClRCO2EtRCO2EtOHOHClCl220221222126214 Scheme 3-1. Four possible reduction products of -chloro--keto esters: (2S-3S)-white, (2R-3S)-black, (2R-3R)-black/white lines, (2S-3R)-black dashes individually, so that enzymes yielding homochiral products can be uncovered. 95,96 Moreover, after a reductase with the desired properties has been identified, whole Escherichia coli cells expressing the same protein can be employed for bioconversions on preparative scales using glucose fed-batch conditions. 97 Cellular metabolic pathways supply NADPH, and the whole cells display very high stereoselectivities because they overexpress only a single yeast reductase. Results and Discussion A series of five -chloro--keto esters was used in this study (Scheme 3-1). Eighteen yeast reductases were isolated as fusion proteins with glutathione S-transferase using previously-described methods. 96 The collection of enzymes included members of the aldose reductase, D-hydroxyacid dehydrogenase, medium chain dehydrogenase and short chain dehydrogenase superfamilies. Each -chloro--keto ester was tested as a substrate for each reductase in the presence of NADPH, which was supplied by a cofactor regeneration system. For comparison, parallel reductions were also carried out with commercial bakers yeast cells for the two cases where literature data were not available. 98

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43 Since we were concerned with the enantiomeric and diastereomeric excess values for each reduction, we had to develop analytical techniques to resolve the enantiomers. Our general approach is to generate a mixture of all four possible products by sodium borohydride. The products are then analyzed by chiral gas chromatography (chiral GC) (Scheme 3-2). ROEtClOOROEtOHOROEtOHOROOHOROOHOClClClClNaBH4 / AcOHSeparable by Chiral Gas Chromatography EtEt Scheme 3-2. Synthesis of all 4 diastereomers by sodium borohydride, which can be separated by chiral gas chromatography This strategy typically works for most alcohols; however, occasionally full separation can not be achieved. This was found during the attempt to separate chlorohydrin 218. To solve this problem, the alcohol 218 was acetylated using acetic anhydride affording derivatives that can be fully resolved on a chiral gas chromatography column (Scheme 3-3). Individual stereoisomers were linked to the appropriate GC peak by isolating alcohols from enzymatic conversions that afforded only single products. Where literature data were available, optical rotation values were used to determine absolute stereochemistry; these assignments were consistent with those made by NMR in all cases.

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44 OEtOOHClOEtOOClDMAP / acetic anhydrideAll Four Diastereomers areSeparable by Gas ChromatographyCH3O218 219 Scheme 3-3. Derivatization technique used to separate all 4 diastereomers for chlorohydrin 218 Comparing the outcomes of reactions using whole bakers yeast cells with those employing isolated yeast reductases clearly demonstrates the utility of examining individual biocatalysts (Figure 3-1). Not only did the purified yeast reductases deliver higher stereoselectivities in most cases, they also produced diastereomers not observed in reductions employing commercial yeast cells. This may result from low expression of some reductases under the physiological conditions prevailing in commercial bakers yeast, and this highlights an important advantage of using isolated reductases, rather than relying on whole yeast cells. Alternative methods to increase expression levels of desirable reductases, such as adding specific enzyme inhibitors, are more difficult to optimize and control. 93,99,100 It should also be noted that the screening reactions could be carried out rapidly, and a complete data set was typically obtained for each substrate within 48 h. The smallest substrate, 220, was accepted by all of the yeast reductases examined, although the stereoselectivities of these reactions were relatively poor except for YOR120w and YGL157w, which afforded (2S, 3S) and (2R, 3S) configuration as the major products, respectively. In all cases, however, only L

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45 Yeast Gene CH3OEtOOCl220 OEtOOClCH3221 OEtOOClCH3222 OEtOOCl126 OEtOOCl214 YJR096w ---a --YDL124w ----YBR149w ----YOR120w ------YHR104w --------YDR368w --YGL185c ----YNL274c ----YPL275w --------YPL113c ------YLR070c --------YAL060w ----YGL157w --YDR541c ------YGL039w YNL331c --YCR107w ----YOL151w --------Yeast Cells b b b Figure 3-1. Biocatalytic reductions of -chloro--keto esters. Yeast enzymes are referred to by their systematic names and grouped by superfamilies. Product compositions from reactions that proceeded to at least 20% conversion within 24 hr are shown in pie charts (2S-3S)-white, (2R-3S)-black, (2R-3R)-black/white lines, (2S-3R)-black dashes

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46 alcohols were formed. This behavior parallels our earlier observations from reactions in which ethyl acetoacetate was used as a substrate for the same collection of yeast fusion proteins. 96 The behavior of higher homolog 221 provides an interesting contrast. In four cases, D-alcohols were the major products. This is significant because D-alcohols are observed much less commonly in biocatalytic reductions and enzymes that deliver this enantioselectivity are correspondingly important. Six enzymes examined accepted 126 as a substrate: four afforded only (2S, 3R) configuration while the remaining two produced mainly (2R, 3S). Benzyl-substituted -keto ester 214 was reduced by three enzymes, with very high stereoselectivities in two cases. Taken together, our results have demonstrated that reductase enzymes uncovered by an analysis of the yeast genome can deliver important chiral building blocks for organic synthesis. At least two of the four possible -chloro--hydroxy ester diastereomers could be produced in high optical purities in most cases. The major deficiency in the present collection is a lack of stereoselective reductases with D-specificities. Biocatalysts with these properties might be identified by including enzymes from additional organisms in our collection of fusion proteins and the increasing pace of genome sequencing project bodes well for expanding the utility of our chemo-enzymatic approach.

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CHAPTER 4 SYNTHESIS OF THE C-13 TAXOL SIDE CHAIN Racemic Synthesis Our approach to the synthesis of the Taxol side chain began with a racemic synthesis. This was developed to test the feasibility of important chemical steps, and also to optimize the reaction conditions without consuming homochiral starting material. This synthesis began with a Darzens reaction of benzaldehyde 85 and methyl chloroacetate 116 to yield trans-methyl 3-phenylglycidate 117 42 (Scheme 4-1). Treatment of trans-117 with dry hydrochloric acid for several hours allowed a highly stereoselective ring opening to afford chlorohydrin 118 42 which was reacted with potassium carbonate to yield cis-epoxide 119. 89 The racemic epoxide 119 underwent a Ritter reaction with benzonitrile, catalyzed by boron trifluoride etherate to form cyclic product 60. 101,102 Treatment of trans-oxazoline 60 with 0.5 M hydrochloric acid yielded the racemic Taxol side chain 41 with an overall yield of 16%. 41,103 H79%OClOMeONaOMeOOMeOHCl99%OMeOClOHK2CO352%OOMeOBenzonitrile / BF3-OEt255%85116(+/-)-117(+/-)-118(+/-)-119 47

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48 ONOMeO0.5 M HCl72%OMeNHOOOH(+/-)60(+/-)-41 Scheme 4-1. Racemic synthesis of the Taxol side chain Once the racemic synthesis was complete, we began our work on the chiral route. Our first step was the chlorination of ethyl benzoylacetate 125, which can be achieved by reaction with tetrabutylammonium bromide and chlorotrimethylsilane 104 (Scheme 4-2). However, we found that this chlorination technique gave side products that were difficult to separate from the desired product 126. After several failed attempts, an alternate literature procedure was found using sulfuryl chloride 105-108 giving high yields and allowing a simple distillation for purification. OOOEtOOOEtClSO2Cl298%OOOEtOOOEtClBu4NBr / Me3SiCllow conversion / difficultypurifying productsimple distillation126125125126 Scheme 4-2. Two chlorination methods for -keto esters Biotransformation Strategy As discussed in chapter 3, we recently published the reduction results for several -chloro--keto esters using our library of purified bakers yeast

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49 reductases. 109 This strategy of using purified reductases works well for the small scale screening of substrates; however, due to the high cost of NADPH, this method is not practical for gram scale syntheses. A simple and economical reduction method is the use of whole cells, utilizing the cells cofactors for the reaction. Our strategy was to implement a scaleable synthesis of the Taxol side chain using an enzymatic reduction as the key step, thus we needed to utilize whole-cell biotransformations. Adam Walton and Parag Parekh initiated our groups research on whole-cell catalyzed reactions using E. coli with overexpressed GRE2 97 a known yeast reductase. They established that the cells kept their reducing capabilities longer in a non-growing nitrogen deprived environment, compared to reactions in complete growth media. Using ethyl acetoacetate as their substrate, they were able to reach a product concentration of 250 mM over a 30 hour period (Figure 4-1). The key step in our route to the Taxol side chain involves the enzymatic reduction of -chloro--keto ester 126, using a similar whole-cell approach as developed by Walton and Parekh. After analyzing the results presented in Chapter 3, we chose two reductases for our synthesis of the Taxol side chain: YDL124w, which affords (2S-3R)-chlorohydrin 127 and YGL039w, which affords

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50 05010015020025030001020304Time (hrs)Concentration (mM) 0 Figure 4-1. Production of (S)-ethyl 3-hydroxybutyrate by engineered E. coli cells under non-growing conditions (2R-3S)-chlorohydrin ent-127 (Scheme 4-3). By using these enzymes, we can synthesize both enantiomers of the optically pure Taxol side chain. OEtOOHClOEtOOHClOEtOClOOEtOClOYDL124wYGL039wOEtONHOHOOEtONHOHO126127130ent-130ent-127126 Scheme 4-3. The Taxol side chain and its enantiomer can be synthesized by utilizing two different enzymes; YDL124w and YGL039w, respectively Optimization of Our Whole-cell System A review from Saluta and Bell reports conditions that can potentially effect protein overexpression by a T7 promoter such as glucose concentration, induction optical density, induction temperature, and induction time. 110 Before we

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51 began our biotransformation studies, these conditions were optimized. In their review, these authors recommend certain parameters to be followed for general protein overexpressions. First, it is known that the overexpression plasmid can be leaky, thus a catabolite repressor should be used to inhibit the protein expression in the growing phase. They recommend supplementation of the growth media with 2% glucose for this reason. Second, they reported that the most advantageous optical density for inductions in these systems are A 600 = 0.5 1.0. The recommended glucose concentration and optical density for induction were used in our system. It is known that the optimal induction temperature can vary when trying to overexpress a protein in its active form. A common complication in the overexpression of proteins is the formation of insoluble-misfolded peptides called inclusion bodies, which are usually caused by the expression at high temperatures (~37 C). 110 This was most likely the case in our early attempts to use YGL039w in our whole-cell biotransformations. SDS-PAGE confirmed the overexpression of our protein of interest; however, reduction attempts were unsuccessful (Figure 4-2).

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52 1marker 2YGL039w 45 31 1 2 3 4 5 6 7 67 116 97 200 3t =0 hr 4t =1 hr 5t =2 hr 6t =3 hr 7t =4 hr Figure 4-2. SDS-Page of the overexpression of YGL039w over a 4-hour time period. The arrow marks the expected position of the YGL039w fusion protein Whole-cell Assays To investigate the inclusion body theory, experiments were developed to test the cells reducing activity when expressing protein at 37 C, 30 C, and 24 C. Three separate cell batches were grown at 37 C until they reached an O.D. = 0.6, then these were cooled to the corresponding temperatures. Once the cells reached their final temperature, they were induced with IPTG (0.1 mM final concentration). Aliquots were taken at various time points and the cell suspension was lysed by sonication, followed by centrifugation to remove the cellular debris. The activity of soluble reductases was screened by the addition of NADPH and ethyl acetoacetate (an excellent substrate for YGL039w). This

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53 solution was monitored at 340 nm over 2 minutes to observe the loss of NADPH, which is directly proportional to the reduction of ethyl acetoacetate. The specific activity for each aliquot was calculated as described in Appendix B, and the activities were plotted versus the time after induction (Figure 4-3). Specific activity vs. Time00.010.020.030.040.050.060.070.080.090510152025Time (Hrs)Specific activity (umol/min-mg) 24 30 37 Spec cells induced at varying temperatures ific activity vs. Time for Figure 4-3. Specific activity of the E. coli cells overexpressing YGL039w, which have been grown under different induction temperatures (37 C, 30 C, and 24 C) From this experiment we concluded that overexpression of YGL039w at 24 C gave the highest activity per cell. In addition, it confirmed our suspicion that YGL039w overexpressed at 37 C formed inclusion bodies, which explained why it was unable to reduce the ketone substrate. These experiments also answered an additional question. It was first considered important to stop the growth of the induced cells after 4 hours. However, this experiment ran for 24 hours, and we saw no evidence for a significant loss of specific activity from the cells up to this point.

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54 In addition to the induction temperature, we were also curious about the effect of the GST tag on the activity of the protein. Therefore, YGL039w was overexpressed both with and without the GST tag, and the same methods to evaluate reductase activity described above were used to compare the activities (Figure 4-4). The results implied that the GST tag, at least in this case, does not adversely affect the activity of the reductase. GST Effect on Specific Activity00.20.40.60.811.21.41.60510152025Time (hr)specific activity (umol/min-mg) GST No GST Figure 4-4. Whole cell activity of an overexpressed YGL039w with a GST tag and YGL039w without a GST tag versus time Whole-cell Reduction of 126 After the optimization of protein expression and activity, the whole-cell reductions of the -chloro--keto esters could begin. The reduction of 126 was run on a 1 liter scale fermentation reaction in a nitrogen-free phosphate buffer. Unfortunately, we encountered a problem while conducting this experiment: 126 was found to decompose while dissolved in water. After analyzing its stability at different pH values, the molecule was found to be reasonably stable at a working

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55 pH under 6.0. Second, we noticed that some -chloro--keto ester will undergo reductive dechlorination, which Bertau and Jorg 90,111,112 suggested was due to a reaction with free glutathione in the cell. In our whole-cell reductions of 126, we found that the amount of dechlorinated product can widely vary for each reaction, and we do not have a strong hypothesis to why there is such a vast inconsistency. Our last problem observed in the whole-cell reduction of 126 was its high toxicity and inhibitory effect towards the cells. Two actions were taken to minimize these toxicity effects. First, we slowed substrate feeding to keep the starting material concentration at a minimum. Second, a non-polar adsorbing Amberlite XAD-4 resin was added to the fermentation reaction. This resin adsorbs the product from the aqueous phase, thereby lowering its inhibitory effect on the cells. On average, we have seen a 20 25 % increase of isolated product for 127 and ent-127 using these tactics. After optimization of both the growing conditions and the conditions for the whole-cell reduction, we were able to achieve a final product concentration of ~6 mM. These concentrations were calculated by GC using the ratio of product peak area versus internal standard peak area. The next step, extraction of the product, proved to be difficult because of the formation of an emulsion caused by cellular debris. To avoid the emulsion, we centrifuged the cells and extracted the supernate with an organic phase. However, product was found in the cell pellet, resulting in a significant loss in

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56 yield. We therefore developed a gentle extraction method in which the aqueous phase was slowly circulated through methylene chloride (Figure 4-6). Concentration of Product012345670510152025Time (hr)Concentration (mM) YDL124w YGL039w Figure 4-5. Concentration of the product for the biotransformation using YDL124w and YGL039w Figure 4-6. Diagram of our gentle extraction technique

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57 After purification by flash chromatography, we were able to achieve a 91% and 85% yield of 127 and ent-127 from the whole-cell reduction with YDL124w and YGL368w, respectively (Scheme 4-4). OEtOOHClOEtOOHClOEtOClOOEtOClOYDL124wYGL039w99% d.e. / 99% e.e.76% d.e. / 99% e.e.91%85%1.33 g/L isolated product1.39 g/L isolated product containing10% anti-chlorohydrin126127126ent-127 Scheme 4-4. Final results for the whole-cell biotransformations after purification Base Catalyzed Ring Closure Our next step required a base catalyzed ring closure to form the corresponding glycidic ester 89 (Scheme 4-5). This reaction can be directed to the cis or trans product by adjusting the strength of the base. Reaction of chlorohydrin 223 and 225, with a weak base will yield exclusively the kinetic product for this reaction 224 and 226, respectively. Treatment of the same two chlorohydrins with a strong base will selectively yield trans-epoxide 226. ROEtOHClOROEtOHClOOROEtOOROEtOK2CO3 / H2OK2CO3 / H2O223224225226

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58 ROEtOHClOROEtOHClOOROEtOOROEtONaOEt / EtO-Na+NaOEt / EtO-Na+223226225226 Scheme 4-5. Results for the ring closure of chlorohydrins: Potassium carbonate results in the kinetic product, whereas sodium ethoxide affords the thermodynamic product. The Azerad lab reported 89 that the reaction involving ethoxide ion preceded by initial epimerization of the chlorohydrin via the formation of the enolate prior to cyclization (Scheme 4-6). Once epimerized to the more thermodynamically favored anti conformation, the chlorohydrin can undergo closure to trans-epoxide 226. ROEtOHClOROEtOHClO-ROEtOHClONaOEtOOEtOR223227225226 ROHHClHCO2EtROHHHClCO2EtNaOEt / EtOHThermodynamically morestable chlorohydrin223225 Scheme 4-6. Mechanism for the sodium ethoxide promoted epoxidation and a Newman projection describing syn versus anti configuration of chlorohydrins Epoxide formation using potassium carbonate required some water to afford glycidic ester 128 with a 99% yield, and the amount of added water proved critical (Scheme 4-7). We found that 1 2 equivalents of water would result in a slow reaction, and more than 3 equivalents of water would often lead to by

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59 products. It was also necessary to use chlorohydrin purified by column chromatography, because the impurities found with the reduction product led to a drastic loss in yield. OEtOHClOOOEtOK2CO3 (3eq) / H2O (3 eq)DMF99%127128 Scheme 4-7. Base promoted formation of cis-glycidic ester 128 Ritter Reaction Once the synthesis of glycidic ester 128 was achieved, it underwent a Ritter reaction with benzonitrile to afford predominately trans-oxazoline 129 with a 55 % yield (Scheme 4-8). This reaction also yielded a small amount of cis-oxazoline 227 which decreased the yield of the desired product. Fortunately, the cis isomer was separable by flash chromatography allowing pure trans-oxazoline 129 to be isolated. OOEtOBenzonitrile / BF3-OEt255%ONOEtOONOEtOMajor ProductMinor ProductSeperable by Flash Chromatography128129227 Scheme 4-8. The Ritter reaction of glycidic ester 128 and benzonitrile An attempt was made to increase selectivity for the trans oxazoline by varying the temperature, solvent polarity, and strength of the Lewis acid. Different temperatures and solvents were found to have no effect on the reaction selectivity. A variety of Lewis acids were also screened to see if they could

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60 increase the trans: cis ratio (Figure 4-7). These attempts were unsuccessful and the original conditions were found to be optimal. Lewis AcidConversionRatio (Trans: Cis)Lithium Bromide0% ---Lithium Chloride0% ---Ytterbium Triflate99% 5: 1Tin (II) Ethyl Hexanoate0% ---Aluminum Chloride0% ---p-Toluene Sulfonic Acid99% 5: 1Triflic Acid99% 5: 1BF3-etherate99% 5: 1 Figure 4-7. Effect of Lewis acids on the Ritter reaction Ring Hydrolysis to the Taxol Side Chain Oxazoline 129 has been used by Bristol Myers-Squibb as a protected form of the Taxol side chain 113 that can be coupled to the terpene core of Taxol (Scheme 1-9). With this said, we were able to synthesize the protected Taxol side chain in 4 steps with an overall yield of 49 %. For academic reasons, and to compare the optical rotation of our material with that synthesized previously, we treated oxazoline 129 and ent-129 with aqueous acid to afford the Taxol side chain 130 and ent-130 as their ethyl esters. Optical rotations for the Taxol side chain 130, and its enantiomer ent-130 were [] D = -11.6 (c = 2.0, CHCl 3 ) and [] D = +12.3, (c = 1.0, CHCl 3 ), respectively. As expected, the 1 H NMR spectra of 130 and ent-130 were found to overlap (Appendix B). In addition, to confirm the enantiopurity of the final products 130 and ent-130, were derivatized with (S)--methoxy--phenylacetic (MPA) and the spectra of the two derivatives were compared (Appendix B). The spectra of ent

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61 130 was found to be 99% enantiomerically pure. A small amount of enantiomer was found in 130, which can be attributed to a small amount of trans-epoxide that was not separated in the ring closure step. However, since ent-130 was synthesized in a 99% ee, it confirmed that subsequent chemical steps in our reaction scheme did not provoke any racemization. For the final step, it is essential that the reaction be carried out under mildly acidic conditions. It has been reported that treatment with weak aqueous acid will result in the hydrolysis product from attack at the C-4 position 103 (Scheme 4-9). However, treatment of oxazoline 129 with 6 M hydrochloric acid will result in amide hydrolysis resulting in compound 228. In addition, we found that storing the oxazoline in the open air at room temperature would result in the hydrolysis product 130 after several weeks. NOCO2EtONHOEtOOHOHOHONH2 Scheme 4-9. Hydrolysis of oxazoline 129 under mildly acidic conditions versus strongly acidic conditions

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CHAPTER 5 SYNTHESIS OF BESTATIN Over the past 30 years, there have been several strategies reported for the synthesis of Bestatin 131 (Chapter 2). Most of these utilize amino acids or other naturally occurring chiral compounds for their starting material. Other strategies utilize the separation of enantiomers by chiral resolution; however, this process can be very difficult and often inefficient. It was our goal to develop a novel synthesis for AHPA 137 and Bestatin 131 through our simple homochiral glycidic ester approach. Syntheis of -Keto Ester 213 Our approach to Bestatin began by synthesizing -keto ester 213 by a literature procedure using Meldrums acid 212 and phenylacetyl chloride 21 114 (Scheme 5-1). The product of this reaction 213 can be easily purified by a simple vacuum distillation. OClOOOO1) pyridine (2.5 eq)2) EtOHOOOEt86%211212213 Scheme 5-1. Synthesis of -keto ester 213 Chlorination of -Keto Ester 213 The chlorination of compound 213 with sulfuryl chloride was found to be difficult, because performing this reaction with stoichiometric amounts of starting material resulted in a large amount of dichlorinated byproduct 229 accompanying 62

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63 the desired product 214 (Scheme 5-2). These compounds were inseparable by distillation, and difficult to separate by flash chromatography. This problem was solved by lowering the reaction temperature and decreasing the ratio of sulfuryl chloride to -keto ester, thus simplifying the purification process. OEtOOSO2Cl2 (1eq)OEtOOOEtOOClClClOEtOOSO2Cl2 (0.6 eq)OEtOOClVery Difficult to SeperateEasily Separated fromStarting Material50 oC25 oCOEtOO213214229213214213 Scheme 5-2. Two different approaches to chlorinate 213 with sulfuryl chloride Enzymatic Reduction of 214 The key step in our approach to AHPA 137 and Bestatin 131 is the whole-cell reduction of compound 214, using an overexpressed bakers yeast reductase in E. coli. This process allows us to introduce two chiral centers into our reaction scheme, and we hoped to achieve this on a gram scale. We recently published the reduction results for several -chloro--keto esters using our library of bakers yeast reductases 109 (Chapter 3). Benzenebutanoic acid, -chloro--oxo-ethyl ester 214 was shown to be a substrate for three bakers yeast reductases; YDR368w, YGL157w, and YGL039w (Scheme 5-3).

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64 OEtOOClOEtOClOHOEtOClOHOEtOClOHYDR368wYGL157w 99% e.e. / 99% d.e.99% e.e. / 99% d.e.41%e.e. / 98%d.e.214215230230 Scheme 5-3. Three bakers yeast reductases that accept 214 as a substrate; YDR368w, YGL039w, and YGL157w Previously, for compounds 127 and ent-127 (Chapter 4), we were able to use literature data to determine the absolute stereochemistry; unfortunately, no literature data was found for compounds 215 and 250. Comparison of the 1 H NMR of these compounds confirmed that YDR368w and YGL157w afforded syn and anti chlorohydrins, respectively (Appendix B). However, since literature data did not exist, another method was used to define the absolute stereochemistry for 215 and 250. We first attempted X-ray crystallography; however, we were unable to grow diffraction-quality crystals from these compounds. Our next approach was NMR analysis by derivatizing the chlorohydrins with (R)and (S)--methoxy--phenylacetic (MPA) acid. Dr. Ion Ghivirigia analyzed these derivatized compounds for their 1 H1 H, 1 H13 C one-bond and 1 H13 C long-range couplings to determine the absolute stereochemistry 115-118 (Appendix B). The results from NMR experiments were used to assign the (2R, 3S) configuration to the YDR368w product, and the (2S, 3S) configuration to the

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65 YGL157w and YGL039w products. Fortunately, the (2R-3S)-chlorohydrin 215, is the intermediate needed for our approach to AHPA 137 and Bestatin 131. Whole-cell Reduction of 214 The reduction of 214 was performed in a 1 liter fermentation reaction in a nitrogen-free phosphate buffer. The growth and biotransformation conditions for the reduction of compound 214 followed those optimized for YGL039w in our Taxol side chain synthesis (Chapter 4). As seen before, this whole-cell reduction also yielded dechlorinated product that formed in the beginning of the reaction (Scheme 5-4). Typically, the first five percent of the starting material would suffer this dechlorination reaction. Occasionally there would be vast variations in the extent of dechlorination, and this phenomenon has yet to be fully understood. OEtOOClOEtOClOHYDR368wOEtOOH214215231 Scheme 5-4. The whole-cell biotransformation resulted in the chlorohydrin 215 and dechlorinated product 231 Substrate 214, much like compound 126, was highly toxic toward the E. coli cells. To lessen this effect, two actions were taken. First, we fed the cells small portions of starting material every hour, thereby keeping the concentration of the toxic starting material at a minimum. Second, a non-polar adsorbing XAD-4 resin was added to the fermentation system. This resin would slowly adsorb the toxic compounds from the reaction system, thus allowing the cells to further reduce substrates and consume glucose and oxygen.

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66 The whole-cell reduction of 214 by cells overexpressing the reductase YDR368w yielded a final product concentration of 5 mM. Using the previously described gentle extraction method, we were able to obtain 1.1 g of product after purification by flash chromatography with an overall yield of 82% (Scheme 5-5). OEtOOHClOEtOClOYDR368w99% d.e. / 99% e.e.82%1.1 g/L isolated product215214 Scheme 5-5. Results for our optimized whole-cell reduction of 214 Base Catalyzed Ring Closure of 215 Treatment of chlorohydrin 215 with potassium carbonate cleanly yielded cis-glycidic ester 216 89 with no need for further purification (Scheme 5-6). Since DMF was used as the solvent for this reaction, it was important to wash the organic phase with several small portions of water to remove any residual DMF. In addition, the amount of water was critical to the success of this reaction. We found that 1 2 equivalents of water would result in a slow reaction, and that more than 3 equivalents of water would often lead to by-products. OHOOEtClK2CO3 (3eq) / H2O (3eq)OOEtO99%DMF215216 Scheme 5-6. Base promoted ring closure for glycidic ester 216 Ritter Reaction Epoxide 216 was opened by benzonitrile and borontrifluoride diethyl etherate to afford the protected form of AHPA 217 101,102 (Scheme 5-7). Unlike

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67 the Ritter reaction in our synthesis of the Taxol side chain (Scheme 4.8), this reaction was completely selective for trans-oxazoline 217, with an overall yield of 78%. OOEtOBenzonitrile / BF3-OEt278%ONOEtO216217 Scheme 5-7. The Ritter reaction of 216 and benzonitrile only afforded the trans-oxazoline 217 Synthesis of Bestatin from 217: First Generation In our first attempt to complete the synthesis of Bestatin, oxazoline 217 was saponified to produce acid 232 (Scheme 5-8). This was subjected to a DCC coupling with L-Leu-OBn in an attempt to form the protected form of Bestatin 233. The final deprotection step was expected to utilize hydrogenolysis of both oxazoline and the benzyl ester, thus yielding Bestatin directly. While the coupling with L-Leu-OBn appeared to have been successful, a number of attempts to hydrogenolyze the oxazoline failed, and we found that the oxazoline could only be deprotected by reflux in the presence of 6 M HCl. Such conditions would also cleave the peptide bond, and this strategy was therefore abandoned.

