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Enzymatic oxidation of aromatic substrates via toluene Dioxygenase and catechol dehydrogenase

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Title:
Enzymatic oxidation of aromatic substrates via toluene Dioxygenase and catechol dehydrogenase application to total synthesis of combretastatins A-1 and B-1
Creator:
Bui, Vu Phong
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English
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xviii, 225 leaves : ill. ; 29 cm.

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Biotransformation ( jstor )
Catechols ( jstor )
Chemicals ( jstor )
Enzymes ( jstor )
Fermentation ( jstor )
Metabolites ( jstor )
Oxidation ( jstor )
Polymers ( jstor )
Stereochemistry ( jstor )
Tetrahedrons ( jstor )
Chemistry thesis, Ph.D ( lcsh )
Dissertations, Academic -- Chemistry -- UF ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Vu Phong Bui.

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University of Florida
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ENZYMATIC OXIDATION OF AROMATIC SUBSTRATES VIA TOLUENE
DIOXYGENASE AND CATECHOL DEHYDROGENASE: APPLICATION TO
TOTAL SYNTHESIS OF COMBRETASTATINS A-I AND B-1














By

VU PHONG BUI


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
































Dedicated to my grandmother, Nguyen Thi Sau














ACKNOWLEDGMENTS

I would like to take this opportunity to express my gratitude to everyone who

helped me arrive where I am today. First I sincerely thank my advisor, Professor Tomas

Hudlicky, for his guidance, support, and encouragement throughout my graduate studies

at the University of Florida. I am forever indebted to him for giving me the opportunity

and assistance to develop into the chemist that I am today. Special thanks go to Dr. Josie

Reed and John Patterson for their help and kindness. I am grateful to Dr. Louise

Greenberg and Dr. Cyrus Greenberg for their kind friendship and helpful advice

throughout my undergraduate and graduate school years and for their help in

proofreading this document. I also would like to thank Professors William Dolbier,

Steven Benner, Dennis Wright, and Lonnie Ingram for their helpful advice, and for

serving on my committee. I am grateful to Dr. Dennis Wright for his fruitful suggestions

and advice (on countless occasions) for solving many of my research-related difficulties.

I am grateful to the University of Florida and the Department of Chemistry for the

opportunity to further my education. Special thanks go to professor James Deyrup and

Lori Clark for their help and advice throughout my time at the University of Florida.

Throughout my career in the Hudlicky research group, I have had the opportunity

to work with a great group of people. I would like to take this moment to thank all past

and present group members for their contribution to my educational career. Special

thanks go to Drs. Mary Ann Endoma, David Gonzales, and Bennet Novak for passing on

their expertise and knowledge of the whole-cell biotransformation processes used in our








group. I am indebted to Dr. Kofi Oppong, Dr. Stefan Schilling, Dr. Jerremy Willis, Stan

Freeman, and Minh Nguyen for their help and friendship throughout my career here. I

also thank Drs. Duan Caiming, Dean Frey, Pierce Kerry, and Dr. Martin Mandel for

lending their vast chemical knowledge and laboratory techniques to my research

endeavors. I thank the current group members Uwe Rinner, Josef Zezula, Kevin Finn,

David Adams, and Heather Hillebrenner for their friendship and for sharing with me the

daily duties of keeping the lab functioning smoothly throughout our time together in the

group.

Lastly and most importantly, I would like to acknowledge my family for supporting

me throughout my time at the University of Florida.














TABLE OF CONTENTS
page

ACKNOW LEDGM ENTS...................... .................................................................iii

LIST OF ABBREVIATIONS .............. ............. .......... ................iii

LIST OF TABLES ..................... ............................... xi

LIST OF FIGURES....................................... ............................... xiii

LIST OF SCHEMES.. .................................................. xiv

ABSTRACT......................... .................................xviii

CHAPTERS

I INTRODUCTION ................ ........ .......................... ......... 1

2 HISTORICAL..................... ...... ..... .................................... 5

B iotransform ations............................. .................................. ........................ 5
O xygenation R actions .................................................................... .... .............. 13
Monooxygenases Catalyze Biotransformation ............................ ....... ......... 17
Hydroxylation of Alkanes....................... ..................... 18
Hydroxylation of Arenes ..................................................... 20
Epoxidation of Alkenes .............................................. ...... 22
Baeyer-Villiger Reactions............................ ......................................... 24
Dioxygenases Catalyze Biotransformation....................... .................. 29
Pseudomonas putida F39D.................................................... 32
Escherichia coli JM109 (pDTG601) .............................. ..................... 35
Toluene Dioxygenase Substrate Specificity ............................... ........... 35
Toluene Dioxygenase Regioselectivity ...................... ....................... 37
Toluene Dioxygenase Stereoselectivity........................ ..................... 42
Synthetic Applications of Dihydrodiols................... ...................... 49
Substituted C atechols................................................................... ...................... 56
Catechol Dehydrogenases Escherichia coli JM109 (pDTG602)......................... 59
C om bretastatins.................................. ........ ........ ..................................... 60
Isolation and Biological Activity ......................... .. ..................... 60
Combretastatins Syntheses .... ............... ......... .................. ....65
Combretastatin A-I and B- .............................................. 65
Com bretastatin A-4.................................. ........... ..... ....... 67








Combretastatin D-1 and D-2........................ .........................69


3 RESULTS AND DISCUSSION.............................................. ...................... 74

Pseudomonas putida F39D and E. coli JM109 (pDTG601) oxidation of 1-phenyl-1-
ethanol ........................................................................... ............................... 74
Biotransformation and Absolute Stereochemistry of New Metabolites ..................78
Phenylcyclohexene .................................. .... ........................ 78
Phenylcyclohexane ................... ................... ......................... 80
2-Phenylcyclohexene............................ ...................... 81
2-phenylcyclohexanol ......................................................... 81
2-Phenylcyclohexanone................................... ................................ 83
Diphenylmethane ....................... ....................... ............ 85
Biooxidation of Cyclopropylbenzene .................................. ........................ 86
M echanistic Investigation .......................................................... ................. 87
Absolute Stereochemistry Correlation of Cyclopropyldiol 383..................... 89
Synthetic Application Potential of Cyclopropylbenzene Metabolite................. 90
Substituted Catechols............................. ................................................................... 91
Chemoenzymatic approach to combretastatins A-1, and B-.................................... 99
Polyhydroxylated Chiral Polymer................................................................ 105


4 CON CLU SION S............................. ............................................................ 1

5 EXPERIMENTAL SECTION ................................ ... .............. ...... 112

General M ethods ....................................................................................... ............. 112
Large Scale Biotransformation E. coli JM109 (pDTG601) ............................... 113
Screening for new substrates ...................................................... 114
Preparation of catechols .................................................................................... 114
General procedure for diimide potassium azodicarboxylate (PAD) reduction of
the diene diols ............................... .... .... 114
Experimentals ...................................................... .......................................... ..... 115
TDO-catalyzed dihydroxylation of arenes..................................................... 115
Cyclopropylbenzene project......................................... .......... .... 131
Substituted Catechol Project....................................................... .......... 135
Chemoenzymatic approach to combetastatin A-I and B-1 project ................ 141
Polyhydroxylated Chiral Polymer Project..................................................... 146


6 SELECTED SPECTRAL DATA.............................................. ... 152

LIST OF REFERENCES ........................................ 202

BIOGRAPHICAL SKETCH ....................................... ....... ...................... 225














LIST OF ABBREVIATIONS

AIBN azoisobutyronitrile

aq. aqueous

BDO benzene dioxygenase

Bn benzyl

borax sodium borate-decahydrate

BPDO biphenyl dioxygenase

BVMO Baeyer-Villiger Monooxygenase

Bz benzoyl

BZDO benzoate dioxygenase

CHMO cyclohexanone monooxygenase

CSP-HPLC chiral stationary phase-high pressure liquid chromatography

DEAD diethylazodicarboxylate

DIBAL diisobutyl aluminium hydride

DMAP N,N'-dimethyl-4-amino pyridine

DMDP 2,5-dideoxy-2,5-diimino-D-mannitol

DMF dimethylformamide

DMP 2,2-dimethoxypropane

DMS dimethylsulfide

DMSO dimethylsulfoxide

E. coli Escherichia coli








ee enantiomeric excess

EtOAc ethyl acetate

Eu(hfc)3) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorato]

europium (II),

HPLC high-performance liquid chromatography

HRMS high resolution mass spectroscopy

IPTG iso-propyl-p-D-thiogalacto-pyranoside

KOH potassium hydroxide

LAH lithiumaluminum hydride

LDA lithium diisopropylamide

L-DOPA 3,4-dihydroxy-phenyl alanine

m-CPBA meta-chloroperbenzoic acid

MOMCI methoxymethyl chloride

MPB 2-(l-methoxyethyl)benzeneboronic acid

MPLC medium-pressure liquid chromatography

MsCI methylsulfonyl chloride

MTPA R-or S-a-methoxy-a-(trifluoromethyl)phenylacetic acid

NDO naphthalene dioxygenase

NMNO N-methylmorpholine-N-oxide

NOEDS nuclear overhauser effect difference spectroscopy

OD optical density

P-450 cytochrome P-450

PAD potassium azodicaboxylate








PAHs

PCBs

PCC

P. putida

pyr.

TBDPSCI

TBDMSCI

TBSCI

TBTH

TDDH

TDII

TDO

TFA

THF

TLC

TsCI

p-TsOH

UV

VIS


polycyclic aromatic hydrocarbons

polychlorinated biphenyls

pyridinium chlorochromate

Pseudomonas putida

pyridine

chloro-tert-butyldiphenylsilane

t-butyldimethylsily chloride

chloro-tert-butyldimethylsilane

tributyl-tin hydride

toluene cis-dihydrodiol dehydrogenase

N,N'-thiocarbonyldiimidazole

toluene dioxygenase

trifluoroacetic acid

tetrahydrofuran

thin-layer chromatography

para-toluenesulfonyl chloride

para-toluenesulfonic acid

ultraviolet

visible














LIST OF TABLES


Table p

Table 2-1. Examples of commercial steroid processes................... ........... ..... ..... 11

Table 2-2. Some representative industrial whole-cells biotransformations ................. 12

Table 2-3. Microbial epoxidation of alkenes .............................. ........................ 23

Table 2-4. Epoxidation of branched alkenes by Nocardia corallina........................... 24

Table 2-5. Enzymatic Baeyer-Villiger oxidation of prochiral ketones........................ 27

Table 2-6. Microbial Baeyer-Villiger oxidation of monocyclic ketones....................... 28

Table 2-7. TDO-catalyzed biotransformation of substrate containing heterocyclic ring
system........................ .................................................................................. 41

Table 2-8. Preferred absolute configuration of cis-dihydrodiol metabolites of
monosubstituted benzenes .................................................. 46

Table 2-9. cis-Dihydrodiols from ortho-substituted benzenes....................................... 47

Table 2-10. cis-Dihydrodiols from meta-substituted benzenes.................................... 47

Table 2-11. cis-Dihydrodiols from para-substituted benzenes......................................... 48

Table 2-12. cis-dihydrodiols from polycyclic aromatic hydrocarbons.......................... 49

Table 2-13. The use of cis-dienediols in synthesis............................................. 53

Table 2-14. Combretastatin A-I and B-l 'H-NMR* ............................... ............ 61

Table 2-15. Combretastatin A-I and B-I 13C-NMR*.............................. ............ 62

Table 2-16. Inhibition of microtubule assembly and biding of colchicine to tubulin by
combretastatin A-1 and B- ................... ........................ 64

Table 2-17. Effect of tested compounds on tubulin polymerization.............................. 70

Table 3-1. Biotransformation of phenylethanol by JM109 (pDTG601) and Pseudomonas
putida F39/D ................................................................................................. 76








Table 3-2. Catechols From Fermentation of Corresponding Arenes with JM109
(pD TG 602)........................................ .... .............................................. 94

Table 3-3. Comparison of efficiency of the enzymatic vs. non-enzymatic preparation of
3-bromo-, 3-methyl-, 3-iodocatechols ......................... ..................... .. 96














LIST OF FIGURES


Figure page

Figure 2-1. Pasteur's product of the first resolution reaction............................... ............ 6

Figure 2- 2. Microbial hydroxylation of steroids...................... ......... ............ .. 20

Figure 2-3. Preferred regioselectivity of mono-and polycyclic arene cis-dihydrodiols... 40

Figure 2-4. The used of Mosher's acid, MTPA, to determine optical purity of 1-
bromonaphthalene diol. .................... .. .. ...................... 44

Figure 2-5. The preferred absolute stereochemistry of metabolites obtained from
biotransformation of mono-substituted aromatic substrates............................. 46

Figure 2-6. Preferred absolute configuration of cis-dihydrodiol metabolites of
disubstituted benzenes ...................................................................................... 48

Figure 2-7. Local bond-forming sites in the cis-dienediols ........................................... 50

Figure 2-8. Examples of primary synthons................................ ...................51

Figure 2-9. Example of secondary synthons................................... ....................... 52

Figure 2-10. combretastatin A-1 and B ............... ................................................... 62

Figure 2-11. Crystal structure of combretastatin A-1 ............................. ............. 63

Figure 2-12. Structural variation in Combretastatins......................................... .... 65

Figure 2-13. Water soluble phospate derivative of combretastatin A-4 ........................ 65

Figure 2-14. Combretastatin D derivatives ......................................... ....................... 69

Figure 3-1. Exploration of reactivity of oxygenated cyclopropylhexanyl systems ......... 90

Figure 3-2. Oxygenated natural products containing a catechol subunit....................... 92

Figure 3-3. Some commercially available catechols................................................... 93

Figure 3-4. Chemoenzymatic approach to 3,4-cis-dihydrodiols via directing................. 96














LIST OF SCHEMES


Schemeage

Scheme 1-1. Aromatic degradation pathway of Pseudomonas putida F39D ................ 3

Schem e 2-1. V vinegar production....................................................... ..................... 6

Scheme 2-2. Reaction catalyzed by Bacterium xylinum................... .......... ........... 8

Scheme 2-3. Saccharomyces cerevisiae catalyzes acyloin condensation as the key step in
the production of L-Ephedrine.................... ................. ..................... 9

Scheme 2-4. Microbial dehydrogenation of D-sorbitol to L-sorbose as the key step in the
Reichstein-Grussner synthesis of L-ascorbic acid............................................ 9

Scheme 2-5. Two-stage fermentation for the production of 2-keto-L-gulonic acid from
D -glucose ............................. ........................................................................ 10

Scheme 2-6. Microbial 11-a-hydroxylation of progesteron.......................... ....... .... 11

Scheme 2-7. Chemical synthesis of cortisone................................... ...................... 11

Scheme 2-8. Monooxygenase cytochrome P450s catalytic cycle................................. 14

