UNIVERSITY of CALIFORNIA, SAN DIEGO Investigations Into the Biosynthesis of Salinosporamide A
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Investigations into the biosynthesis of salinosporamide A: new insights on PKS extender units and the origin of a nonproteinogenic amino acid A Dissertation submitted in partial satisfaction of the Requirements for the degree Doctor of Philosophy in Oceanography by Yuan Liu Committee in charge: Professor Bradley S. Moore, Chair Professor William Fenical Professor William Gerwick Professor Yoshihisa Kobayashi Professor Emmanuel Theodorakis 2010 © Yuan Liu, 2010 All rights reserved. The Dissertation of Yuan Liu is approved, and it is acceptable in quality and form for publication on microfilm: Chair University of California, San Diego 2010 iii To my wife Crystal and our daughter Hannah and To my parents, Fu-ru Liu and Xue-ying Gao Thank you for your encouragement and love iv TABLE OF CONTENTS Signature Page…………………………………………………………………... iii Dedication……………………………………………………………………….. iv Table of Contents………………………………………………………………… v List of Abbreviations…………………………………………………………….. ix List of Figures…………………………………………………………………..... xiii List of Tables…………………………………………………………………….. xvii Acknowledgments……………………………………………………………….. xviii Vita………………………………………………………………………………. xx Abstract…………………………………………………………………………... xxi CHAPTER ONE: INTRODUCTION……………………………………………. 1 1.1 Natural products as a source of therapeutic agents……………………... 1 1.1.1 The history of natural products as drugs……………………….. 1 1.1.2 Natural products as clinical agents …….……………………… 3 1.1.3 Genome mining for natural products discovery……………….. 6 1.2 Polyketide and nonribosomal peptide natural products ………………... 9 1.3 Proteasome and its inhibitors………………………………………….... 13 1.3.1 Structure and function of the proteasome……………………… 13 1.3.2 Proteasome inhibitors……………………….…………………. 16 1.3.2.1 Synthetic inhibitors: peptide aldehyde, vinyl sulfones, and boronates………………………… 17 1.3.2.2 Natural product inhibitors…………………................ 20 1.3.2.2.1 α’, β’-epoxyketones and TMC-95s………...... 20 1.3.2.2.2 β-lactones………………………………….…. 22 1.4 Salinosporamide A: a potent anticancer agent………………………….. 25 1.4.1 Salinosporamide A producer Salinispora……………………… 25 1.4.2 The mechanism of action against the 20S proteasome………… 27 v 1.5 Total synthesis of salinosporamide A…………………………………... 30 1.5.1 Corey’s first total synthesis …………………………………..... 30 1.5.2 Second total synthesis by Danishefsky……….……………….. 33 1.5.3 Pattenden’s racemic total synthesis……..……………………… 35 1.5.4 Romo’s racemic synthesis via a bis-cyclization process……….. 36 1.5.5 Machela’s enantioselective total synthesis……………………... 37 1.5.6 Hatakeyama’s asymmetric synthesis…………………………… 39 1.6 References...……………………………………………………………... 42 CHAPTER TWO: Characterization of an unexpected and unprecedented chlorination pathway in the biosynthesis of salinosporamide A……………………………… 51 2.1 Introduction…….………………………………………………... 51 2.1.1 Feeding experiments with stable isotopes ………………. 53 2.1.2 Analysis of the sal gene cluster………………………….. 55 2.2 Results……………………………………….…………………… 59 2.2.1 Feeding experiments with 4-chlorobutyric-d6 and its thioester in the wild type……………………………... 59 2.2.2 The chloroethylmalonyl-CoA pathway.…………………. 60 2.2.3 Gene inactivation and chemical complementation………. 66 2.2.3.1 Synthesis of putative intermediates………….. 66 2.2.3.2 Chemical complementation………………….. 70 2.2.4 Accumulation of 5-ClR in S. tropica salM mutant……… 74 2.3 Discussion…………………………….…………………………. 76 2.4 Acknowledgments………………………………………………. 82 2.5 Experimental……….……………………………………………. 82 2.5.1 Materials and methods…………………………………... 82 2.5.2 Feeding experiment methods……………………………. 84 2.5.3 Synthetic methods………………………………………. 84 2.6 References……………………………………………………….. 93 APPENDIX ONE: The spectra relevant to chapter two………………… 97 vi CHAPTER THREE: Biosynthesis of salinosporamides: four new PKS extender units……………………………………………………. 101 3.1 Introduction……………………………………………………... 101 3.1.1 Biosynthesis of salinosporamide B from (2S)-ethylmalonyl-CoA ………………………………… 104 3.2 Results ………………………………………………………….. 106 3.2.1 Biosynthesis of salinosporamide D from (2S)-methylmalonyl-CoA……………………………….. 106 3.2.2 Biosynthesis of salinosporamide E from a novel PKS extender unit (2S)-propylmalonyl-CoA………….... 107 3.2.3 Biosynthesis of bromosalinosporamide from a novel PKS extender unit (2S)-bromoethylmalonyl-CoA.……… 109 3.2.4 Biosynthesis of fluorosalinosporamide from a novel PKS extender unit (2S)-fluoroethylmalonyl-CoA………. 111 3.3 Discussion……………………………………………..………… 115 3.4 Acknowledgments………………………………………………. 121 3.5 Experimental…………………………………………………….. 121 3.5.1 Materials and Methods………………………………….. 121 3.5.2 Feeding experiment methods…..………………………... 122 3.5.3 Synthesis of 4-bromocrotonic acid and 4-fluorocrotonic acid……………………………….. 