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68 NOOEtOK2CO3NOOHODCC / L-Leu-OBnNOHNOOEtOXH2NOHHNOOOH217232233131 Scheme 5-8. First attempt for the synthesis of Bestatin Synthesis of Bestatin from 217: Second Generation Our second attempt to convert oxazoline 217 to Bestatin involved nucleophilic attack on the ring carbon to yield an sp 3 center that would be more susceptible to deprotection (Scheme 5-9). We chose a nickel-catalyzed addition of PhMgBr to afford Grignard product 234 119 We attempted a DCC coupling with L-Leu-OtBu and crude 234, but unfortunately the products of the Grignard reaction and the Leucine coupling reaction were very difficult to dissolve in a variety of solvents, thus making these intermediates extremely difficult to characterize. Since Bestatin was not observed at the end of this sequence, this route was also abandoned. NOOHOPhMgBr / dppNiCl2HNOOHODCC / L-Leu-OtBu232234

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69 H2NOHHNOOOHHNOHNO3 N HClOtBuOSeparation and Isolation w asnotPracticalX235131 Scheme 5-9. Second attempt for the synthesis of Bestatin Synthesis of Bestatin from 217: Third Generation Based on the difficulties encountered in converting oxazoline 217 directly to Bestatin 131, a simpler approach was taken. As mentioned in Chapter 2, Bestatin is composed of two main parts, (2S, 3R)-3-Amino-2-hydroxy-4-phenylbutanoic acid 137 (AHPA) and L-leucine. The most difficult challenge for the synthesis of Bestatin 131 is the synthesis of AHPA 137. Our final approach was to synthesize AHPA by simply deprotecting oxazoline ester 217 with 6 M HCl (Scheme 5-10). Since literature methods can be used to convert AHPA 137 to Bestatin 131, this completes a formal total synthesis of this molecule. NOOEtOH2NOHOHO6M HClH2NOHHNOOOH1) BocCl2) DCC / L-Leu-OBz3) H2 / Pd/C217137131 Scheme 5-10. Final Strategy to AHPA 137 and Bestatin 131 Hydrolysis of 217 proceeded smoothly and after a simple purification by a Dowex cation exchange resin, we were able to isolate AHPA with a 40% overall yield. As a confirmation, commercially available AHPA was purchased and compared with our synthetic material. The 1 H NMR spectrum of the

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70 commercially available AHPA and our synthesized AHPA were identical (Appendix B). In addition, the optical rotation for our synthetic AHPA was [] D = 23.2 (c = 1.3, 1 M HCl), AHPA (Sigma-Aldrich) [] D = 23.4 (c = 1.0, 1 M HCl). The overlap of spectral data and optical rotation values 67 confirms that our previous definition of absolute stereochemistry was correct using NMR.

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CHAPTER 6 SYNTHETIC APPROACH TO CHUANGXINMYCIN Introduction Chuangxinmycin 286 is a natural product isolated from Actinoplanes tsinanensis (Figure 6-1). Initial studies suggested that it has in vitro antibacterial activity against a variety of gram-negative and gram-positive bacteria. In addition, it has shown antimicrobial activity against Escherichia coli and Shigella dysenteriae in mouse models 120 Clinical results have shown successful treatment for septicaemia and for urinary and binary infections cause by E. coli. Presently, Chuangxinmycins mode of action is not completely understood; however, it is reported that this drug has an affect on the tryptophan biosynthetic pathway. NHSCO2HCH3236 Figure 6-1. Chuangxinmycin 236 The Akita labs Approach to Chuangxinmycin In 1997, the Akita group published synthesis of racemic Chuangxinmycin 121 by coupling (+/-)-(2,3)-syn-epoxy butanoate 243 and 4-iodoindole 242 (Scheme 6-1). This synthesis began with 2-amino-6-nitrotoluene 237, which was treated 71

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72 with CH(OEt) 3 to afford 238. Imidate ester 238 was reacted with potassium ethoxide followed by a palladium catalyzed hydrogenation to yield 4-amino indole derivative 241. Diazotization of 241 with sodium nitrite and subsequent treatment with potassium iodide resulted in 4-iodoindole derivative, which was converted into 4-iodoindole 242 by hydrolysis. Reaction of racemic glycidic ester 243 and 242 in the presence of tin (IV) chloride afforded (+/-)-4-iodoindolmycenate 244. Treatment of 244 with methanesulfonyl chloride in pyridine gave thioacetoxy ester 245, which was deacetylated under weak alkaline conditions. Treatment of 246 with Pd(PPh 3 ) 4 gave methyl ester 247 that was treated with aqueous base to result in Chuangxinmycin 236. NO2CH3NH2NO2CH3NOEtNO2NHCH(OEt)3 / TsOH(COOEt)2 / KOEt89%77%NaH / ClCOOMe93%NO2NCH3ONH2NCH3OH2 / Pd/C73%1) NaNO2-H+ /KI2) NaOMe / MeOH80%INHOH3CCO2Me32%SnCl4INHOMeH3COHO70%1) MsCl / pyridine2) CsSAc237238239240241242243244

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73 INHOMeH3CSO90%K2CO3 / MeOHINHOMeH3CSHOPd(PPh3)4 / Et3N74%CH3ONHSCO2MeCH3NHSCO2HCH3NaOH / MeOH32%245246247236 Scheme 6-1. Akitas synthesis of (+/-)-Chuangxinmycin 236 Enzymatic Reduction of 220 This publication 121 described a method to synthesize racemic Chuangxinmycin through trans-glycidic ester 243. However, to achieve the absolute stereochemistry at the C-4 and C-5 positions, one must develop a chiral synthesis to this ester. We recently disclosed that reductase YOR120w afforded the (2R, 3S) chlorohydrin by reduction of 220 with a >98% e.e and >98% d.e. 109 Using this chlorohydrin, we proposed a practical, gram-scale synthesis to make (2R, 3S)-epoxy butanoate 243 with a whole-cell biotransformation using an overexpressed bakers yeast reductase as the key step (Scheme 6-2). H3COEtOOHClYOR120wH3COEtOOClOH3COONaOEt / EtOH220248243 Et Scheme 6-2. Proposed scheme to the Chuangxinmycin intermediate (2R, 3S)-epoxy butanoate 243

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74 Whole-cell Reduction of 220 The whole-cell reduction of 2-chloro ethylacetoacetate 220 was carried out in a 1 liter fermentation vessel in a nitrogen deprived phosphate buffer, using conditions similar to those described earlier (Chapter 4). 97 This reduction was able to achieve a much larger final concentration when compared to the whole-cell reductions of 126 and 214. Nonetheless, this reaction fell short in final product concentration when compared to the reduction of ethyl acetoacetate by GRE2 (Figure 4-1). Between these cases, there was about a ten-fold decrease for 2-chloro ethylacetoacetate 220 (Figure 6-2). This is most likely due to the increased toxicity toward the cells from the chlorine functionality on substrate 220. Whole-cell reduction of 2-chloro ethylacetoacetate did yield an approximately four-fold higher final concentration when compared to the whole-cell reduction of 214. We believe this is because 220 is less hydrophobic than 214, and thus has a smaller inhibitory effect on the cells. H3COEtOHOH3COEtOHOClOEtOHOCl32.2 g/L4.4 g/L1.1 g/L249248215 Figure 6-2. Final product concentrations for 249, 248, and 215 by the corresponding engineered E. coli The reduction of 220 was catalyzed by E. coli cells containing overexpressed reductase YOR120w. To lessen the toxicity to the cells, the substrate was added in small increments over 24 hours, and a one liter reaction afforded 4.4 grams of product with an 89% overall yield. As was also seen in

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75 other reductions of chlorinated -keto esters, there was a small dechlorination by-product. In this case, however, we found only a small percentage of this by-product. While separation was simpler due to the small amount of dechlorinated by-product, we did encounter a problem while trying to extract 248 from the reaction mixture. Even using our gentle extraction technique (Chapter 4), we were unable to achieve complete extraction of product from the aqueous phase, even after 5 days of extraction. This can be attributed to the high solubility of the reduction product 248 in water. As a result, the organic layer was replaced with fresh methylene chloride three times to allow an adequate extraction. Base Catalyzed Ring Closure of 248 To synthesize the homochiral glycidic ester, the chlorohydrin 248 was treated with sodium ethoxide to afford predominately trans-glycidic ester 243. Unfortunately, this reaction did not follow the trend seen in the literature 89 which reported the trans-epoxide as the only product formed. Our ring closure reported a 5 : 1 (trans : cis) product ratio and we were unable to get adequate separation of these diastereomers (Scheme 6-4). H3COEtOOHClOH3COEtONaOEtOH3COOMajorMinorDifficult to Separate243250248 Et Scheme 6-4. Ring closure promoted by sodium ethoxide This observation can be explained by the size of the -carbon chain. The reaction of chlorohydrins with sodium ethoxide is directed by the thermodynamic

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76 stability of the anti versus the syn conformation (Scheme 4.6). Due to its small size, the methyl group does not adequately direct the steric course of the reaction. We also investigated ring closure of 248 by treatment with a weak base. Chlorohydrin 248 was reacted with potassium carbonate (3 equiv) and a catalytic amount of water (3 equiv) over a 5 hour period (Scheme 6-5). Gas chromatography and NMR analysis confirmed that this epoxidation followed the general trend, affording only the cis-glycidic ester. H3COEtOOHClH3COEtOOClOH3COOYOR120w89%K2CO3 (3eq) / H2O (3eq)85%220248250 Et Scheme 6-5. Ring closure of chlorohydrin 248 using potassium carbonate and water We were generally unsuccessful in the synthesis of (2R, 3S)-epoxy butanoate 243. A simple solution to this problem is to find a reductase that will result anti-chlorohydrin 251 with a high d.e. and e.e. This will then allow for the epoxidation using potassium carbonate, which should afford only the trans-glycidic ester 243 (Scheme 6-6). H3COEtOOHClH3COEtOOClOH3COOreductaseK2CO3 / H2O220251243 Et Scheme 6-6. Proposed synthesis to (2R, 3S)-epoxy butanoate 243

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CHAPTER 7 CONCLUSIONS AND FUTURE WORK Studying our collection of purified bakers yeast reductases has shown that they can be utilized to afford highly synthetically useful intermediates with high stereoselectivities. In addition, these reductions can be carried out with engineered whole-cells to yield products on gram scales. We have also shown that these reduction products can be easily transformed into glycidic esters, which are popular intermediates for a variety of pharmaceutical drugs. Our reduction library allowed us to make the (2S-3R)-127 and (2R-3S)-chlorohydrins ent-127 needed for synthesizing both Taxol side-chain antipodes with high enantiomeric excess. Using these intermediates, we were able to react these chlorohydrins with a weak base to afford the corresponding glycidic esters. The glycidic esters underwent a Ritter reaction with benzonitrile to form the protected Taxol side chain with an overall yield of 49% and its enantiomer with an overall yield of 38%. This is advantageous because it eliminates any additional steps that are needed to take the Taxol side chain to a form in which it can be coupled to Baccatin III. Benzenebutanoic acid, -chloro--oxo-, ethyl ester 214 was shown to be a substrate for YDR368w, thus affording (2R, 3S)-chlorohydrin 215 in high enantiopurity. This result allowed us to synthesize oxazoline 217 through our glycidic ester and Ritter reaction route, thus resulting in a protected form of AHPA. Treatment of oxazoline 217 with strong acidic conditions resulted in 77

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78 AHPA 137 in 6 steps with an overall yield of 42%. This completes a formal total synthesis of Bestatin 131. The whole-cell reductions of three key -chloro--keto esters resulted in final concentrations of 1.1 g for 215, 1.39 g for 127, and 4.4 g for 248. This trend is a direct result of the product toxicity to the cells. This toxicity is probably due to the hydrophobicity of the compounds, along with the addition of the chlorine moiety. Future work for these reductions would focus on the optimization of these biotransformations. For example, the dechlorination of the starting material may be eliminated by incubating the cells with a chlorinated compound that is commercially available and easily removed. Additional work would focus on engineering at the genomic level, thus making cells more resistant to product toxicity and/or more efficient at reducing substrates. The two pharmaceutical routes reported in this thesis are only two examples from a wide range of possibilities. If we increase our library of purified bakers yeast reductases, or expand the library with reductases from other organisms, it may help in expanding our synthetic potential. As seen in Chapter 6, if a reductase is found to yield the (2S, 3S)-chlorohydrin 251, we will be able to synthesize the (2R, 3S)-epoxy butanoate 243, which is an intermediate to Chuangxinmycin 236. Other possible pharmaceutical intermediates that can be formed through homochiral glycidic esters are, but not limited to: Diltiazem 252, KRI-1230 253, Amistatin ent-253, and Indolmycin 254 (Figure 7-1)

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79 SONMe2NH2CH2COAcOCH3NHOH3CONNHCH3ONOOHisHNHOOO252253254 Figure 7-1. Other pharmaceutical drugs that can be synthesized from homochiral glycidic ester intermediates: Diltiazem 252, KRI-1230 253, Amistatin ent-253, and Indolmycin 254

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APPENDIX A EXPERIMENTAL General Methods and Instrumentation Standard media and techniques for growth and maintenance of E. coli were used, and Luria-Bertani (LB) medium contained 1% Bacto-Tryptone, 0.5% Bacto-Yeast Extract, and 1% NaCl. Synthetic reactions were carried out under argon atmosphere, with the exception of water containing reactions. Reactions were monitored by TLC (silica, 60 A) or by GC using a DB-17 column (0.25 mm x 25 m x 0.25 m thickness) with a flame ionization detector, and for 137 reversed-phase HPLC (4.6 250 mm C 18 column) using a water-CH 3 CN solvent system (both solvents containing 0.1% trifluoracetic acid) was used. For chiral separation, GC was used with a Chirasil-Dex CB column (0.25 mm x 25 m x 0.25 m thickness) or a Chirasil-L-Val (0.25 mm x 25 m x 0.25 m thickness) with a flame ionization detector. NMR spectra for 1 H and 13 C were recorded on Varian 300 MHz instruments. Chemical shifts are reported at 25 C in ppm relative to TMS. Optical rotations were measured in CHCl 3 at room temperature (Perkin-Elmer 241 digital polarimeter) unless otherwise stated. Elemental analysis was performed by Atlantic Microlab, Inc. in Atlanta, Georgia. Racemic alcohols were prepared from the corresponding ketones by reduction with sodium borohydride. 80

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81 Whole-cell Activity Assays for Inductions Carried Out at Different Temperatures A 35 mL solution of LB broth (supplemented with 30 g/mL kanamycin) was inoculated with a single colony of E. coli (BL21(DE3)(pIK6)) and shaken overnight at 37 C. The preculture (10 mL) was diluted (1: 100) into 1 L of LB (supplemented with 30 g/mL kanamycin and 4 g/L glucose) in three 2 L baffled flasks. The cultures were grown for 2 hours at 37 C with shaking at 400 rpm until they reached an O.D. 600 = 0.6. One flask was placed at 24 C, one at 30 C and the other was left at 37 C. The cells were allowed to shake at 400 rpm for 15 minutes and then reductase overproduction was induced with isopropylthio-D-galactoside at a final concentration of 0.1 mM. The cells were kept under the same conditions and aliquots (100 mL) were taken at various times. Cells were collected by centrifugation (6000 g for 10 min at 4 C), resuspended in phosphate buffer (3 mL, 100 mM, pH = 7), then PMSF was added to a final concentration of 1.5 mM. The aliquots were stored at 4 C and then sonicated for 10 seconds. The cell suspension was centrifuged (6000 g for 10 min at 4 C) to remove cellular debris, decanted, and the supernate was stored on ice. A premixed solution of phosphate buffer (10 mM, pH = 7) and ethyl acetoacetate (5 mM) was maintained at 30 C. The premixed solution (1 mL) was added to a quartz cuvette, followed by 10 L of NADPH solution (20 mM) and the cellular supernate (volume varied). The cuvette was gently mixed and immediately monitored at 340 nM (120 sec at 20 sec intervals). The slope was calculated and used to find the specific activity (Appendix B).

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82 Procedures and Data OOOEtCl126 Ethyl benzoylacetate 125 (6.0 g, 28.0 mmol) was added to chloroform (170 mL). The reaction mixture was purged with argon at 50 C and then sulfuryl chloride (2.5 mL, 28.0 mmol, 1 equiv) was added over 15 minutes. The reaction mixture was stirred at 50 C for 3 hours and then allowed to cool to room temperature. Water (200 mL) was added to the mixture and the aqueous layer was extracted with methylene chloride (3 x 100 mL). The combined organic layers were dried with magnesium sulfate and concentrated. The resulting residue was purified by vacuum distillation (2 mm Hg, 120 C) to afford 6.2 g as a colorless oil 126 in 98% yield. 1 H NMR: (CDCl 3 ) : 8.00 (d, 2H), 7.60 (m, 3H), 5.62 (s, 1H), 4.29 (q, 2H, J = 6.9), 1.24 (t, 3H, J = 6.9). 13 C NMR: (CDCl 3 ) : 188.6, 165.6, 134.7, 133.8, 129.6, 129.3, 63.6, 58.4, 14.3. IR (neat): v(cm -1 ): 2984.5, 1763.8, 1691.7, 1268.3, 1182.8. OEtOHClO127 A 45 mL solution of LB broth (supplemented with 30 g/mL kanamycin) was inoculated with a single colony of E. coli (BL21(DE3)(pIK8)) and shaken overnight at 37 C. The preculture (40 mL) was diluted (1: 100) to 4.0 L of LB (supplemented with 30 g/mL kanamycin and 4 g/L glucose) in a New Brunswick M19 fermenter. The culture was grown for 2 hours at 37 C with a stir rate of 800

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83 rpm and an air flow of 0.5 vessel volumes per minute (vvm) until it reached O.D. 600 = 0.6. The cell suspension was cooled to 28 C over 15 minutes and then reductase overexpression was induced with isopropylthio--D-galactoside at a final concentration of 0.1 mM. The cells were kept under the same conditions for 6 hours and then collected by centrifugation (6000 g for 10 min at 4 C). Half of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KP i (pH = 5.6) containing 4 g/L glucose. The bioconversion was carried out in a Braun Biostat B fermenter at 30 C with the pH maintained at 5.6 using 3 M NaOH. The dissolved oxygen was maintained at 75% saturation using a fixed air flow of 0.25 vvm and variable stirring rate. After the addition of the XAD-4 resin (0.5 g), portions of neat 126 (0.2 mL) were added approximately every hour over a total of 12 hours to provide a final concentration of 6 mM. Portions of glucose were added after 3.0 hours and 6.0 hours to maintain the glucose concentration at approximately 4 g/L. Consumption of 126 and glucose slowed significantly after 8 hours. After 24 hours, the reaction was gently extracted with methylene chloride (2 x 300 mL) to avoid an emulsion. The combined organic layers were dried with magnesium sulfate and concentrated under vacuum. The crude oil was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 1.33 g of 127 as a colorless oil with a 91% yield after recovered starting material. [] D = -3 (c = 0.68, CHCl 3 ), Lit. 88 [] D = -3 (c = 1.7, CHCl 3 ) 1 H NMR: (CDCl 3 ) : 7.25 (m, 5H), 5.00 (d, 1H, J = 6.3), 4.35 (d, 1H, J = 6.3), 3.99 (q, 2H, J = 6.9), 1.02 (t, 3H, J = 6.9). 13 C NMR: (CDCl 3 ) : 168.4, 138.7, 129.1, 128.9, 127.2, 77.1, 75.1, 63.4, 62.7, 14.2. IR (neat): v(cm -1 ): 3475.3, 2982.2, 1745.8, 1373.1,

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84 1258.9, 1095.7. Anal. Calcd for C 11 H 13 O 3 Cl: C, 57.78; H, 5.73. Found: C, 57.92; H, 5.86. OEtOHClOent-127 A 45 mL solution of LB broth (supplemented with 30 g/mL kanamycin) was inoculated with a single colony of E. coli (BL21(DE3)(pIK6)) and shaken overnight at 37 C. The preculture (40 mL) was diluted (1: 100) to 4.0 L of LB (supplemented with 30 g/mL kanamycin and 4 g/L glucose) in a New Brunswick M19 fermenter. The culture was grown for 2 hours at 37 C with a stir rate of 800 rpm and an air flow of 0.5 vessel volumes per minute (vvm) until it reached an O.D. 600 = 0.6. The cell suspension was cooled to 28 C over 15 minutes and then reductase overexpression was induced with isopropylthio-D-galactoside at a final concentration of 0.1 mM. The cells were kept under the same conditions for 6 hours and then collected by centrifugation (6000 g for 10 min at 4 C). Half of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KP i (pH = 5.6) containing 4 g/L glucose. The bioconversion was carried out in a Braun Biostat B fermenter at 30 C with the pH maintained at 5.6 using 3 M NaOH. The dissolved oxygen was maintained at 75% saturation using a fixed air flow of 0.25 vvm and variable stirring rate. After the addition of the XAD-4 resin (0.5 g), portions of neat 126 (0.2 mL) were added approximately every hour over a total of 12 hours to provide a final concentration of 6 mM. Portions of glucose were added after 3.0 hours and 6.0 hours to maintain the glucose concentration

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85 at approximately 4 g/L. Consumption of 126 and glucose slowed significantly after 8 hours. After 24 hours, the reaction was gently extracted with methylene chloride (2 x 300 mL) to avoid an emulsion. The combined organic layers were dried with magnesium sulfate and concentrated under vacuum. The crude oil was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 1.39 g of ent-127 as a colorless oil with an 85% yield after recovered starting material. [] D = +4 (c = 0.68, CHCl 3 ) 1 H NMR: (CDCl 3 ) : 7.25 (m, 5H), 5.00 (d, 1H, J = 6.3), 4.35 (d, 1H, J = 6.3), 3.99 (q, 2H, J = 6.9), 1.02 (t, 3H, J = 6.9). 13 C NMR: (CDCl 3 ) : 168.4, 138.7, 129.1, 128.9, 127.2, 77.1, 75.1, 63.4, 62.7, 14.2. IR (neat): v(cm -1 ): 3475.3, 2982.2, 1745.8, 1373.1, 1258.9, 1095.7. OCO2Et128 Chlorohydrin 127 (1.3 g, 5.7 mmol) was added to DMF (28 mL) and stirred at room temperature. Potassium carbonate (2.2 g, 17.1 mmol, 3 equiv) and water (525 L) were added to the reaction mixture and stirred for 5 hours. The resulting mixture was diluted with water (75 mL), and then the aqueous layer was extracted with diethyl ether (3 x 75 mL). The organic layer was then washed with water (6 x 5 mL) to remove residual DMF. The combined organic layers were dried with magnesium sulfate and concentrated under reduced pressure to yield 1.08 g of 128 as a colorless oil in a 99% yield. [] D = +24 (c = 1.5, CHCl 3 ) Lit. 89 [] D = +25 (c = 1.1, CHCl 3 ) 1 H NMR: (CDCl 3 ) : 7.31 (m, 5H), 4.27 (d, 1H, J = 4.8), 4.00 (m, 2H), 3.82 (d, 1H, J = 4.8), 1.01 (t, 3H, J = 6.9). 13 C NMR: (CDCl 3 )

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86 : 167.0, 133.3, 129.0, 128.8, 128.4, 127.0, 126.2, 61.6, 57.8, 56.1, 14.3. IR (neat): v(cm -1 ): 2982.2, 1753.0, 1729.6, 1245.0, 1053.6, 698.7. Anal. Calcd for C 11 H 12 O 3 : C, 68.74; H, 6.29. Found: C, 68.34; H, 6.57. ent-128 was prepared the same way and spectral data matched that reported above, except for the optical rotation which was [] D = -28.9 (c = 2.9, CHCl 3 ). NOCO2Et129 Glycidic ester 128 (0.50 g, 2.6 mmol) was added to benzonitrile (4 mL) and cooled to 0 C under argon atmosphere. Boron trifluoride diethyl etherate (330 L, 2.6 mmol, 1 equiv) was slowly added to the reaction mixture over a 10 minute period. The reaction was allowed to stir and warm to room temperature over 3 hours. Saturated sodium bicarbonate (4 mL) was then added to the reaction and stirred for 2 hours. The reaction mixture was diluted with water (20 mL) followed by extraction with methylene chloride (3 x 20 mL). The combined organic layers were separated and dried with magnesium sulfate, followed by purification by flash chromatography (silica, Cyclohexane: Ether 85: 15) affording 0.42 g of 129 as a colorless oil with a 55% yield. [] D = +11 (c = 1.1, CHCl 3 ) 1 H NMR: (CDCl 3 ) : 8.02 (d, 2H), 7.30 (m, 8H), 5.37 (d, 1H, J = 6.3), 4.81(d, 1H, J = 6.3), 4.25 (m, 2H), 1.26 (t, 3H, J = 7.2). 13 C NMR: (CDCl 3 ) : 170.3, 141.4, 132.1, 129.1, 128.9, 128.7, 128.6, 128.2, 126.7, 74.9, 62.0, 14.4. IR (neat): v(cm -1 ):

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87 2982.2, 1753.0, 1729.6, 1245.0, 1053.6, 698.7. ent-129 was prepared the same way and spectral data matched that reported above, except for the optical rotation which was [] D = -12 (c = 1.7, CHCl 3 ). ONHOEtOOH130 A solution of compound 129 (0.32 g, 1.10 mmol) in 0.5 M HCl (2.5 mL) and ethanol (7 mL) was held at reflux for 6 hours. After the solvent was removed under reduced pressure, the residue was dissolved in methylene chloride (10 mL) and washed with water (2 x 7.5 mL). The combined organic layers were dried with magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, Hexane: EtOAc 50: 50) to afford 0.3 g of 130 as a white solid in an 85% yield. Mp = 162 163 C. [] D = -12 (c = 2.0, CHCl 3 ) 1 H NMR: (CDCl 3 ) : 7.76 (d, 2H), 7.30 (m, 8H), 6.98 (d, 1H, J = 8.3), 5.76 (d, 1H, J 1 = 2.1, J 2 = 9.3), 4.62 (q, 1H, J = 2.1), 4.30 (m, 2H), 3.30 (d, 1H, J = 4.2), 1.30 (t, 3H, J = 6.9). 13 C NMR: (CDCl 3 ) : 173.1, 167.1, 138.9, 134.4, 131.9, 128.9, 128.8, 128.1, 127.2, 127.1, 62.9, 60.6, 55.0, 14.4. IR (neat): v(cm -1 ): 3416.0, 3352.3, 1718.0, 1637.9, 1528.6. ent-130 was prepared the same way and spectral data matched that reported above, except for the optical rotation, which was [] D = +12 (c = 1.0, CHCl 3 )