Scheme 2-9. Catalytic cycle of flavin-dependent mono-oxygenases........................... 16

Scheme 2-10. Enzymatic oxidation reactions............................. ...................... 17

Scheme 2-11. Monooxygenase catalyzed reactions............................ ....................... 18

Scheme 2-12. Microbial hydroxylation of 1,4-cineole ............................. ........... 19

Scheme 2-13. Possible mechanism in biodegradation of aromatics by eukaryotic cells. 21

Scheme 2-14. Microbial hydroxylation of aromatics.......................... ................. 21

Scheme 2-15. Mechanism of the Baeyer-Villiger reaction........................................... 25

Scheme 2-16. Progesterone metabolism by BVMOs in Cylindrocarpon radicicola....... 26

Scheme 2-17. Chemoenzymatic syntheses of various useful target molecules involving
the biotransformation by CHMO of bicyclo[3.2.0]-type ketones....................... 27








Scheme 2-18. Degradation of aromatics by microbial dioxygenases ............................ 30

Scheme 2-19. Dihydroxylation of aromatic hydrocarbon by TDO.............................. 30

Scheme 2-20. M odes of catechol cleavage ................................... ....................... 31

Scheme 2-21. Catabolic pathway for toluene used by Pseudomonas putida Fl. Genes
designations for individual proteins are shown in parentheses......................... 33

Scheme 2-22. Regioselectivity of dioxygenases in hydroxylation of aromatic substrates37

Scheme 2-23. Regioselectivity of TDO in biooxidation of 1,4-disubstituted aromatic
substrates.............................. ................................................... .............. 38

Scheme 2-24. Regioselectivity of TDO in biooxidation of 1,2-disubstituted substrates. 38

Scheme 2-25. Chemoenzymatic approach to 3,4-cis-dihydrodiols............... ......... .. 39

Scheme 2-26. Regioselectivity of 1,3-disubstituted aromatic substrates....................... 39

Scheme 2-27. Biotransformation of 2-bromonaphthalene ............................................. 43

Scheme 2-28. Preparation of Mosher ester 153...................... ......... .............. 43

Scheme 2-29. Preparation of di-MTPA esters 158 for determination of cis-dienediol 2
absolute stereochemistry................................... ................................ 45

Scheme 2-30. Preparation of boronic ester 160 for determination of cis-dienediol
absolute stereochemistry............................ ....... ......................... 45

Scheme 2-31. Historically important application of arene cis-diols in synthesis ............ 50

Scheme 2-32. Synthesis of methylcatechol from furfural...................... ..................... 57

Scheme 2-33. Synthesis of bromocatechol ............................... .................... 57

Scheme 2-34. Synthesis of bromocatechol from quaiacol........................................... 58

Scheme 2- 35. Synthesis of 3-fluorocatechol............................ .. ...................... 58

Scheme 2-36. Synthesis of 3-methoxyatechol ...................................................... 59

Scheme 2-37. Total synthesis of combretastatins A-1 and B-I ..................................... 66

Scheme 2-38. Suzuki approach to combretastatin A-4................................................. 67

Scheme 2-39. Synthesis of combretastatin A-4 via Suzuki cross-coupling reaction....... 68

Scheme 2-40. Synthesis of combretastatin A-4 ...................... ................... 68








Scheme 2-41. Total synthesis of combretastatin D-2...................... ....... .............. 71

Scheme 2-42. Total synthesis of combretastatin D-1 ............................. ............ 72

Scheme 3-1. Biooxidation of substrates containing benzylic chiral centers....................74

Scheme 3-2. Biooxidation of racemic phenylethanol by JM109 (pDTG601) ............... 77

Scheme 3- 3. Chemoenzymatic approach to morphine synthesis--introduction.............. 78

Scheme 3-4. Biooxidation of 1-phenylcyclohexene ............................... ............ 78

Scheme 3- 5. Absolute stereochemistry correlation of metabolite 346.......................... 79

Scheme 3-6. Biooxidation of phenylcyclohexane..................... ..................... 80

Scheme 3- 7. Chemical proof of the absolute stereochemistry of metabolite 354........... 80

Scheme 3-8. Biooxidation of 2-phenylcyclohexene and absolute stereochemistry of new
metabolite........................................................................ 81

Scheme 3-9. Biooxidation of trans-2-phenylcyclohexanol 343..................................... 82

Scheme 3-10. Derivatization of triols 365 for separation ............................................. 82

Scheme 3-11. Absolute stereochemistry of new metabolites obtained from................... 83

Scheme 3-13. Biotransformation of enantiomerically pure 2-phenylcyclohexanone...... 84

Scheme 3-14. Absolute stereochemistry of metabolites obtained from biotransformation85

Scheme 3-15. Biooxidation of diphenylmethane..................... ........ ............ .. 86

Scheme 3-16. Proposed reaction sequence for the formation of cis-dienediol................ 86

Scheme 3-17. Mechanistic possibilities for cis-dihydroxylation................................. 87

Scheme 3- 18. Biotransformation of cyclopropylbenzene............................................ 88

Scheme 3-19. Absolute stereochemistry correlation of metabolite obtain from
biotransformation of cyclopropylbenzene .................................................... 90

Scheme 3- 20. Synthetic potential of cyclopropyl epoxide 389.....................................91

Scheme 3-21. An environmentally benign approach to 3,4-catechols through ............... 97

Scheme 3- 22. Transformations of 3,4-bromocatechol.............................................. 98

Scheme 3- 23. Retrosynthetic design of catechol (or arene-cis-diol) synthons............... 99








Scheme 3-24. Chemoenzymatic retrosynthetic possibilities to combretastatins............ 101

Scheme 3-25. Sonogashira coupling approach to combretastatins.............................. 102

Scheme 3- 26. Suzuki approach to combretastatins...................... ..................... 102

Scheme 3-27. Enzymatic generation of catechol for approach to combretastatin A-l and
B .................................................... .......................................................... 10 3

Scheme 3-28. Chemoenzymatic approach to combretastatin analogs ......................... 105

Scheme 3-29. Chemoenzymatic approach to polyhydroxylated chiral polymer ........... 106

Scheme 3-30. Synthesis of monomer bis-allyl ether 438............................................ 108

Scheme 3-31. Polymerization of 452 with Grubbs' First-generation catalyst.............. 108

Scheme 3-32. Generation and polymerization of pentenylether 451........................... 109

Scheme 3-33. Chemoenzymatic approach to fully hydroxylated chiral polymer.......... 109














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

ENZYMATIC OXIDATION OF AROMATIC SUBSTRATES VIA TOLUENE
DIOXYGENASE AND CATECHOL DEHYDROGENASE: APPLICATION TO
TOTAL SYNTHESIS OF COMBRETASTATINS A-I AND B-I

By

VU PHONG BUI

May 2003

Chair: Tomas Hudlicky
Major Department: Chemistry

Toluene dioxygenase (TDO)-carrying microorganisms, the Pseudomonas putida

F39/D and the genetically engineered Escherichia coli JM109 (pDTG601), were used to

investigate the enzyme's kinetic resolution power in resolving aromatic substrate with

remote chiral center. In the biotransformation of 1-phenyl-ethanol with P. putida F39/D,

the S-enantiomer was consumed at faster rate than its R-counter part. However, when the

same substrate was oxidized using E. coli JM109 (pDTG601), both enantiomers were

oxidized indiscriminately at the same rate. Similar substrates with larger substituents at

the stereogenic center were also oxidized without any preference for either enantiomer by

JM109 (pDTG601). Biotransformation of cyclopropylbenzene was carried out to

investigate whether or not the biotransformation process goes through a radical

intermediate. New metabolites obtain in these studies were isolated, characterized, and

their absolute stereochemistry were established through conventional chemical

manipulations.








A series of substituted catechols were made using E. coli JM109 (pDTG602),

which carries the toluene cis-dihydrodiol dehydrogenase (TDDH), as the source of

biocatalyst. 4-Bromoanisole catechol obtained through biotransformation of

corresponding arene, 4-bromoanisole, with JM109 (pDTG602) was used in the total

syntheses of Combretastatins A-i and B-1. (IS, 2S)-3-bromocyclohexa-3,5-diene-1,2-

diol was also used as chiral synthon for synthesis of polyhydroxylated chiral polymer.













CHAPTER 1
INTRODUCTION

Aromatic compounds are ubiquitous in the environment and originate from both

natural and anthropogenic sources. The latter has become an issue of public concern for

human health and environment, as more and more of these compounds find their ways

into the ecosystem through misuse, accidental spillage, and improper disposal.'Although

many aromatic hydrocarbons are biodegradable by existing microorganisms,2 there are

many man-made xenobiotics, such as polychlorinated biphenyls (PCBs) that are

extremely stable and some are non-biodegradable.3'5 As a result, bioaccumulation of

these recalcitrant chemicals has occurred and has given rise to a whole host of biological

problems including mortality, edema, hepatotoxicity, teratogenicity, immunotoxicity,

reproductive toxicity, and promotion of cancer, affecting organisms at all levels.6'7

The toxic effect of aromatic hydrocarbons on living systems brought about

awareness of the need for prevention and cleanup of already polluted areas. In this effort,

bioremediation technologies have recently emerged as economical and practical viable

alternatives to conventional physicochemical methods. Because of microorganisms'

ability to adapt quickly to a changing environment, a very diverse collection of

microorganisms now can be found in many kinds of extreme environments. These

microorganisms are capable of metabolizing a variety of environmental contaminants,

ranging from heavy metals to recalcitrant aromatic hydrocarbons. While a detailed

review on bioremediation technology is beyond the scope of this discussion, it is evident








that microorganisms possess great potential that remains to be exploited for scientific

endeavors.

In recent years, microbes have been recognized as indispensable biocatalysts for

manufacturing enantiomerically pure materials in the chemical and pharmaceutical

industries. Biocatalysts have been used to simplify and enhance the production process

of complex chemicals and drug intermediates. They can add stereospecificity to the

process, eliminating the need for complicated separation and purification steps.

In the field of organic synthesis, the application of enzymes has led to significant

developments in stereoselective approaches to well-known reactions, such as aldol-

condensations,814 ketone-reductions,15'6 Baeyer-Villiger oxidations,17.18 and acyloin

condensation. 1- Although the introduction of chirality is one of the main aims, the use

of microbial enzymes as catalysts for the production of chemically useful compounds

such as alcohols, acids, and amides has been the focus of attention for many years. In

most cases, enzymes have been used for reactions difficult to achieve by purely chemical

methods, e.g., regioselective functionalization of a non-activated carbon center or

regioselective conversion of one out of several similar functional groups in a molecule.

These tasks are facilitated by the mild conditions under which enzymatic reactions take

place.

Recognizing the great potential of microorganisms as tools in synthesis, the last

two decades have witnessed a steady growth of activity within the field.-28 Research

groups from across the globe continue to explore for useful applications of microbes in

their synthetic designs.








The Hudlicky research program is heavily involved in using the toluene

dioxygenase-carrying microorganism, Pseudomonas putida F39D, as the source of

biocatalyst for generation of chiral synthons for application toward numerous synthetic

targets. The P. putida F39D was discovered by Gibson and colleagues in the late 1960s,

and is noteworthy for its ability to metabolize aromatic hydrocarbons to afford

synthetically useful chiral synthons of type 2 (Scheme 1-1).29 With the disclosure of

racemic pinitol synthesis by Ley in 1987,30 the practical utility of TDO in synthesis

wild strain of P. putida


R R R
toluene cis-dihydrodiol O further
dioxygenase dehydrogenase ": metabolisms
Sacetate
OH -" H
1 2 3

mutant strain R=H, alkyl, aryl, halogen ...
P. putida 39/0
Scheme 1-1. Aromatic degradation pathway of Pseudomonas putida F39D

was realized. Since then, metabolites obtain through biooxidation of aromatic substrates

with P. putida F39D have been used in various enantioselective syntheses, including

polyphenylene,3'32 prostaglandin E2a,33-35 and indivir36.37 that are of industrial and

medical significant.

The historical chapter of this dissertation gives some important developments in the

realm of biotransformation. The use of molecular oxygen as oxidizing agent by

monooxygenases and dioxygenases-carrying microorganisms for the conversion of

alkanes, alkenes, and aromatic to more polar identities is also discussed. A section of this

chapter is designated for a brief review of TDO properties, including the enzyme's

substrate specificity, regioselectivity, and stereoselectivity. Selected syntheses of highly

oxygenated natural products and targets of industrial significant are highlighted to








illustrate the versatility as well as the practicality of TDO as a biocatalyst for synthesis.

The syntheses of a few substituted catechols by conventional chemical methods as well

as by enzymatic means are included. The last part of the historical chapter is saved for

reviewing a class of natural products, combretastatins, including their discovery,

biological activities, and syntheses because combretastatins A-i and B-1 are among the

synthetic targets of this work.

The results and discussion chapter of this document is devoted to explore the

synthetic utility of toluene dioxygenase (TDO) and the second enzyme, toluene cis-

dihydrodiol dehydrogenase (TDDH), of Pseudomonas putida F39D through gaining

better understanding of the enzymes' properties and functions. The kinetic resolution

capacity of TDO in resolving aromatic substrates with proximal and distal stereogenic

centers and the possibility of the enzyme going through a radical intermediate during the

catabolism of aromatic substrates are among the main topics of this chapter. The utility

of TDDH to generate substituted catechols also will be discussed as an environmental

benign method to generate synthons for approaches to combretastatins A-I and B-l. The

synthesis of polyhydroxylated chiral polymer is the last part of this chapter.













CHAPTER 2
HISTORICAL

Biotransformations

Throughout the history of mankind, microorganisms have been of tremendous

social and economic importance. Without us even being aware of their existence,

microorganisms were used in food and beverage production very early in history.

Sumerians and Babylonians practiced beer brewing before 6000 B.C. Egyptians,

Hebrews, Greeks, and later Gauls and Iberians, began using yeast to produce fermented

food products such as bread, wine, and beer as early as 4000 B.C. It was only in the 19th

century that scientific progress revealed the secrets behind yeast fermentation. The

pioneering work of French chemist, Louis Pasteur, in the mid-1800s, led to a scientific

understanding of yeast fermentation that eventually became industrially important in

producing chemical commodities such as alcohols and organic material by fermentation.

Lactic acid was probably the first optically active compound to be produced industrially

by fermentation, in 1880.38

In the course of time, it was discovered that microorganisms could modify certain

compounds by simple, chemically well-defined reactions catalyzed by enzymes.39 Today,

these processes are termed "biotransformations." The essential difference between

fermentation and biotransformation is that there are several catalytic steps between

substrate and product in fermentation; while there are only one or two steps in

biotransformation. Another distinction is that the chemical structures of the substrate and

the product are similar in biotransformation, but not necessarily in fermentation.