125 3.6 References………………………………………………..……… 128 APPENDIX TWO: Spectra relevant to chapter three………………………….. 131 vii CHAPTER FOUR: Analysis of the novel biosynthetic building block cyclohexenyl- alanine of salinosporamide A…………………………………………………… 135 4.1 Introduction………………….………………………………….. 135 4.1.1 Incorporation experiments with 13C-labeled precursors… 136 4.1.2 Engineered biosynthesis of salinosporamides with varied C-5 substituents………………………...…... 138 4.2 Results…………………………………………………………… 143 4.2.1 To synthesize 3-hydroxy-3-(cyclohex-2-enyl)alanine…... 144 4.2.1.1 Coupling reaction with 2-cyclohexenyl zinc chloride…………………………………. 144 4.2.1.2 Ugi reaction approach ……………..………… 145 4.2.2 To synthesize 3-(cyclohex-2-enyl)alanine……………….. 152 4.3 Experimental……………………………………………………... 156 4.3.1 Materials and methods………………………………….... 156 4.3.2 Feeding experiment methods…………………………….. 157 4.3.3 Synthetic procedures …..………………………………… 158 4.4 References……………………………………………………….. 169 APPENDIX THREE: Spectra relevant to chapter four…………………………. 171 CHAPTER FIVE: Discussion and outlook ……………………………………… 172 5.1 References………………………………………………………... 180 viii LIST OF ABBREVIATIONS A adenylation Ac acetyl, acetate AcOH acetic acid ACP acyl carrier protein Ar aryl Arg arginine Asn asparagine AT acyl transferase ATP adenosine triphosphate Bn benzyl BOC tert-butyloxycarbonyl BOPCl bis(2-oxo-3-oxazolidinyl)phosphinic chloride bp boiling point br broad C condensation calcd calculated CAN ceric ammonium nitrate cat. catalytic amount CCR crotonyl-CoA carboxylase/reductase ClCA 4-chlorocrotonic acid ClCCR 4-chlorocrotonyl-CoA carboxylase/reductase 5’-ClDA 5’-chloro-5’-deoxyadenosine 5-ClR 5-chloro-5-deoxy-D-ribose 5-ClRI 5-chlororibonate 5-ClRL 5-chloro-5-deoxy-D-ribono-1,4-lactone 5-ClRP 5-chloro-5-deoxy-D-ribose-1-phosphate CoA coenzyme A CSA camphorsulfonic acid ix CT-L chymotrypsin-like d doublet or day d.r. diastereomeric ratio Da daltons DAHP 3-deoxy-D-arabinoheptulosonic acid-7-phosphate DCC dicyclohexylcarbodiimide DH dehydrates DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide E epimerization EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ee enantiomeric excess EI electron impact equiv. (eq.) equivalent ER enoylreductase Et ethyl EtClHB ethyl 4-chloro-3-hydroxybutyrate 5’-FDA 5’-fluoro-5’-deoxyadenosine 5-FRP 5-fluoro-5-deoxy-D-ribose-1-phosphate Gly glycine h hour HMPA hexamethylphosphoramide Hz hertz IC inhibitor concentration i-Pr iso-propyl IR infrared (spectrum) KR ketonereductase KS ketonesynthase LDA lithium diisopropylamine x Leu leucine LiHMDS lithium bis(trimethylsilyl)amide m multiplet Me methyl min minutes mM milliomolar mol mole Ms methanesulfonyl (mesyl) MT methyltransferase NAC N-acetyl cysteine NMM N-methylmorpholine NMO N-methylmorpholine N-oxide NMR nuclear magnetic resonance NRP nonribosomal peptide NRPS nonribosomal peptide synthetase [O] oxidation PCC pyridinium chlorochromate PCP peptidyl carrier protein Pd/C palladium on charcoal Ph phenyl PhH benzene PK polyketide PKS polyketide synthase PMB para-methyoxybenzyl ppm parts per million Pro proline p-TsOH para-toluenesulfonic acid Py pyridine quant. quantitative RT room temperature xi s singlet SAM S-adenosyl-L-methionine SAR structure-activity relationship t triplet TBAF tetrabutylammonium fluoride TBS tertiary-butyldimethylsilyl t-Bu tertiary-butyl TCM Traditional Chinese Medicine TE thioesterase Tf trifluoromethanesulfonyl TFA trifloroacetic acid THF tetrahydrofuran Thr1 N-terminal threonine Thr1Oγ β-hydroxyl group of N-terminal threonine TLC thin-layer chromatography TMS trimethylsilyl TPAP tetrapropylammonium perruthenate vs vinyl sulfone xii LIST OF FIGURES Figure 1.1 Examples of natural product drugs……………………….… 4 Figure 1.2 Examples of PK, NRP, and PK-NRP hybrid natural product …………………..……………………………. 10 Figure 1.3 Domain structures of PKS and NRPS...………………… 11 Figure 1.4 Typical structures of 20S proteasome and 26S proteasom. 15 Figure 1.5 Mechanism of peptide hydrolysis by 20S proteasome…... 16 Figure 1.6 Synthetic peptide proteasome inhibitors and the mechanism of the inhibition………………………….. 19 Figure 1.7 Natural product proteasome inhibitors……………………. 21 Figure 1.8 Structures of β-lactone inhibitors, their inhibition mechanism, and β5 subunit of 20S proteasome in complex with omuralide……………………………….. 24 Figure 1.9 Structures and mechanism of action of salinosporamide A, B, and omuralide against 20S proteasome…………………………………………… 26 Figure 1.10 The crystal structure of salinosporamide A in complex with 20S proteasome………………………… 28 Figure 1.11 Corey’s total synthesis of salinosporamide A…………..... 32 Figure 1.12 Second total synthesis of salinosporamide A by Danishefsky……………………..…………………….. 34 Figure 1.13 Pattenden’s racemic synthesis……………………..……... 35 Figure 1.14 Romo’s racemic total synthesis…….…………………….