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88 OOOEt213 Meldrums acid 212 (13.2 g, 91.6 mmol) was added to methylene chloride (170 mL) under an argon atmosphere. This solution was cooled to 0 C, and then phenylacetyl chloride 211 (14.16 g, 91.6 mmol, 1 equiv) was added to the reaction mixture over a 15 minute period. The reaction was allowed to reach room temperature and then stirred overnight. The reaction mixture was followed by a wash with 10% HCl (2 x 25 mL) and water (1 x 25 mL). The combined organic layers were then dried with magnesium sulfate and concentrated. The oily residue was dissolved in 100 mL of ethanol and held at reflux for 3 hours. The solution was concentrated and then distilled under vacuum (80 C, 0.3 mmHg) to afford 16.2 g of 213 as a colorless oil in a 86% yield. 1 H NMR: (CDCl 3 ) : 7.30 (m, 5H), 4.14 (q, 2H, J = 6.1 Hz), 3.79 (s, 2H), 3.43 (s, 2H), 1.23 (t, 3H, J = 6.1 Hz). 13 C NMR: (CDCl 3 ) : 167.5, 129.9, 129.8, 129.6, 129.3, 129.1, 128.9, 127.8. IR (neat): v(cm -1 ): 2984.3, 1742.3, 1730.0, 1315.9, 1030.4. OOOEtCl214 -Keto ester 213 (15.5 g, 75 mmol) was added to chloroform (170 mL) and stirred under argon at 25 C, then sulfuryl chloride (5.7 mL, 60 mmol, 0.6 equiv) was slowly added over a 15 minute period. The reaction mixture was stirred

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89 under argon for 3 hours or until the reaction was confirmed complete by TLC. Water (100 mL) was added to the mixture and the aqueous layer was extracted with methylene chloride (3 x 50 mL). The combined organic layers were dried with magnesium sulfate and concentrated yielding a light pink oil. The oil was separated by flash chromatography (silica, Hexanes: Toluene 1:1) to yield 13.2 g of 214 as a pink oil in 92% yield with recovered starting material. 1 H NMR: (CDCl 3 ) 7.32 (m, 5H), 4.87 (s, 1H), 4.22 (q, 2H, J = 6.3 Hz), 4.01 (d, 2H, J = 4.5 Hz), 1.27 (t, 3H, J = 6.1 Hz). 13 C NMR: (CDCl 3 ) : 196.5, 165.1, 132.7, 129.9, 129.0, 127.7, 63.4, 60.6, 45.9, 14.1. IR (film): (cm -1 ): 2987, 1764, 1692, 1269, 1185. OHOOEtCl215 A 45 mL solution of LB broth (supplemented with 30 g/mL kanamycin) was inoculated with a single colony of E. coli (BL21(DE3)(pIK4)) and shaken overnight at 37 C. The preculture (40 mL) was diluted (1: 100) into 4.0 L of LB (supplemented with 30 g/mL kanamycin) in a New Brunswick M19 fermenter. The culture was grown for 2 hours at 37 C with a stir rate of 800 rpm and an air flow of 0.5 vessel volumes per minute (vvm) until it reached O.D. 600 = 0.6. The cell suspension was cooled to 28 C over 15 minutes and then reductase overexpression was induced with isopropylthio--D-galactoside to a final concentration at 0.1 mM. The cells were kept under the same conditions for 6

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90 hours and then collected by centrifugation (6000 g for 10 min at 4 C). Half of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KP i (pH = 5.6) containing 4 g/L glucose. The bioconversion was carried out in a Braun Biostat B fermenter at 30 C with a pH maintained at 5.6 using 3 M NaOH. The dissolved oxygen was maintained at 75% saturation using a fixed air flow of 0.25 vvm and variable stirring rate. After the addition of the XAD-4 resin (0.5 g), portions of neat 214 (0.2 mL) were added approximately every hour over a total of 12 hours to provide a final concentration of 5 mM. Portions of glucose were added after 3.0 hours and 6.0 hours to maintain the glucose concentration at approximately 4 g/L. Consumption of 214 and glucose slowed significantly after 7 hours. After 24 hours, the reaction was gently extracted with methylene chloride (2 x 300 mL) to avoid an emulsion. The combined organic layers were dried with magnesium sulfate and concentrated under vacuum. The crude oil was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 1.1 g of 215 as a colorless oil with an 82% yield. [] D = +23.7 (c = 0.7, CHCl 3 ). 1 H NMR: (C 6 D 6 ) 7.06 (m, 5H), 4.24 (ddd, 1H, J 1 = 3.3 Hz, J 2 = 6.6 Hz, J 3 = 7.1 Hz), 4.05 (d, 1H J = 3.3 Hz), 3.78 (q, 2H, J = 7.2 Hz), 2.82 (dd, 1H, J 1 = 7.1 Hz, J 2 = 13.8 Hz), 2.72 (dd, 1H, J 1 = 6.6 Hz, J 2 = 13.8 Hz), 2.4 (br s, 1H), 0.79 (t, 3H, J = 7.2 Hz). 13 C NMR: (CDCl 3 ) 169.1, 137.1, 129.8, 129.2, 127.4, 73.4, 63.0, 61.2, 40.4, 14.4. IR (film): (cm -1 ): 3483, 1740, 1300, 1183, 1025. Anal. Calcd for C 12 H 15 ClO 3 : C, 59.39; H, 6.23. Found: C, 59.75; H, 6.72.

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91 OOEtO216 Chlorohydrin 215 (1.0 g, 4.1 mmol) was added to DMF (19 mL) and stirred at room temperature. Potassium carbonate (1.7 g, 12.4 mmol, 3 equiv) and water (390 L) was added to the reaction mixture and stirred for 5 hours. The resulting mixture was diluted with water (50 mL), and the aqueous layer was extracted with ether (3 x 50 mL). The combined organic layers were then washed with water (6 x 5 mL) to remove residual DMF. The organic layer was dried with magnesium sulfate and purified by flash chromatography (silica, Cyclohexanes: Ether 85: 15) to afford 0.84 g of 216 as a colorless oil in a 99% yield. [] D = +37.1 (c = 3.0, CHCl 3 ). 1 H NMR: (CDCl 3 ) : 7.10 (m, 5H), 3.87 (dd, 2H, J = 2.7 Hz, J = 6.9), 3.17 (d, 1H, J = 4.2 Hz), 3.00 (dq, 2H, J 1 = 6.3, J 2 = 14.8), 0.83 (t, 3H, J = 7.2 Hz) 13 C = NMR: (CDCl 3 ) : 168.6, 137.0, 129.3, 129.1, 127.2, 62.0, 58.1, 53.3, 34.2, 14.7. IR (neat): v(cm -1 ): 2983.6, 2917.8, 1748.8, 1197.8, 1032.0. Anal. Calcd for C 12 H 14 O 3 : C, 69.89; H, 6.84. Found: C, 70.10; H, 6.91. NOOEtO217

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92 Glycidic ester 216 (100 mg, 0.49 mmol) was added to benzonitrile (900 L) and cooled to 0 C under an argon atmosphere. Boron trifluoride diethyl etherate (61.5L, 0.49 mmol, 1 equiv) was then slowly added to the reaction mixture over 10 minutes. The reaction was allowed to stir and warm up to room temperature over a 3 hour period. Saturated sodium bicarbonate (2 mL) was then added to the reaction and stirred for 2 hours. The reaction mixture was diluted with water (10 mL) followed by extraction with methylene chloride (3 x 15 mL). The organic layers were combined and dried with magnesium sulfate, followed by purification by flash chromatography (silica, Cyclohexane: Ether 9: 1) affording 0.12 g of 217 as a colorless oil with a 78% overall yield. [] D = -57.1 (c = 2.0, CHCl 3 ). 1 H NMR: (C 6 D 6 ) 8.37 (m, 2H), 7.28 (m, 8H), 4.90 (ddd, 1H, J 1 = 6.3 Hz, J 2 = 6.3 Hz, J 3 = 6.9 Hz), 4.78 (d, 1H, J = 6.3 Hz), 3.93 (m, 2H), 3.18 (dd, 1H, J 1 = 6.3 Hz, J 2 = 13.8 Hz), 2.92 (dd, 1H, J 1 = 6.9 Hz, J 2 = 13.8 Hz), 0.91 (t, 3H, J = 7.2 Hz). 13 C NMR: (C 6 D 6 ) 170.2, 163.2, 131.8, (other aromatic signals obscured by solvent), 80.3, 73.7, 61.2, 42.1, 14.0. IR (film): (cm -1 ): 3029, 2982, 1752, 1655, 1206, 1027, 695. Anal. Calcd for C 19 H 19 O 3 N: C, 73.77; H, 6.19; N, 4.53. Found: C, 73.63; H, 6.14; N, 4.56. NH2OHOHO137 Oxazoline 217 (323 mg, 1.04 mmol) was dissolved in ethanol (8 mL) and water (6 mL) and then concentrated HCl (15 mL, 12 M) was added. This solution

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93 was heated at reflux for 8 hours and then concentrated under reduced pressure. The residue was resuspended in water (3 mL) and ethanol (1.5 mL) and then a Dowex 50WX8-100 resin (5 g) was added and gently swirled by hand for 5 minutes. The suspension was filtered and rinsed with water (15 mL), followed by a rinse with ethanol (15 mL). The beads were then rinsed with 2 M ammonium hydroxide and the filtrate was collected and lyophilized to afford 0.16 g of 137 as a white powder. The solid was dissolved in 6 M HCl (4 mL) and the solution was lyophilized to afford 0.20 g of the HCl salt with an 83% overall yield. [] D = +23.2 (c = 1.3, 1 M HCl). (Sigma-Aldrich) [] D = +23.4 (c = 1.0, 1 M HCl) Lit 67 [] D = +26.8 (c = 0.7, 1 M HCl) 1 H NMR: (D2O) : 7.39 (m, 5H), 4.37 (d, 1H, J = 3.0 Hz), 3.98 (dt, 1H, J 1 = 3.0 Hz J 2 = 7.5 Hz), 3.10 (dq, 2H, J 1 = 7.5 Hz, J 2 = 15 Hz). 13 C NMR: (D2O) : 174.6, 135.3, 129.8, 129.6, 128.1, 68.8, 54.8, 35.5. IR (KBr): v(cm -1 ): 3385.1, 2916.9, 2848.6, 1731.5, 1604.6, 1496.8, 1029.4. Melting Point: 201 203 C. Melting Point: (Sigma-Aldrich) 203 204 C. H3COOOHCl248 Et A 45 mL solution of LB broth (supplemented with 30 g/mL kanamycin) was inoculated with a single colony of E. coli (BL21(DE3)(pIK30)) and shaken overnight at 37 C. The preculture (40 mL) was diluted (1: 100) to 4.0 L of LB (supplemented with 30 g/mL kanamycin) in a New Brunswick M19 fermenter. The culture was grown for 2 hours at 37 C with a stir rate of 800 rpm and an air

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94 flow of 0.5 vessel volumes per minute (vvm) until it reached O.D. 600 = 0.6. The cell suspension was cooled to 28 C over 15 minutes and then reductase overexpression was induced with isopropylthio--D-galactoside to a final concentration at 0.1 mM. The cells were kept under the same conditions for 6 hours and then collected by centrifugation (6000 g for 10 min at 4 C). Half of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KP i (pH = 5.6) containing 4 g/L glucose. The bioconversion was carried out in a Braun Biostat B fermenter at 30 C with a pH maintained at 5.6 using 3 M NaOH. The dissolved oxygen was maintained at 75% saturation using a fixed air flow of 0.25 vvm and variable stirring rate. Portions of commercially available ethyl 2-chloroacetoacetate 220 (0.25 mL) were added approximately 4 times an hour over a total of 8 hours to provide a final concentration of 24 mM. Portions of glucose were added after 2.0 hours and 5.0 hours to maintain the glucose concentration at approximately 4 g/L. Consumption of 220 and glucose slowed significantly after 6 hours. After 12 hours, the reaction was gently extracted with methylene chloride (4 x 300 mL) to avoid an emulsion. The combined organic layers were dried with magnesium sulfate and concentrated under vacuum. The crude oil was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 4.4 g of 248 as a colorless oil with an 89% yield. [] D = +10.8 (c = 1.9, CHCl 3 ). Lit. 107 [] D = +12.4 (c = 1.0, CHCl 3 ). 1 H NMR: (C 6 D 6 ) : 4.00 (m, 1H, J = 5.7 Hz), 3.88 (d, 1H J = 4.7 Hz), 3.82 (q, 2H, J = 7.2 Hz), 2.33 (bs, 1H), 1.02 (t, 3H, J = 6.3), 0.84 (t, 3H, J = 7.2). 13 C NMR: (C 6 D 6 ) : 168.6, 68.6, 63.5,

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95 62.3, 19.7, 13.9. IR (neat): v(cm -1 ): 3458.3, 2984.3, 1747.1, 1182.6, 944.1, 878.4. OH3COO250 Et Chlorohydrin 248 (100 mg, 0.6 mmol) was added to DMF (3.2 mL) and stirred at room temperature. Potassium carbonate (249 mg, 1.80 mmol, 3 equiv) and water (62 L, 3 equiv) was added to the reaction mixture and stirred for 5 hours. The resulting mixture was dissolved in water (30 mL), and the aqueous layer was extracted with diethyl ether (3 x 30 mL). The organic layer was then washed with water (6 x 3 mL) to remove residual DMF. The combined organic layers were dried with magnesium sulfate and purified by flash chromatography (silica, Cyclohexanes: Ether 85: 15) to afford 0.66 g of 250 as a colorless oil in an 85% yield. [] D = -13.8 (c = 1.4, CHCl 3 ). 1 H NMR: (C 6 D 6 ) : 3.91 (q, 2H, J = 7.2 Hz), 3.19 (d, 1H J = 4.2 Hz), 2.73 (m, 1H, J = 4.5 Hz), 1.11 (d, 3H, J = 5.4), 0.90 (t, 3H, J = 7.2). 13 C NMR: (C 6 D 6 ) : 168.0, 60.9, 53.0, 52.8, 14.2, 12.9. IR (neat): v(cm -1 ): 3418.5, 2929.5, 1663.3, 1389.9, 1258.8, 1098.5. OH3COO243 Et Chlorohydrin 248 (500 mg, 3.01 mmol) was dissolved in ethanol (12 mL) and cooled to 0 C. A small chunk of excess sodium (213 mg, 9.00 mmol) was slowly added to the reaction mixture and allowed to stir for three hours at 0 C.

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96 The solution was concentrated under reduced pressure and then 0.5 M HCl (20 mL) added to the residue. The aqueous mixture was extracted with diethyl ether (3 x 20 mL) and the organic layers were combined and dried by magnesium sulfate. The combined organic layers were concentrated under reduced pressure to afford a mixture of cis and trans isomers that could not be fully separated. Spectral data was not obtained because 243 was not obtained in pure form.

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APPENDIX B ADDITIONAL INFORMATION NMR Analysis for Absolute Configuration of Compound 215 and 230 NMR spectra were recorded on a Varian Inova spectrometer equipped with a 5 mm indirect detection probe, operating at 500 MHz for 1 H and at 125 MHz for 13 C. Chemical shifts at 25C are reported in ppm relative to TMS. In this case benzene-d 6 was used as the solvent to analyze compounds 215 and 230 to remove some signal overlap. Complete 1 H and 13 C chemical shifts assignments for the compound were based on the 1 H1 H, 1 H13 C one-bond and 1 H13 C long-range couplings seen in the proton and the G-BIRD-HSQMBC spectra. The relative configuration of the reduction product was determined by NMR through the combined use of 3 J(H-H) homonuclear and n J(C-H) long range heteronuclear coupling constants 115,116 Of the six staggered rotamers of the erythro (anti) and threo (syn) configurations, presented in Figure B-3, four can be identified by the pattern of coupling constants. The other two rotamers, A3 and B3, display the same pattern of coupling constants. Fortunately, in none of the pairs of diastereomers that we analyzed was threo in the A3 conformation and the erythro in the B3. The long range coupling constants presented in Figure B-1 were measured in f2 slices of the G-BIRD-HSQMBC spectra, 122 with a precision of 0.5 Hz. The patterns of coupling constants indicate that the preferred conformation is A1 for the threo configuration and B3 for the erythro, i.e. the conformation in which the 97

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98 R and the COOEt groups are anti. These are the only conformations for 215 and 230. OEtOOHCl215 YDR368w 3 J (Hz) Size OEtOOHCl230 YGL157w 3 J (Hz) Size OEtOOHCl230 YGL039w 3 J (Hz) Size 3 J(H 2 -H 3 ) 3.4 small 7.8 large 7.8 large 3 J(H 2 -C 4 ) 1.2 small 2.5 small 2.5 small 3 J(H 3 -C 1 ) 2.3 small 3.1 small 3.1 small 2 J(H 2 -C 3 ) 2.1 small 5.7 large 5.7 large conformer A1 B3 B3 Stereochemistry threo erythro erythro Figure B-1. Long range coupling constants (Hz) for the major enzymatic reduction product of YDR368w, YGL157w, and YGL039w Absolute configurations were determined based on the differences in chemical shifts between the (R)and (S)--methoxy--phenylacetic (MPA) esters 117,118. The esterification was carried out in the NMR tube, by adding one equivalent of a mixture consisting of 2 parts (R)-MPA and 1 part (S)-MPA to the alcohol, followed by the addition of DCC (1.5 equiv.) and DMAP (0.5 equiv.). The chemical shifts difference between the Rand S-MPA esters demonstrate that 215 is the 2R,3S ester and 230 is the 2S,3S ester (Figure B-2).

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99 S-threoYDR368wH2'H1'H1'aH2H3H4aH4bHorthoalcohol1.334.284.284.284.393.022.957.29R-MPA ester1.254.164.164.325.622.912.836.98S-MPA ester1.023.843.704.275.623.073.117.26R-MPA alcohol-0.08-0.12-0.120.041.23-0.11-0.12-0.31S-MPA alcohol-0.31-0.44-0.58-0.011.230.050.16-0.03RS0.230.320.460.050.00-0.16-0.28-0.28S-erythroYGL157wH2'H1'H1'aH2H3H4aH4bHorthoalcohol1.244.184.184.284.193.052.807.20R-MPA ester1.304.154.154.325.572.952.956.82S-MPA ester1.153.873.804.275.573.173.03nmR-MPA alcohol0.06-0.03-0.030.041.38-0.100.15-0.38S-MPA alcohol-0.09-0.31-0.38-0.011.380.120.23RS0.150.280.350.050.00-0.22-0.08YGL039wH2'H1'H1'aH2H3H4aH4bHorthoalcohol1.244.184.184.094.193.052.807.20R-MPA ester1.274.144.144.365.552.952.956.82S-MPA ester1.123.873.804.235.563.173.03nmR-MPA alcohol0.03-0.04-0.040.271.36-0.100.15-0.38S-MPA alcohol-0.12-0.31-0.380.141.370.120.23RS0.150.270.340.13-0.01-0.22-0.08 Figure B-2. The chemical shifts for the major reduction products of YDR368w, YGL157w, and YGL039w

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100 OHHRC2HClEtO2C OHHRC2HClEtO2C OHHRC2HClEtO2C 3 J(H 2 -H 3 ) small small large 3 J(H 2 -C 4 ) small large small 3 J(H 3 -C 1 ) small large small 2 J(H 2 -C 3 ) small large large conformer A1 A2 A3 Stereochemistry threo threo threo OHHRC2HClEtO2C OHHRC2HClEtO2C OHHRC2HClEtO2C 3 J(H 2 -H 3 ) small small large 3 J(H 2 -C 4 ) large small small 3 J(H 3 -C 1 ) small large small 2 J(H 2 -C 3 ) large small large conformer B1 B2 B3 Stereochemistry erythro erythro erythro Figure B-3. Coupling constants in the staggered rotamers of threo and erythreo diastereomers of reduction products large small 3 J(H 2 -H 3 ) 8 to 11 1 to 4 3 J(H 2 -C 4 ) 6 to 8 1 to 3 3 J(H 3 -C 1 ) 6 to 8 1 to 3 2 J(H 2 -C 3 ) -5 to -7 0 to -2 Figure B-4. Range of values for the large and small coupling constants (Hz)

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101 Specific Activity Calculations A = bc = L / mol-cm b = cm c = mol / L Velocity (mol / L-min) = [A / t] / b = c / t Activity (mol / min-mL) = (moles NADPH consumed / unit) / Volume of protein used Specific activity (mol / min-mg) = activity / protein concentration Protein concentration was calculated by a traditional Bradford assay using BSA, monitored at 595 nm (Figure B-5). Bradford Assayy = 0.0369x + 0.0217R2 = 0.986400.10.20.30.40.5051015[BSA] ug/mLAbs 595 nm ABS Linear (ABS) Figure B-5. Line equation for the Bradford assay

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102 NMR Spectra NH2OHOHO137 Figure B-6. NMR of (+)-AHPA synthesized by our strategy

PAGE 118

103 NH2OHOHO137 Figure B-7. NMR spectra of authentic (+)-AHPA

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104 ONHOEtOOH130 Figure B-8. NMR spectra of the Taxol side chain 130

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105 ONHOEtOOHent-130 Figure B-9. NMR spectra of the Taxol side chain enantiomer ent-130

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106 Figure B-10. 1 H NMR for derivatized 130 and ent-130: Top spectra is the (S)-MPA ester of ent-130; bottom spectra is the (S)-MPA ester of the Taxol side chain 130

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107 OEtOOHClOEtOOHCl215230 Figure B-11. 1 H NMR spectra of syn product from YDR368w (top) and anti product from TGL157w (bottom)

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LIST OF REFERENCES (1) Noble, R. L.; Beer, C. T.; Cutts, J. H. Annals of the New York Academy of Sciences, 76, 882-894. (2) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665-667. (3) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. Journal of the American Chemical Society 197, 93, 2325-2327. (4) Holton, R. A.; Somoza, C.; Kim, H. B.; Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; et al. Journal of the American Chemical Society 1994, 116, 1597-1598. (5) Holton, R. A.; Kim, H. B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; et al. Journal of the American Chemical Society 1994, 116, 1599-1600. (6) Nicolaou, K. C.; Nantermet, P. G.; Ueno, H.; Guy, R. K.; Couladouros, E. A.; Sorensen, E. J. Journal of the American Chemical Society 1995, 117, 624-633. (7) Nicolaou, K. C.; Liu, J. J.; Yang, Z.; Ueno, H.; Sorensen, E. J.; Claiborne, C. F.; Guy, R. K.; Hwang, C. K.; Nakada, M.; Nantermet, P. G. Journal of the American Chemical Society 1995, 117, 634-644. (8) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Nantermet, P. G.; Claiborne, C. F.; Renaud, J.; Guy, R. K.; Shibayama, K. Journal of the American Chemical Society 1995, 117, 645-652. (9) Nicolaou, K. C.; Ueno, H.; Liu, J. J.; Nantermet, P. G.; Yang, Z.; Renaud, J.; Paulvannan, K.; Chadha, R. Journal of the American Chemical Society 1995, 117, 653-659. (10) Danishefsky, S. J.; Masters, J. J.; Young, W. B.; Link, J. T.; Snyder, L. B.; Magee, T. V.; Jung, D. K.; Isaacs, R. C. A.; Bornmann, W. G.; et al. Journal of the American Chemical Society 1996, 118, 2843-2859. (11) Wender, P. A.; Marquess, D. G.; McGrane, L. P.; Taylor, R. E. Chemtracts: Organic Chemistry 1994, 7, 160-171. 108

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109 (12) Mukaiyama, T.; Shina, I.; Iwadare, H.; Sakoh, H.; Tani, Y.-I.; Hasegawa, M.; Saitoh, K. Proceedings of the Japan Academy, Series B: Physical and Biological Sciences 1997, 73B, 95-100. (13) Mukaiyama, T.; Shiina, I.; Iwadare, H.; Saitoh, M.; Nishimura, T.; Ohkawa, N.; Sakoh, H.; Nishimura, K.; Tani, Y.-I.; Hasegawa, M.; Yamada, K.; Saitoh, K. Chemistry--A European Journal 1999, 5, 121-161. (14) Kusama, H.; Hara, R.; Kawahara, S.; Nishimori, T.; Kashima, H.; Nakamura, N.; Morihira, K.; Kuwajima, I. Journal of the American Chemical Society 2000, 122, 3811-3820. (15) Bisaria, V.; Panda, A. Current opinion in biotechnology :199, 370-374. (16) Brodelius, P.; Deus, B.; Mosbach, K.; Zenk, M. H. Enzyme Engineering 1980, 5, 373-381. (17) Reinhard, E.; Alfermann, A. W. Advances in Biochemical Engineering 1980, 16, 49-83. (18) Deus-Neumann, B.; Zenk, M. H. Planta Medica 1984, 50, 427-431. (19) Hall, R. D.; Yeoman, M. M. Journal of Experimental Botany 1987, 38, 1391-1398. (20) Schripsema, J.; Verpoorte, R. Planta Medica 1992, 58, 245-249. (21) Chang, S. H.; Ho, C. K.; Chen, Z. Z.; Tsay, J. Y. Plant Cell Reports 2001, 20, 496-502. (22) Stierle, A.; Strobel, G.; Stierle, D. Science :1993, 260, 214-216. (23) Huang, Q.; Roessner, C. A.; Croteau, R.; Scott, A. I. Bioorganic & Medicinal Chemistry 2001, 9, 2237-2242. (24) Walker, K.; Long, R.; Croteau, R. Proceedings of the National Academy of Sciences of the United States of America :2002, 99, 9166-9171.. (25) Jennewein, S.; Croteau, R. Applied Microbiology and Biotechnology :2001 Oct, 57, 13-19. (26) Jennewein, S.; Wildung Mark, R.; Chau, M.; Walker, K.; Croteau, R. Proceedings of the National Academy of Sciences of the United States of America :2004, 101, 9149-9154. (27) Chau, M.; Croteau, R. Archives of Biochemistry and Biophysics 2004, 427, 48-57.

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110 (28) Chau, M.; Jennewein, S.; Walker, K.; Croteau, R. Chemistry & biology :2004, 663-672.:. (29) Chauviere, G.; Guenard, D.; Picot, F.; Senilh, V.; Potier, P. Comptes Rendus des Seances de l'Academie des Sciences, Serie 2: Mecanique-Physique, Chimie, Sciences de l'Univers, Sciences de la Terre 1981, 293, 501-503. (30) Denis, J. N.; Greene, A. E.; Serra, A. A.; Luche, M. J. Journal of Organic Chemistry 1986, 51, 46-50. (31) Holton, R. A. In U.S. Patent 5,015,744: United States, 1989. (32) Deng, L.; Jacobsen, E. N. Journal of Organic Chemistry 1992, 57, 4320-4323. (33) Wang, Z.-M.; Kolb, H. C.; Sharpless, K. B. Journal of Organic Chemistry 1994, 59, 5104-5105. (34) Lee, K.-Y.; Kim, Y.-H.; Park, M.-S.; Ham, W.-H. Tetrahedron Letters 1998, 39, 8129-8132. (35) Borah, J. C.; Gogoi, S.; Boruwa, J.; Kalita, B.; Barua, N. C. Tetrahedron Letters 2004, 45, 3689-3691. (36) Lee, D.; Kim, M.-J. Tetrahedron Letters 1998, 39, 2163-2166. (37) Kayser, M. M.; Mihovilovic, M. D.; Kearns, J.; Feicht, A.; Stewart, J. D. Journal of Organic Chemistry 1999, 64, 6603-6608. (38) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C. Journal of Organic Chemistry 1998, 63, 2351-2353. (39) Hamamoto, H.; Mamedov, V. A.; Kitamoto, M.; Hayashi, N.; Tsuboi, S. Tetrahedron: Asymmetry 2000, 11, 4485-4497. (40) Mandai, T.; Oshitari, T.; Susowake, M. Synlett 2002, 1665-1668. (41) Castagnolo, D.; Armaroli, S.; Corelli, F.; Botta, M. Tetrahedron: Asymmetry 2004, 15, 941-949. (42) Srivastava, R. P.; Zjawiony, J. K.; Peterson, J. R.; McChesney, J. D. Tetrahedron: Asymmetry 1994, 5, 1683-1688. (43) Zhou, Z.; Mei, X. Synthetic Communications 2003, 33, 723-728. (44) Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.; Takeuchi, T. Journal of Antibiotics 1976, 29, 97-99.