The story of microbial biotransformations is closely connected with vinegar


biocatalyst 0OH 20
--OH C02 OH+ H20
4 5
Scheme 2-1. Vinegar production

production, dating back to 2000 years B.C. Vinegar production, perhaps the oldest

example of microbial oxidation, shows important developments in the field of

biotransformation by living cells (Scheme 2-1). However, much of the scientific

evidence for this important microbial transformation did not emerge until the mid-1800s.

The ability of a microbe to carry out kinetic resolution of racemic compounds was

first recognized by Pasteur in 1858 when (+)-tartaric acid was resolved with Penicilium

COaH
HOtH
H -OH
CO2H
(-)-(S,S)-tartaric acid 6
Figure 2-1. Pasteur's product of the first resolution reaction.

glaucum.40 He showed that the organism resolved racemic tartaric acid by consuming

only the (+)-tartaric acid, while leaving the (-)-enantiomer 6 untouched (Figure 2-1).41

From his work in this area, Pasteur became interested in fermentation and the concept of

spontaneous generation, a widely held belief that living organisms can somehow arise

spontaneously from nonliving matter.

Over the next few years, Pasteur became deeply involved with the French wine

industry investigating the "I'amer" condition, which turned wine sour, and destroyed

large quantities of the best Burgundy every year. At the time, fermentation was thought

to be a straightforward chemical breakdown of sugar into alcohol, carbon dioxide and

water (as a result of inherent instability of sugar). Scientists had found yeast cells in

fermenting vats of wines and recognized them as being living organisms, but believed








they were a product of fermentation. During his investigation of the "I'amer" condition

for the French wine industry, Pasteur examined specimens of wines under a microscope

and saw plump yeast cells in good wine and rod-like shapes in wine with too much acid.

Analysis of the wine showed that organic, or biological compounds were formed during

the fermentation, products that sugar alone could not produce. From his previous

research, Pasteur knew these compounds had to be formed by living cells. Pasteur

realized that the fermentation process was conducted by yeast and that other

microorganisms were responsible for turning the fermentation sour.

Pjalcr e eCnualjl, identified and isolated specific microorganisms responsible for

normal and abnormal fermentations of wine, beer and vinegar, and gave birth to the

science of bacteriology. He also showed that by heating each of the above liquids, the

microorganisms were killed and the wine was protected from further contamination.

With the identification of undesirable bacterial contamination of wines, volatile acidity

was eliminated and better quality wines were more routinely produced.

Pasteur's pioneer work in fermentation in 1862 conclusively disproved the theory

of spontaneous generation and led to the germ theory of infection. His work on wine,

vinegar, and beer resulted in the development of the pasteurization process.

In 1886, Brown confirmed Pasteur's findings and named Bacterium xylinum as the

agent responsible vinegar production.42 He also found that this organism could oxidize

propanol 7 to propionic acid 8 and mannitol 9 to fructose43 10 (Scheme 2-2).

In 1897, Eduard Buchner,44 a German chemist who also had been interested in the

problems of alcoholic fermentation since the 1880s, argued that enzymes were the









OH Bacterium AOH
7 xylinum 8
CH2OH CH2OH
H- -OH H- -OH
H- OH Bacterium H- -OH
HO H xylinum HO H
HO H O=
CH2OH CH2OH
9 10

Scheme 2-2. Reaction catalyzed by Bacterium xylinum

causative agents responsible for conversion of alcohol to acid and not whole yeast cells as

was originally thought. This view was not in line with the conventional belief at that

time, namely, that without life there could be no fermentation. Despite of this, Buchner

and his brother Hans Buchner in 1897 succeeded in preparing a cell-free liquid extract

that was capable of converting sugar into ethanol and carbon dioxide, just as though

whole yeast cells had been present. He named the causative agent as zymase.45 Buchner

succeeded in the proving this concept where many others, including Liebig and Pasteur,

had failed. He did this by grinding yeast cells with sand and then filtering the crude

preparation for enzyme isolation. Eventually, it became clear that the conversion of

sugar into alcohol by means of yeast juice entails a series of stepwise reactions, and that

zymase really is a mixture of several enzymes. Buchner's discovery of zymase then led

to establishment of the enzyme theory of fermentation. This gave birth to the science of

biochemistry, in which where much effort over the years has been invested in gaining a

thorough understanding of biological systems.

As biochemical knowledge of microorganisms and their environmental function

become more evident, microorganisms are being used more frequently for the production

of fine chemicals. Neuberg and Hirsch4647 in 1921 showed that acyloin ,.ioden jlI,'.r-








22,48 of benzaldehyde 11 and 2-oxo-propionic acid 12 could be achieved to furnish

levorotatory l-hydroxy-l-phenylproan-2-one 13, when the substrates are added to a

carbohydrate solution undergoing active yeast fermentation. This process was later

modified to use acetaldehyde directly instead of 2-oxo-propionic acid to afford a much

better yield of a-hydoxy ketone 13 which is used industrially in the production of L-

ephedrinel449'50 (Scheme 2-3).



Y"H + y cerevisiae ,r IY chemical
O0 0 ~ HNH
11 12 13 14
CO2
Scheme 2-3. Saccharomyces cerevisiae catalyzes acyloin condensation as the key step in
the production of L-Ephedrine

The ability of microorganisms to carry out biotransformations such as acyloin

condensation mentioned above, was soon recognized as essential in the manufacture of

CH2OH CH2OH CH2OH
HO-" HO- Acetobacter 0
HO Catalytic HO suboxydans HO
OH OH -OH
HO H HO- NAD NADH2 HO-
CHO CH2OH CH2OH 0
D-glucose 15 D-sorbitol 16 L-sorbose 17 2
Chemical
O 0 CO2Na CO2H oxiation
HO Acid NaO 0= HO
HO treatment NaO- HO-
OH OH
HO- HO- H2 HO-
CH2OH CH2OH CH20H
L-ascorbic acid 20 2-keto-L-gulonic acid/ 2-keto-L-gulonic acid 18
sodium salt 19
Scheme 2-4. Microbial dehydrogenation of D-sorbitol to L-sorbose as the key step in the
Reichstein-Grussner synthesis of L-ascorbic acid








many biologically active compounds. The ability of Acetobacter suboxvdans5' to

oxidize D-sorbitol 16 to L-sorbose 17 was discovered in 1928 and used as the key step in

the Reichstein-Grussner synthesis of L-ascorbic acid 20 in 193452 (Scheme 2-4). The

importance of L-ascorbic acid in the production of vitamins and its use as an antioxidant

in the manufacture of food accounts for its worldwide production of about 40,000 tons

each year.

Today, in addition to the Reichstein-Grussner synthesis (Scheme 2-4), the

commercial production of L-ascorbic acid is carried out using Erwinia sp. and Coryne

bacterium sp. in two fermentation steps to provide approximately I kg of L-ascorbic acid

for every 2 kg of glucose used (Scheme 2-5).53

CHO CO2H CO2H
H- -OH 0= O=
HO H Erwinia sp. HO--H Coryne- HO--H L-a a
H OH H--OH bacterium sp. H- OHacid
H--OH O= HO- -H
CH2OH CH20H CH2OH
D-glucose 2,5-diketo-D-gluconic acid 2-Keto-L-gulonic acid
21 22
Scheme 2-5. Two-stage fermentation for the production of 2-keto-L-gulonic acid from
D-glucose

Another industrially important microbial transformation was disclosed in 195254.55

for the conversion of that progesterone 23 to 11 -a-hydroxyprogesterone 24 by the molds

Rhizopus arrhius.56 The significance of this microbial transformation was soon realized

when the hydroxylated metabolite 24 was used in the synthesis of therapeutic steroids

and their derivatives.57 The microbial hydroxylation step simplified and improved the

efficiency of many approaches to synthesis of corticosteroid hormones and their

derivatives. An example of such an application is illustrated in the production of

prednisone 25, from 11 -hydroxyprogesterone 24 (Scheme 2-6). Although the











Rhizopusp
arrhius H P

progesterone 23 11-a-hydroxyprogesterone 24 prednisone 25

Scheme 2-6. Microbial 1 l-a-hydroxylation of progesteron

chemical syntheses of similar steroids were available from deoxycholic acid 26, they

were deemed lengthy, complicated and uneconomical, as in the case of cortisone 27

synthesis, which involves 31 steps (Scheme 2-7).58 The microbial II-a-hydroxylation

CO2H o OH
OH
0 -OH
chemical transformations
H 31 steps I
HO' O
deoxycholic acid 26 cortisone 27
Scheme 2-7. Chemical synthesis of cortisone

of progesterone quickly made the commercialization of steroid derivatives such as

cortisone a reality by reducing the price of this commodity from $200 to $6 per gram.

Table 2-1. Examples of commercial steroid processes
Substrate Product Microorganism Manufacturer
Progesterone la -Hydroxy- Rhizopus nigricans Upjohn company
progesterone
Component S Cortisol Curvularia lunata Pfizer, Inc.,
Gist-Brocades
9a-Fluoro- 9a-Fluoro-16a- Streptomyces E. R. Squibb & Sons
cortisol hydroxycortisol roseochromogens Lederle Laboratories
Cortisol Prednisolone Arthrobacter simplex Schering Corp.
Progesterone I-Dehydro- Cylindrocarpon E. R. Squibb & Sons
testololactone radicicola
3 -Sitosterol Androstadiendione Mycobacterium sp., G. D. Searle & Co.
M. fortuitum mutants Upjohn company








Further improvements of this biotransformation process have led to a current price of less

than $1 per gram of cortisone.59

The microbial reaction steps listed in Table 2-1 are of great economic significance

for the commercial production of steroids. For example, the transformation of

progesterone to a C19-steroid is used industrially in testosterone and estrogen production

and the microbial dehydration of ring A is also used in estrogen production.57

In the 1950s, the DNA double helix structure the genetic code of heredity and

the chemical nature of RNA were discovered. This discovery can be regarded as a major

milestone among this century's main scientific achievements. It led to the synthesis of

recombinant DNA and gave impetus to genetic engineering in the sixties and seventies.

This development quickly made recombinant DNA technology an integral part of

Table 2-2. Some representative industrial whole-cells biotransformations.
Products Biocatalyst Since Company
Vinegar bacteria 1823 Various
L-2-Methylamino-l- yeast 1930 Knoll AG, Germany
phenylpropan-1-ol
L-Sorbose Acetobacter suboxvdans 1934 Various
Prednisolone Arthrobacter simplex 1955 Schering AG, Germany

L-Aspartic acid Escherichia coli 1958 Tanabe Seiyaku
Co., Japan
7-ADCA Bacillus megaterium 1970 Asahi Chemical
Industry, Japan
L-Malic acid Brevibacterium 1974 Tanabe Seiyaku
ammoniagenes Co., Japan
D-p-Hydroxy- Pseudomonas striata 1983 Kanegafuchi, Chemical
phenylglycine Co., Japan
Acrylamide Rhodococcus sp. 1985 Nitto Chemical
Ltd, Japan
D-Aspartic acid Pseudomonas 1988 Tanabe Seiyaku
and L-Alanine dacunhae Co., Japan
L-Carnitine Agrobacterium sp. 1993 Lonza, Czech. Rep.
2-Keto-L-gluconic Acetobacter sp. 1999 BASF, Merck,
acid Cerestar, Germany








industrial microbial transformation. Recombinant technology which is widely used

today, allows over-expression of desired proteins such as biocatalysts, thereby resulting

in higher yields and diminished biological waste. Almost all chemical and

pharmaceutical companies rely on one or more forms of this technology to meet the

challenges of their trade. Table 2-2 lists some representative industrial whole-cells

biotransformations for the production of specialty chemicals.60

Oxygenation Reactions

Molecular oxygen is a powerful oxidizing agent essential for life's functions in

aerobic organisms. Oxygen interacts with a variety of biological systems including

oxidases, peroxidases and oxygenases that are important in cellular energy metabolism

and respiration.61 These enzymes utilize transition metals, most commonly Fe and Cu, or

heteroaromatic systems such as pteridium62 or flavin to activate dioxygen into highly

reactive singlet and hydroxyl radical species for electron transfer and oxidation of organic

molecules.

One of the most widely distributed enzyme systems among living organisms that

utilize oxygen is the iron-dependent cytochrome P-450 monooxygenases.63-66 These

enzymes are particularly abundant in detoxification organs such as the liver, but they are

also found in microorganisms. This class of enzymes was discovered in 194767'68 and has

been found to be involved in a variety of biochemical pathways including steroid

metabolism,6971 assimilation of non-carbohydrate carbon sources72'73 and detoxification

of xenobiotics. Numerous studies have been devoted to this enzyme system and the

cyclic mechanism for P-450 catalyzed oxygenation reactions was first postulated in

1971,74 and the crystalline structure of the enzyme has been elucidated in the presence75







and absence of substrate.76 A summary of the catalytic cycle catalyzed by cytochrome P-

450 is depicted in Scheme 2-8.

Sub Sub
O O, H20 SubO OH2 SubO H 0 Sub
/;Fe / /;Fe / N N IFI/ F
N- N N-1N N- N- I-
S S N S S-N
Cysa33b 33' Cys Cys-S 2s 9


( r6
S Sub Sub
0--- 0--0 Sub
N-I-N N-I-N N-I-N
N- I-N N-j-N Nil-*
S S. S
Cys32 e 31a Cys 02 30 Cys

Sub O

or
N-:-N

S
31b Cys
Scheme 2-8. Monooxygenase cytochrome P450s catalytic cycle

In the resting stage, the iron species is coordinated equatorially by a heme moiety

with water or hydroxide as an exchangeable axial ligand trans to the thiolate of a cysteine

residue, 28. The binding of substrate is accompanied by the desolvation of the binding

pocket and expulsion of the quao axial ligand77 to give rise to species 29. This process

alters the enzyme's configuration and increases its redox potential by 130 mV.78 These

changes allow the enzyme to accept an electron from NAD(P)H to give species 30 onto

which a molecular oxygen is bound to give a Cytochrome P-450 dioxygen complex that

can be represented by two resonances forms, 31a and 31b. The addition of a second








electron to 31 yields a ferric peroxide adduct 32, which can be protonated to give a

hydroperoxide complex. A second protonation of the same oxygen then leads to

heterolytic 0-0 bond cleavage, releasing water and generating the proposed oxo-ferryl

(O=Fev) porphyring radical 33a intermediate that is sometimes represented as (FeO)3*

33b. One atom of oxygen is expelled as water while the other forms the reactive

intermediate (FeO)31 which oxidizes the substrate to form the product. The expulsion of

the product, SubO, regenerates the initial ferric iron porphyrin species79and closes the

catalytic cycle. The mechanistic representation of Cytochrome P-450 above is illustrative

of how an enzyme system can utilize oxygen in the catabolism of organic matter.