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111 (45) Aoyagi, T., Suda, H., Nagai, J. Arch. Biochem., Biophys. 1976. (46) Suda, H.; Takita, T.; Aoyagi, T.; Umezawa, H. Journal of Antibiotics :1976 Jan, 29, 100-101.:. (47) Nakamura, H.; Suda, H.; Takita, T.; Aoyagi, T.; Umezawa, H. Journal of Antibiotics, 1976, 102-103. (48) Suda, H.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. Archives of Biochemistry and Biophysics, 1976, 196-200. (49) Harbeson, S. L.; Rich, D. H. Biochemistry,1988,, 7301-7310. (50) Abe, F.; Ino, K.; Bierman, P. J.; Talmadge, J. E.; Sawada, H.; Ogino, T.; Ishizuka, M.; Aoyagi, T. International Congress Series 2001, 1218, 155-159. (51) Ishizuka, M.; Masuda, T.; Kanbayashi, N.; Fukasawa, S.; Takeuchi, T.; Aoyagi, T.; Umezawa, H. Journal of Antibiotics, 1980, 642-652. (52) Aozuka, Y.; Koizumi, K.; Saitoh, Y.; Ueda, Y.; Sakurai, H.; Saiki, I. Cancer Letters, 2004, 216, 35-42. (53) Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.; Takeuchi, T. Journal of Antibiotics 1976, 29, 97-99. (54) Bourinbaiar, A. S. In U.S.; (Metatron, Inc., USA). Us, 2001, p 21. (55) Pulido-Cejudo, G. In PCT Int. Appl.; (Canbreal Therodiagnostics Canada Holding Corp., Can.). Wo, 2000, p 43 pp. (56) Pulido-Cejudo, G.; Conway, B.; Proulx, P.; Brown, R.; Izaguirre, C. A. Antiviral Research 1997, 36, 167-177. (57) Bourinbaiar, A.; Lee-Huang, S.; Krasinski, K.; Borkowsky, W. Biomedicine & Pharmacotherapy 1994, 48, 55-61. (58) Pulido-Cejudo, G.; Conway, B.; Proulx, P.; Brown, R.; Izaguirre, C. A. Antiviral Research :1997, 36, 167-177. (59) Bourinbaiar, A. S.; Lee-Huang, S.; Krasinski, K.; Borkowsky, W. Biomedicine & Pharmacotherapy, 1994, 48, 55-61. (60) Suda, H.; Takita, T.; Aoyagi, T.; Umezawa, H. Journal of Antibiotics 1976, 29, 600-601. (61) Nishizawa, R.; Saino, T.; Suzuki, M.; Fujii, T.; Shirai, T.; Aoyagi, T.; Umezawa, H. Journal of Antibiotics :1983, 36, 695-699.

PAGE 127

112 (62) Pearson, W. H.; Hines, J. V. Journal of Organic Chemistry 1989, 54, 4235-4237. (63) Norman, B. H.; Morris, M. L. Tetrahedron Letters 1992, 33, 6803-6806. (64) Palomo, C.; Aizpurua, J. M.; Cuevas, C. Journal of the Chemical Society, Chemical Communications 1994, 1957-1958. (65) Koseki, K.; Ebata, T.; Matsushita, H. Bioscience, Biotechnology, and Biochemistry 1996, 60, 534-536. (66) Bergmeier, S. C.; Stanchina, D. M. Journal of Organic Chemistry 1999, 64, 2852-2859. (67) Seki, M.; Nakao, K. Bioscience, Biotechnology, and Biochemistry 1999, 63, 1304-1307. (68) Semple, J. E.; Owens, T. D.; Nguyen, K.; Levy, O. E. Organic Letters 2000, 2, 2769-2772. (69) Lee, B. W.; Lee, J. H.; Jang, K. C.; Kang, J. E.; Kim, J. H.; Park, K.-M.; Park, K. H. Tetrahedron Letters 2003, 44, 5905-5907. (70) Kudyba, I.; Raczko, J.; Jurczak, J. Tetrahedron Letters 2003, 44, 8685-8687. (71) Wasserman, H. H.; Petersen, A. K.; Xia, M. Tetrahedron 2003, 59, 6771-6784. (72) Gentile, A.; Giordano, C.; Fuganti, C.; Ghirotto, L.; Servi, S. Journal of Organic Chemistry 1992, 57, 6635-6637. (73) Furui, M.; Furuya, T.; Seko, H. In Jpn. Kokai Tokkyo Koho; (Tanabe Seiyaku Co, Japan). Jp, 1996, p 14. (74) Furutani, T.; Furui, M.; Mori, T.; Shibatani, T. Applied Biochemistry and Biotechnology 1996, 59, 319-328. (75) Furui, M.; Shibatani, T. Maku 1996, 21, 41-48. (76) Furui, M.; Furutani, T.; Shibatani, T.; Nakamoto, Y.; Mori, T. Journal of Fermentation and Bioengineering 1996, 81, 21-25. (77) Cane, D. E.; Sohng, J. K.; Williard, P. G. Journal of Organic Chemistry 1992, 57, 844-851. (78) Wang, Z.-X.; Shi, Y. Journal of Organic Chemistry 1997, 62, 8622-8623.

PAGE 128

113 (79) Wang, Z.-X.; Miller, S. M.; Anderson, O. P.; Shi, Y. Journal of Organic Chemistry 1999, 64, 6443-6458. (80) Armstrong, A.; Ahmed, G.; Dominguez-Fernandez, B.; Hayter, B. R.; Wailes, J. S. Journal of Organic Chemistry 2002, 67, 8610-8617. (81) Aggarwal, V. K.; Hynd, G.; Picoul, W.; Vasse, J.-L. Journal of the American Chemical Society 2002, 124, 9964-9965. (82) Corey, E. J.; Choi, S. Tetrahedron Letters 1991, 32, 2857-2860. (83) Imashiro, R.; Kuroda, T. Tetrahedron Letters 2001, 42, 1313-1315. (84) Imashiro, R.; Yamanaka, T.; Seki, M. Tetrahedron: Asymmetry 1999, 10, 2845-2851. (85) Jacobsen, E. N.; Deng, L.; Furukawa, Y.; Martinez, L. E. Tetrahedron 1994, 50, 4323-4334. (86) Anand, N.; Kapoor, M.; Koul, S.; Taneja, S. C.; Sharma, R. L.; Qazi, G. N. Tetrahedron: Asymmetry 2004, 15, 3131-3138. (87) Cabon, O.; Larcheveque, M.; Buisson, D.; Azerad, R. Tetrahedron Letters 1992, 33, 7337-7340. (88) Carbon, O.; Buisson, D.; Larcheveque, M.; Azerad, R. Tetrahedron: Asymmetry 1995, 6, 2199-2210. (89) Carbon, O.; Buisson, D.; Larcheveque, M.; Azerad, R. Tetrahedron: Asymmetry 1995, 6, 2211-2218. (90) Jorg, G.; Bertau, M. Chembiochem : a European journal of chemical biology :2004 Jan 3, 5, 87-92. (91) Shieh, W.; Gopalan, A. S.; Sih, C. J. Journal of the American Chemical Society 1985, 107, 2993-2994. (92) Shieh, W. R.; Sih, C. J. Tetrahedron: Asymmetry 1993, 4, 1259-1269. (93) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. Journal of Organic Chemistry 1991, 56, 4778-4783. (94) Martzen, M. R.; McCraith, S. M.; Spinelli, S. L.; Torres, F. M.; Fields, S.; Grayhack, E. J.; Phizicky, E. M. Science, 1999, 286, 1153-1155. (95) Kaluzna, I.; Andrew, A. A.; Bonilla, M.; Martzen, M. R.; Stewart, J. D. Journal of Molecular Catalysis B: Enzymatic 2002, 17, 101-105.

PAGE 129

114 (96) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. Journal of the American Chemical Society 2004, 126, 12827-12832. (97) Walton Adam, Z.; Stewart Jon, D. Biotechnology Progress :2004, 20, 403-411. (98) Seebach, D.; Sutter, M. A.; Weber, R. H.; Zueger, M. F. Organic Syntheses 1985, 63, 1-9. (99) Nakamura, K.; Inoue, K.; Ushio, K.; Oka, S.; Ohno, A. Chemistry Letters 1987, 679-682. (100) Nakamura, K.; Miyai, T.; Nagar, A.; Oka, S.; Ohno, A. Bulletin of the Chemical Society of Japan, 1989, 62, 1179-1187. (101) Fotadar, U.; Becu, C.; Borremans, F. A. M.; Anteunis, M. J. O. Tetrahedron 1978, 34, 3537-3544. (102) Smith, J. R. L.; Norman, R. O. C.; Stillings, M. R. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) 1975, 1200-1202. (103) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C. Synlett 1999, 1727-1730. (104) Tomisawa, K.; Kameo, K.; Matsunaga, T.; Saito, S.; Hosoda, K.; Asami, Y.; Sota, K. Chemical & Pharmaceutical Bulletin 1986, 34, 701-712. (105) Martin-Kohler, A.; Widmer, J.; Bold, G.; Meyer, T.; Sequin, U.; Traxler, P. Helvetica Chimica Acta 2004, 87, 956-975. (106) Liu, C.-L.; Li, L.; Li, Z.-M. Bioorganic & Medicinal Chemistry 2004, 12, 2825-2830. (107) Matsuki, K.; Sobukawa, M.; Kawai, A.; Inoue, H.; Takeda, M. Chemical & Pharmaceutical Bulletin 1993, 41, 643-648. (108) Inoue, H. In Jpn. Kokai Tokkyo Koho; (Tanabe Seiyaku Co., Ltd., Japan). Jp, 1991, p 5 pp. (109) Kaluzna, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga, I.; Stewart, J. D. Journal of Organic Chemistry 2005, 70, 342-345. (110) Saluta, M., Bell, P.A. In Life Science News Online, 1998. (111) Jorg, G.; Bertau, M. Analytical Biochemistry 2004, 328, 22-28. (112) Chesney, J. A.; Eaton, J. W.; Mahoney, J. R., Jr. Journal of Bacteriology 1996, 178, 2131-2135.

PAGE 130

115 (113) Gennari, C.; Carcano, M.; Donghi, M.; Mongelli, N.; Vanotti, E.; Vulpetti, A. Journal of Organic Chemistry 1997, 62, 4746-4755. (114) Oikawa, Y.; Sugano, K.; Yonemitsu, O. Journal of Organic Chemistry 1978, 43, 2087-2088. (115) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. Journal of Organic Chemistry 1999, 64, 866-876. (116) Williamson, R. T.; Marquez, B. L.; Sosa, A. C. B.; Koehn, F. E. Magnetic Resonance in Chemistry 2003, 41, 379-385. (117) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915-2925. (118) Seco, J. M.; Quinoa, E.; Riguera, R. Chemical Review, 2004, 104, 17-117. (119) Wenkert, E.; Han, A. Heterocycles, 1990, 30, 929-937. (120) Brown, M. J.; Carter, P. S.; Fenwick, A. E.; Fosberry, A. P.; Hamprecht, D. W.; Hibbs, M. J.; Jarvest, R. L.; Mensah, L.; Milner, P. H.; O'Hanlon, P. J.; Pope, A. J.; Richardson, C. M.; West, A.; Witty, D. R. Bioorganic & Medicinal Chemistry Letters 2002, 12, 3171-3174. (121) Kato, K.; Ono, M.; Akita, H. Tetrahedron Letters 1997, 38, 1805-1808. (122) Marquez, B. L.; Gerwick, W. H.; Williamson, R. T. Magnetic Resonance in Chemistry 2001, 39, 499-530.

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BIOGRAPHICAL SKETCH Brent Derek Feske was born in Joliet, Illinois, on June 28 th 1978. He began his initial research working with Dr. John Allen on the synthesis of ion selective electrodes to monitor nitrate levels in local wetlands. He then worked for Dr. Newton Hilliard on the isolation of the proteins involved for the unusual metabolic pathway in Thiobacillus neapolitanus. Brent also spent two summers at Albemarle Corporation. His first summer was spent in the analytical department working with HPLC and GC. His second summer was spent in the applications department working with flame retardants in polyurethane foam. In 2000, Brent graduated cum laude from Southeastern Louisiana University and then moved his scientific career to the University of Florida. His graduate research was on the synthesis of chiral molecules and pharmaceuticals using E. coli engineered to overexpress a single bakers yeast reductase. He will further this research, along with teaching undergraduate chemistry, at Armstrong Atlantic State University in Savannah, Georgia. 116


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Title: Synthetic Applications of Homochiral Glycidic Esters Derived from Enzymatic Reductions
Physical Description: Mixed Material
Copyright Date: 2008

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SYNTHETIC APPLICATIONS OF HOMOCHIRAL GLYCIDIC ESTERS DERIVED
FROM ENZYMATIC REDUCTIONS













By

BRENT DEREK FESKE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005
































Copyright 2005

by

Brent Derek Feske
































This document is dedicated to my loving parents.















ACKNOWLEDGMENTS

First, I would like to thank my advisor, Dr. Jon D. Stewart, for his guidance

and tutelage. He was always there to answer my questions and was willing to

assist me with learning new techniques. In addition, I would like to thank him for

taking me on my favorite bike ride. I would also like to thank Dr. Thomas Lyons

for his friendship, helpful advice, and a listening ear. I thank the rest of my

committee, Dr. Nigel Richards, Dr. Dolbier, and Dr. Madeline Rasche, for their

variety of contributions to my education.

I would like to thank all of the Stewart group members for their time, advice,

scientific help and laughs. I thank Kavitha Vedha-Peters for "taking me under her

wing" and teaching me the basics of organic chemistry. Many thanks go to

Iwona Kaluzna for her research collaboration in the laboratory and also her

friendship. I thank Brian Kyte for his help in molecular biology, proofreading, and

being a good friend. I would also like to acknowledge Despina Bougioukou for

giving me her advice EVERYDAY, whether I wanted it or not. In addition, I would

like to thank Neil Stowe, Dimitri Daschier, Heather Hillebrenner, Magdalena

Swiderska, and Parag Parekh for their combined assistance.

I am also grateful for the help I have received from Luke Koroniak with his

thorough knowledge of synthetic chemistry. I thank Ion Ghivirigia for his help in

determining absolute structures by NMR and other related problems. I also









acknowledge Lori Clark, Dr. Ben Smith, and Dr. Jim Deyrup for their continuous

guidance, assistance, and friendship.

I would like thank my loving fiancee Valerie for her supportiveness and for

her understanding during the periods that I lived in the laboratory. Lastly and

most importantly, I want to thank my parents for their support during this process.

They have continually made sacrifices for me throughout my life and this could

never have been achieved without them.















TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................... ........................ ...............iv

LIST O F FIG URES ................................................................ viii

LIST O F SCHEM ES ................................................................ x

A B S T R A C T ............. ................................................................ ...... xiv

CHAPTER

1 HISTORICAL BACKGROUND OF TAXOL ........................................... 1

Discovery of Taxol ..................................................... ...... ....... 1
Synthetic Strategies for T axol ....................................................... ........ 3
Total Synthesis of Taxol ................................................................... 4
Isolation of Taxol from Plant Tissue Cultures ............... ........... 7
Taxol Producing Fungus ............................ ...... ........... ................. 7
Semi-synthesis of Taxol: Synthesis of the Taxol Side Chain ............. 10
Asymmetric metal catalysis ...................... ....... ........... 11
Enzyme catalysis .................... ..... .......... ......... 15
Resolution of enantiomers............................ ............. 22

2 HISTORICAL BACKGROUND OF BESTATIN..................................... 27

Discovery of Bestatin .................. ............................_.. ....... ....... .... 27
Synthetic Approaches to Bestatin ............. .......... .................. 28

3 STEREOSELECTIVE, BIOCATALYTIC REDUCTIONS OF a-CHLORO-3-
KETO ESTERS ........... ........... ........... ............... ............... 41

In tro d u c tio n ......... ....... .. ................................... ..... ............... 4 1
Results and Discussion ..... ........................... ...... 42

4 SYNTHESIS OF THE C-13 TAXOL SIDE CHAIN .................... ............... 47

R acem ic S ynthesis......................................... ................ ............ 47
Biotransformation Strategy.......... .............. ......... .................. 48
Optimization of Our Whole-cell System .......... ......... .................. 50









W hole-cell Assays ............. .... ............................. 52
Whole-cell Reduction of 126..... ........................... 54
Base Catalyzed Ring Closure ...... ................. ............... 57
Ritter Reaction ...... ..................................... 59
Ring Hydrolysis to the Taxol Side Chain ................................................... 60

5 SYNTHESIS OF BESTATIN ...... ................................. 62

Syntheis of 3-Keto Ester 213 ........... ......... .................. ... ............... 62
Chlorination of 3-Keto Ester 213 .......... ........... .. ....... ............. 62
Enzymatic Reduction of 214 ........... ........... .......... .................... 63
Whole-cell Reduction of 214 ...... .......... ................ 65
Base Catalyzed Ring Closure of 215 ............... ...... ... ............... ........ 66
R hitter Reaction .................... ......... ................. ............. 66
Synthesis of Bestatin from 217: First Generation .................... ............... 67
Synthesis of Bestatin from 217: Second Generation............................. 68
Synthesis of Bestatin from 217: Third Generation.................. .......... 69

6 SYNTHETIC APPROACH TO CHUANGXINMYCIN................................ 71

Introduction ............ ......... ...... .................. ................ 71
The Akita lab's Approach to Chuangxinmycin......... ...... .. ........... 71
Enzymatic Reduction of 220 ...... .... ............ .................... ............... 73
Whole-cell Reduction of 220 ...... ......... ........................ 74
Base Catalyzed Ring Closure of 248 ........... .......................... ............... 75

7 CONCLUSIONS AND FUTURE WORK ................. ............. ............... 77

APPENDIX

A EXPERIMENTAL ........................................... ............... 80

B ADDITIONAL INFORMATION................ ............................. 97

LIST OF REFERENCES .......... ... ....................... ................................ 108

BIOGRAPHICAL SKETCH ...... .... ............... ................... ........ ....... 116















LIST OF FIGURES


Figure page

1-1 Natural products with anticancer activity: Vinblastine 1, Vincristine 2,
Taxol 3, Camptothecin 4. .............. ............ ......... ..... ........... 1

1-2 Taxol bound to a tubulin a,3 dimer ................ ... ................... ......... 2

2-1 Structure of Bestatin 131 (ubenimex)4647........................ ...... ......... 27

3-1 Biocatalytic reductions of a-chloro-3-keto esters ...... ............. .......... 45

4-1 Production of (S)-ethyl 3-hydroxybutyrate by engineered E. coli cells
under non-grow ing conditions ............................................ ............. .. 50

4-2 SDS-Page of the overexpression of YGL039w over a 4-hour time period.
The arrow marks the expected position of the YGL039w fusion protein .... 52

4-3 Specific activity of the E. coli cells overexpressing YGL039w, which have
been grown under different induction temperatures (37 C, 30 C, and 24
C) .............................................. .......... 53

4-4 Whole cell activity of an overexpressed YGL039w with a GST tag and
YGL039w without a GST tag versus time .......................... ... ............... 54

4-5 Concentration of the product for the biotransformation using YDL124w
and Y G L039w ................. ..................... ............... .............. 56

4-6 Diagram of our gentle extraction technique .......... ...... ...... ............ 56

4-7 Effect of Lewis acids on the Ritter reaction ........ ................ ............. 60

6-1 Chuangxinmycin 236 ............................ ......... .............. 71

6-2 Final product concentrations for 249, 248, and 215 by the corresponding
e n g in e e re d E co li ............... .......................................... ............... 7 4

7-1 Other pharmaceutical drugs that can be synthesized from homochiral
glycidic ester intermediates: Diltiazem 252, KRI-1230 253, Amistatin ent-
253, and Indolmycin 254 ................... .... .. .. ....................... 79









B-1 Long range coupling constants (Hz) for the major enzymatic reduction
product of YDR368w, YGL157w, and YGL039w................................. 98

B-2 The chemical shifts for the major reduction products of YDR368w,
YGL157w, and YGL039w .......................... ................... .............. 99

B-3 Coupling constants in the staggered rotamers of threo and erythreo
diastereomers of reduction products................................... ................ 100

B-4 Range of values for the large and small coupling constants (Hz) ........... 100

B-5 Line equation for the Bradford assay ...... ...... .. .... .................. 101

B-6 NMR of (+)-AHPA synthesized by our strategy............... ..... ........ 102

B-7 NMR spectra of authentic (+)-AHPA ....... ..... ..................................... 103

B-8 NMR spectra of the Taxol side chain 130 ............................ ............... 104

B-9 NMR spectra of the Taxol side chain enantiomer ent-130 .................... 105

B-10 1H NMR for derivatized 130 and ent-130: Top spectra is the (S)-MPA
ester of ent-130; bottom spectra is the (S)-MPA ester of the Taxol side
chain 130 ............. .. ............. ............... ................... .......... 106

B-11 H NMR spectra of syn product from YDR368w (top) and anti product
from TGL157w (bottom) ............... ............................................ 107















LIST OF SCHEMES


Scheme page

1-1 Summary of Holton's approach..................... ............. ............. 4

1-2 Summary of Nicolaou's approach ............... ........... ...... ............... 5

1-3 Summary of Danishefsky's approach ................................... ............... 5

1-4 Sum mary of W ender's approach........................ .. ........................ 6

1-5 Summary of Mukaiyama's approach................ ......... ......... ............ ... 6

1-6 Summary of Kuwajima's approach......................................... 7

1-7 The biosynthetic pathway to Baccatin III............... ................................. 9

1-8 The biosynthetic pathway of Taxol side chain and its coupling to Baccatin
III .................................... .................. .......... 9

1-9 Semi-synthesis of Taxol: Coupling of Baccatin III to the Taxol side chain 11

1-10 Greene's Strategy to the Taxol side chain ............................. ............... 12

1-11 Jacobsen's strategy to the Taxol side chain ................. ............. ........ 13

1-12 The Sharpless strategy to the Taxol side chain ............... ................... 13

1-13 Ham's strategy to the Taxol side chain ................................... ............. .. 14

1-14 Barua's strategy to the Taxol side chain ................................ ............... 15

1-15 Kim's strategy to the Taxol side chain............................. ... ............ 17

1-16 Kayser and Stewarts's first strategy to the Taxol side chain using bakers'
yeast .................................................................. .... ...... ........ 18

1-17 Kayser and Stewart's second strategy to the Taxol side chain ................ 18

1-18 Cardillo's strategy to the Taxol side chain....................... ........... .... 19

1-19 Hamamoto's strategy to the Taxol side chain .......................................... 20









1-20 Mandai's strategy to the Taxol side chain .............................. ......... 21

1-21 Botta's strategy to the Taxol side chain ................................................. 22

1-22 McChesney's strategy to the Taxol side chain......................................... 23

1-23 Zhou's strategy to the Taxol side chain........................... ... ............ 24

1-24 Our proposed synthesis of the Taxol side chain .................. ............. 25

1-25 Dynam ic Kinetic Resolution ...................................... ........ ............... 26

2-1 Suda's strategy to Bestatin ................................... ........... .................. 29

2-2 U m ezaw a's strategy to Bestatin........................................... .... ................. 30

2-3 Pearson and Hine's strategy to Bestatin ....................... ....... .............. 31

2-4 Norman and Moris's strategy to Bestatin ...................... .................. 32

2-5 Palom o's strategy to Bestatin.......................... ......................... ...... 32

2-6 Koseki's strategy to Bestatin ...................... ........... ...... .. .............. 33

2-7 Bergmeier and Stanchina's strategy to Bestatin ....................... .... 34

2-8 S eki's strategy to B estatin ............................................... .... ................. 35

2-9 Sem ple's strategy to Bestatin.......................... ......................... ...... 36

2-10 Park's strategy to Bestatin .............................................. 37

2-11 Jurczak's strategy to Bestatin ...... ................... .............. 38

2-12 W asserm an's strategy to Bestatin................................... ..................... 39

2-13 Our proposed synthesis of AHPA and Bestatin................... ........... 40

3-1 Four possible reduction products of a-chloro-3-keto esters: (2S-3S)-
white, (2R-3S)-black, (2R-3R)-black/white lines, (2S-3R)-black dashes.... 42

3-2 Synthesis of all 4 diastereomers by sodium borohydride, which can be
separated by chiral gas chromatography ............... ............................ 43

3-3 Derivatization technique used to separate all 4 diastereomers for
chlorohydrin 218 ........... ......... ......... ... ........... ............... 44

4-1 Racemic synthesis of the Taxol side chain ............... ........................ 48









4-2 Two chlorination methods for 3-keto esters ............... ... ............. 48

4-3 The Taxol side chain and its enantiomer can be synthesized by utilizing
two different enzymes; YDL124w and YGL039w, respectively.................. 50

4-4 Final results for the whole-cell biotransformations after purification........... 57

4-5 Results for the ring closure of chlorohydrins: Potassium carbonate
results in the kinetic product, whereas sodium ethoxide affords the
therm odynam ic product. .................................. ................................. 58

4-6 Mechanism for the sodium ethoxide promoted epoxidation and a
Newman projection describing syn versus anti configuration of
chlorohydrins .................................... ........... 58

4-7 Base promoted formation of cis-glycidic ester 128............................... 59

4-8 The Ritter reaction of glycidic ester 128 and benzonitrile................... .... 59

4-9 Hydrolysis of oxazoline 129 under mildly acidic conditions versus
strongly acidic conditions ................... ............. ..................... ......... 61

5-1 Synthesis of 3-keto ester 213 .............................................. 62

5-2 Two different approaches to chlorinate 213 with sulfuryl chloride ........... 63

5-3 Three bakers' yeast reductases that accept 214 as a substrate;
YDR368w, YGL039w, and YGL157w .......... .. ...... .... .............. 64

5-4 The whole-cell biotransformation resulted in the chlorohydrin 215 and
d e ch lo rinate d p ro d uct 23 1 ......................................................................... 6 5

5-5 Results for our optimized whole-cell reduction of 214....... ........................ 66

5-6 Base promoted ring closure for glycidic ester 216 .................... ..... 66

5-7 The Ritter reaction of 216 and benzonitrile only afforded the trans-
oxazoline 217...................... ............ ................... ............... 67

5-8 First attempt for the synthesis of Bestatin ....................... ... .. ............ 68

5-9 Second attempt for the synthesis of Bestatin............... ........................ 69

5-10 Final Strategy to AHPA 137 and Bestatin 131 .................. ................ 69

6-1 Akita's synthesis of (+/-)-Chuangxinmycin 236 ............... .............. .... 73

6-2 Proposed scheme to the Chuangxinmycin intermediate (2R, 3S)-epoxy
butanoate 243 ............. ............................... ............... .............. 73









6-4 Ring closure promoted by sodium ethoxide ................................. .. 75

6-5 Ring closure of chlorohydrin 248 using potassium carbonate and water... 76

6-6 Proposed synthesis to (2R, 3S)-epoxy butanoate 243........................... 76















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYNTHETIC APPLICATIONS OF HOMOCHIRAL GLYCIDIC ESTERS DERIVED
FROM ENZYMATIC REDUCTIONS

By

Brent Derek Feske

December 2005

Chair: Jon D. Stewart
Major Department: Chemistry

A library of eighteen known bakers' yeast reductases has been screened

for their ability to reduce several a-chloro-3-keto esters. By using these enzymes

in whole-cell biotransformations an easily scaleable method to synthesize gram

quantities of homochiral chlorohydrins can be achieved. A stereoselective, base

catalyzed ring closure can be used to transform these compounds into

enantiopure glycidic esters, which are useful intermediates to several biologically

active molecules.

Using a whole-cell reduction and a Ritter reaction as key steps, we were

able to develop a new route to both antipodes of the C-13 Taxol side chain and a

formal total synthesis of (-)-Bestatin. We synthesized the protected form of the

Taxol side chain in four steps with an overall yield of 49%. (2S, 3R)-3-Amino-2-

hydroxy-4-phenylbutyric acid was synthesized in six steps with an overall yield of

42%, thus completing a formal synthesis of (-)-Bestatin.









Depending on the substrate, we were able to consistently yield 1 to 4.5 g/L

of product for our whole-cell biotransformations. Thus, the overexpression of

bakers' yeast reductases in whole-cells proved to be an adequate and scaleable

method for the synthesis of homochiral chlorohydrins.















CHAPTER 1
HISTORICAL BACKGROUND OF TAXOL

Discovery of Taxol

In 1960, the National Cancer Institute established a program that purified

organic molecules from biological samples and screened the compounds for their

pharmaceutical activities. This program was triggered by the early success of

Beer et al., who found the antileukemic agents vinblastine 1 and vincristine 2 in

periwinkle leaves from Madagascar.1 Throughout this program's 22 year tenure,

only two compounds were found to show medicinal potential, Taxol 3 (paclitaxel)

and camptothecin 4 (Figure 1-1).

OH OH

N

SCOOCH3 COOCH3

H3CO/ .,,/ H3CO / ,/
N OCOCH3 N OCOCH3
1H COOCH3 H HCOOCH3
I O 2 H30 OH
H

0
SAcO N 0

0 .A .., HC
C OH
\ HcH 4 /4 OHO
3 OAc 4

Figure 1-1. Natural products with anticancer activity: Vinblastine 1, Vincristine 2,
Taxol 3, Camptothecin 4.

Taxol was isolated from the bark of the Pacific Yew tree (Taxus brevifolia)

and shown to have anti-tumor activity in 1962. Even though Taxol showed








promising anti-cancer activity, further testing slowed because it was highly

insoluble in water, which would make it almost impossible to deliver to patients.

In addition, as the demand for Taxol increased, the supply of Pacific Yew trees

and thus Taxol was diminishing.

In 1979, there was a breakthrough in understanding Taxol's anti-cancer

activity. Susan B. Horwitz, a molecular pharmacologist, along with Schiff et al.

found that Taxol's mode of action was completely different from those of

traditional cancer drugs.2 Many of these drugs destabilize a cell's ability to make

microtubules, which are essential for cell replication. Taxol's mode of action is

actually the opposite: it stabilizes the microtubules during cell replication,

preventing their separation (Figure 1-2). Due to the cells' inability to divide they

will eventually grow so large as to trigger their own death.


tubulin c,3 dimer taxol GDP
GTP









minus *~ plus
end T end

E (5 PDB 1JFF


Figure 1-2. Taxol bound to a tubulin a,3 dimer









This newly discovered mode of action accelerated the clinical trials of Taxol.

After many years of thorough testing, the Food and Drug Administration (FDA)

approved Taxol for the treatment of ovarian and breast cancer in 1992. This

newly approved and promising cancer drug quickly developed a strong

commercial demand. This led to a problem, however, since the slow growing

Pacific Yew tree was considered near the brink of extinction and this precluded

an adequate supply of Taxol for the clinical use by isolating the natural product.

To solve this problem, chemists were given the task of producing what would

become the largest selling cancer drug ever placed on the pharmaceutical

market, yielding sales in the billions of dollars.

Synthetic Strategies for Taxol

The isolation of Taxol from the Pacific Yew tree has low yields, so auxiliary

strategies have been developed to obtain this drug. These include total

synthesis, plant tissue cultures, engineering of a Taxol producing fungus, and the

coupling of Baccatin III (which contains the ring structure of Taxol) with the Taxol

side chain. Due to low yields and the high number of steps, the total synthesis of

Taxol is unlikely to become an option for commercial production. Ongoing work

on the engineering of plant tissue cultures continues, but many scientists believe

that plant cells are too difficult to manipulate for high Taxol yields. As a result,

engineering the Taxol producing fungus and semi-synthesis by coupling of

Baccatin III to the side-chain are considered the primary strategies for

commercial production.









Total Synthesis of Taxol

In 1971, the Wani et al. group published the structure of Taxol 3, which is

composed of a complex poly-oxygenated diterpene and a phenylisoserine side-

chain.3 As a result, synthetic chemists were intrigued by the challenge of

synthesizing such a complex organic molecule. It took nearly 20 years, but in

1994 the Holton group and Nicolaou group almost simultaneously reported the

total synthesis of Taxol. Since that time, four more total syntheses have been

completed on Taxol.

The Holton lab approach4'5 used (-)-camphor 5 as the starting material for a

strategy in which the A and B rings of Taxol were created first, then this unit was

fused to the C ring (Scheme 1-1). The oxetane (D) ring was subsequently

formed through a tosylate intermediate. This synthesis was composed of 41

steps with an overall yield of 2%.

OP PO
Op -- ~ op
HWRH 3
0 0 POp HO/
5 6 7

Scheme 1-1. Summary of Holton's approach

The Nicolaou group6-9 first constructed the A and C ring systems from

hydrazone 8 and bicyclic aldehyde 9, followed by a McMurry cyclization to form

the ABC ring system intermediate 11 (Scheme 1-2). The D ring was added

through the formation of a triflate silyl ether intermediate and treated under mildly

acidic conditions to make the oxetane. This total synthesis of Taxol was

completed in fifty-one steps with an unreported overall yield.











OTBS 0
C HO OH
OTH HO OH OBn

OBn 0 IH pO 0 HI "'0 /
TPSO OBn 0 0 H

Hi,. 0 0
H "o10 11

9

Scheme 1-2. Summary of Nicolaou's approach

The Danishefsky group1 used the Wieland Miescher ketone 12 and

trimethylcyclohexane-1,3-dione 13 as starting materials for the synthesis of the C

and D rings (Scheme 1-3). They then used an intramolecular Heck reaction to

fuse the AB rings to the CD ring structure. This synthesis required 47 steps and

resulted in an overall yield of less than 0.1%.



01- AcO O
12
+ HO,', .. 3

HOBz H C O
O O OAc
14

13

Scheme 1-3. Summary of Danishefsky's approach

The Wender group1 utilized a-pinene 15 as a matrix to make the A and B

rings through a fragmentation technique with an epoxy alcohol (Scheme 1-4).

The C ring was added via an aldol condensation and then formation of the D ring

following in several steps by direct closure of the diol. This synthesis of Taxol

was accomplished in 37 steps with an unreported overall yield.










O AcO 0
S --o R- 3

OH HB 0z OR

15 16 17

Scheme 1-4. Summary of Wender's approach

In 1997, the Mukaiyama group12'13 reported the total synthesis of Taxol

utilizing L-serine 18 as the starting material (Scheme 1-5). In their strategy, the

BC ring system was synthesized via a pinacol coupling cyclization; then addition

of the A ring followed. In subsequent steps they added the D ring and the Taxol

side-chain, thereby affording Taxol with an unreported yield.

AcO OTES
BnO O OH
0 TBSO 3
HO OH TESO" --
NH2 H -
PMBO OBn O
0
18 19 20

Scheme 1-5. Summary of Mukaiyama's approach

The Kuwajima group's14 synthesis started with a Peterson olefination to

afford dienol silyl ether 21 to form the A ring (Scheme 1-6). Next, 2-

bromobenzaldehyde dibenzylacetal 22 was used to synthesize the C-ring

fragment. The coupling of the A and C rings and subsequent cyclization to form

the B ring resulted in the tricarbocycle. This was converted to Taxol after several

additional steps in an unreported overall yield.









OTIPS

HO .SPh AcO
0=3-- X AcO 0

+ TBS
H \ ,3^/ OTroc
CH(OBn)2 HOAc
Br
23

22

Scheme 1-6. Summary of Kuwajima's approach

Isolation of Taxol from Plant Tissue Cultures

Large-scale plant cell cultures have been shown to be useful sources of

certain natural products.15-17 This usually requires a cell selection with medium

optimization, genetic engineering, elicitation of enzyme systems, precursor

feeding, and overall process optimization. Research on the optimization of

Taxol producing plant cells is still ongoing; however, due to the difficulty of

engineering plant cells it is not expected to be a practical route for the production

of Taxol.18-20

As an example, Chang et al. recently collected tissues from Taxus mairei, a

plant found in Taiwan at an altitude of 2000 m above see level, and discovered

that the amount of Taxol and Taxol derivatives found in this plant were higher

than the pacific yew and other Taxus species.21 However, when using callus

cells from Taxus mairei, an optimized cell line was only able to produce 200 mg/L

of Taxol after a six week incubation period.

Taxol Producing Fungus

In 1993, Stierle et al. found that Taxol is produced by the fungus

Taxomyces andreanae.22 Unfortunately, Taxol is only produced at










concentrations of 25-50 ng/L in this organism. On the other hand, fungal cells

can be engineered more easily than plant cells, so the future of using engineered

cells for the production of Taxol is promising.

The Croteau lab has taken on the challenge of fully deciphering the Taxol

biosynthetic pathway in Taxomyces andreanae.2328 His group has found that

there are 19 enzymatic steps from the basic geranylgeranyl diphosphate used as

the isoprenoid precursor. The biosynthesis of Taxol begins with the 2-C-methyl-

D-erythritol phosphate pathway (MEP) (Scheme 1-7). Isopentenyl diphosphate

24 and dimethylallyl diphosphate 25 are then combined to form geranylgeranyl

diphosphate 26 (GGPP) from geranylgeranyl diphosphate synthase. The next

step is the cyclization of GGPP to taxadiene 27 by taxadiene synthase. Over the

next three steps, taxadiene is decorated with an alcohol and acetate functionality.

The order of additional oxygenations beyond this point has not been totally

deciphered.


'i'opp
24 IPP /
MEP GGPPS
Pathway P IPPI

'OPP 26
25 DMAPP


taxadiene cyctochrome P450 1) taxa-4(20), 11(12)-diene-
synthase taxadiene 5a-o-acetyl transferase
*..__ ^ 5a-hydroxylase ,,
H "' /OH 2) cytochromeP450 taxane-
H H 10B-hydroxylase
27 28













^ nHO' H
H :Z HCz H o
29 30

Scheme 1-7. The biosynthetic pathway to Baccatin III

Once Baccatin III 30 has been formed, the C-13 Taxol side chain is added.

The side chain begins from a-phenylalanine 31, which is converted to 3-

phenylalanine 32 by phenylalanine aminomutase PAM (Scheme 1-8). In the next

step, 32 is activated as the corresponding CoA thioester followed by an aroyl

transfer to Baccatin III resulting in 33. Lastly, the side chain is hydroxylated at

the C-2 position and then N-benzoylated to afford the biosynthetic product, Taxol

3.

AcO
0 NH2 O OH
PAMH
OHH zOH HH O O

OAc
31 32 30


AcO
1) CoA thioester NH2 0 OH
ligation w 1) 2' hydroxylation
2) aroyl transfer 2) N-benzoylation
HOBzd' =--
OAc
33

Scheme 1-8. The biosynthetic pathway of Taxol side chain and its coupling to
Baccatin III

The strategy for engineering the Taxol-producing fungus is to locate the

bottlenecks in the pathway and overexpress the genes responsible for the slow

steps. This plan also includes knocking out competitive pathways that may lead

to undesirable products; this can be done by adding elicitors or by gene









knockout. If these two strategies are successful, it would increase the

concentration of the final product and also allow for easier purification.

Semi-synthesis of Taxol: Synthesis of the Taxol Side Chain

Taxol's path to becoming a commercial drug was an uphill battle, but in the

late 1980's it became evident that Taxol would soon become FDA approved.

Since a commercial source had not yet been found, Pierre Potier et al. began to

study this problem. They found and extracted a compound from the Pacific Yew

bush (Taxus baccata), which contains the terpene core of Taxol.29 This

compound, 10-deacetylbaccatin III 34, can be isolated from the leaves of the

bush in high yield. In addition, only the leaves are removed from the bush, which

can be regenerated by the plant, allowing it to be a renewable source. Upon

isolation of 10-deacetylbaccatin III 34, it can be coupled to the protected form of

the Taxol side chain 35 and/or 36 yielding the full structure of Taxol 3 (Scheme 1-

9). Greene et al. developed a side chain synthesis and coupling procedure;

however, it resulted in an enantiomeric excess (e.e.) of only 78%.30 Due to the

low enantiomeric excess, this synthesis did not meet the purity standards set by

the FDA.














HO [I .AG
OH AcO
HO," + 35 NH 0 OH

H 0 OH zH
OAc HOBzd" H 0
34 TESO, 3 OAc

0o

36

Scheme 1-9. Semi-synthesis of Taxol: Coupling of Baccatin III to the
Taxol side chain

To answer the commercial demand for Taxol, Dr. Robert Holton developed

a metal alkoxide process for the Taxol semi-synthesis in the early 1990's with a

74% overall yield.31 This patent was licensed by Bristol-Myers Squibb and has

been used for the commercial production of Taxol since its approval in 1993.

Since then, a number of different approaches to the Taxol side chain have been

reported. These approaches are listed within three categories: asymmetric metal

catalysis, enzymatic catalysis, and enantiomer separation.

Asymmetric metal catalysis

The main challenge for the synthesis of the Taxol side chain is to define the

stereochemistries of the C-2 and C-3 positions with high selectivities. Popular

solutions to this product involve asymmetric metal catalysts that afford the

desired enantiomer in good to high enantiomeric excess.

In 1986, The Greene lab was the first to publish the synthesis of the Taxol

side chain using this approach (Scheme 1-10).30 He applied the Sharpless

epoxidation methodology to cis-cinnamyl alcohol 37 to afford chiral epoxy alcohol










38 in 78 % e.e., which was then oxidized and esterified by diazomethane to yield

glycidic ester 39. Ring opening of the 39 with azidotrimethylsilane yielded azide

40. Reaction with benzoyl chloride under Schotten-Baumann conditions followed

by hydrogenation resulted in the Taxol side chain 41.

t-BuOOH / Ti(OiPr)4 H 0 H 1) RuCl3 / NalO4 H ,
H2(+) -DT 2) CH2N2 OMe
70% CH2H 90% 0
78% e.e.
37 38 39

1) C6HsCOCI /
Me3SiN3 2) H2 / Pd NH 0
0 ^4 89% "
84% OMe 89% OMe
SOHOH

40 41

Scheme 1-10. Greene's Strategy to the Taxol side chain

The Jacobsen group synthesis started by a Lindlar reduction of ethyl

phenylpropiolate 42 (Scheme 1-11).32 The key step used (salen) Mn (III)

complex 48 as an inorganic asymmetric catalyst resulting in the glycidic ester 44.

Ring opening with ammonia and hydrolysis of the amide resulted in a-hydroxy-3-

amino acid 46. Addition of benzoyl chloride and treatment with aqueous

hydrochloric acid afforded the Taxol side chain 47.

H2 NaOCI
Lindlar cat. [ \ (R,R)-1 (6 mol%)
CO2Et 84% --CO2Et 56% CO2Et
96% e.e.
42 43 44










NH 0 1) Ba(OH)2 NH2 0 1) BzCI / NaOH/
NH3 / EtOH 2) H2S04 2) HCI NH 0
f Y NH2 'OH II
65% OH 92% OH 74% OH
6- OH
45 46 47



t-B u N O' N--H
t Mn, t-Bu

t-Bu 0
48 t-Bu

Scheme 1-11. Jacobsen's strategy to the Taxol side chain

Sharpless et al. used an inorganic catalyst approach to carry out the

asymmetric dihydroxylation of trans-methyl cinnamate 49 (Scheme 1-12).33 The

syn-diol 50 was converted to acetoxy bromo ester 51 by reaction with trimethyl

orthoacetate in the presence of a catalytic amount of p-TsOH. After reacting 51

with sodium azide, the acetoxyazide ester was hydrogenated giving N-acetyl-3-

phenylisoserine 52. The amide ester was then hydrolyzed to afford the acid and

benzoylated to the Taxol side chain 47.

S 0.5 mol% (DHQ)2-PHAL OH 1) CH3C(OCH3)3
0.2 mol% K20sO2(OH)4 p-TsOH
OCH3 0---OCH3 2) AcBr/CH2Cl2
3 NMO (60% in water)
t-BuOH OH 60%
72% 99% e.e.
49 50


Br O ) NaN3/DMF Me O 1) 10% HCI (aq) O
2) H2/10% Pd/C NH 2) BzCI NaOH NH 0
OCH3 I |J_____ ~-
OCH3 74% OCH3 72% OH
O OH K- OH

51 52 47

Scheme 1-12. The Sharpless strategy to the Taxol side chain

Ham et al. synthesis began with the protection of L-phenylglycine 53,

followed by treatment with N, O-dimethylhydroxylamine hydrochloride to afford










Weinreb amide 54 (Scheme 1-13).34 Reduction of 54 by lithium aluminum

hydride followed by a Wittig reaction resulted in ester 55. Treatment of 55 with

DIBAL gave alcohol 56, which was acetylated over a three step process resulting

in 57. The key step was a palladium catalyzed oxazoline formation yielding only

trans-compound 58. This was then oxidized to acid 59 by sodium periodate.

The resulting acid 59 was reacted with diazomethane resulting in the protected

Taxol side chain 60.

1) (Boc)20 / NaOH 1) LAH / ether
S 2) HNCH3(OCH3)-HCI 2) (CH30)2POCH2CO2CH3
OH 81% 80%
NH2 BocNH
53 54

1)3 N HCI
2) BzCI Et3N
DIBAL / BF30Et2 3) Ac2 / pyridine
OCH3 89% OH 78%
BocNH BocNH
55 56




iAc 55% Pd(PPh3)4/ K2CO3 N RuCI3/ NalO4
78% N 0 78%
Bz' NH


57 58




N CH2N2 / ether
99%
OH 99% OMe
0 0

59 60

Scheme 1-13. Ham's strategy to the Taxol side chain

The Barua lab began by reacting benzyl alcohol 61 and epichlorohydrin to

afford epoxide 62 (Scheme 1-14).35 Treatment of 62 with 30% HCIO4 gave the










diol, which was oxidized to the aldehyde 63 with Pb(OAc)4. The key step of this

synthesis was a Shibasaki asymmetric Henry reaction with aldehyde 63 and

phenylnitromethane 64 yielding alcohol 65 with a 90% e.e. Simple acetylation of

the Henry adduct, followed by a hydrogenation resulted in 67. After oxidation of

the alcohol using CrO3, 68 was benzoylated with benzoyl chloride and then the

acetate was hydrolytically removed to the C-13 Taxol side chain 47.

epichlorohyrdrin
50% aq. NaOH 1) 30% HCIO4 NO2
OHOH TBAB O 2) Pb(OAc)4 O H
^ 80% ^ 0 68% 0 -
61 62 63 64

NO2 NO2 NH2
La-(R)-BINOL Ac20/Py 10% H2 Pd/C
-. OBn -- OBn -OH
80% OH O 93% 80% H

65 66 67


NH2 0 1) BzCI / NaOHNH
CrO3 /AcOH /H20 2) Et2NMe-H20 H
-_I I I OH -OH
82% OAc 75%
OH
68 47

Scheme 1-14. Barua's strategy to the Taxol side chain

Enzyme catalysis

Another strategy to afford the proper stereochemistry at the C-2 and C-3

positions of the Taxol side chain involves the use of enzymes. Typically,

acylases or lipases are the enzymes of choice for organic chemists. These

enzymes can be an efficient source for chirality; however, the maximum yield

possible from the racemate is fifty percent. Despite these problems, there have

been several published syntheses using enzymes as a key step in their

synthesis.










The Kim lab's Taxol side chain synthesis began with a kinetic resolution of

racemic diol 69, using the lipase from Pseudomonas cepacia (Scheme 1-15).36

Unlike other kinetic resolutions, in which there is at least a fifty percent loss in

yield, Kim's synthesis utilized both lipase products as precursors for the Taxol

side chain. After the enzymatic reaction, 70 was tosylated, which allowed for ring

closure by the addition of a weak base. Glycidic ester 39 was then reacted with

sodium azide to form 40. The other enzymatic product, 72, was brominated by

hydrobromic acid and acetic acid to afford 73. Reaction of 73 with sodium azide

and then sodium acetate also gave compound 40. These compounds were

combined and treated with benzoyl chloride, followed by a palladium catalyzed

hydrogenation resulting in the Taxol side chain 41.

OH 0
OMe
.^- OH
69

54% 44%
LPS/vinyl
OH 0 acetate OH
Y OMe Ac+ OMe
SOAc OH

70 1 TsCl/ 72
87% EtN 72 89% HBr-AcOH



OAc O Br 0
rOMe OMe
S OTs OAc
71 73











p-TsOH / 1) NaN3
83% K2CO3 85% 2) AcONa


0 N3 0
OMe NaN3 -OMe
C 0 94% OH
39 40
1) BzCI / Et3N
88% 2) H2 Pd-C



N ^- OH
41

Scheme 1-15. Kim's strategy to the Taxol side chain

In 1999, Kayser and Stewart et al. also used an enzymatic approach to

synthesize the Taxol side chain.37 They developed two syntheses, both utilizing

stereoselective ketone reductions by bakers' yeast.

In the first strategy, racemic glycidic ester 74 was opened with sodium

azide, then the resulting alcohol was oxidized by Jones reagent to yield 75

(Scheme 1-16). Incubation of racemic 75 with bakers' yeast gave the

diastereomeric reduction products 76. syn-Azido alcohol 40 was purified by

column chromatography and the Greene strategy (Scheme 1-10) was used to

complete the Taxol side chain 41.

0 1)NaN3 N3 0 N3 0
OMe 2) Jones reagent O bakers' yeast Me
S27% 0 67% OH
7 : 3 (syn: anti)
74 75 76









N3 1) C6HsCOCI O
chromatography 2) H2 / Pd NH 0
OH 89% OMe
OH
40 41

Scheme 1-16. Kayser and Stewarts's first strategy to the Taxol side chain using
bakers' yeast

The second approach began with the LDA-mediated addition of 77 and 78,

resulting in racemic 3-lactam 79 (Scheme 1-17). The ketal was hydrolyzed with

concentrated sulfuric acid, then the ketone was reduced by incubation with

bakers' yeast resulting in three of the four possible diastereomers. syn-Alcohols

81 and 82 were separated from 83 by chromatography to yield a protected form

of the Taxol side chain with an enantiomeric excess of 82%.


OEt N PMP LDA H2SO4 0N
EtO J OEt NPMP A EtO /CL
O 91% TN 85%
0 0 PMP 0 PMP
77 78 79 80


bakers'yeast HO, HO HO, HO
N N N
S PMP 0 PMP 0 PMP 0 PMP
41% 4% 8% 0%
81 82 83 84

Scheme 1-17. Kayser and Stewart's second strategy to the Taxol side chain

The Cardillo group synthesized racemic 88 by reaction of benzaldehyde 85

with malonic acid 86 and ammonium acetate 87, which was subsequently

benzoylated with benzoyl chloride (Scheme 1-18).38 Incubating 89 with penicillin

G acylase resulted in formation of homochiral 3-amino acid 91. This was

benzoylated and reacted with thionyl chloride and methanol, resulting in methyl










ester 92. The addition of LiHMDS produced the lithium dianion, which after

reacting with iodine, led to rearrangement product 60. Refluxing oxazoline 60

with weak aqueous hydrochloric acid afforded the Taxol side chain 41.

O NH2O
S H 0 N4 EtOH / reflux
H + HO1- -OH + AO- NH4+ 75% OH

85 86 87 88

0 NH2 0
BzCIl Et3N NH 0 Penicillin G acylase NH 0
75% OH 100% OH


89 90 91


NH2 1)Et3N / BzCI 1) LiHMDS
2 2) SOCl2 / MeOH 2) 12
OH 70% OMe 95%


91 92


0

N 0 1M HCI / MeOH NH 0
OMe 85% OMe
o 0OH

60 41

Scheme 1-18. Cardillo's strategy to the Taxol side chain

The Hamamoto lab's synthesis approach began with the Darzens

condensation of 85 and 93 to produce a-keto-3-chloro ester 94 (Scheme 1-19).39

KS-Selectride reduced 94 to give predominately anti-chlorohydrin 95. Lipase

then resolved 95 to afford homochiral chlorohydrin 96, which was reacted with

sodium azide to afford 40. Reaction of 40 with benzoyl chloride followed by a

palladium catalyzed hydrogenation afforded the Taxol side chain 41.










O 0 Cl 0O
NaOMe KS-Selectride
"H + Cl O e OMe -
H OMe 91% 81%
- Cl O
85 93 94


Cl 0 Lipase Cl O
SJ OAc I NaN3
OMe 4Ac OMe NaN
46% 82%
K OH U1_ OH
99% e.e.
95 96


N3 O 1) BzCI /DMAP /
-.- ^2) H2 Pd/C NH 0
OMe -
SOH 70% OMe
OH OH
40 41

Scheme 1-19. Hamamoto's strategy to the Taxol side chain

The Mandai group began by reacting phenylacetic acid 97 with LDA to form

the lithium dienolate, which was reacted with acrolein, then stirred with 3 N

hydrochloric acid to afford acid 98 (Scheme 1-20).40 In the next step, 98 was

esterified with allyl alcohol to give 99, which was reacted with Chirazyme in 2-

propenyl acetate and toluene to give 100 and 101. The latter was transformed

into cyclic carbamate 102, via the Curtius rearrangement of the free acid

produced by the palladium-catalyzed hydrogenolysis of the allyl ester.

Carbamate 102 was then protected with (Boc)20 and oxidized with ruthenium

oxide and sodium periodate to afford acid 103. Treatment with 2 M sodium

hydroxide resulted in ring opening, and trifluoroacetic acid was used to remove

the Boc protecting group. In the last step, benzoyl chloride was used to afford

the Taxol side chain 47.