In contrast, flavin-dependent mono-oxygenases use a different mechanism, which

involves a flavin cofactor, Scheme 2-9.80-82 First, NADPH reduces the Enz-FAD complex

thus destroying aromaticity. The FADH2 so formed is oxidized via Michael-type

addition of molecular oxygen yielding a hydroperoxide species (FAD-4a-OOH).

Deprotonation of the latter affords a peroxide anion, which undergoes a nucleophilic

attack on the carbonyl group of the substrate, usually an aldehyde or a ketone. The

tetrahedral intermediate thus formed, collapses via rearrangement of the carbon-

framework forming the product ester or lactone. Finally, water is eliminated from the

FAD-4a-OH species to reform FAD.

The cytochrome P450 mechanism resembles the chemical oxidation by hypervalent

transition metal oxidants through an electrophilic attack, while the FAD-dependent

mechanism parallels the oxidation of organic compound by peroxides or peracids, which

act as nucleophiles.83








H NADPH DP H H
N NH NH NH


34 O Sub 35 H O
[Enz-FAD] [Enz-FADH2-Sub]
S u b O O 0

H20
H H
-- N.-N C A N. /NN. N


I oxidation step I
N NH 0 N NH
37 H HO 36 H O0
OH J HO
[Enz-FAD-4a-OH-SubO] O [Enz-FAD-4a-OOH-Sub]




Scheme 2-9. Catalytic cycle of flavin-dependent mono-oxygenases

Enzymatic oxygenation reactions are particularly intriguing since direct

oxyfunctionalization of unactivated organic compounds are difficult to attain through

conventional chemical methods. Thus transformations of cheap inert raw materials, such

as alkanes, alkenes and aromatics into oxidized products of greater value such as

aldehydes, ketones, alcohols, carboxylic acids, by means of biocatalysts are appealing

alternatives to industry. The highly regio- and enantiospecific properties of many

biocatalyst systems made them ideal for the precise manufacturing of specialty

chemicals. The enzymatic oxygenation of organic molecule may go through one of the

following processes depending upon the enzyme system involved, Scheme 2-10.

In reactions catalyzed by monooxygenases, one oxygen atom from molecular

oxygen is incorporated into the substrate and the other one is reduced to form water by

means of a cofactor usually NADH or NADPH. While in reactions catalyzed by

dioxygenases, the enzymes simultaneously incorporate both oxygen atoms into the








substrate. These two processes are among the most common and important in aromatic

hydrocarbon oxidation.

Dehydrogenases
SubH2 + Donor Sub + DonorH2

I cofactor-recycling
Oxygenases
SubH2 + Donor + 02 Monooxygenases Sub + DonorH2 + H20

cofactor-recycling

Sub + 02 Di-Oxygenases SubO2
Oxidases
SubH2 + 02 Sub + H202
2 2H+
02 +2e -- 022 H202

02 + 4e 202 + 2H20
Peroxidases
2 SubH + H202 2 Sub- + 2 H20--- Sub-Sub
Sub+ H202 SubO + H20
Scheme 2-10. Enzymatic oxidation reactions

Monooxygenases Catalyze Biotransformation

Monooxygenases are ubiquitous in nature and serve many important biological

functions including detoxification of xenobiotics. They have a broad range of substrates

and can tolerate a very wide range of functional groups (Scheme 2-11).

Although the reaction mechanisms of various monooxygenases differ greatly

depending upon the sub-type of enzyme, their mode of oxygen-activation is the same in

term of their requirement of NAD(P)H and oxygen for function. In the following

sections, a series of monooxygenases that catalyze the mono-hydroxylation of alkanes,

arenes, and alkenes as well as enzymes that performed Baeyer-Villiger reactions are

described.








Sub +02 + H + NAD(P)H Monooxygenases SubO + NAD(P)+ + H20


H OH H OH


R R-O O0
R,-X R,-X=O RI, R2 R O-R2
X=N,S, Se, P

Substrate Product Type of reaction Type of cofactor
alkane alcohol hydroxylation metal-dependent
aromatic phenol hydroxylation metal-dependent
alkene epoxide epoxidation metal-dependent
ketone ester/lactone Baeyer-Villiger flavin-dependent
heteroatoma heteroatom-oxide heteroatom oxidation flavin-dependent
Scheme 2-11. Monooxygenase catalyzed reactions

Hydroxylation of Alkanes

Hydroxylation of unactivated hydrocarbon is a subject of great interest within the

synthetic community.8486 However, despite the invested effort, functionalization of

unactivated hydrocarbon remains an unresolved challenge. Available approaches to the

problem are limited8789 and problematic with little regio- and stereoselectivity control.

By contrast, biochemical hydroxylation of unactivated hydrocarbon is common and

is among the most useful processes9094 with great synthetic utility. Biooxidation of the

hydrocarbon of steroids and terpenoids, for example, are industrially important processes.

In this aspect, methane oxygenases play many important roles. This class of enzyme

specializes in the conversion of alkane to alcohol in the presence of NADH.95 The

enzymes from two obligate methylotrophs, Methylococcus capsulatus (Bath) 9697 and

Methylosinus tricosporium OB3b98 have been isolated, purified, and studied. They have

been shown to have a very broad substrate range and are capable of oxidizing a variety of








alkanes, alkenes, aromatic, and heterocyclic compounds," sometimes with little

regioselectivity control. Complex substrates have been converted into more than one

oxidation product as in the case of hydroxylation of 1,4-cineole 38 by Streptomyces

griseusl" and Bacillus cereusi1o (Scheme 2-12). When Streptomyces griseus was used,

8-hydroxycineole 41 was observed to be the major product along with trace amounts of

exo- and endo-2-hydroxy derivatives with low optical purity. On the other hand, when

Bacillus cereus was used, (2R)-exo- and (2R)-endo-hydroxycineoles were obtained,

exclusively in a ratio of 7:1, both in excellent enantiomeric excess.

aj HO 0 0
2 Microorganism 2 + 2
OH 8
HOH
1,4-cineole 2R-exo 2R-endo
38 39 40 41
Microorganism e.e. [%] exo e.e. [%] endo exo/endo ratio
Streptomyces griseus 46 74 1:1.7a
Bacillus cereus 94 94 7:1
8-hydroxycineole was the major product
Scheme 2-12. Microbial hydroxylation of 1,4-cineole

However there are monooxygenase systems that are extremely precise in regio-

selectivity and give rise to only one oxidation product. The importance of such

biocatalyst systems have been realized and used industrially for hydroxylating of the 9a-

and 16a-carbons the steroid framework in the production of therapeutic agents.102'103

Today, almost all carbon centers of the steroid skeleton can be selectively hydroxylated

by using an appropriate microorganism.'4 For example, the Il a-hydroxylation of

progesterone 42 can be achieved with Rhizopus arrhizus55 or Aspergillus niger105 and is

used industrially in manufacturing of 1 la-hydroxyprogesterone (Figure 2-2). The 73-

hydroxylation of lithiocholic acid 43 via Fusarium equisetil6 is another good example of








methane oxygenase transformation to generate drugs capable of dissolving cholesterol

and which also can be used for therapeutic treatment of gallstones (Figure 2-2).

0
ACO2H



H 7P
O H O\" -
progesterone lithiocholoic acid
42 43
Rhizopus arrhizus Fusarium equiseti
Aspergillus niger
Figure 2- 2. Microbial hydroxylation of steroids

Hydroxylation of Arenes

Aromatic hydrocarbons are stable identities and are notoriously inert to most

chemical manipulations. Harsh conditions, which are often associated with low

selectivity and uncontrollable formation of byproducts, are often required for

functionalization of the aromatic moiety. The difficulties associated with such methods

make them unappealing and impractical for synthetic applications.107109

By contrast, selective hydroxylation of aromatic compounds can be achieved

through enzymatic means. Monohydroxylation of aromatic substrates by

monooxygenases has been the subject of many studies. Most monooxygenases require

NAD(P)H to reduce the flavin to a 4a-hydroxyperoxyflavin required for hydroxylation of

the aromatic ring. The transformation is generally very regioselective and the

hydroxylating sites tend to be either ortho-l10'11 or para112,113- to the existing substituent

of the aromatic ring. Meta-hydroxylation on the other hand is rarely observed.14

Mechanistic studies of monooxygenases in eukaryotic cells such as fungi, yeasts

and higher organisms suggested that the process proceeds predominantly via epoxidation

of the aromatic species to generate unstable arene-oxides 45 as the first step in the








biotransformation process. The instability of arene-oxides has been shown to be

responsible for toxic and mutagenic effect by reacting with DNA, RNA and proteins.115

Arene-oxide can rearrange easily via hydride anion (NIH-shift)"6 migration to form the

phenolic product17 49 or hydrolyzed to a trans-diol 46 by epoxide hydrolase (Scheme 2-

13). Phenolic components are often further oxidized by polyphenol oxidases to

corresponding catechols18 which can undergo further metabolism.

R R
epoxide OH OH
hydrolase D further
R R "OH OH metabolism
D nD H20 46 47
mono ,fhS
Soxygenase R R
44 45 NIH-shift 0 thOHOO
44 45 NIH-shift 0 aromatize OH further
-" I metabolism
C -- "t([D(H)
48 H 49
Scheme 2-13. Possible mechanism in biodegradation of aromatics by eukaryotic cells

Enzymatic hydroxylation of aromatic substrates is a well-established industrial

process for the production of therapeutic agents. The production of L-DOPA (3,4-

dihydroxy-phenyl alanine)"9 for treatment of Parkinson's Diseasel20 and 6-

hydroxynicotinic acid 51 from nicotinic acid by either Pseudomonas or Bacillus sp. are

just two examples of monooxygenase-catalyzed aromatic hydroxylation reactions used

CO2H Pseudomonas CO2H
or Bacillus sp. J..
N 7 HO N
50 51
02 6-hydroxynicotinic acid

OH H Cunninghamella OH
N O, O N echinulata H O N

52 02 HO 53
prenalterol
Scheme 2-14. Microbial hydroxylation of aromatics








industrially (Scheme 2-14). The production of the pharmacologically important 3-

blocker, racemic prenalterol 53, via selective p-hydroxylation of a simple aromatic

precursor, using Cunninghamella echinulata, 12is another good example of whole-cell

biotransformation.122-126

Epoxidation of Alkenes

Chiral epoxides are extensively employed as high-value intermediates in the

synthesis of chiral compounds. In recent years much research has been devoted to the

development of catalytic methods for their production.127 The Katsuki-Sharpless

methods for the asymmetric epoxidation of allylic alcohols128 and the asymmetric

dihydroxylation of alkenes are now widely applied as reliable procedures. Catalysts for

the epoxidation of non-functionalized olefins have been developed more recently.129,130

Although high selectivity has been achieved for the epoxidation of cis-alkenes, the

selectivity achieved with trans- and terminal olefins were less selective and continue to

be a challenge within the synthetic community.

By contrast, monooxygenases-catalyzed epoxidation can be achieved with excellent

stereoselectivity from a wide range of alkenes that are not accomplishable via traditional

methods.'31"32 Despite the wide distribution of mono-oxygenases within all types of

organisms, only alkane and alkene utilizing bacteria, and to a lesser extent fungi, have

been shown to be capable of epoxidizing alkenes.4,91"133

The most intensively studied microbial epoxidizing agent is the co-hydroxylase

system of Pseudomonas oleovorans.134 This consists of three protein components: (i)

rubredoxin, (ii) NADH-dependent rubredoxin reductase and (iii) an oo-hydroxylase (a

sensitive non-heme iron protein), and catalyzes not only the hydroxylation of alphatic C-

H bonds, but also the epoxidation of alkenes.'35








Besides Pseudomonas oleovorans, numerous bacteria have been shown to

epoxidize alkenes.'36 As shown in Table 2-3, the optical purity of epoxides depends upon

the strain used, although the absolute configuration is usually R.137 This concept has been

recently applied to the synthesis of chiral alkyl and aryl gycidyl ethers.38'139 The latter

are of interest for the preparation of 3-substituted I -alkylamino-2-propanols, which are

widely used as adrenergic receptor blocking agents.140

Table 2-3. Microbial epoxidation of alkenes
R,~ 2 microof anism R 0

02 H20


Microorganism
Pseudomonas
oleovorans



Corynebacterium equi

Mycobacterium sp.


Xanthobacter Py2

Nocardia sp. IP


RI
n-CsHi
n-C7HI5
H
NH2CO-CH2-C6H4-O
CH.OCit-i .C, H.-0
CH3
n-C.3H27.
H
CH3
Ph-O
Cl
CH3
Cl
CH3


R2 Config. e.e. 3]
H R 70-80
H R 60
H R 86
H S 97
H S U.
H R 70
H R -100
H R 98
CH3 R,R 74
H S 80
H S 98
.... .................. K ........................ 8 ..............
CH3 R,R 78
H S 98
H R 98


More recently, other microorganisms also have been found to perform epoxidation

of alkenes with excellent yield and optical purity. As can be seen from Table 2-4, non-

terminal alkenes can be epoxidized by a Mycobaterium or Xanthobacter sp.,'41 while

Nocardia coralline has been reported to convert branched alkenes into the corresponding

(R)-epoxides with good optical purities.








Table 2-4. Epoxidation of branched alkenes by Nocardia corallina


R Nocard 0rallina )
56 57R
02 H20

R e.e. [%]
n-C.H7- 76
n-C4H9- 90
n-C5HI,- 88

However, the disadvantages associated with mono-oxygenase catalyzed

epoxidation reactions include the enzyme's instability, its dependency upon cofactors and

the precise coordination requirement of its subunits for proper function. These factors

discourage the exploitation of isolated enzymes as biocatalysts for epoxidation of

alkenes. Instead, whole-cell fermentations are generally employed. The drawback of this

approach is often linked to the epoxides tendency to react with cellular proteins and

enzymes leading to cell death. If the problem of product toxicity is surmounted, the

microbial epoxidation of alkenes also will be feasible on an industrial scale.142143 The

toxic effects of the epoxide formed and its further (undesired) metabolism by epoxide

hydrolases can be reduced by employing biphasic media'44 for the production of chiral

epoxides. Despite such difficulties, enzymatic epoxidation of alkenes continues to be a

reliable alternative for numerous synthetic applications, an example of which is the

generation of the epoxy-phosphonic acid derivative, 'fosfomycin',145 whose

enantiospecific synthesis by classical methods would have been extremely difficult. This

was accomplished by a microbial epoxidation reaction using Penicillim spinulosum.