1) LDA(2.1 eq) OH
CO2H 2) THF / acrolein H2SO4 / CH2CI2
70% HO2C 85% AllylO2C
OH OH
97 98 99


Chirazyme O
CH2=C(OAc)Me
AllylO2C t AllylO2C + AllylCO2
toluene
OH OAc OH
48% (98.5% e.e) 49% (99% e.e)
99 100 101


1) NaOH / MeOH
[ 1) Pd(OAc)2 /PPh3 0 1) (Boc)20 / Et3N DMAP 0 2) TFA O
2) DPPA/ Et3N 2) RuO2 / Nal4 3) BzCI NaHCO3
IN HN O BocN O HN
80% 88% 71% CO2H
AllylO22C C O2H H
OH (D 6 OH
101 102 103 47

Scheme 1-20. Mandai's strategy to the Taxol side chain

The Botta group began by adding the Grignard salt of acetylene to

benzaldehyde 85 (Scheme 1-21).41 Next, the racemic alcohol 104 was subjected

to a Ritter reaction with acetonitrile and sulfuric acid. Alkyne 105 was then

hydrogenated in the presence of Lindlar catalyst, and deacetylated by aqueous

hydrochloric acid to yield 107. The enantioselective acetylation of 107 using

Candida antartica lipase resulted in 108 and 109. Amide 109 was deacetylated

with aqueous acid to afford 110, which was then benzoylated with benzoyl

chloride. The addition of Os04 and NMO to 111 oxidized the alkene to a mixture

of alcohol diastereomers. After oxidation with Jones reagent, L-Selectride

yielded predominately 114. The Deoxo-Flour reagent was added which resulted

in the ring closure product, oxazoline 115. Oxidation of 115 with PCC and then

acid catalyzed hydrolysis resulted in the Taxol side chain 41.











0 OH
SMgBr MeCN / H2SO4
S IH -f -
'IlKe


0
0NH



105


Candida antartica
NH2 lipase / NH2
HCI AcOEt
38%

43% / 98% e.e.
107 108


NH2
HCI
32%


0
BzCI/Py/DMAP O NH
99%


109 110 111

0 0 0
ONH Jones reagent ONH L-selectride ONH
rOTBDPS 84% rY[OTBDPS 99% OOTBDPS
112H 13 OH
112 113 114


Ph
Deoxo-Fluor
94% O


1) PCC Ph O
,, HCI 0
2) CH2N2 N 0O 0 -NH 0
50% ,OMe 85%OM
I 4 OMe
0 4-1 OH

60 41


Scheme 1-21. Botta's strategy to the Taxol side chain


Resolution of enantiomers

As seen above, popular published methods of the Taxol side chain use


either an asymmetric metal catalyst or an enzymatic catalyst to afford the proper


stereochemistry. However, some other strategies involve the racemic synthesis


of the Taxol side chain, followed by the separation and purification of


enantiomers.


0
NH





106

0
ANH
cr.-_


H2/ Lindlars
99%


0
ANH



46% / 98% e.e.
109


1) OsO4/ NMO
2) TBDPSCI
Imidazole
48%










The McChesney lab's approach utilized the Darzen reaction of methyl

chloroacetate 116 and benzaldehyde 85 (Scheme 1-22).42 Reaction of 117 with

dry hydrochloric acid opened the epoxide with retention of configuration at C-3

affording chlorohydrin 118. Ring closure using basic Amberlite 400 resin gave

cis-glycidic ester 119. Ring opening with ammonia followed by benzoylation

afforded 120. Reaction of 120 with the acidic Amberlite resin 120 in methanol

resulted in the racemic Taxol side chain. This was then resolved by entrainment

to afford 41 with an enantiomeric excess of 95%.

0 0
J H + CI\OMe NaOMe / MeOH HCI / benzene
H + CI "'CO2Me
0 73% 70%

85 116 117

CI OH O
H,, VCO2Me Amberlite 400 (OH) NH3/MeOH H2
H 60% --- CO2Me 69% NH2
60% 69% OH
118 119 45


/ O 1) Amberlite 120 (H+) / \
BzCI/ NaOH NH 0 2) Resolution NH 0
74% NH2 65% OMe
SOH H OH

120 41

Scheme 1-22. McChesney's strategy to the Taxol side chain

The Zhou group began with ammonolysis of the glycidic ester 121 to yield

isoserineamide 122 (Scheme 1-23)43. Benzoylation provided 123, and acid

catalyzed methanolysis gave the methyl ester 124. The use of thionyl chloride

and hydrochloric acid inverted the C-2 hydroxyl to produce 41, which was

subsequently hydrolyzed to the racemic acid. This was resolved with R-(+)-a-









methyl benzylamine to provide the Taxol side chain 47 with an unreported

enantiomeric excess.

0 NH2 0H

81% OH 75% NH2
SNH42 OH
121 122 123

o0 0
/\ f\^ 01) K2CO3 /H20Y
TsOH/MeOH NH 0 SOCI2 /HCI NH 0 2) Resolution NH 0
67% OMe 49% OMe 78% OH
OH 6 OH OH
124 41 47

Scheme 1-23. Zhou's strategy to the Taxol side chain

As seen above, there are numerous approaches to the synthesis of the

Taxol side chain. Many of these routes are often limited by the use of unsafe

chemicals and/or conditions that are difficult for industrial scale-up. In addition,

sometimes the synthetic routes do not yield a product with a high enantiomeric

excess (e.e.) or diastereomeric excess (d.e.), which is essential for the

commercial sale of pharmaceuticals. Many strategies to the Taxol side chain use

reactions with lipase or the resolution of enantiomers, both of which can be very

time consuming, thus inhibiting an industrial high-throughput process. With this

said, it was our goal to develop an efficient and easily scaleable reaction for the

homochiral C-13 Taxol side chain.

We proposed a five step synthesis for the Taxol side chain, which begins

with the reaction of ethyl benzoylacetate 125 with sulfuryl chloride to afford a-

chloro-3-keto ester 126 (Scheme 1-24). Ester 126 can be added to Escherichia

coli (E. coli) that have overexpressed a single bakers' yeast reductase, to yield









homochiral chlorohydrin 127. Treatment of 127 with a weak base should yield

the optically pure cis-glycidic ester 128. Subsequent treatment of the epoxide

with benzonitrile and a catalytic Lewis acid can result in trans-oxazoline 129.

Oxazoline 129 can then be treated under mildly acidic conditions to form the

optically pure Taxol side chain 130. This reaction scheme utilizes mild conditions

and reagents that can be used on a large scale.

O 0 0 0 OH 0
OEt S02C 2 OEt YDL124w OEt
CI CI
125 126 127


K2CO3 0 BF3-OEt2/ PhCN H30+
CO2Et N 0 ONH
"'CO2Et OEt
0' OH

128 129 130

Scheme 1-24. Our proposed synthesis of the Taxol side chain

As seen in the proposed synthesis of the Taxol side chain we plan to utilize

the reduction products of a-chloro-3-keto esters using a single bakers' yeast

reductase. The enzymatic product has four possible diastereomers, given by a

dynamic kinetic resolution (Scheme 1-25). This is made possible through the low

pKa of the a-proton in 3-keto esters. The average pKa for this functionality is

10, which allows the a-carbon to quickly epimerize. Thus, if a reductase exhibits

a preference for either substrate enantiomer, the rapid racemization re-

establishes the equilibrium. This allows all of the starting material to be

converted to a single diastereomer product. Once the substrate has entered the

active site, the reductase will transfer a hydride from NADPH to the 3-carbon









typically resulting in a highly stereoselective reduction. This characteristic is vital

to our strategy, because it inserts two chiral centers in our scheme in a highly

stereoselective manner.

o o 0 0
Fast
R 'OEt -Fast R OEt
H CI Ci H


[H] [H]

H OHO HOH 0
R OEt R OEt
Scheme 1-25. Dynamic Kinetic Resolution
Scheme 1-25. Dynamic Kinetic Resolution














CHAPTER 2
HISTORICAL BACKGROUND OF BESTATIN

Discovery of Bestatin

In 1975, The Umezawa group discovered and isolated an antitumor and

antimicrobial agent from Streptomyces olivoreticuli named Bestatin 131

(ubenimex) (Figure 2-1).44 Bestatin was found while screening cultures of

actinomycetes for their ability to inhibit aminopeptidase B. This screening was

ignited by the recent findings that exopeptidases have a strong effect on

mammalian cell surfaces.45 At that time, the research started as a hypothesis,

but after 30 years, several aminopeptidase inhibitors are now used to treat a

variety of cancers and antibiotic infections.



NH2OH

OH O
131

Figure 2-1. Structure of Bestatin 131 (ubenimex)46'47

The function of Aminopeptidase B is to hydrolyze the N-terminal lysyl and

arginyl residues from peptide substrates.44'48 Aminopeptidase B is also thought

to play a role in processing various peptide signals and precursor enzymes, by

binding to membrane macrophages and lymphocytes through membrane

aminopeptidases.49'50 This binding induces a cascade of responses like

increased cytokines, colony stimulating factors, and cell apoptosis.50-52









Presently, Bestatin is used as an oral medication for the treatment of cancer

and bacterial infection in Japan. In addition, Bestatin is often used in conjunction

with other antibiotics and anticancer drugs because it causes the proliferation of

T cells thus enhances the immune response.50 Bestatin also shows potential as

an anti-inflammatory agent and for the treatment of HIV.53-59

In 1976, shortly after its discovery, the Nakamura lab's published the crystal

structure of Bestatin, confirming that the compound was the dipeptide [(2S, 3R)-

3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine.47 Upon verification of the

correct configuration, Suda et al. began the first synthesis of this challenging

molecule that contains three asymmetric centers. Since this publication, there

have been several documented asymmetric syntheses of this popular anticancer

and antimicrobial agent, which will be introduced in chronological order.

Synthetic Approaches to Bestatin

The Suda lab's60 1976 strategy began with Boc-protected D-phenylalanine

132, which was coupled with pyrazole to form pyrazolide 133 (Scheme 2-1). This

was reacted with 2 equivalents of lithium aluminum hydride to afford aldehyde

134. The diastereomeric mixture of bisulfite adducts 135 was then reacted with

sodium cyanide resulting in acyl cyanide 136. Hydrolysis of 136 with 6 M

hydrochloric acid gave the diastereomeric acid mixture, which was separated by

chromatography to afford the natural occurring amino acid (2S, 3R)-(3-amino-2-

hydroxy-4-phenylbutanoic acid (AHPA) 137. This was reprotected with Boc-CI

before it was reacted with benzoyl protected L-leucine. A simple hydrogenation

of 139 afforded Bestatin 131 in an overall yield of 14%.










Boc Boc Boc
o NH pyrazole / DCC NH LiAIH4 NH
OH H3/ N ^- N- N A H
83% 1,"" N-
0 0 0
132 133 134

Boc Boc 1) 6 N HCI
2eq NaHS03 | NH NaCN NH 2) chromatography
74% SO3Na 94% CN
OH OH
135 136

Boc
H2 0 BocCI H L-leu-OBz DCC
Sv OH 78% v OH 34%
OH OH
137 138


Boc )-- \ -
-^ NH 0 ( H2 Pd/C -\ NH20
N OBz 91% N OH
O HO OH H
139 131

Scheme 2-1. Suda's strategy to Bestatin

The Umezawa lab's61 synthesis of Bestatin commenced by reacting N-acyl-

a-aminoacetophenone 140 and glyoxylic acid 141 to yield 142 (Scheme 2-2).

This was then subjected to a palladium catalyzed hydrogenation, which reduced

the benzylic carbon to afford 143. The racemate of 143 was resolved with S-(-)-

a-methylbenzylamine to give the optically pure salt 144. Refluxing 144 in

aqueous hydrochloric acid gave the free amine 136 that was Boc-protected. The

protected peptide 145 was formed by the DCC coupling of benzyl protected L-

leucine. A simple hydrogenation resulted in Bestatin 131 in an overall yield of

10%.










0 OH
0 H 0 o HO OOH
N, 4yOH NaHC03__I_ H2 / Pd/C
K H 62% U- NH 0 82%
0 0 0=

140 141 142

OH OH
OH S(-)MBA OH OH
resolution HCI OH
SNH 0 43% NH 0 64%
0=o 0= 12 O

143 144 136

OH 0 OH 0
1) (Boc)20 H O OH H O
2) L-leu-OBz / DCC N OBn 1)H2 /Pd/C N OH
86%o NH 0 85% NH2 0
Boc

145 131

Scheme 2-2. Umezawa's strategy to Bestatin

Pearson and Hines'62 synthesis began with dioxalanone 146, which

underwent an aldol condensation with phenyl acetaldehyde resulting in the

mixture of diastereomers 147 (Scheme 2-3). The diastereomers were separated

by column chromatography and homochiral 147 was reacted with

diphenylphosphoryl azide to form azido compound 148. Refluxing 148 in

aqueous hydrochloric acid resulted in ester 149. The ethyl ester was saponified,

followed by a DCC coupling of the acid and the benzyl protected L-leucine. The

peptide 150 was then deprotected by a palladium catalyzed hydrogenation

resulting in Bestatin 131.



1) LiHMDS
Ph- O 0 2) phenyl acetaldehyde H Ph3P / DEAD / DPPA N3
S56% Ph Oh 79% Ph 0


146 147 148










S1)LiOH/H20O
EtOH / HCI 3 0 2) L-leu-OBn NH2 0
----- ^-A ------- N ^ ^ -Bn
OEt 60% N O n
OH OH 0

149 150


H2 /Pd/C NH
86% N J/OH
OH 0
131

Scheme 2-3. Pearson and Hine's strategy to Bestatin

Norman and Morris's63 synthesis began with the diesterification of L-malic

acid 151 resulting in 152 (Scheme 2-4). This was benzylated under basic

conditions and the adduct was saponified to diacid 153. This was selectively

esterified by forming the cyclic anhydride intermediate, which was opened

regioselectively by ethanol to form 154. A base catalyzed Curtius

rearrangement with diphenylphosphoryl azide resulted in the protected

compound 155. Ester 155 was saponified with lithium hydroxide, which was

coupled using EDC with L-leucine methyl ester affording 156. This was

deprotected with 1 M sodium hydroxide to yield Bestatin 131.


0 0 1) LHMDS / PhCH2Br
HO JO EtOH / H+ EtO- OEt 2)1 M NaOH a o
S OH t OEt 70% HO Y A
0 OH 0 OH OH
0 OH
151 152 153


O 1) LiOH
1) TFAA/ EtN 2) Leu-OCH3 / NMM/
2) EtOH o 1)DPPA/Et3N O NH / EDC/HOBt
97% HO OEt 65% EtO 64%
0 OH 0
154 155










0
O o0 LNH O0 HO NH2

100%

156 131

Scheme 2-4. Norman and Moris's strategy to Bestatin

The Palomo group64 started their approach by coupling 157 and 158 to form

3-lactam 159 (Scheme 2-5). This was followed by a two-step dehydroxylation of

the benzyl carbon yielding 160. The 3-lactam 160 was protected with (Boc)20

and then reacted with L-leucine and sodium azide to form adduct 162. The

addition of TFA followed by hydrogenation gave the deprotected compound 131.

OTBDMS OH 1) NaH I Mel
STiC4 / TEA Bn H H 2) n-Bu3SnH / Et3B
N PyS 79% 60%
PMP 0 PMP

157 158 159


H1) (NH4)2Ce(NO3)6
Bn2) (Boc)20 / DMAP Bn L-Leu-OBn / NaN3
73% 88%
0 PMP 0 Boc
160 161


S 1) TFA
2) H2/Pd/C 0 O
Boc-N N COBn 98% H2N N CO2H
OBn OH

162 131

Scheme 2-5. Palomo's strategy to Bestatin

The Koseki group65 commenced their synthesis of Bestatin from 2,3-

isopropylidene-D-ribose 163 (Scheme 2-6). The addition of phenyl magnesium

bromide to the sugar gave diastereomeric mixture of Grignard adducts 164. The

addition of sodium periodate gave the cyclic product 165, which was purified and










reacted with Jones reagent to form cyclic ester 166. A hydrogenation followed by

a DCC coupled addition of benzyl protected L-leucine afforded amide 167. Diol

168 underwent a two-step process mediated by 1-methyl-2-fluoropyridine to

afford azide 169, which was hydrogenated to afford Bestatin 131.

HO0 OH OH OH
S PhMgBr / THF HO'- --- NalO4 / ether
020 020

163 164


H 1) chromatography 1) H2 Pd/C
2) Jones reagent O 2) L-leu-OBn / DCC
o- 83%


165 166


S o H H I 1) 1-methyl-2-fluoropyridine
N TFA N OBn 2) NaN3HMPA
OOBn
S Bn 43% OH 58%


167 168

OH 0 OH 0
N B H2 Pd/C N OH
OBn OH
SN3 0 2 NH2

169 131
Scheme 2-6. Koseki's strategy to Bestatin

Bergmeier and Stanchina66 began with the protection of mannitol 170,

which was oxidized to acid 172 (Scheme 2-7). This was reduced by sodium

borohydride to form aldehyde 173, which was subjected to a Wittig reaction, and

subsequent acid treatment to afford allylic alcohol 175. This was selectively

monosilylated with tert-butyldiphenylsilyl chloride, followed by reaction with CDI

and sodium azide to form 177, which was then heated in a sealed tube to yield

cyclized product 178. Aziridine 178 was reacted sequentially with phenyllithium










and TBAF to form 179, which was oxidized and coupled with protected L-leucine

to provide dipeptide 180. The next three steps resulted in Bestatin 131 with an

overall yield of 6% in 18 steps.


OH OH Me2C(OMe)2 / OH 1) NaOl4 / NaHCO3
O OH SnCl2 O 2) KMnO4/KOH 0
HO 77% 85% 0 OH
OH OH OH O
/ 0
170 171 172


1) (COCI)2
2) NaBH4 O0 Ph3PCH2 Dowex(H+) OH
80% O H OJL 91% HO -
0
173 174 175


TBDPS-CI/ 1) CDI 0 109 C / CH2CI2 0
imidazole OH 2) NaN3 seaed tube
91% TBDPSO,, 41% TBDPSO 3 TBDPSO B
H
176 177 178

O O
1) PhLi I CuCN O 1) RuCl3 / NalO4 O -O
2) nBu4NF N H 2) L-Leu-OtBu / TBTU H NH (BOC)20
HOJ"/NH t-BuO "N _OO
50% 83% 95%
0


179 180

0
0 H OH
0 O 1) LIOH HH
t-BuO N 2)TFA HO Nt
t-BuO O NH2
0 68%

181 131
Scheme 2-7. Bergmeier and Stanchina's strategy to Bestatin

The Seki lab's67 approach began with protected L-aspartic acid 182, which

was converted in three steps to oxazolidinone 183 (Scheme 2-8). Treatment of

183 with benzyl bromide afforded 184. The lithium enolate of 184 generated by

LiHMDS and subsequent treatment with 3-phenyl-2-(phenylsulfonyl)oxaziridine

gave the chiral alcohol 185. Hydrogenation of 185 resulted in amino alcohol 186,











which was then treated with benzyl chloroformate to protect the free amine.


Alcohol 187 was reacted with DEAD and formic acid resulting in format 188.


This 189 was reacted with L-leucine, then deprotected with hydrogenolysis to


yield Bestatin 131.

1) PCI / PhH/AIC3 O OBn
MeO2CNH 2) PhSiHMe2 /TFANH | 1
NH 3) NaOMe/THF 0 NH O BnBr/Ag2CO3 O N 0
HO OEt 34% 1- -. 83% ur-n-v


0


182


1) LiHMDS
2) phenylsulonyl oxazaridine
62%


OBn

0- N 0
Me
18OH
185


Cbz
Cbz-CI O NH 0
85% 7 Y OMe
OH

187


1) NH3 Cbz
2) NaOH NH 0 L

70% O'-Me
OH


H2/Pd/C / HCI H2
86% O OMe
OH

186


Cbz
DEAD/ HCO2H Obe 'NH 0

60% OMe
OCHO

188


-leu-OBn / DCC
99%


H2 /Pd/C N H

90% N fOH
OH H

131

Scheme 2-8. Seki's strategy to Bestatin


The Semple group68 approached their synthesis by utilizing the coupling of


192 and 194 (Scheme 2-9). The synthesis began with N-a-Cbz-D-Phe-H that


was reduced over two steps to form aldehyde 192. Next, benzyl protected L-


e










leucine 193 was reacted over two steps to form isonitrile 194. After the addition

of trifluoroacetic acid, 192 and 194 were reacted to form the diastereomeric

mixture of dipeptides 195. After a simple hydrogenation, the diastereomers of

131 were separated by liquid chromatography, resulting in pure Bestatin 131 in a

13% overall yield.



OH 0
CbzHN HCI-H2N
191 193
1) BF3-THF 1) CH3CO2CHO/ Et3N
83% 2) Pyr-SO3 / Et3N 84% 2) C13CO2CCI/ NMM


__ -^ f^~O^ (,iy^ TEA / pyridine j-, 1 0 .,.

CbzHN HNH CN 65% H2N )N OBn
0 0 OH 0
192 194 195
1) H2 / Pd/C
2) HPLC separation
I 0
29% H
H2N NH'N
OH O
131

Scheme 2-9. Semple's strategy to Bestatin

The Park group69 synthesis began with an alkyne Grignard reaction on (R)-

phenylalaninal resulting in predominately syn-197 (Scheme 2-10). O-Benzylation

of 197 afforded 199, and the alkyne was subsequently oxidized to acid 200. This

underwent a DCC coupling to produce 201, which was subjected to a two-step

deprotection process that resulted in Bestatin 131.










0 OH OH
H 9M MgBr

S NHPf 96% NHPf NHPf
9.5:1 Major 9.5:1 Minor
196 197 198


OH OBn OBn
S BnBr/NaH KMnO4/HOAc
97% 87% NHPfO

197 199 200


L-Leu-OCHT3/ HOBt OBn 1) LIOH OH H
DCC TOH2) H eu-OCH3 2 Pd/C N C OH
91%. HNHPfO 93% NH2 0

201 131

Scheme 2-10. Park's strategy to Bestatin

In 2003, the Jurczak lab's70 began their synthesis with aldehyde 202. They

utilized a nitro aldol reaction with 1-nitro-2-phenylethane 203 (Scheme 2-11).

The aldol product was purified by chromatography to yield homochiral 204. A

Nitro group reduction by Raney-Ni hydrogenation, followed by the addition of a

Boc protecting group resulted in 205. This was cyclized in the presence of DMP

to the protected form of p-amino a-hydroxy acid 206. Addition of L-leucine to 206

resulted in protected dipeptide 207. This was followed by two deprotection steps

that yielded optically pure Bestatin 131.










Ph 1)Al203 Ph
H NO2 2) Chromatography 0
0 + 0-I
H 81%
0 OH
202 203 204


1) H2/Raney-Ni Ph 1)DMP / TsOH
2) (Boc)20 HN 2) NaOMe B N
0 I I I BocN
93% 78% OMe
OH /0
205 206


BocN O O 1) TsOH H2N OH O
L-Leu-OMe H 2) 1N HCI H
N OMe OH
95% OMe 70%


207 131

Scheme 2-11. Jurczak's strategy to Bestatin

The Wasserman group's71 synthesis began with N-Boc-D-phenylalanine

132 which was coupled with (cyanomethylene) triphenylphosphorane 208 to

afford 209 (Scheme 2-12). This was reacted with ozone and L-Leu-OBn to form

the doubly protected dipeptide 210. A stereoselective reduction with zinc

borohydride resulted in 145 with a diastereomeric excess of 86%. Refluxing with

aqueous hydrochloric acid followed by a palladium catalyzed hydrogenation

resulted in Bestatin 131.

Boc, Boc 1) 03 / CH2C12
NH PPh3 EDCI/ DMAP NH PPh3 2) L-Val-OBn
OH + CN 88% OV-CN 62%
O 0
132 208 209

Soc oc
\Bn 1) Zn(BH4)2 Boc, NH 0 ,E
oN 85% .. OBn










1)2M HCI H O
2) H2 / Pd/C H
92% OH H 0

131

Scheme 2-12. Wasserman's strategy to Bestatin

Over the past 30 years, there have been several documented strategies for

the synthesis of Bestatin. Most of these utilize amino acids or other naturally

occurring chiral compounds for their starting material. Other strategies require

enantiomer separation by chiral resolution; however, this process can be very

difficult and often inefficient. It was our goal to develop a novel synthesis for

AHPA and Bestatin through our simple glycidic ester approach.

Our proposed synthesis begins with a literature procedure using meldrum's

acid 212 and phenylacetyl chloride 211 to yield 213 (Scheme 2-13). Reaction of

3-keto ester 213 with sulfuryl chloride affords a-chloro-3-keto ester 214.

Compound 214 can be added to E. coli that have overexpressed a bakers' yeast

reductase, affording (2R-3S)-chlorohydrin 215 as the homochiral reduction

product. Treatment of chlorohydrin 215 with potassium carbonate will result in

cis-glycidic ester 216. Epoxide 216 can be treated with borontrifluoride diethyl

etherate and benzonitrile to afford the rearrangement product 217. Reflux with 6

M HCI should result in hydrolysis product (2S, 3R)-(3-amino-2-hydroxy-4-

phenylbutanoic acid (AHPA) 137. Following the Suda lab's synthetic strategy,

AHPA 137 can be coupled with L-leucine over three steps to yield Bestatin 131.















C21

211


O





212


0 0

Ot
CI


YDR368w


1) pyridine (2.5 eq)
2) EtOH


-O'OEt


f OOH

OEt
Cl


K2CO3


Benzonitrile / BF3OEt2
O


0
217


Boc
BocCI NH 0


OH


6 M HCI


1) DCC/ L-Leu-OBz
2) H2 / Pd/C


H2N OH

S-OH
0


137


H2N OH O

H OH
0 H


Scheme 2-13. Our proposed synthesis of AHPA and Bestatin


SO2C12


O
OEt
0














CHAPTER 3
STEREOSELECTIVE, BIOCATALYTIC REDUCTIONS OF a-CHLORO-3-KETO
ESTERS

Introduction

Homochiral glycidic esters are versatile intermediates that can be converted

into a variety of high-value products. Optically active glycidates can be prepared

by a number of routes including asymmetric Darzens reactions, chiral alkene

oxidation methodologies and by ring closure of homochiral a-halo-3-hydroxy

esters.72-86 We were particularly interested in the last strategy because

asymmetric reductions of a-chloro-3-keto esters might afford each of the four

possible glycidate precursors via dynamic kinetic resolution processes from

single, inexpensive starting materials (Scheme 3-1). Here, we explore the

potential of individual reductase enzymes from baker's yeast (Saccharomyces

cerevisiae) as solutions to the problem of obtaining homochiral glycidate

precursors.

Reductions of a-chloro-3-keto esters by whole cells of commercial baker's

yeast generally produce disappointing mixtures of alcohol diastereomers.87-90

Recent work has revealed that the yeast genome encodes a large number of

reductases and it seemed likely that their simultaneous participation was mainly

responsible for the modest stereoselectivities commonly observed in yeast-

mediated ketone reductions.91-93 In response, we have adapted a fusion protein

strategy94 that allows the properties of yeast reductases to be assessed









OH OH
R CO2Et R rCO2Et
CI CI
Cl Cl
0 0
At t A [H]
R OEt
CI
220 R=Me
221 R = Et
222 R = n-Pr
126 R=Ph O OH
214 R = Bn R CO2Et R CO2Et
Cl CI

Scheme 3-1. Four possible reduction products of a-chloro-3-keto esters: (2S-
3S)-white, (2R-3S)-black, (2R-3R)-black/white lines, (2S-3R)-black
dashes

individually, so that enzymes yielding homochiral products can be uncovered.95'96

Moreover, after a reductase with the desired properties has been identified,

whole Escherichia coli cells expressing the same protein can be employed for

bioconversions on preparative scales using glucose fed-batch conditions.97

Cellular metabolic pathways supply NADPH, and the whole cells display very

high stereoselectivities because they overexpress only a single yeast reductase.

Results and Discussion

A series of five a-chloro-3-keto esters was used in this study (Scheme 3-1).

Eighteen yeast reductases were isolated as fusion proteins with glutathione S-

transferase using previously-described methods.96 The collection of enzymes

included members of the aldose reductase, D-hydroxyacid dehydrogenase,

medium chain dehydrogenase and short chain dehydrogenase superfamilies.

Each a-chloro-3-keto ester was tested as a substrate for each reductase in the

presence of NADPH, which was supplied by a cofactor regeneration system. For

comparison, parallel reductions were also carried out with commercial bakers'

yeast cells for the two cases where literature data were not available.98









Since we were concerned with the enantiomeric and diastereomeric excess

values for each reduction, we had to develop analytical techniques to resolve the

enantiomers. Our general approach is to generate a mixture of all four possible

products by sodium borohydride. The products are then analyzed by chiral gas

chromatography (chiral GC) (Scheme 3-2).

OH O OH O
R QOEt R OEt
R I NaBH4/AcOH Cl Cl
R OEt
Cl OH O OH O
R =-" OEt R OEt
Cl Cl
Separable by Chiral Gas Chromatography

Scheme 3-2. Synthesis of all 4 diastereomers by sodium borohydride, which can
be separated by chiral gas chromatography

This strategy typically works for most alcohols; however, occasionally full

separation can not be achieved. This was found during the attempt to separate

chlorohydrin 218. To solve this problem, the alcohol 218 was acetylated using

acetic anhydride affording derivatives that can be fully resolved on a chiral gas

chromatography column (Scheme 3-3).

Individual stereoisomers were linked to the appropriate GC peak by

isolating alcohols from enzymatic conversions that afforded only single products.

Where literature data were available, optical rotation values were used to

determine absolute stereochemistry; these assignments were consistent with

those made by NMR in all cases.











CH3

n OH 0 DMAP / acetic anhydride OEt Et
Cl Cl
218 219
All Four Diastereomers are
Separable by Gas Chromatography

Scheme 3-3. Derivatization technique used to separate all 4 diastereomers for
chlorohydrin 218

Comparing the outcomes of reactions using whole bakers' yeast cells with

those employing isolated yeast reductases clearly demonstrates the utility of

examining individual biocatalysts (Figure 3-1). Not only did the purified yeast

reductases deliver higher stereoselectivities in most cases, they also produced

diastereomers not observed in reductions employing commercial yeast cells.

This may result from low expression of some reductases under the physiological

conditions prevailing in commercial bakers' yeast, and this highlights an

important advantage of using isolated reductases, rather than relying on whole

yeast cells. Alternative methods to increase expression levels of desirable

reductases, such as adding specific enzyme inhibitors, are more difficult to

optimize and control.93'99'100 It should also be noted that the screening reactions

could be carried out rapidly, and a complete data set was typically obtained for

each substrate within 48 h.

The smallest substrate, 220, was accepted by all of the yeast reductases

examined, although the stereoselectivities of these reactions were relatively poor

except for YOR120w and YGL157w, which afforded (2S, 3S) and (2R, 3S)

configuration as the major products, respectively. In all cases, however, only L-










Yeast CH3 OEt CH3 OEt CH3 OEt OEt OEt
Gene cl a Cl 0 ci ci
220 221 222 126 214
YJR096w ______ ---a

YDL124w --- ---

YBR149w --- ---

YOR120w --- --- ---

YHR104w --- --- --- ---

YDR368w --



YNL274c --- ---

YPL275w --- --- --- ---

YPL113c --- --- ---

YLR070c --- --- --- ---

YALO60w --- ---

YGL157w ---

YDR541c --- --- ---

YGLO39w

YNL331c ---

YCR107w --- ---

YOL151w --- --- --- ---
Yeast KJ 3| ^ ,, r
YaCellsb GIII b b


Figure 3-1. Biocatalytic reductions of a-chloro-3-keto esters. Yeast enzymes are
referred to by their systematic names and grouped by superfamilies.
Product compositions from reactions that proceeded to at least 20%
conversion within 24 hr are shown in pie charts (2S-3S)-white, (2R-
3S)-black, (2R-3R)-black/white lines, (2S-3R)-black dashes









alcohols were formed. This behavior parallels our earlier observations from

reactions in which ethyl acetoacetate was used as a substrate for the same

collection of yeast fusion proteins.96 The behavior of higher homolog 221

provides an interesting contrast. In four cases, D-alcohols were the major

products. This is significant because D-alcohols are observed much less

commonly in biocatalytic reductions and enzymes that deliver this

enantioselectivity are correspondingly important. Six enzymes examined

accepted 126 as a substrate: four afforded only (2S, 3R) configuration while the

remaining two produced mainly (2R, 3S). Benzyl-substituted 3-keto ester 214

was reduced by three enzymes, with very high stereoselectivities in two cases.

Taken together, our results have demonstrated that reductase enzymes

uncovered by an analysis of the yeast genome can deliver important chiral

building blocks for organic synthesis. At least two of the four possible a-chloro-3-

hydroxy ester diastereomers could be produced in high optical purities in most

cases. The major deficiency in the present collection is a lack of stereoselective

reductases with D-specificities. Biocatalysts with these properties might be

identified by including enzymes from additional organisms in our collection of

fusion proteins and the increasing pace of genome sequencing project bodes

well for expanding the utility of our chemo-enzymatic approach.















CHAPTER 4
SYNTHESIS OF THE C-13 TAXOL SIDE CHAIN

Racemic Synthesis

Our approach to the synthesis of the Taxol side chain began with a racemic

synthesis. This was developed to test the feasibility of important chemical steps,

and also to optimize the reaction conditions without consuming homochiral

starting material.

This synthesis began with a Darzen's reaction of benzaldehyde 85 and

methyl chloroacetate 116 to yield trans-methyl 3-phenylglycidate 11742 (Scheme

4-1). Treatment of trans-117 with dry hydrochloric acid for several hours allowed

a highly stereoselective ring opening to afford chlorohydrin 11842, which was

reacted with potassium carbonate to yield cis-epoxide 119.89 The racemic

epoxide 119 underwent a Ritter reaction with benzonitrile, catalyzed by boron

trifluoride etherate to form cyclic product 60.101,102 Treatment of trans-oxazoline

60 with 0.5 M hydrochloric acid yielded the racemic Taxol side chain 41 with an

overall yield of 16%.41'103


H + ClI,'OMe NaOMe 0 0 HCI
0 79% 7 '/-OMe 99%
/u 0

85 116 (+/-)-117


CI OH 0
-. O e K2CO3 1 Benzonitrile / BF3-OEt2
'f OMe OMe -
52% 55%

(+/-)-118 (+/-)-119












0.5 M HCI NH 0
N 0 72%
-Om 72% OMe
O6
0

(+/-)- 60 (+/-)41

Scheme 4-1. Racemic synthesis of the Taxol side chain

Once the racemic synthesis was complete, we began our work on the chiral

route. Our first step was the chlorination of ethyl benzoylacetate 125, which can

be achieved by reaction with tetrabutylammonium bromide and

chlorotrimethylsilane104 (Scheme 4-2). However, we found that this chlorination

technique gave side products that were difficult to separate from the desired

product 126. After several failed attempts, an alternate literature procedure was

found using sulfuryl chloride105108, giving high yields and allowing a simple

distillation for purification.

O 0 0 0
Bu4NBr / Me3SiCI
OEt -- OEt
Cl
125 126
low conversion / difficulty
purifying product

O 0 0 0
OEt S02C2 OEt
98%

125 126
simple distillation

Scheme 4-2. Two chlorination methods for P-keto esters

Biotransformation Strategy

As discussed in chapter 3, we recently published the reduction results for

several a-chloro-p-keto esters using our library of purified bakers' yeast









reductases.109 This strategy of using purified reductases works well for the small

scale screening of substrates; however, due to the high cost of NADPH, this

method is not practical for gram scale syntheses. A simple and economical

reduction method is the use of whole cells, utilizing the cell's cofactors for the

reaction.

Our strategy was to implement a scaleable synthesis of the Taxol side

chain using an enzymatic reduction as the key step, thus we needed to utilize

whole-cell biotransformations. Adam Walton and Parag Parekh initiated our

group's research on whole-cell catalyzed reactions using E. coli with

overexpressed GRE297, a known yeast reductase. They established that the

cells kept their reducing capabilities longer in a non-growing nitrogen deprived

environment, compared to reactions in complete growth media. Using ethyl

acetoacetate as their substrate, they were able to reach a product concentration

of 250 mM over a 30 hour period (Figure 4-1).

The key step in our route to the Taxol side chain involves the enzymatic

reduction of a-chloro-3-keto ester 126, using a similar whole-cell approach as

developed by Walton and Parekh. After analyzing the results presented in

Chapter 3, we chose two reductases for our synthesis of the Taxol side chain:

YDL124w, which affords (2S-3R)-chlorohydrin 127 and YGL039w, which affords











300

250

200
c
.0
150
C
U 100
o
0
50

0
0 10 20 30 40
Time (hrs)




Figure 4-1. Production of (S)-ethyl 3-hydroxybutyrate by engineered E. coli cells
under non-growing conditions

(2R-3S)-chlorohydrin ent-127 (Scheme 4-3). By using these enzymes, we can

synthesize both enantiomers of the optically pure Taxol side chain.

0 0 OH 0 O
.JYDL124w A
OEt YDLw OEt NH 0
I OEt
SOH
126 127 130


0 0 OH 0 0
OEt YGL039w OEt ___ NH 0


xy OH

126 ent-127 ent-130

Scheme 4-3. The Taxol side chain and its enantiomer can be synthesized by
utilizing two different enzymes; YDL124w and YGL039w, respectively

Optimization of Our Whole-cell System

A review from Saluta and Bell reports conditions that can potentially effect

protein overexpression by a T7 promoter such as glucose concentration,

induction optical density, induction temperature, and induction time.110 Before we









began our biotransformation studies, these conditions were optimized. In their

review, these authors recommend certain parameters to be followed for general

protein overexpressions. First, it is known that the overexpression plasmid can

be 'leaky', thus a catabolite repressor should be used to inhibit the protein

expression in the growing phase. They recommend supplementation of the

growth media with 2% glucose for this reason. Second, they reported that the

most advantageous optical density for inductions in these systems are A600 = 0.5

- 1.0. The recommended glucose concentration and optical density for induction

were used in our system.

It is known that the optimal induction temperature can vary when trying to

overexpress a protein in its active form. A common complication in the

overexpression of proteins is the formation of insoluble-misfolded peptides called

inclusion bodies, which are usually caused by the expression at high

temperatures (~37 oC).110 This was most likely the case in our early attempts to

use YGL039w in our whole-cell biotransformations. SDS-PAGE confirmed the

overexpression of our protein of interest; however, reduction attempts were

unsuccessful (Figure 4-2).












1 2 3 4 5 6
1- marker

2- YGL039w
116
3- t =0 hr 97

4-t=1 hr 67 .

5- t =2 hr
45
6- t =3 hr

7- t =4 hr
31







Figure 4-2. SDS-Page of the overexpression of YGL039w over a 4-hour time
period. The arrow marks the expected position of the YGL039w fusion
protein

Whole-cell Assays

To investigate the inclusion body theory, experiments were developed to

test the cells' reducing activity when expressing protein at 37 C, 30 C, and 24

C. Three separate cell batches were grown at 37 C until they reached an O.D.

= 0.6, then these were cooled to the corresponding temperatures. Once the cells

reached their final temperature, they were induced with IPTG (0.1 mM final

concentration). Aliquots were taken at various time points and the cell

suspension was lysed by sonication, followed by centrifugation to remove the

cellular debris. The activity of soluble reductases was screened by the addition

of NADPH and ethyl acetoacetate (an excellent substrate for YGL039w). This










solution was monitored at 340 nm over 2 minutes to observe the loss of NADPH,

which is directly proportional to the reduction of ethyl acetoacetate. The specific

activity for each aliquot was calculated as described in Appendix B, and the

activities were plotted versus the time after induction (Figure 4-3).


Specific activity vs. Time for cells
induced at varying temperatures
0.09
E 0.08 -
S0.07
S0.06 *
E 124
0.05 -
.30
0.04 -
.> 37
5 0.03
.2 0.02 -
S0.01 -
0 ,-"-"- -
0 5 10 15 20 25
Time (Hrs)



Figure 4-3. Specific activity of the E. coli cells overexpressing YGL039w, which
have been grown under different induction temperatures (37 C, 30 C,
and 24 C)

From this experiment we concluded that overexpression of YGL039w at 24

C gave the highest activity per cell. In addition, it confirmed our suspicion that

YGL039w overexpressed at 37 C formed inclusion bodies, which explained why

it was unable to reduce the ketone substrate. These experiments also answered

an additional question. It was first considered important to stop the growth of the

induced cells after 4 hours. However, this experiment ran for 24 hours, and we

saw no evidence for a significant loss of specific activity from the cells up to this

point.










In addition to the induction temperature, we were also curious about the

effect of the GST tag on the activity of the protein. Therefore, YGL039w was

overexpressed both with and without the GST tag, and the same methods to

evaluate reductase activity described above were used to compare the activities

(Figure 4-4). The results implied that the GST tag, at least in this case, does not

adversely affect the activity of the reductase.


GST Effect on Specific Activity

& 1.6
1.4 -
= 1.2
o0
E 1
-a GST
S0.8 -
1 0 No GST
S0.6 *
o
0.4 -
U 0.2 "
o -*=*
CL 0 *
0 5 10 15 20 25
Time (hr)




Figure 4-4. Whole cell activity of an overexpressed YGL039w with a GST tag
and YGL039w without a GST tag versus time

Whole-cell Reduction of 126

After the optimization of protein expression and activity, the whole-cell

reductions of the a-chloro-3-keto esters could begin. The reduction of 126 was

run on a 1 liter scale fermentation reaction in a nitrogen-free phosphate buffer.

Unfortunately, we encountered a problem while conducting this experiment: 126

was found to decompose while dissolved in water. After analyzing its stability at

different pH values, the molecule was found to be reasonably stable at a working









pH under 6.0. Second, we noticed that some a-chloro-3-keto ester will undergo

reductive dechlorination, which Bertau and Jorg90'111'112 suggested was due to a

reaction with free glutathione in the cell. In our whole-cell reductions of 126, we

found that the amount of dechlorinated product can widely vary for each reaction,

and we do not have a strong hypothesis to why there is such a vast

inconsistency.

Our last problem observed in the whole-cell reduction of 126 was its high

toxicity and inhibitory effect towards the cells. Two actions were taken to

minimize these toxicity effects. First, we slowed substrate feeding to keep the

starting material concentration at a minimum. Second, a non-polar adsorbing

Amberlite XAD-4 resin was added to the fermentation reaction. This resin

adsorbs the product from the aqueous phase, thereby lowering its inhibitory

effect on the cells. On average, we have seen a 20 25 % increase of isolated

product for 127 and ent-127 using these tactics.

After optimization of both the growing conditions and the conditions for the

whole-cell reduction, we were able to achieve a final product concentration of ~6

mM. These concentrations were calculated by GC using the ratio of product

peak area versus internal standard peak area.

The next step, extraction of the product, proved to be difficult because of

the formation of an emulsion caused by cellular debris. To avoid the emulsion,

we centrifuged the cells and extracted the supernate with an organic phase.

However, product was found in the cell pellet, resulting in a significant loss in










yield. We therefore developed a gentle extraction method in which the aqueous

phase was slowly circulated through methylene chloride (Figure 4-6).


Concentration of Product


7
6
5
0 4
3
U 2
r-

02


0


5 10 1
Time (hr)


*YDL124w
* YGL039w


5 20


Figure 4-5. Concentration of the product for the biotransformation using
YDL124w and YGL039w


Product extraction apparatus

Peristaltic pump
(ca. 10 mL/ min)


Cell suspension *

0.5 L Filter flask *

CH2CI2,


SCell suspension
(1 L beaker)


t
Magnetic stir bar


Figure 4-6. Diagram of our gentle extraction technique


*



II-------------
*. :
i U .ii










After purification by flash chromatography, we were able to achieve a 91%

and 85% yield of 127 and ent-127 from the whole-cell reduction with YDL124w

and YGL368w, respectively (Scheme 4-4).

O 0 OH
YDL124w
fr i y OEt 91% OEt

126 127
99% d.e. / 99% e.e.
1.33 g/L isolated product

O 0 OH
O YGLO39w -
OE- OEt
OEt 85% OEt

126 ent-127
76% d.e. / 99% e.e.
1.39 g/L isolated product containing
10% anti-chlorohydrin

Scheme 4-4. Final results for the whole-cell biotransformations after purification

Base Catalyzed Ring Closure

Our next step required a base catalyzed ring closure to form the

corresponding glycidic ester89 (Scheme 4-5). This reaction can be directed to the

cis or trans product by adjusting the strength of the base. Reaction of

chlorohydrin 223 and 225, with a weak base will yield exclusively the kinetic

product for this reaction 224 and 226, respectively. Treatment of the same two

chlorohydrins with a strong base will selectively yield trans-epoxide 226.

OH O 0 OH 0 0
K2003 I H20 RK2003 / H20 R
R OEt K2C3/ H2 OEt R -OEt K2CO3/ H20 R OEt
CI 0 CI 0
223 224 225 226











OH 0 0 OH 0 0
R O NaOEt / EtO Na+ R"OEt R O NaOEt/ EtO-Na+ R '
R- O ROEt R--Y'AOR OEt -- 7OEt
Cl 0 Cl 0
223 226 225 226

Scheme 4-5. Results for the ring closure of chlorohydrins: Potassium carbonate
results in the kinetic product, whereas sodium ethoxide affords the
thermodynamic product.

The Azerad lab reported89 that the reaction involving ethoxide ion preceded

by initial epimerization of the chlorohydrin via the formation of the enolate prior to

cyclization (Scheme 4-6). Once epimerized to the more thermodynamically

favored anti conformation, the chlorohydrin can undergo closure to trans-epoxide

226.

OH O OH O OH 0 0
R OEt NaOEt R OEt R =YR OEt R R' OEt
Cl Cl Cl 0
223 227 225 226


CO2Et CO2Et
H OH H OH


NaOEt / EtOH
H Cl Cl H

R R
223 225
Thermodynamically more
stable chlorohydrin

Scheme 4-6. Mechanism for the sodium ethoxide promoted epoxidation and a
Newman projection describing syn versus anti configuration of
chlorohydrins

Epoxide formation using potassium carbonate required some water to

afford glycidic ester 128 with a 99% yield, and the amount of added water proved

critical (Scheme 4-7). We found that 1 2 equivalents of water would result in a

slow reaction, and more than 3 equivalents of water would often lead to by-










products. It was also necessary to use chlorohydrin purified by column

chromatography, because the impurities found with the reduction product led to a

drastic loss in yield.

OH 0 K2CO3 (3eq) / H20 (3 eq)
OEt DMF OEt
OEt 99%

127 128

Scheme 4-7. Base promoted formation of cis-glycidic ester 128

Ritter Reaction

Once the synthesis of glycidic ester 128 was achieved, it underwent a Ritter

reaction with benzonitrile to afford predominately trans-oxazoline 129 with a 55 %

yield (Scheme 4-8). This reaction also yielded a small amount of cis-oxazoline

227 which decreased the yield of the desired product. Fortunately, the cis isomer

was separable by flash chromatography allowing pure trans-oxazoline 129 to be

isolated.


0
t Benzonitrile / BF3-OEt2
0 55% N 0 + N O
SOEt OEt
0 0

128 129 227
Major Product Minor Product
Seperable by Flash Chromatography

Scheme 4-8. The Ritter reaction of glycidic ester 128 and benzonitrile

An attempt was made to increase selectivity for the trans oxazoline by

varying the temperature, solvent polarity, and strength of the Lewis acid.

Different temperatures and solvents were found to have no effect on the reaction

selectivity. A variety of Lewis acids were also screened to see if they could









increase the trans: cis ratio (Figure 4-7). These attempts were unsuccessful and

the original conditions were found to be optimal.

Lewis Acid Conversion Ratio (Trans: Cis)
Lithium Bromide 0% ---
Lithium Chloride 0% ---
Ytterbium Triflate 99% 5: 1
Tin (II) Ethyl Hexanoate 0% ---
Aluminum Chloride 0% ---
p-Toluene Sulfonic Acid 99% 5:1
Triflic Acid 99% 5:1
BF3-etherate 99% 5: 1

Figure 4-7. Effect of Lewis acids on the Ritter reaction

Ring Hydrolysis to the Taxol Side Chain

Oxazoline 129 has been used by Bristol Myers-Squibb as a protected form

of the Taxol side chain113 that can be coupled to the terpene core of Taxol

(Scheme 1-9). With this said, we were able to synthesize the protected Taxol

side chain in 4 steps with an overall yield of 49 %.

For academic reasons, and to compare the optical rotation of our material

with that synthesized previously, we treated oxazoline 129 and ent-129 with

aqueous acid to afford the Taxol side chain 130 and ent-130 as their ethyl esters.

Optical rotations for the Taxol side chain 130, and its enantiomer ent-130 were

[a]D = -11.6 (c = 2.0, CHCl3) and [a]D = +12.3, (c = 1.0, CHCl3), respectively. As

expected, the 1H NMR spectra of 130 and ent-130 were found to overlap

(Appendix B). In addition, to confirm the enantiopurity of the final products 130

and ent-130, were derivatized with (S)-a-methoxy-a-phenylacetic (MPA) and the

spectra of the two derivatives were compared (Appendix B). The spectra of ent-









130 was found to be 99% enantiomerically pure. A small amount of enantiomer

was found in 130, which can be attributed to a small amount of trans-epoxide that

was not separated in the ring closure step. However, since ent-130 was

synthesized in a 99% ee, it confirmed that subsequent chemical steps in our

reaction scheme did not provoke any racemization.

For the final step, it is essential that the reaction be carried out under mildly

acidic conditions. It has been reported that treatment with weak aqueous acid

will result in the hydrolysis product from attack at the C-4 position103 (Scheme 4-

9). However, treatment of oxazoline 129 with 6 M hydrochloric acid will result in

amide hydrolysis resulting in compound 228. In addition, we found that storing

the oxazoline in the open air at room temperature would result in the hydrolysis

product 130 after several weeks.




N 0

O co2Et

NH2 0 07 0/
OH O NH 0
OH
OHV OEt
OH

Scheme 4-9. Hydrolysis of oxazoline 129 under mildly acidic conditions versus
strongly acidic conditions














CHAPTER 5
SYNTHESIS OF BESTATIN

Over the past 30 years, there have been several strategies reported for the

synthesis of Bestatin 131 (Chapter 2). Most of these utilize amino acids or other

naturally occurring chiral compounds for their starting material. Other strategies

utilize the separation of enantiomers by chiral resolution; however, this process

can be very difficult and often inefficient. It was our goal to develop a novel

synthesis for AHPA 137 and Bestatin 131 through our simple homochiral glycidic

ester approach.

Syntheis of p-Keto Ester 213

Our approach to Bestatin began by synthesizing 3-keto ester 213 by a

literature procedure using Meldrum's acid 212 and phenylacetyl chloride 21114

(Scheme 5-1). The product of this reaction 213 can be easily purified by a

simple vacuum distillation.

0 1) pyridine (2.5 eq)
O.)-) 0 etOH oElf 0 0
ci 2) E 86% OEt

211 212 213

Scheme 5-1. Synthesis of 3-keto ester 213

Chlorination of p-Keto Ester 213

The chlorination of compound 213 with sulfuryl chloride was found to be

difficult, because performing this reaction with stoichiometric amounts of starting

material resulted in a large amount of dichlorinated byproduct 229 accompanying










the desired product 214 (Scheme 5-2). These compounds were inseparable by

distillation, and difficult to separate by flash chromatography. This problem was

solved by lowering the reaction temperature and decreasing the ratio of sulfuryl

chloride to 3-keto ester, thus simplifying the purification process.

0 0 0 S02CI2(1eq) 0 0 0 0
OEt 50 2C Oeq)OEt Et
Cl Cl Cl
213 214 229
Very Difficult to Seperate


S 0 0jOt S02C12 (0.6 eq) O O 0 0
OEt 25 C OEt 0Et
Cl
213 214 213
Easily Separated from
Starting Material

Scheme 5-2. Two different approaches to chlorinate 213 with sulfuryl chloride

Enzymatic Reduction of 214

The key step in our approach to AHPA 137 and Bestatin 131 is the whole-

cell reduction of compound 214, using an overexpressed bakers' yeast reductase

in E. coli. This process allows us to introduce two chiral centers into our reaction

scheme, and we hoped to achieve this on a gram scale. We recently published

the reduction results for several a-chloro-3-keto esters using our library of

bakers' yeast reductases109 (Chapter 3). Benzenebutanoic acid, a-chloro-3-oxo-

ethyl ester 214 was shown to be a substrate for three bakers' yeast reductases;

YDR368w, YGL157w, and YGL039w (Scheme 5-3).










0 YDR368w I YGL157w
OEt -OEt v -. OEt
Cl CI CI
215 214 230
99% e.e. / 99% d.e. 99% e.e. / 99% d.e.






OEt
Cl
230
41% e.e. / 98% d.e.

Scheme 5-3. Three bakers' yeast reductases that accept 214 as a substrate;
YDR368w, YGL039w, and YGL157w

Previously, for compounds 127 and ent-127 (Chapter 4), we were able to

use literature data to determine the absolute stereochemistry; unfortunately, no

literature data was found for compounds 215 and 250. Comparison of the 1H

NMR of these compounds confirmed that YDR368w and YGL157w afforded syn

and anti chlorohydrins, respectively (Appendix B). However, since literature data

did not exist, another method was used to define the absolute stereochemistry

for 215 and 250. We first attempted X-ray crystallography; however, we were

unable to grow diffraction-quality crystals from these compounds. Our next

approach was NMR analysis by derivatizing the chlorohydrins with (R)- and (S)-

a-methoxy-a-phenylacetic (MPA) acid. Dr. Ion Ghivirigia analyzed these

derivatized compounds for their 1H-1H, 1H-13C one-bond and 1H-13C long-range

couplings to determine the absolute stereochemistryl15-118 (Appendix B).

The results from NMR experiments were used to assign the (2R, 3S)

configuration to the YDR368w product, and the (2S, 3S) configuration to the









YGL157w and YGL039w products. Fortunately, the (2R-3S)-chlorohydrin 215, is

the intermediate needed for our approach to AHPA 137 and Bestatin 131.

Whole-cell Reduction of 214

The reduction of 214 was performed in a 1 liter fermentation reaction in a

nitrogen-free phosphate buffer. The growth and biotransformation conditions for

the reduction of compound 214 followed those optimized for YGL039w in our

Taxol side chain synthesis (Chapter 4). As seen before, this whole-cell reduction

also yielded dechlorinated product that formed in the beginning of the reaction

(Scheme 5-4). Typically, the first five percent of the starting material would suffer

this dechlorination reaction. Occasionally there would be vast variations in the

extent of dechlorination, and this phenomenon has yet to be fully understood.

o 0 YDR368w .^ OH 0 + OH 0
a OEt YD +Y3 ~ OEt O+ OEt
CI CI
214 215 231

Scheme 5-4. The whole-cell biotransformation resulted in the chlorohydrin 215
and dechlorinated product 231

Substrate 214, much like compound 126, was highly toxic toward the E. coli

cells. To lessen this effect, two actions were taken. First, we fed the cells small

portions of starting material every hour, thereby keeping the concentration of the

toxic starting material at a minimum. Second, a non-polar adsorbing XAD-4 resin

was added to the fermentation system. This resin would slowly adsorb the toxic

compounds from the reaction system, thus allowing the cells to further reduce

substrates and consume glucose and oxygen.