Baeyer-Villiger Reactions

The Baeyer-Villiger oxidation of ketone with a peroxy acid to produce

corresponding lactone/ester was first reported by Baeyer and Villiger over a century








ago.146 Fifty years later, the mechanism of this reaction was deciphered by Criegee'47

and is still accepted in its original formulation (Scheme 2-15). The reaction proceeds via

0 0
R R+ HS05 R p OR, + HS04
58 62

0
X-COOH
59

R 0-H 0
-VY --- X-Co+
R fOO R OR' X-cO
60 0 61
"Criegee-intermediate"
Scheme 2-15. Mechanism of the Baeyer-Villiger reaction

the formation of an intermediate resulting from addition of the peracid to the carbonyl

group to form the tetrahedral "Criegee-intermediate' 60 followed by migration of one of

the substituents on the peroxy oxygen and formation of the product. The migration step

is concerted, rate determining and product determining. The regiochemistry of oxygen-

insertion of the chemical and the enzymatic Baeyer-Villiger reaction can usually be

predicted by assuming that the carbon atom best able to support a positive charge will

migrate preferentially.148 The applications of the Baeyer-Villiger reaction in organic

synthesis are widespread and have been reviewed.91,133,148152

In biological systems, the Baeyer-Villiger reaction is carried out by

monooxygenases. Enzymatic Baeyer-Villiger oxidation is a flavin-dependent process

that catalyzes the breakdown of carbon structures containing a ketone moiety.

Mechanistically, the process is closely related to the purely chemical process80' 53

proposed by Griegee. Of the enzymes studied to date, almost without exception all have

been found in bacteria and fungi.154








A number of bacterial enzymes have been purified and characterized,155 however,

the best characterized Baeyer-Villiger Monooxygenase (BVMO) to date is

cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. The

complete sequence of the enzyme is available,'56 and the enzyme is believed to be

monomeric with only one active site. 57

The importance of BVMOs was first recognized over 40 years ago during the initial

era of steroid biotransformation when the biotransformation of progesterone 42 by

0
020 0 OH



42 XH 2 6
progesterone XH2 H 63
Baeyer-Villiger

0 H20 02



0 66 H2 XH2 65
testololactone Baeyer-Villiger
Scheme 2-16. Progesterone metabolism by BVMOs in Cylindrocarpon radicicola

Cylindrocarpon radicicola'58 was used in the synthesis of testololactone 66 (Scheme 2-

16). Over the intervening years, the scope of the reaction was vigorously studied. The

enzyme is found to be capable of oxidizing a variety of substrates with excellent regio-

and stereoselectivity,'59-16' making it an ideal synthetic tool for chemoenzymatic

approaches to synthesis of a wide range of useful compounds.'62

Biotransformations of various unsubstituted bicyclic ketones containing a furan or

pyran ring have been used to yield chiral synthons for chemoenzymatic syntheses of








useful compounds, such as multifiden 70 and viridiene 71,163 the antibiotic sarkomycin A

75,164 and the insect antifeedant clerodin 80 (Scheme 2-17).165


06
67 68 69 Multifidene Viridiene
0 0 0 0
O HMO OI

72 0 7 74 75 nCO2H
Sarkomycin A 0 H
0 0 0
CHMO 0 .' 0

76 + ,, o O
6 77 78 79

0 / OAc
6AcO
AcO Clerodin
Scheme 2-17. Chemoenzymatic syntheses of various useful target molecules involving
the biotransformation by CHMO of bicyclo[3.2.0]-type ketones


BVMOs are also an excellent source of catalyst for desymmetrization of prochiral

ketones to yield the corresponding lactones.'1766 As depicted in Table 2-5, CHMO

catalyzed oxygen insertion occurred on both sides of the ketone, depending upon the


Table 2-5. Enzymatic Baeyer-Villiger oxidation of prochiral ketones
O O O
BVMO (0)

R R R
81 82 83
R Configuration e.e. [%]
CH3-O- S 75
Et- S >98
n-Pr- S >98
t-Bu- S >98
n-Bu- R 52








substituent in the 4-position, to give predominantly the (S)-configuration products. A

switch to the R-lactone was observed with 4-n-butylcyclohexanone. Simple models were

recently developed which allow the prediction of the stereochemical outcome of Baeyer-

Villiger oxidation catalyzed by cyclohexanone mono-oxygenase of Acinetobacter and

Pseudomonas sp by determining which group within the Criegee-intermediate is prone to

migration.167,168

Table 2-6. Microbial Baeyer-Villiger oxidation of monocyclic ketones
0 0
R Acinetobacter sp. o + .,R

84 85 86
R e.e. of lactone [%]
n-CsHII 97
n-C7Hi5 95
n-C9H19 85
n-CliH23 73

Racemic ketones can be resolved by BVMOs, in which only one enantiomer gets

oxidized and its counterpart remains unchanged,'69 Table 2-6. For example, a-substituted

cyclopentanones were stereospecifically oxidized by an Acinetobacter sp. to form the

corresponding (S)-configurated 8-lactones,170 substances which constitute valuable

components of various fruit flavors.

Bicyclic halo-ketones, which are used for the synthesis of anti-viral 6'-fluoro-

carbocyclic nucleoside analogues, were resolved by using the same technique.17' Both

enantiomers were obtained with >95% optical purity. The exquisite enantioselectivity of

the microbial oxidation is due to the presence of the halogen atoms, since the de-

halogenated bicycle[2.2.1 ]hepan-2-one was transformed with low selectivity.








Above-mentioned Baeyer-Villiger oxidations are not trivial to perform on a

preparative scale. Whole-cell transformations are difficult to perform, as some strains

such as Acinetobacter calcoaceticus are pathogenic and suffer from low yields due to

over-metabolism and/or side reactions catalyzed by other active enzymes in the

microorganisms. On the other hand, the use of isolated mono-oxygenases for Baeyer-

Villiger oxidations is not trivial, as these enzymes are linked to NADPH-recycling, which

is a notoriously difficult process.

The difficulties posed by BVMOs have led investigators to seek other looked

elsewhere for alternatives. In this context, using yeast genetically engineered to contain

the cyclohexanone monooxygenase of Acinetobacter calcoaceticus, has been an excellent

and safe substitute for preparative applications. 72175

Dioxygenases Catalyze Biotransformation

The benzene nucleus originating from both natural and anthropogenic sources is

ones of the most abundant chemical structures in the biosphere. This class of compounds

has attracted a lot of attention from the scientific communities due to its adverse health

affects. The relationship between cancer and exposure to this class of chemical was first

suggested in 1761176 and further supported by evidence presented in 1775,177 showing a

high incidence of scrotal skin cancer in chimney sweeps through exposure to coal-tar.

The link between exposure to aromatic hydrocarbons and cancer was unequivocally

proven in 1930. 78'179 Within the last few decades there has been a virtual explosion of

scientific reports on aromatic hydrocarbons as carcinogenic agents that interfere with a

multitude of biological processes.180 Such investigations have contributed to the

realization that aromatic hydrocarbons are among the main causes of human cancers.181

The impetus drive for a better understanding of the biodegradation aromatic








hydrocarbons biodegradation is therefore essential, since such understanding may allow

development of bioremediation technology and production of chiral precursors for

synthetic applications.

The degradation of aromatic hydrocarbons is carried out almost exclusively by

dioxygenase-carrying microorganisms.182 The initial step of the metabolic process is

often the cycloaddition of oxygen to the aromatic moiety forming an unstable

dioxetane183 which is then enzymatically reduced to yield synthetically useful cis-glycols.

This process constitutes the key step in the aromatic hydrocarbon degradation pathway

and is a prerequisite for further fission and catabolism of the aromatic compounds,

Scheme 2-18.


S + 02 di-oxygenase reductase

91 92
dioxetane cis-glycol


oxidative OH dihydrodiol
further ring-cleavage dehydrogenase
metabolism ( OH

NAD(P)+ NAD(P)H
Scheme 2-18. Degradation of aromatics by microbial dioxygenases

Dioxygenases are involved in several stages of the aromatic hydrocarbon

degradation pathway. Some diooxygenases, such as TDO, Scheme 2-19, are responsible

for hydroxylation of the aromatic ring, while other dioxygenases, such as catechol

CH3 CH3

02 + NADH + H TDO
94 95
Scheme 2-19. Dihydroxylation of aromatic hydrocarbon by TDO








dioxygenases, 84 are known to be involved in the ring fission process of transforming

catechol precursors into aliphatic products, as illustrated in Scheme 2-20. Generally, the

catechol dioxygenases require no cofactor and can cleave the catechol moiety via either

an intradiol or extradiol fashion, as depicted in Scheme 2-20. The intradiol-cleaving

enzymes utilize Fe(m) to give metabolite 96, while the extradiol-cleaving enzymes

utilize Fe(II)185.186 and in a few cases, Mn(II)187189 to give metabolite 97.

R
O COOH intradiol
R \. COOH cleavage
A OH 96
I _cis, cis-muconic acid
OH R
02 < O0 extradiol
0I OOH cleavage
OH
97
Scheme 2-20. Modes of catechol cleavage

There are diverse classes of dioxygenases existing in nature that are capable of

oxidizing a variety of organic substrates. However, the breakdown of the aromatic

nucleus has received the most attention. Numerous studies have been launched to probe

for a better understanding of this important metabolic process. As a result, there is a

great deal of knowledge known about dioxygenases. They are multicomponent enzyme

systems involving several non-heme iron proteins and require NADH for function.184"190

192 To date there are a number of microorganisms that have been found to be capable of

utilizing a wide range of carbon sources for energy and growth and these enzymes have

been classified according to the type of substrate they oxidize. The following are some of

the most common dioxygenases found to date: benzene dioxygenase (BDO), toluene

dioxygenase (TDO), naphthalene dioxygenase (NDO), benzoate dioxygenase (BZDO),

and biphenyl dioxygenase (BPDO). The overlapping in substrate specificity among the








enzymes is common and therefore it is difficult to provide definitive information on the

most appropriate type of dioxygenase for a particular type of biotransformation. In

general terms, however, the TDO system is most suitable for cis-dihydroxylation of

substituted benzene substrates and bicyclic arenes, NDO is particularly useful for bi- and

tri-cyclic arenes, BPDO is more appropriate for larger polycyclic aromatic hydrocarbons

and BZDO is required for benzoic acid substrates. More than twelve types of

multicomponent ring-hydroxylating dioxygneases have been identified and purified.'93

A number of mutant dioxygenases also have been identified and characterized and

these have been instrumental in providing cis-dihydrodiols chiral synthons for numerous

synthetic applications. For example, Pseudomonas putida mutant strain F39D190,194 and

UV431 provide a source of TDO, while JT103,'95 B13,196 and BG1197 are a source of

BZDO and 9816/11192 has been identified as the mutant strain for NDO. A mutant strain

of a Beijerinckia species, Spingomonas yanoikuyae B8/36'98 has been identified as the

source of BPDO and Alcaligenes eutrophus 335/B9 for BZDO.199 Since, these mutant

strains carry an ineffective cis-dihydrodiol dehydrogenase gene, the aromatic

hydrocarbon metabolic pathways is terminated after the initial cis-dihydrodioxylation

process. As a result, the cis-dihydrodiol metabolite accumulates. The genes encoding the

TDO components in both F39D and UV4 strains have been cloned and expressed in

Escherichia coli (pDTG601)200and (pKST 11),201 respectively. The expression of these

dioxygenases in E. coli hosts has been extremely useful in facilitating the production of

chiral synthons for synthetic applications.

Pseudomonasputida F39D

In 1968 after a series of studies on microbial oxidation of aromatic hydrocarbons

by soil bacteria, Gibson and colleagues discovered a strain of the bacteria, Pseudomonas








putida Fl (PpFI)194 that could utilize benzene, toluene, and ethylbenzene as sources of

carbon for energy and growth. Subsequent studies showed that the organism would not

grow if the aromatic hydrocarbons were added directly to the growth media. However,

good growth was observed when the substrate was added as a vapor. Toluene appeared

to be the best substrate for cell growth. Cells that were induced with toluene were found

to be able to metabolize a variety of other substrates. Isopropylbenzene was oxidized at

approximately half the rate observed with toluene. Propylbenzene and butylbenzene

were metabolized even slower. This evidence led investigators to believe that the

bulkiness of the substituent on the aromatic ring has an inverse effect on the metabolic

rate of the substrates. Oxygen uptake experiments202 indicated that the organism

consumed 1.0 mole of oxygen per mole of substrate and the oxidation process was

initiated by incorporating both atoms of molecular oxygen into the aromatic nucleus to

form (+)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene.23'204 This metabolic

transformation catalyzed by a multicomponent mononuclear non-heme iron



Toluene dioxygenase
94
ISPToL Ferredoxino ReductaseroL NAD+
02 (todCIC2) (todB) (todA)

ISPTL FerredoxinT ReductaseTOL NADH+ H+

NAD+ NADH + H+ COO
OH OH 02 OH20 OH

SOH Toluene OH3-methylcatechol OH HOHD
95 dihydrodiol 98 2,3-dioxygenase 99 hydrolase CH3COO
dehyroxygenase (todE) (todF)
(todD)
Scheme 2-21. Catabolic pathway for toluene used by Pseudomonas putida F Genes
designations for individual proteins are shown in parentheses








oxygenase system, designated toluene dioxygenase (TDO).205 The individual components

of toluene dioxygenase have been purified and are organized as shown in Scheme 2-21.

The enzyme is believed to go through the following sequence of actions during the

catabolism of aromatic substrate. Electrons are initially accepted from NADH by a

flavoprotein, reductase OL 206 and transferred them to a small iron-sulfur protein,

ferredoxinToL207 FerredoxinToL then reduces the terminal dioxygenase, which is a large

iron-sulfur protein that has been designated ISPTOL208 The reduced oxygenase catalyzes

the addition of both atoms of molecular oxygen to toluene to form cis-toluene

dihydrodiol. The further metabolism of cis-toluene dihydrodiol involves NAD'-

dependent dehydrogenation reaction mediated by cis-toluene dihydrodiol dehydrogenase

to form 3-methylcatechol.209 Extradiol cleavage at the 2,3 position by 3-methylcatechol

2,3dioxygenase yields 2-hydroxy-6-oxo-2,4 -hepatadienoate, which is further

metabolized to 2-hydroxypenta-2,4-dienoate and acetate.

The enzyme system of toluene dioxygenase has been extensively studied since its

first disclosure in 1968. Individual enzymes within the metabolic pathway depicted in

Scheme 2-21 have been purified and characterized.206 Mutant P. putida with defective in

each of the bacteria genes also have been identified and isolated.210'21 A mutation that

occurred at the todD gene resulted in a mutant strain P. putida 39/D. This mutant strain

would no longer grow on toluene or benzene as the sole source of carbon. When P.

putida 39/D was grown on glucose, in the presence of benzene, cis-benzenediol

accumulated in the culture medium suggesting that the organism no longer carried a fully

functional enzyme system as disclosed in Scheme 2-21. It appears that the toluene

dihydrodiol dehydrogenase (todD) gene was disabled so that the metabolic pathway of








the P. putida 39/D is terminated at the cis-dienediol stage, Scheme 1-1. The early

termination in this metabolic pathway makes this organism a valuable tool for the

generation of synthetically useful chiral synthons.

Escherichia coli JM109 (pDTG601)

In order to study the individual enzymes, namely toluene dioxygenase, toluene

dihydrodiol dehydrogenase, and 3-methylcatechol 2,3-dioxygenase, of the P. putida Fl's

metabolic pathway in detail, Gibson and colleagues constructed three clones that over-

expressed the first three enzymes of the metabolic pathway shown in Scheme 2-21. The

genes todC C2BA that encode for toluene dioxygenase (reductaseToL, ferredoxinToL, and

ISPToL) was cloned into a plasmid designated as pDTG601 and the expression of the

genes was placed under control by a tac promoter which can be chemically induced by

isopropyl-p-D-thiogalactopyranoside (IPTG). The plasmid was then placed in an E. coli

host named JM109 (pDTG601).200 The development of this recombinant microorganism

has many advantageous over the P. putida F39D strain in generating chiral metabolites

for synthetic applications. Since handling and growing JM109 (pDTG601) is much

easier than handling and growing P. putida F39D, JM109 (pDTG601) has enabled

toluene dioxygenase to become much more accessible for scientific research. Large

amount of JM109 (pDTG601) can be grown easily through fermentation processes and

induced to over-express TDO for used in manufacturing of chiral synthons for various

synthetic applications. Today the use of JM109 (pDTG601) has become routine and is an

integral part of many synthetic ventures.

Toluene Dioxygenase Substrate Specificity

The broad substrate specificity of toluene dioxygenase has allowed for the

generation of a diverse group of metabolites with varying degrees of substitutions.








Several key features of TDO-catalyzed cis-dihydroxylation processes have been

elucidated, which have helped shed light into the nature of the active sites of toluene

dioxygenase enzyme system.

For example, substrates with charged or very polar substituents, such as, hydroxy,

amino, carboxy, nitro, sulfonic acid and sulfone, directly linked to the benzene ring have

been found to be resistant to TDO dihydroxylation. In spite of these limitations, the

dioxygenase enzymes are capable of accepting arene substrates with a wide range of

substituents.

The degree of substitution and types of substituents on the aromatic rings also are

key components in the TDO's catabolism processes of aromatic substrates. The

likelihood a substrate is accepted by TDO decreases as the degree of substitution

increases. When multiple electron-withdrawing substituents such as halogens are

present, they reduce electron density from the aromatic ring thus hindering the activity of

the aromatic moiety. On the other hand, when electron-donating groups are present, the

reactivity of the substrates are increased; however, electron rich substituents tend to

destabilize the cis-dihydrodiol metabolite and in many cases metabolites containing such

substituents have been found to decompose spontaneously during the biotransformation

or purification procedure. For the disubstituted substrates, a 1,3-arrangement of

substituents suffer the so-called 'meta effect' that would result in an increased inhibitory

effect on the ring dihydroxylation rate in comparison with the substrates with substituents

at the 1,2 or 1,4 positions.

The size of substituents on the aromatic ring also plays a role in determining the

ability of TDO to accept the substrates. The smaller the substituent on the arene, the








better the substrate it is for the enzyme. For example, in the cis-dihydrodiols of

alkylbenzenes, the rate of metabolism for substrate with a methyl substituent was

observed to be twice as fast as for a substrate with n-butyl-substituent. The catabolism

rate for t-butylbenzene was even slower and the cis-dihydrodiol metabolites of higher

homologues are unknown.194 Both naphthalene and biphenyl are good substrates for

TDO enzymes, perhaps indicating that flat, rigid molecules fit better into the enzyme's

active site than molecules with bulky, flexible, substituents.

Toluene Dioxygenase Regioselectivity

The TDO-catalyzed cis-dihydroxylation of monosubstituted aromatic substrates is

very regioselective. With few exceptions of monohydroxylation, sulfoxidation,

dealkylation and desaturation of substituents, the cis-dihydroxylation of monosubstituted

benzenes by TDO occurred primarily at the benzene 2,3-bond. The hydroxylation at the

1,2-bond is the sole example of benzoic acid oxidation catalyzed by BZDO and no

examples of dioxygenase-catalyzed cis-dihydroxylation at a 3,4-bond have been found

(Scheme 2-22).

ROH
BZDO OH
1,2-bond
oxidation 1
R R
2 TDO OH
/ 2,3-bond H
S oxidation 2
1 R


3,4-bond'
oxidation 1 OH
OH
102
Scheme 2-22. Regioselectivity of dioxygenases in hydroxylation of aromatic substrates








Regioselectivity of TDO-catalyzed cis-dihydroxylation of disubstituted benzene

substrates is restricted to unsubstituted arene bonds. For para-disubstituted benzene

substrates of type 102, the oxidation of the aromatic moiety will occur at bond b to afford

cis-dienediol 103 if R>R' and give diol 104 if R'>R, (Scheme 2-23).

R R
OH ,OH
b TDO or
-Y OH ."" OH
R' R' R'
102 103 104
If: R>R' R'>R
Scheme 2-23. Regioselectivity of TDO in biooxidation of 1,4-disubstituted aromatic
substrates


For ortho-disubstituted benzene substrates, Scheme 2-24, TDO-catalyzed oxidation

will occur at either bonds b or d depending upon the size of the substituents on the ring.

With a preference being shown for b hydroxylation if substituent R was larger than R'

the oxidation will occur primary at bond b giving rise to metabolite a-hydroxylated

metabolite. On the other hand, if R' was larger than R, oxidation at bond d will be

preferred.212'213 When both substituents are of similar size, a mixture of cis-dihydrodiols

formed due to oxidation at both bonds b and d.

R R R
R' N 0 R OOH r R

R R OH or HO"
105 106 1076H
If: R>R' R'>R
Scheme 2-24. Regioselectivity of TDO in biooxidation of 1,2-disubstituted substrates

The directing effect of the substituents has practical implications and has been

exploited for the preparation of cis-dienediols of type 110 and 113 (Scheme 2-25) that are

not obtainable through conventional biotransformation. Ortho- or meta-substituted








iodobenzenes can be oxidized to the corresponding enantiopure cis-dihydrodiols and the

metabolites was then subjected to catalytic hydrogenation would give rise to a new range

of 3,4-cis-dihydrodiols unattainable by direct enzyme-catalyzed asymmetric

dihydroxylation, (Scheme 2-25).213


X TDO X H2/Pd/C X H
S----OH OH
108 109 110
I I
f D TDO H/Pd/C 0H
X X OH X H
111 112 113
Scheme 2-25. Chemoenzymatic approach to 3,4-cis-dihydrodiols

Regioselectivity for meta-disubstituted benzenes in TDO-catalyzed

biotransformation is similar to that for ortho-disubstituted benzene substrates. There is a

preference for attack at bonds b if R>R' to afford cis-dienediol type 115. On the other

hands, dihydroxylation of bond c will be predominant if R'>R to afford metabolite 116 as

the major product (Scheme 2-26).

R R R

Sdioxygenase OH o
oH R '."OH
114 115 1160H
If: R>R' R'>R
Scheme 2-26. Regioselectivity of 1,3-disubstituted aromatic substrates

Dihydroxylation of tri-substituted benzene substrates by TDO is rare and follows a

pattern of regioselectivity similar to that observed in the oxidation of di-substituted

benzene substrates by TDO. The broad substrate specificity of TDO allows for

hydroxylation of bicyclic arenes, such as naphthalene and heteroarenes.'83214

Dihydroxylation of polycyclic arene of higher homologues of naphthalene also occur but








are carried out primarily by dioxygenases such as NDO and BPDO215.216 that are capable

of accommodating larger substrates. A strong preference for dihydroxylation at the bay-

region bond is observed with a minor proportion of oxidation occurring at non-K

positions (Figure 2-3). It is noteworthy that little evidence has been obtained for attack at

the region of highest electron density in the angular arenes, i.e. the K-region, with either

the NDO or BPDO enzymes.




OH OH OH OH

OH OH OH OH
e 118 119 120 121
dioxygenases

117 I I


OH OH OH

OH N OH OH
122 123 124
Figure 2-3. Preferred regioselectivity of mono-and polycyclic arene cis-dihydrodiols

The presence of one or more heteroatoms in the arene ring has been found to exert

a strong influence on the regioselectivity of dioxygenase-catalyzed dihydroxylation. For

bicyclic heteroarenes quinoline,217 isoquinoline,217 quinoxaline,217 quinazoline,217

benzothiophene,218 2-methylbenzo-thiophene,219 benzofuran,220 and 2-

methylbenzofuran,2'9.220 the cis-dihydroxylation of the carbocyclic arene ring is generally

preferred over the heterocyclic ring. In some cases, mixture of metabolites have been

observed as a result of oxidation at both carbocyclic and heterocylic rings as in the case

of the benzothiophene and benzofuran, Table 2-7.218'219 However, where no carbocyclic








alternative exists, dihydroxylation occurs at the heteroarene ring itself, as in the case of

thiophene and 3-methylthiophene.219

Table 2-7. TDO-catalyzed biotransformation of substrate containing heterocyclic ring
system.
Substrates Metabolites
OH OH

OH N OH
NN N
N12 N126 127

..- N O ^ OH N_ OH
N +
128 129 130
OH
N -N : OH

131 132
OH
N N OH
133 134
OH OH OH

O+ /OH + O
135 136 137 138
OH OH
OH

+ _O H
139 140 141
OH

C (^OH
142 143
"b OH


144 145



Electron rich functional groups such as cyano-, vinyl-, alkoxy- and alkylthio-,

appear to have a relatively strong directing effect. However, predictions of








regioselectivity of multi-substituted substrates are more difficult because of (1)

uncertainty about the effective size of substituents in different conformations, (2) product

instability and (3) further dioxygenase-catalyzed oxidation of the substituent.

Toluene Dioxygenase Stereoselectivity

The ability of toluene dioxygenase to oxidize a wide range of aromatic substrates

while maintaining a high degree of optical purity is a remarkable biological process. The

combination of such properties has no doubt made TDO an attractive catalyst for the

production of synthetically useful chiral synthons. Indeed, the practicality of TDO in

organic synthesis continues to fuel much of the interest within the chemoenzymatic

synthesis arena. Today, many research groups from across the globe continue to devote

their time and energy to gain better understanding of the enzyme's properties as well as

its synthetic potential. Thanks to their efforts, more than three hundred cis-dihydrodiol

metabolites of arenes have been reported and much information about the properties of

the enzyme are known.

As the use of cis-dienediols for synthesis becoming more routine, the search for

general methods for determining the optical purity and absolute stereochemistry of new

metabolites is underway.221'222 Traditional methods employed for such purpose have

included the synthesis of these metabolites,220 their chemical transformation to known

compounds, conversion of the metabolites to diastereomers and derivatives,24 X-ray

crystallography,223 stereochemical correlation5.68 circular dichroism,224 and NMR

spectroscopy.221.222.225

For example, the use of Mosher's chiral acids, R-or S-a-methoxy-a-

(trifluoromethyl)phenylacetic acid (MTPA), to form NMR-distinguishable

diastereoisomeric esters in the presence of homochiral lanthanide shift reagent such as,







tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorato] europium (lII), Eu(hfc)3),

has been useful in the determination of optical purity and absolute configuration of new

metabolites. For instance, this approach has been used in determining the optical purity

of metabolites obtained from biooxidation of 1-bromonaphthalene and 2-

bromonaphthalene. In this effort, metabolites 147 and 148, obtained from the

biooxidation of corresponding 1-bromonaphthalene, were separated and converted into

bis-MPTA protected ester 150 via catalytic hydrogenation and acylation of the cis-

hydroxyl groups with MPTA (Scheme 2-27).

Br OH OH OMPTA
N OH OH
Br HPd/C +)-R-MPTA DMFMPTA
S MeOH DCCCH2C,2 CID
TDO 147 149 150
146 Br OH OMPTA
HPd/C R-MPA;DMF OMPTA
"IOH CC 12T
148 OH 149 150
Scheme 2-27. Biotransformation of 2-bromonaphthalene

'H-NMR analysis of 150 derived from 147 and 148 in presence of an equal molar

quantity of Eu(hfc)3 showed a single signal for benzylic methane at 8 = 6.70 ppm that

matched with the standard sample of (-)-cis-tetrahydronaphthalene diol obtained from

hydrogenation of the product of microbial dihydroxylation of naphthalene. By contrast,

analysis of the 'H-NMR of the racemic Mosher esters 153 obtained from chemical

OH OMTPA
OsO4, NMO O, (+-R-MTPA, DMAP OMTPA
acetone/H20 DCC, CH2C 2
151 152
153
Scheme 2-28. Preparation of Mosher ester 153








manipulation of 1,2-dihydronaphthalene 151, Scheme 2-28, showed well-resolved signals

for benzylic methines (6 a-isomer = 6.85 ppm and p-somer = 6.70 ppm) of both ester isomers.

The combination of methods used above led to the establishment for enantiomeric excess

of diol 147 and 148 to be >98%.


i ~OMTPA OH
(b) OMTPA OH

J150 155
S) OMTPA Br OH
d. OMTPA OH
J MT (b)p O O
150 147
OMTPA Br
C.1so: ) OMTPOA
150 | 14 S 'OH
OH
d. OMTPA OH
(d "1OMTpA -1




(e)
OMTPA

8 7 6 5 < II
b(ppm) (+) 153 151
Reagents: i) H2/Pd/C; ii) MTPA; iii) OsO

Figure 2-4. The used of Mosher's acid, MTPA, to determine optical purity of 1-
bromonaphthalene diol.

The formation of syn-cycloadducts between the cis-dihydrodiol metabolite of type

2 and 4-phenyl-1,2,4-triazoline-3,5-dione 156, followed by protection of the free diols to

form corresponding di-MTPA esters 158 provided another general method for

enantiopurity determination 'H-NMR analysis (Scheme 2-29). The advantages of this

method include: (1) the formation of crystalline products, which allow absolute

configurations to be confirmed by X-ray crystallography, (2) good resolution of signals

using 9F NMR analysis and (3) ready availability of both R and S-enantiomers of MTPA.