The whole-cell reduction of 214 by cells overexpressing the reductase

YDR368w yielded a final product concentration of 5 mM. Using the previously

described gentle extraction method, we were able to obtain 1.1 g of product after

purification by flash chromatography with an overall yield of 82% (Scheme 5-5).

i 0 0 YDR368w OH 0
OEt 82% oEt
Cl Cl
214 215
99% d.e. / 99% e.e.
1.1 g/L isolated product

Scheme 5-5. Results for our optimized whole-cell reduction of 214

Base Catalyzed Ring Closure of 215

Treatment of chlorohydrin 215 with potassium carbonate cleanly yielded

cis-glycidic ester 21689, with no need for further purification (Scheme 5-6). Since

DMF was used as the solvent for this reaction, it was important to wash the

organic phase with several small portions of water to remove any residual DMF.

In addition, the amount of water was critical to the success of this reaction. We

found that 1 2 equivalents of water would result in a slow reaction, and that

more than 3 equivalents of water would often lead to by-products.

_HO K2C03(3eq)/ H20 (3eq)
EDMF .,, OEt
Ot 99% s9I
cl 0
215 216

Scheme 5-6. Base promoted ring closure for glycidic ester 216

Ritter Reaction

Epoxide 216 was opened by benzonitrile and borontrifluoride diethyl

etherate to afford the protected form of AHPA 217101'102 (Scheme 5-7). Unlike









the Ritter reaction in our synthesis of the Taxol side chain (Scheme 4.8), this

reaction was completely selective for trans-oxazoline 217, with an overall yield of

78%.



.,/ O. Benzonitrile / BF3-OEt2
"* QrOEt _________
78% OEt
0 Et
0
216 217

Scheme 5-7. The Ritter reaction of 216 and benzonitrile only afforded the trans-
oxazoline 217

Synthesis of Bestatin from 217: First Generation

In our first attempt to complete the synthesis of Bestatin, oxazoline 217 was

saponified to produce acid 232 (Scheme 5-8). This was subjected to a DCC

coupling with L-Leu-OBn in an attempt to form the protected form of Bestatin

233. The final deprotection step was expected to utilize hydrogenolysis of both

oxazoline and the benzyl ester, thus yielding Bestatin directly. While the coupling

with L-Leu-OBn appeared to have been successful, a number of attempts to

hydrogenolyze the oxazoline failed, and we found that the oxazoline could only

be deprotected by reflux in the presence of 6 M HCI. Such conditions would also

cleave the peptide bond, and this strategy was therefore abandoned.











K2CO3 DCC / L-Leu-OBn
N 0 N O
'~ OEt / OH
O 0
217 232


H2N OH
N 0 X OH
0 -
C j' N 1OEt 0
OOt
0 -

233 131

Scheme 5-8. First attempt for the synthesis of Bestatin

Synthesis of Bestatin from 217: Second Generation

Our second attempt to convert oxazoline 217 to Bestatin involved

nucleophilic attack on the ring carbon to yield an sp3 center that would be more

susceptible to deprotection (Scheme 5-9). We chose a nickel-catalyzed addition

of PhMgBr to afford Grignard product 234119. We attempted a DCC coupling with

L-Leu-OtBu and crude 234, but unfortunately the products of the Grignard

reaction and the Leucine coupling reaction were very difficult to dissolve in a

variety of solvents, thus making these intermediates extremely difficult to

characterize. Since Bestatin was not observed at the end of this sequence, this

route was also abandoned.



S PhMgBr /dppNiC2 DCC / L-Leu-OtBu
N^ ZO n HN 0 OH
OH .,OH
n 0










3NHCI H2N OH O
HN -O ,, 0 H N
N H / OH
OtBu 0


235 131
Separation and Isolation
was not Practical

Scheme 5-9. Second attempt for the synthesis of Bestatin

Synthesis of Bestatin from 217: Third Generation

Based on the difficulties encountered in converting oxazoline 217 directly to

Bestatin 131, a simpler approach was taken. As mentioned in Chapter 2,

Bestatin is composed of two main parts, (2S, 3R)-3-Amino-2-hydroxy-4-

phenylbutanoic acid 137 (AHPA) and L-leucine. The most difficult challenge for

the synthesis of Bestatin 131 is the synthesis of AHPA 137. Our final approach

was to synthesize AHPA by simply deprotecting oxazoline ester 217 with 6 M

HCI (Scheme 5-10). Since literature methods can be used to convert AHPA 137

to Bestatin 131, this completes a formal total synthesis of this molecule.

1) BocCI
H2N OH 2) DCC/ L-Leu-OBz H2N OH 0
6M HCI OH 3) H2 / Pd/C OH
Nj\O_ 0 OH O
/OEt O
0
217 137 131

Scheme 5-10. Final Strategy to AHPA 137 and Bestatin 131

Hydrolysis of 217 proceeded smoothly and after a simple purification by a

Dowex cation exchange resin, we were able to isolate AHPA with a 40% overall

yield. As a confirmation, commercially available AHPA was purchased and

compared with our synthetic material. The 1H NMR spectrum of the






70

commercially available AHPA and our synthesized AHPA were identical

(Appendix B). In addition, the optical rotation for our synthetic AHPA was [a]D =

23.2 (c = 1.3, 1 M HCI), AHPA (Sigma-Aldrich) [a]D = 23.4 (c = 1.0, 1 M HCI).

The overlap of spectral data and optical rotation values67 confirms that our

previous definition of absolute stereochemistry was correct using NMR.














CHAPTER 6
SYNTHETIC APPROACH TO CHUANGXINMYCIN

Introduction

Chuangxinmycin 286 is a natural product isolated from Actinoplanes

tsinanensis (Figure 6-1). Initial studies suggested that it has in vitro antibacterial

activity against a variety of gram-negative and gram-positive bacteria. In

addition, it has shown antimicrobial activity against Escherichia coli and Shigella

dysenteriae in mouse models120. Clinical results have shown successful

treatment for septicaemia and for urinary and binary infections cause by E. coli.

Presently, Chuangxinmycin's mode of action is not completely understood;

however, it is reported that this drug has an affect on the tryptophan biosynthetic

pathway.

S CO2H
S CH3

N
H
236


Figure 6-1. Chuangxinmycin 236

The Akita lab's Approach to Chuangxinmycin

In 1997, the Akita group published synthesis of racemic Chuangxinmycin121

by coupling (+/-)-(2,3)-syn-epoxy butanoate 243 and 4'-iodoindole 242 (Scheme

6-1). This synthesis began with 2-amino-6-nitrotoluene 237, which was treated










with CH(OEt)3 to afford 238. Imidate ester 238 was reacted with potassium

ethoxide followed by a palladium catalyzed hydrogenation to yield 4-amino indole

derivative 241. Diazotization of 241 with sodium nitrite and subsequent

treatment with potassium iodide resulted in 4-iodoindole derivative, which was

converted into 4-iodoindole 242 by hydrolysis. Reaction of racemic glycidic ester

243 and 242 in the presence of tin (IV) chloride afforded (+/-)-4'-

iodoindolmycenate 244. Treatment of 244 with methanesulfonyl chloride in

pyridine gave thioacetoxy ester 245, which was deacetylated under weak alkaline

conditions. Treatment of 246 with Pd(PPh3)4 gave methyl ester 247 that was

treated with aqueous base to result in Chuangxinmycin 236.

NO2 N02 N02
CH CH(OEt)3 / TsOH CH3 (COOEt)2 / KOEt
89% 77%
NH2 N OEt H

237 238 239

NO2 NH2
N0 1) NaNO2-H /KI
NaH /CICOOMe H2 / Pd/C 2) NaOMe / MeOH
93% N 73% N 80%
O CH3 0 CH3

240 241

0
0 H3C, OMe 1) MsCI/ pyridine
0 SnCl4 2) CsSAc
SN H3C 'CCO2Me 32% 0H 70% /
H N
H


242 243










0 0
H3C OMe H3C. OMe
I H OMe K2C03 / MeOH I H OMe Pd(PPh3)4 / Et3N
\ 90% 'SH 74%
N CH3 N
H H
245 246

C02Me C02H
CH3 NaOH / MeOH \ CH3
32%
N N
H H

247 236

Scheme 6-1. Akita's synthesis of (+/-)-Chuangxinmycin 236

Enzymatic Reduction of 220

This publication121 described a method to synthesize racemic

Chuangxinmycin through trans-glycidic ester 243. However, to achieve the

absolute stereochemistry at the C-4 and C-5 positions, one must develop a chiral

synthesis to this ester. We recently disclosed that reductase YOR120w

afforded the (2R, 3S) chlorohydrin by reduction of 220 with a >98% e.e and

>98% d.e.109 Using this chlorohydrin, we proposed a practical, gram-scale

synthesis to make (2R, 3S)-epoxy butanoate 243 with a whole-cell

biotransformation using an overexpressed bakers' yeast reductase as the key

step (Scheme 6-2).

0 0 OH 0 0
3C Ot YOR120w 3C ,, Et NaOEt / EtOH A3C
H3C O-t H3C O 3C"
Cl Cl 0
220 248 243

Scheme 6-2. Proposed scheme to the Chuangxinmycin intermediate (2R, 3S)-
epoxy butanoate 243









Whole-cell Reduction of 220

The whole-cell reduction of 2-chloro ethylacetoacetate 220 was carried out

in a 1 liter fermentation vessel in a nitrogen deprived phosphate buffer, using

conditions similar to those described earlier (Chapter 4).97 This reduction was

able to achieve a much larger final concentration when compared to the whole-

cell reductions of 126 and 214. Nonetheless, this reaction fell short in final

product concentration when compared to the reduction of ethyl acetoacetate by

GRE2 (Figure 4-1). Between these cases, there was about a ten-fold decrease

for 2-chloro ethylacetoacetate 220 (Figure 6-2). This is most likely due to the

increased toxicity toward the cells from the chlorine functionality on substrate

220. Whole-cell reduction of 2-chloro ethylacetoacetate did yield an

approximately four-fold higher final concentration when compared to the whole-

cell reduction of 214. We believe this is because 220 is less hydrophobic than

214, and thus has a smaller inhibitory effect on the cells.


OH 0 OH O OH O
H3C- Et H3C --OEt O Et
CI CI
249 248 215
32.2 g/L 4.4 g/L 1.1 g/L



Figure 6-2. Final product concentrations for 249, 248, and 215 by the
corresponding engineered E. coli

The reduction of 220 was catalyzed by E. coli cells containing

overexpressed reductase YOR120w. To lessen the toxicity to the cells, the

substrate was added in small increments over 24 hours, and a one liter reaction

afforded 4.4 grams of product with an 89% overall yield. As was also seen in









other reductions of chlorinated 3-keto esters, there was a small dechlorination

by-product. In this case, however, we found only a small percentage of this by-

product.

While separation was simpler due to the small amount of dechlorinated by-

product, we did encounter a problem while trying to extract 248 from the reaction

mixture. Even using our gentle extraction technique (Chapter 4), we were unable

to achieve complete extraction of product from the aqueous phase, even after 5

days of extraction. This can be attributed to the high solubility of the reduction

product 248 in water. As a result, the organic layer was replaced with fresh

methylene chloride three times to allow an adequate extraction.

Base Catalyzed Ring Closure of 248

To synthesize the homochiral glycidic ester, the chlorohydrin 248 was

treated with sodium ethoxide to afford predominately trans-glycidic ester 243.

Unfortunately, this reaction did not follow the trend seen in the literature89, which

reported the trans-epoxide as the only product formed. Our ring closure reported

a 5 : 1 (trans : cis) product ratio and we were unable to get adequate separation

of these diastereomers (Scheme 6-4).

OH 0 0
H3CEt NaOEt H3C -OEt + H3C OEt
Cl 0
248 243 250
Major Minor
Difficult to Separate

Scheme 6-4. Ring closure promoted by sodium ethoxide

This observation can be explained by the size of the y-carbon chain. The

reaction of chlorohydrins with sodium ethoxide is directed by the thermodynamic









stability of the anti versus the syn conformation (Scheme 4.6). Due to its small

size, the methyl group does not adequately direct the steric course of the

reaction.

We also investigated ring closure of 248 by treatment with a weak base.

Chlorohydrin 248 was reacted with potassium carbonate (3 equiv) and a catalytic

amount of water (3 equiv) over a 5 hour period (Scheme 6-5). Gas

chromatography and NMR analysis confirmed that this epoxidation followed the

general trend, affording only the cis-glycidic ester.

O O OH 0 0
YOR120w K2CO3 (3eq) / H20 (3eq) -
H3C OEt 89% H3C OEt 8% H3C' r-OEt
Cl Cl 0

220 248 250

Scheme 6-5. Ring closure of chlorohydrin 248 using potassium carbonate and
water

We were generally unsuccessful in the synthesis of (2R, 3S)-epoxy

butanoate 243. A simple solution to this problem is to find a reductase that will

result anti-chlorohydrin 251 with a high d.e. and e.e. This will then allow for the

epoxidation using potassium carbonate, which should afford only the trans-

glycidic ester 243 (Scheme 6-6).

O O OH O 0
OEt reductase H3C OEt K2CO3 / H20
H3C- OEt H3C-" OEt H3C"-'- oEt
CI CI 0
220 251 243

Scheme 6-6. Proposed synthesis to (2R, 3S)-epoxy butanoate 243














CHAPTER 7
CONCLUSIONS AND FUTURE WORK

Studying our collection of purified bakers' yeast reductases has shown that

they can be utilized to afford highly synthetically useful intermediates with high

stereoselectivities. In addition, these reductions can be carried out with

engineered whole-cells to yield products on gram scales. We have also shown

that these reduction products can be easily transformed into glycidic esters,

which are popular intermediates for a variety of pharmaceutical drugs.

Our reduction library allowed us to make the (2S-3R)-127 and (2R-3S)-

chlorohydrins ent-127 needed for synthesizing both Taxol side-chain antipodes

with high enantiomeric excess. Using these intermediates, we were able to react

these chlorohydrins with a weak base to afford the corresponding glycidic esters.

The glycidic esters underwent a Ritter reaction with benzonitrile to form the

protected Taxol side chain with an overall yield of 49% and its enantiomer with

an overall yield of 38%. This is advantageous because it eliminates any

additional steps that are needed to take the Taxol side chain to a form in which it

can be coupled to Baccatin III.

Benzenebutanoic acid, a-chloro-3-oxo-, ethyl ester 214 was shown to be a

substrate for YDR368w, thus affording (2R, 3S)-chlorohydrin 215 in high

enantiopurity. This result allowed us to synthesize oxazoline 217 through our

glycidic ester and Ritter reaction route, thus resulting in a protected form of

AHPA. Treatment of oxazoline 217 with strong acidic conditions resulted in









AHPA 137 in 6 steps with an overall yield of 42%. This completes a formal total

synthesis of Bestatin 131.

The whole-cell reductions of three key a-chloro-3-keto esters resulted in

final concentrations of 1.1 g for 215, 1.39 g for 127, and 4.4 g for 248. This trend

is a direct result of the product toxicity to the cells. This toxicity is probably due

to the hydrophobicity of the compounds, along with the addition of the chlorine

moiety. Future work for these reductions would focus on the optimization of

these biotransformations. For example, the dechlorination of the starting material

may be eliminated by incubating the cells with a chlorinated compound that is

commercially available and easily removed. Additional work would focus on

engineering at the genomic level, thus making cells more resistant to product

toxicity and/or more efficient at reducing substrates.

The two pharmaceutical routes reported in this thesis are only two

examples from a wide range of possibilities. If we increase our library of purified

bakers' yeast reductases, or expand the library with reductases from other

organisms, it may help in expanding our synthetic potential. As seen in Chapter

6, if a reductase is found to yield the (2S, 3S)-chlorohydrin 251, we will be able to

synthesize the (2R, 3S)-epoxy butanoate 243, which is an intermediate to

Chuangxinmycin 236. Other possible pharmaceutical intermediates that can be

formed through homochiral glycidic esters are, but not limited to: Diltiazem 252,

KRI-1230 253, Amistatin ent-253, and Indolmycin 254 (Figure 7-1)











OCH3


S
OAc

Me2NH2CH2C

252


0
N-
0


HO HN-His

0


Figure 7-1. Other pharmaceutical drugs that can be synthesized from homochiral
glycidic ester intermediates: Diltiazem 252, KRI-1230 253, Amistatin
ent-253, and Indolmycin 254














APPENDIX A
EXPERIMENTAL

General Methods and Instrumentation

Standard media and techniques for growth and maintenance of E. coli were

used, and Luria-Bertani (LB) medium contained 1% Bacto-Tryptone, 0.5% Bacto-

Yeast Extract, and 1% NaCI. Synthetic reactions were carried out under argon

atmosphere, with the exception of water containing reactions. Reactions were

monitored by TLC (silica, 60 A) or by GC using a DB-17 column (0.25 mm x 25 m

x 0.25 pm thickness) with a flame ionization detector, and for 137 reversed-

phase HPLC (4.6 x 250 mm C18 column) using a water-CH3CN solvent system

(both solvents containing 0.1% trifluoracetic acid) was used. For chiral

separation, GC was used with a Chirasil-Dex CB column (0.25 mm x 25 m x 0.25

pm thickness) or a Chirasil-L-Val (0.25 mm x 25 m x 0.25 pm thickness) with a

flame ionization detector. NMR spectra for 1H and 13C were recorded on Varian

300 MHz instruments. Chemical shifts are reported at 25 C in ppm relative to

TMS. Optical rotations were measured in CHCI3 at room temperature (Perkin-

Elmer 241 digital polarimeter) unless otherwise stated. Elemental analysis was

performed by Atlantic Microlab, Inc. in Atlanta, Georgia. Racemic alcohols were

prepared from the corresponding ketones by reduction with sodium borohydride.









Whole-cell Activity Assays for Inductions Carried Out at Different
Temperatures

A 35 mL solution of LB broth (supplemented with 30 pg/mL kanamycin) was

inoculated with a single colony of E. coli (BL21(DE3)(plK6)) and shaken

overnight at 37 C. The preculture (10 mL) was diluted (1: 100) into 1 L of LB

(supplemented with 30 pg/mL kanamycin and 4 g/L glucose) in three 2 L baffled

flasks. The cultures were grown for 2 hours at 37 C with shaking at 400 rpm

until they reached an O.D.600 = 0.6. One flask was placed at 24 C, one at 30 C

and the other was left at 37 C. The cells were allowed to shake at 400 rpm for

15 minutes and then reductase overproduction was induced with isopropylthio- 3-

D-galactoside at a final concentration of 0.1 mM. The cells were kept under the

same conditions and aliquots (100 mL) were taken at various times. Cells were

collected by centrifugation (6000 g for 10 min at 4 C), resuspended in phosphate

buffer (3 mL, 100 mM, pH = 7), then PMSF was added to a final concentration of

1.5 mM. The aliquots were stored at 4 C and then sonicated for 10 seconds.

The cell suspension was centrifuged (6000 g for 10 min at 4 C) to remove

cellular debris, decanted, and the supernate was stored on ice.

A premixed solution of phosphate buffer (10 mM, pH = 7) and ethyl

acetoacetate (5 mM) was maintained at 30 C. The premixed solution (1 mL)

was added to a quartz cuvette, followed by 10 pL of NADPH solution (20 mM)

and the cellular supernate (volume varied). The cuvette was gently mixed and

immediately monitored at 340 nM (120 sec at 20 sec intervals). The slope was

calculated and used to find the specific activity (Appendix B).









Procedures and Data

O 0
a-, OEt

126

Ethyl benzoylacetate 125 (6.0 g, 28.0 mmol) was added to chloroform (170

mL). The reaction mixture was purged with argon at 50 C and then sulfuryl

chloride (2.5 mL, 28.0 mmol, 1 equiv) was added over 15 minutes. The reaction

mixture was stirred at 50 C for 3 hours and then allowed to cool to room

temperature. Water (200 mL) was added to the mixture and the aqueous layer

was extracted with methylene chloride (3 x 100 mL). The combined organic

layers were dried with magnesium sulfate and concentrated. The resulting

residue was purified by vacuum distillation (2 mm Hg, 120 C) to afford 6.2 g as a

colorless oil 126 in 98% yield. 1H NMR: (CDCl3) 5: 8.00 (d, 2H), 7.60 (m, 3H),

5.62 (s, 1H), 4.29 (q, 2H, J = 6.9), 1.24 (t, 3H, J = 6.9). 13C NMR: (CDCl3) 5:

188.6, 165.6, 134.7, 133.8, 129.6, 129.3, 63.6, 58.4, 14.3. IR (neat): v(cm-1):

2984.5, 1763.8, 1691.7, 1268.3, 1182.8.

OH 0
C-C OEt

127

A 45 mL solution of LB broth (supplemented with 30 pg/mL kanamycin) was

inoculated with a single colony of E. coli (BL21(DE3)(plK8)) and shaken

overnight at 37 C. The preculture (40 mL) was diluted (1: 100) to 4.0 L of LB

(supplemented with 30 pg/mL kanamycin and 4 g/L glucose) in a New Brunswick

M19 fermenter. The culture was grown for 2 hours at 37 oC with a stir rate of 800









rpm and an air flow of 0.5 vessel volumes per minute (vvm) until it reached

O.D.600 = 0.6. The cell suspension was cooled to 28 C over 15 minutes and

then reductase overexpression was induced with isopropylthio-(3-D-galactoside at

a final concentration of 0.1 mM. The cells were kept under the same conditions

for 6 hours and then collected by centrifugation (6000 g for 10 min at 4 C). Half

of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KPI (pH = 5.6)

containing 4 g/L glucose. The bioconversion was carried out in a Braun Biostat B

fermenter at 30 C with the pH maintained at 5.6 using 3 M NaOH. The

dissolved oxygen was maintained at 75% saturation using a fixed air flow of 0.25

vvm and variable stirring rate. After the addition of the XAD-4 resin (0.5 g),

portions of neat 126 (0.2 mL) were added approximately every hour over a total

of 12 hours to provide a final concentration of 6 mM. Portions of glucose were

added after 3.0 hours and 6.0 hours to maintain the glucose concentration at

approximately 4 g/L. Consumption of 126 and glucose slowed significantly after

8 hours. After 24 hours, the reaction was gently extracted with methylene

chloride (2 x 300 mL) to avoid an emulsion. The combined organic layers were

dried with magnesium sulfate and concentrated under vacuum. The crude oil

was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 1.33

g of 127 as a colorless oil with a 91% yield after recovered starting material.

[a]D = -3 o (c = 0.68, CHCI3), Lit.88 [a]D = -3 o, (c = 1.7, CHCI3) 1H NMR: (CDCI3)

5: 7.25 (m, 5H), 5.00 (d, 1H, J = 6.3), 4.35 (d, 1H, J = 6.3), 3.99 (q, 2H, J = 6.9),

1.02 (t, 3H, J = 6.9). 13C NMR: (CDCI3) 5: 168.4, 138.7, 129.1, 128.9, 127.2,

77.1, 75.1, 63.4, 62.7, 14.2. IR (neat): v(cm-1): 3475.3, 2982.2, 1745.8, 1373.1,









1258.9, 1095.7. Anal. Calcd for C11H1303C: C, 57.78; H, 5.73. Found: C,

57.92; H, 5.86.

OH 0
'NOEt

ent-127
A 45 mL solution of LB broth (supplemented with 30 pg/mL kanamycin) was

inoculated with a single colony of E. coli (BL21(DE3)(plK6)) and shaken

overnight at 37 oC. The preculture (40 mL) was diluted (1: 100) to 4.0 L of LB

(supplemented with 30 pg/mL kanamycin and 4 g/L glucose) in a New Brunswick

M19 fermenter. The culture was grown for 2 hours at 37 oC with a stir rate of 800

rpm and an air flow of 0.5 vessel volumes per minute (vvm) until it reached an

O.D.600 = 0.6. The cell suspension was cooled to 28 oC over 15 minutes and

then reductase overexpression was induced with isopropylthio- 3-D-galactoside

at a final concentration of 0.1 mM. The cells were kept under the same

conditions for 6 hours and then collected by centrifugation (6000 g for 10 min at 4

oC). Half of the cells (25 g wet weight) were resuspended in 1 L of 10 mM KPI

(pH = 5.6) containing 4 g/L glucose. The bioconversion was carried out in a

Braun Biostat B fermenter at 30 oC with the pH maintained at 5.6 using 3 M

NaOH. The dissolved oxygen was maintained at 75% saturation using a fixed air

flow of 0.25 vvm and variable stirring rate. After the addition of the XAD-4 resin

(0.5 g), portions of neat 126 (0.2 mL) were added approximately every hour over

a total of 12 hours to provide a final concentration of 6 mM. Portions of glucose

were added after 3.0 hours and 6.0 hours to maintain the glucose concentration









at approximately 4 g/L. Consumption of 126 and glucose slowed significantly

after 8 hours. After 24 hours, the reaction was gently extracted with methylene

chloride (2 x 300 mL) to avoid an emulsion. The combined organic layers were

dried with magnesium sulfate and concentrated under vacuum. The crude oil

was purified by flash chromatography (Cyclohexane: Ether 85:15) to afford 1.39

g of ent-127 as a colorless oil with an 85% yield after recovered starting material.

[a]D = +4 o(c = 0.68, CHCI3) 1H NMR: (CDCI3) 5: 7.25 (m, 5H), 5.00 (d, 1H, J=

6.3), 4.35 (d, 1H, J = 6.3), 3.99 (q, 2H, J = 6.9), 1.02 (t, 3H, J = 6.9). 13C NMR:

(CDCI3) 5: 168.4, 138.7, 129.1, 128.9, 127.2, 77.1, 75.1, 63.4, 62.7, 14.2. IR

(neat): v(cm-'): 3475.3, 2982.2, 1745.8, 1373.1, 1258.9, 1095.7.

0
Co02Et

128

Chlorohydrin 127 (1.3 g, 5.7 mmol) was added to DMF (28 mL) and stirred

at room temperature. Potassium carbonate (2.2 g, 17.1 mmol, 3 equiv) and

water (525 pL) were added to the reaction mixture and stirred for 5 hours. The

resulting mixture was diluted with water (75 mL), and then the aqueous layer was

extracted with diethyl ether (3 x 75 mL). The organic layer was then washed with

water (6 x 5 mL) to remove residual DMF. The combined organic layers were

dried with magnesium sulfate and concentrated under reduced pressure to yield

1.08 g of 128 as a colorless oil in a 99% yield. [a]D = +24 o(c = 1.5, CHCI3) Lit.89

[a]D = +25 o (c = 1.1, CHCI3) 1H NMR: (CDCI3) 5: 7.31 (m, 5H), 4.27 (d, 1H, J =

4.8), 4.00 (m, 2H), 3.82 (d, 1H, J = 4.8), 1.01 (t, 3H, J = 6.9). 13C NMR: (CDCI3)