Ph 0 0
R O N O N PhN PhNA
NOH 156 R R
O, O OH2 1 H [0 OMTPA
2 157 158
Reagents: i) (R) or (S)-x-methoxy-x-(trifluoromethyl)phenylacetyl chloride.
Scheme 2-29. Preparation of di-MTPA esters 158 for determination of cis-dienediol 2
absolute stereochemistry

More recently enantiopurity of cis-dihydrodiols can be determined using R- and S-

2-(1-methoxyethyl)benzeneboronic acid (MPB) 159 followed by 1 H NMR analysis

(Scheme 2-30).221

R R .
1 O H H :C O M e R M eO

OH (-)-S1 B(OH),
159 160
Scheme 2-30. Preparation of boronic ester 160 for determination of cis-dienediol
absolute stereochemistry

The attractive features of this method include the smaller quantity of cis-

dihydrodiols required and the rapid rate of reaction, which allows less stable metabolites

to be analyzed under milder conditions. The most sensitive and direct method for

enantiopurity determination of cis-dihydrodiols involves the use of chiral stationary

phase-high pressure liquid chromatography (CSP-HPLC). However, this method

requires both enantiomers to be available in order to demonstrate their separation. Since

the majority of cis-dihydrodiol metabolites are rather difficult to obtain in racemic form

and appear to be enantiomerically homogeneous, the CSP-HPLC analysis method212 is

generally used in a complementary manner with the other methods.221'222

In term of stereoselectivity, it is now commonly accepted that biotransformation of

arenes using TDO produces a single enantiomer. The oxidation of monosubstituted








benzenes by TDO, with the exception of fluorobenzene, gives almost exclusively a single

enantiomer of the absolute configuration and with an enantiomeric excess of greater than




&OH
2
Figure 2-5. The preferred absolute stereochemistry of metabolites obtained from
biotransformation of mono-substituted aromatic substrates

98% (Figure 2-5). The size of substituents on the aromatic ring plays a key role in

defining the absolute stereochemistry of the metabolites. The bulkier the substituent, the

greater directing effect it has on the stereochemical outcome of the oxidation process.

When the size of the substituent is too small, as in the case of fluorobenzene, low

stereocontrol was observed with enantiomeric excess ranging from 60-80%. This

phenomenon is probably due to the relatively small size of the fluorine atom, which

would allow more flexibility of the substrate within the active site of the TDO enzyme.

Table 2-8226 provides a few examples of TDO-catalyzed biotransformations of mono-

substituted benzene with excellent enantiomeric excess.

Table 2-8. Preferred absolute configuration of cis-dihydrodiol metabolites of
monosubstituted benzenes
R R

TDOOH

Metabolites R % ee
98 CH3 >98
161 Br >98
162 CH2CH(CH3)2 >98
164 CH=CH2 >98
165 CH2OCH3 >98


For ortho-disubstituted benzene substrates, the bulkiest substituent on the ring

exerts the strongest influence on the hydroxylation site and therefore controls the








stereochemistry of the metabolites. With the exception of ortho-difluorobenzene

substrate,227 the cis-dihydrodiol metabolites obtained from biotransformation of ortho-

disubstituted benzenes appear to be enantiopure. The lower ee values obtained for the

cis-dihydrodiol metabolites of ortho-difluoro-benezene (64%) again may be a result of

the reduced steric requirements of the fluorine atoms (Table 2-9).26

Table 2-9. cis-Dihydrodiols from ortho-substituted benzenes
R R
R' R O
TDO OH

OH
106 107
Metabolites R R' % ee
166 F F 64
167 Me F >98
168 Br F >98
169 CH=CH2 Cl >98
170 I Cl >98


For meta-disubstituted benzene substrates, similar phenomenon of regio- and

stereoselectivity was observed. Again, with the exception of ortho-difluorobenzene,227

which afforded an ee of 64%, the enantiopurity of cis-dihydrodiol metabolites obtained

from TDO-catalyzed oxidation processes are almost always greater than 98%. Table 2-

10, lists a few examples of TDO-catalyzed biooxidation of meta-substituted benzene.226

Table 2-10. cis-Dihydrodiols from meta-substituted benzenes
R R

S TDO OH
__ R' R' OH
Metabolites R R' % ee
171 F F 56
172 I Br >98
173 CF3 F >98
174 I F >98
175 Me F >98








Dioxygenase-catalyzed cis-dihydroxylation of para-disubstituted benzene

substrates, on the other hand, produces cis-dihydrodiols with a wide range of

enantiopurity.212 The relative sizes of the substituents (CF3>I>Br>CI-Me>F>H) provide

an empirical method for both the prediction of regioselectivity and facial selectivity

during TDO-catalyzed cis-dihydroxylation of benzene substrates. The general structure

depicted in Table 2-11226 indicates that the preference for a particular enantiomer will be

related to the differential in size between the large R and small R' groups.

Table 2-11. cis-Dihydrodiols from para-substituted benzenes
R R R
_,To C OH HO,,.*
OH HOY"
R' R' R'
103 104 105
If: R>R' R'>R
Compounds R R' % ee
176 I F 88
177 I Me 80
178 Br Me 37
179 Cl Me 15
180 Br F 56
181 I F 88
182 CF3 I >98
183 CF3 Me >98


groups. The absolute configuration of the preferred cis-dihydrodiol enantiomer from

TDO-catalyzed oxidation for di-substituted benzene substrates is summarized in Figure

2-6. 227

R
R R OH
R' OH : OH OH

R OH R' OH R'
R>R' R>R' R>R'
Figure 2-6. Preferred absolute configuration of cis-dihydrodiol metabolites of
disubstituted benzenes








TDO also is capable of oxidizing a wide range of polycyclic aromatic hydrocarbons

(PAHs) and heterocyclic aromatic hydrocarbons such quinoline, isoquinoline,

quinoxaline, with good yields and excellent enantiomeric excess.217 Table 2-12-6 lists

some of these metabolites to illustrate the structural diversity obtained from this

transformation.

Table 2-12. cis-dihydrodiols from polycyclic aromatic hydrocarbons
Substrates Products ee%


S>98
125 126

N "'OH >98
127OH
OH
N N OH >98

128 129
OH


N OH >98
131 132OH
SOH >98

184 154
OH


OH >98
185 186


Synthetic Applications of Dihydrodiols

The synthetic utility of the new chiral centers derived from the cis-dihydroxylation

of arene via TDO has been extensively reviewed. Despite the important original








disclosure made by Gibson on aromatic degradation by P. putida in 1968, it took almost

20 years for the synthetic application of this discovery to be realized with the syntheses

of racemic pinitol by Ley30 in 1987 and polyphenyene31'32 by ICI (Scheme 2-31).

OH


C ^OH MeO S OH
188 n 92 -OH
polypheenylene H
(-)-pinitol, 187
Scheme 2-31. Historically important application of arene cis-diols in synthesis

Research groups around the world soon followed these initiatives and demonstrated the

diversity of substrate range of the dioxygenase enzymes. To date, several hundred cis-

diols that have been isolated and identified, and new metabolites continue to be

discovered that could eventually lead synthetic chemists toward new applications and

more diverse targets. The rich functionality of the metabolites offers flexibility for

chemical manipulations including: asymmetric Diels-Alder reactions, epoxidation,

photochemical oxygenation, metallation, diol cleavage, diene cleavage, carbene additions

and ozonolysis that are synthetically useful (Figure 2-7).

oxidative cleavage


[2+1] ,OH sigmatropic
[2+2] rearrangements
[2+3]OH
[2+4]
oxidation,
electrophilic addition
Figure 2-7. Local bond-forming sites in the cis-dienediols

Such chemical manipulations have been extremely rewarding in the creation of a

whole new class of "primary synthons" that possess even greater versatility and

applicability than the original metabolites for synthesis of complex synthetic targets








(Figure 2-8).24 Such "primary synthons" can be further manipulated into "secondary

synthons" (Figure 2-9) for synthetic applications that have proved very fruitful.24 In this

X X X


oK HO' O O
0189 190 OH N191
R 191
R = CO2Me, Cbz, Ts

o x x


HO' O ZI OH O
192 OH 193 194
X = CI, Br, I, CN, alkyl, aryl


X X X

& OR
195 196 0 197
R = Cbz, CO2Me R = THS, TBS
Figure 2-8. Examples of primary synthons

manner, cyclohexanoids such as (+)-pinitol228 and D-myo-inositol derivatives229 have

been prepared by making use of the possibility of functionalizing every carbon atom of

the glycol in a stereo-control approach. Cyclopentanoid synthons on the other hand have

been used for the synthesis of prostaglandins and terpenes via ring-opening/closure

sequence." Rare carbohydrates such as D- and L- erythrose230 and L-ribonolactone23'

were obtained from chlorobenzene as were pyrolizidine alkaloids.232 In addition, other

complex natural products such as (-)-zeylena, (+)-lycoricidine, (-)-specionin, and (+)-

pancratistatin also have been synthesized efficiently using cis-dienediols.







x

HO Ox N3 OMe 0
OH OH \ OMe O
198 199 200 Fe(CO)3 201
0 OH 0

Ar QO 0 CO O0
NHTs NNHR NHR OR R= H, TBS
202 203 204 205

X X 9H 0,
) OH ^OH KOH
k^-OH (OH N'
OTBS R R= CO2Me, Cbz, Ts
206 207 208 209
X = CI, Br, I, CN, alkyl, aryl
Figure 2-9. Example of secondary synthons
Hudlicky and his research group have been pioneers in this effort. Most of the

work of the group to date has been conducted with a diverse range of diols derived from

the biotransformation of corresponding arenes by TDO.25 Table 2-13 lists some

representative synthetic products that are illustrative of the applicability and versatility of

TDO as a synthetic tool.

The synthetic potential of dioxygenase enzyme system, such as TDO, provides a

viable research tool that currently is used by a growing number of research groups across

the globe for a variety of synthetic applications, and the activity within the field continues

to grow.










Table 2-13. The use of cis-dienediols in synthesis
Diols Product of asymmetric syntheses

OH OH OH OH
A(OH xOH^ HOx 3^OH HO OH

S OH HO' OH MeO OH
OH NH2 OH OH
211 212 213 214
conduritol C233 conduramineA-1234'235 D-chiro-inositol236 (-)-pinitol237
OAc 0 OH
c D-- o D HO o L .OR
c HD '' "OMe 0
O21 H AD A HO OH HO OH
SD OA y X=CH, NH, 0
215 217 OH 216 Y= NH, OH
deuterated hexose234'235,238 L-ascorbic acid239 hexoses;240 aminohexoses;240
azahexoes;240 carbasugar240


kifunensine241,242


S eciODin biddle


220
(+l-asRicilin244


S2412








OH OH OH
HO OH HO OH OH

O oH O H OH


222 OH 0 223 224
pancratistatin245246 7-deoxypancratistatin246,247 lycoricidine248.249


B,

C: OH
OH
200


225
morphine250


OH OMe H 0


OH OH 0
227
ent-bengamide E252


OH OH
HO.,. HO/ OH

HO"" 0 OH
OH 226 NH2.HCI
amino-inositol dimmer251
OH


HO -
OH H,


HOn o H OH
OCH3 228
Aza-C-disaccharide253


234 235 -\ 236 k 237
zeylena257 Diels-Alder adduct258 Diels-Alder adduct258 Diels-Alder adduct258






















, H ,,,

242 HO ''


243
morphinan261


ent-morphinan261


N CO2
I -

HO" OH
OH 2460H

245 24 OH
shikimic acid262
OH

Br O
0 CbN 'OH
BrjOH A NH

OH 0
248
narciclasine263

OH
HON,, N OH OH
/t \N NN,,,-
t-Bu 0
258 H 250

indinavir3637
Diols Products of non-asymmetric syntheses
















OH OH OH
1Hl HO ^1 OH HO > .,OH HO OH
OH HO vOH HO "OH HO- -Y OH
210 OH OHH
255 256 27H
muco-inositol266,267 neo-inosito1266,267 allo-inosito1266,267
0 H

HO8H N -

H 0
258 259
indigo268


Substituted Catechols

Catechol and its derivatives are common structural features of many natural

products. Many of them are known to possess diverse biological activity, such as

antihyperglycemic,269 antimutagenic,270 antibiotic, and cytotoxic activities.271 For these

reasons, the role of catechol derivatives has become a topic of increased study in recent

years. However, despite their chemical and biological importance, only a limited number

of studies have been devoted to the synthesis of catechols derivatives. Available

synthetic methods for generation of substituted catechols tend to be cumbersome, non-

selective and involve harsh conditions.272'273 The following paragraphs provide a few

examples of how substituted catechols are made through the use of conventional

chemical methods. These examples give details that are illustrative of the difficulties

associated with using currently available methods for the synthesis of such compounds.







The synthesis of toluene catechols, for instance, involves 7 steps with an overall

yield of 5% starting from furfural274 (Scheme 3-32).

CH\ EtOH; HSO4 I_ \ Electrolysis. E -CO2Et
a -CHO --96.5% \- CO2Et in EtOH EtO--
260 261 61.5% 262 OEt
Raney-Ni, H2
CH3 100%
CH3
OH Dioxane, HCI, H3 CH3CH2COgEt
SReflux J fCOCHCO 2Et Na CO2Et
-OH 13% EtO OoEt 67.5% EtO 0 OEt
98 264 263
Scheme 2-32. Synthesis of methylcatechol from furfural

Preparation of bromocatechol 270 was achieved by first converting 2,3-

dimethoxybenzadehyde 265 to 2,3-dimethoxybenzoic acid 266 through permanganate

oxidation.275 The acid was then converted to 2,3-dimethoxybenzamide 267, which was

then subjected to a modified Hoffmann rearrangement process276 to furnish 2,3-

dimethoxyaniline 268. The resulting aniline 268 was converted to 2,3-

dimethoxybromobenzene 269, using a modified Sandmeyer procedure.277 Finally,

demethylation of dimethoxybromobenzene 269 with aluminum trichloride in

chlorobenzene278 produced 3-bromocatechol 270 in 5 steps with an overall yield of 49%

(Scheme 2-33).

COH CO2H CONH2
KMnO4 K' 88%
OMe 84% OMe OMe
265 266 267
83%
Br Br NH2

OH 80% ,: 0W e oAe
rOH OMe &OMe

270 269 268
Scheme 2-33. Synthesis of bromocatechol








Bromination and demethylation of quaiacol is another approach to 3-

bromocatechol. Bromination of quaiacol in a solution of t-BuNH2 and toluene provided

2-bromo-6-methoxyphenol 272 as the major product plus a small amount of 3-bromo-6-

methoxyphenol 273 as well as trace amount of polybrominated products 274. The side-

products were minimized when two or more molar equivalents of quaiacol were used to

give the desired product in a 60% yield. Demethylation of 2-bromo-6-methoxyphenol

272 with boron tribromide in methylene chloride resulted in 3-bromocatechol,279 as

illustrated in Scheme 2-34.

OH OH
MeO .Br HBB O Br
OH CH2CI2 I

1 Br2, tBuNH2 272 270
Toluene +
271 OH OH
MeO MeO ,Br
I B+ I
273 Br Br 274
Scheme 2-34. Synthesis of bromocatechol from quaiacol.

In the synthesis of 3-fluorocatechol 277, 2,3-dimethoxyaniline 268 was treated with

nitric acid and quenched with floroboric acid to give diazonium salt 275.280 The salt was

pyrolyzed to give 2,3-dimethoxyfluorobenzene 276, which was subjected to

demethylated by aluminum chloride to give 3-fluorocatechol 277 in a 29% overall yield

(Scheme 2-35).281

+-
NH2 N2BF4 F F
OMe OMe OMe OH

268 50% 275OMe OMe 58% 2 OH
Scheme 2- 35. Synthesis of 3-fluorocatechol








The synthesis of anisole catechol 261 was accomplished via demethylation of aryl

methyl ether with thioethoxide ion in dimethyl formamide to give an excellent yield of

96% (Scheme 2-36).282

OMe OMe
OMe DMF OH
DMF <
(OMe ( OH
278 279
Scheme 2-36. Synthesis of 3-methoxyatechol

The above examples are representative of chemical methods available for the

syntheses of 1,2-monosubstituted catechols. Most methods involve the use of harsh

conditions such as strong acids, or oxidants to cleave alkylated catechols with poor

selectivity and low yields.

Enzymatic approaches to catechol synthesis are available but under-explored. To

the best of our knowledge, only three or four methods of making catechols through

enzymatic means exist, in addition to the original report by Gibson. The process by

Frost283 involves the use of E. coli AB2834/pKD136/pKD9.069A for conversion of

glucose to the parent catechol. Another method involved the oxidation of substituted 2-

nitrophenols to various catechols with nitrophenol oxygenase via Pseudomonas Sp.

Strain T-12 to generate various substituted catechols.284 Recently, Yoshida and co-

workers reported a new organism capable of oxidizing aromatic substrates to catechols in

one step.285

Catechol Dehydrogenases Escherichia coli JM109 (pDTG602)

Studies of soil bacteria degradation of aromatic compounds led David Gibson in

the late 1960's to elucidate the metabolic pathway as shown in Scheme 1-1. Although

the use of TDO as a means of generating chiral synthons for synthetic applications is well








established, the use of the second enzyme, TDDH, has been limited to explorations in

cloning.29 This is surprising in view of the potential of simple catechols to serve as

synthetic building blocks for various synthetic applications.

In 1989 the gene todCIC2BAD encodes for TDDH, which is responsible for the

transformation of aromatics to corresponding catechols was cloned and expressed into an

E. coli host JM109 (pDTG602).200 Although the organism has been in existence for more

than a decade, there are few reports of the use of the organism for production of

catechols.

Combretastatins

Isolation and Biological Activity

The importance of natural products in biomedical sciences is well established, as

illustrated by the number of scientific publications devoted to this subject. New natural

products are being discovered everyday and their biological affects are routinely being

evaluated. Today, natural products and their derivatives are integral parts of every drug

discovery program. As part of the effort in combating chronic diseases such as cancer,

AIDS, and TB, the worldwide exploratory survey of plants for natural products with

desirable biological activity is an active and promising field, offering hope for combating

chronic diseases.

In this effort, George Pettit and co-workers have traveled to all comers of the world

to explore new sources of natural products from medicinal native plants. In 1981, their

efforts led them to investigate the medicinal potential of an African bush willow tree,

Combretum caffrum, of the Combretaceae family.286 The tree has been used by the

natives for centuries for treatment of leprosy, cancer, and mental illness. 287-289 In 1985, a

large-scale extraction of the bark of the African bush willow tree was initiated, yielding








0.7g of combretastatin A-1 (9.1x10-4 %) and 39.6mg (5.1x 10" %) of combretastatin B-

1.290 Structural analyses of combretastatin A-I and B-1 via IR and UV suggested the

presence of aromatic systems. High resolution EIMS indicated molecular formula

C18H2006 and Cg1H2206 for combretastatin A-I and B-I, respectively. 'H-NMR

spectroscopic data of the natural products exhibited signals for four methoxy group

protons and indicated that combretastatin B-l is a dihydro derivative of A-1. 'H-NMR

data of combretastatin A-I showed four aromatic protons, two of which are magnetically

identical and relatively shielded at 8 6.460. The other two are doublets of an AB spin

system appearing at 8 6.310 and 8 6.691 with a coupling constant (J=8.64 Hz) typical of

two ortho coupled aromatic protons. Another set of AB spin system appears as doublets

at 8 6.453 and 6.523 with coupling constant (J=12.2 Hz) and two exchangeable protons at

6 5.438, suggesting the presence of dihydroxy functionality. The 'H-NMR data for

combretastatin A-I and B-1 is summarized in Table 2-14.

Table 2-14. Combretastatin A-1 and B-l 'H-NMR*
Combretastatin A- Combretastatin B-l
Coupling Coupling
protons ppm Constant (Hz) protons ppm (Hz)
3,5-OCH3 3.597 (6H, s) CH2-CH2- 2.851(4H,m)
4-OCH3 3.760 (3H, s) 4'-OCH3 3.827(3H,s)
4'-OCH3 3.770 (3H, s) 3,5-OCH3 3.831(6H,s)
2',3'-OH 5.438 (2H, s) 4-OCH3 3.856(3H,s)
5'-H 6.310 (1H,d) JAB =8.64 2',3'-OH 5.382(lH,s)
CH=CH 6.453 (lH, d) JA'B'=12.2 5.398(IH,s)
2,6-H 6.460 (2H, s) 5'-H 6.390(1H,d) JAB =8.36
CH=CH 6.523 (IH, d) JBA'=12.2 2,6-H 6.420(2H,s)
6'-H 6.691 (IH, d) JBA =8.6 6'-H 6.577(1H,d) JBA =8.36
*chemical shift assignments relative to TMS in CDC13 solution. For proton-numbering -
see Figure 2-11


Further NMR studies, including 13C-NMR and nuclear overhauser effect difference

spectroscopy (NOEDS), of combretastatin A-1 and B-l, suggested structural features as








MeO MeO

MeO OH MeO OH
OMe OH OMe OH
280 OMe 281 OMe
combretastatin A-1 combretastatin B-1
Figure 2-10. combretastatin A-i and BI

shown in Figure 10. 13C-NMR data of combretastatin A-I and B-I are summarized in

Table 2-15.

Table 2-15. Combretastatin A-I and B-1 '3C-NMR*


Carbon Combretastatin A-I Combretastatin B-I
1 132.49 138.18
2 106.13 105.67
3 152.80 153.05
4 132.67 132.35
5 152.80 153.05
6 106.13 105.67
la 130.21 36.49
1'a 124.06 31.82
1' 117.91 121.55
2' 141.72 142.19
3' 137.42 136.21
4' 146.37 145.40
5' 102.98 102.52
6' 120.17 120.32
3,5-OCH3 55.85 56.12
4-OCH3 60.79 60.18
4'-OCH3 56.16 56.18
*see Figure 2-11 for carbon-numhberin*


Combretastatin A-l's structure was subsequently and unequivocally established by

X-ray crystallography, Figure 2-11.291

In numerous studies of the biological properties of combretastatins A-I and B-1,

the compounds were shown to cause mitotic arrest292 in cultured cells. Competitive

inhibition studies between combretastatins A-I and B-l and the well-known antimitotic

agents podophyllotoxin 282, steganancin 283, colchicines 284, suggested that


._._ _



























Figure 2-11. Crystal structure of combretastatin A-I

combretastatins share a common binding site on tubulin and are competitive inhibitors of

colchicine, as summarized in Table 2-16, Experiment II.293-297 Combretastatin A-I was

found to be capable of displacing 97.8% of colchicine from its binding site, which

suggests its has an affinity for the active site on tubulin. These natural products also were

found to be potent inhibitors of microtubule assembly at 2-3pM (IC5o) concentrations,

Table 2-16, Experiment I.

These biological properties demonstrated for combretastatin A-1 and B-I

encouraged further exploration and biological evaluation of this class of natural products.

Since the disclosure of combretastatin A-1 and B-1, a series of combretastatins have been

reported, Figure 2-12. The majority of the members of this class of compounds have

been shown to be potent against a series of cancer cells lines. They interact with and

disrupt the vascular life support system of tumors, and in the process, starve and prevent

cancerous cells from metastasizing. The results of preclinical studies in which tumor








Table 2-16. Inhibition of microtubule assembly and biding of colchicine to tubulin by
combretastatin A-I and B-1
MeO -MeO

MeO MeOH OH
OMe 1 L OMe
280 OH 281 OH
Combretastatin A-1 OMe Combretastatin B-1 OMe


OMe O-- 283 O-- 284 OMe
Podophyllotoxin Steganacin Colchicine
Experiment I Experiment II
Drug Microtubule Assembly Colchicine Binding
ID5o (pM) % of control
Combretastatin A-1 2 2.2
Combretastatin B-I 3 14
Combretastatin 11 34
Podophyllotoxin 3 13
Steganacin 6 49
Colchicine 6
'Defined as the drug concentration inhibiting the extent of microtubule assembly by
50% (adapted from published work by Pettit et al.)291'298


cells were treated with combretastatins showed that these agents produced tumor

shrinkage and ultimately tumor disappearance. Thus, this class of natural products may

offer a new approach to cancer treatment that is a radical departure from conventional

drugs.

Among the combretastatins known to date, (Figure 2-12), combretastatin A-4 has

been identified as the most active vascular targeting and anti-angiogenesis agent.299 In

1998, clinical trials with its water-soluble phosphate derivative300'30' 291, Figure 2-13,









MeO Meo N Meor N

MeO MeO f O 0 OHO -e
OMe Me O OMe OH OMe O
285 OMe 280 OMe 286 OMe 287 OMe
combretastatin combretastatin A-1 combretastatin A-2 combretastatin A-4
SOH OH

Meoo MeoO yOH
OMe OMe OMe OH 0
288 OMe 281 OMe o 290 O
combretastatin A-5 combretastatin B-1 combretastatin D-1 combretastatin D-2
Figure 2-12. Structural variation in Combretastatins

began302 and are continuing into phase II. Today studies continue to investigate the

structure-activity relationship of substituted stilbene derivatives of this type.303"306

MeO -

MeO 0
OMe ONa
291 OMe ONa
Figure 2-13. Water soluble phospate derivative of combretastatin A-4

From these studies it has been shown that the 3,4,5-trimethoxy aryl unit, a small

group on the 4'-position, and separation of two aryl rings by a two carbon unit, are

important for antimitotic activity, while substitution with a hydroxyl group on the 3'-

position is not essential.303'307-309

Combretastatins Syntheses

Combretastatin A-1 and B-1

Early syntheses of combretastatins and their analogues used the Wittig reaction as

the key step for installing the biaryl moieties flanked by a methylene bridge. In the

synthesis of combretastatins A-1 and B-1, 2,3,4-trihydroxybenzaldehyde 292 was treated

with sodium borate-decahydrate (borax) in water for selective formation of the 2,3-borate








ester, which allowed for specific methylation of the 4-hydroxy group by dimethylsulfate.

The reaction mixture was quenched with concentrated hydrochloric acid to afford

dihydroxybenzadehyde 293,310311 which was then treated with diisopropylethylamine and

t-butyldimethylsily chloride (TBDMSCI) in dimethylformamide (DMF) to form 2,3-t-

butyldimethylsilyl ether 294.312 The synthesis of combretastatin A-I was then

accomplished by coupling TBDMS-protected aldehyde 294 with phosphonium salt 296,

(readily prepared via treating 3,4,5-trimethoxybenzyl bromide 295 with t-BuLi and

followed with triphenylphosphine in THF) to give a 92.5% yield of a 9:1 ratio of Z/E

combretastatin A-I (Scheme 2-37). The products mixture was reduced to via

hydrogenation to arrive at combretastatin B-l. The disadvantage of this approach to

combretastatins is the resulting mixture of products requires separation.

Br+
CHO CHO CHO CH2PPh CH2Br
i, ii, iii
OH OH OSiMe2tBu MeO OMe MeO OMe
OH OMe OMe OMe OMe
292 293 294 296 295


v, vi
OMe OMe
OH OH

OH OH MeO :,

vii MeO OH

MeO OMe MeO OMe 280 OMe
OMe OMe combretastatin A-1
281 297
combretastatin B-1
Conditions: i) Borax, NaOH; ii) (MeO)2SO2; iii) HCI; iv) DMF;EtN(iPr)2,TBDMS-CI; v) BuLi,
THF, -150C-rt; vi) TBAF, THF; vii) H2/C/Pd, MeOH
Scheme 2-37. Total synthesis of combretastatins A-I and B-1








Combretastatin A-4

Since combretastatin A-4 is the most potent anticancer agent among the

combretastatins discovered to date, this drug and its derivatives have been the focus of

many syntheses. As shown in the Scheme 2-38, arylacetylene 302, which can be

fashioned in a few steps, was the key intermediate in an approach developed by Furstner

et al. to combretastatin A-4.298 The key step in this approach involved the coupling of

arylbromide 300 with the lithiated trimethoxyacetylene 301 via a 9-MeO-9-BBN-

mediated Suzuki-type cross coupling reaction to produce internal alkyne 302. Selective

hydrogenation of 302 with a Lindlar's catalyst (5%Pd/C, ethylenediamine) in MeOH,

followed by deprotection of the TBDMS-group with aqueous TBAF provided a 86:14

mixture of combretastatin A-4 and its saturated analogue 303 (Scheme 2-38).


Br Br Br L
Br MCPBA; B TBDMSCI, B 0
I CH2C2 imidazole, THF
CHO 86% OH 83% OSiMe2tBu MeO OMe
OMe OMe OMe
298 299 300 OMe

OMe OMe 9-MeO-9-BBN
MMeO MeO MeO PdCl2(dppf)

MeO MeO 1) TBAF, EtOAc, 70%
+ 2) H2;Pd/C; EDA; MeO -OMe
MeOH, 74% 302
MeO MeO 3) TBAF, H20, EtOAc
OH OH
combretastatin A-4 303
Scheme 2-38. Suzuki approach to combretastatin A-4

Using similar strategy, Lawrence and colleagues disclosed their approach to

combretastatin A-4 with an overall yield of 29% over 5 steps (Scheme 2-39).313 Again,

the key step in the synthesis involved the coupling of the readily available

trimethoxyacetylene 306298 with aryl iodine 307 to give diarylalkyne 308. The internal








Br
CHO Br I
S PPh3; CBr4 nBuLi
CH2CI2 85%
MeO OMe 65% MeO OMe MeO OMe
OMe OMe OMe
304 305 306

(Ph3P)4Pd/Cul,
Meoo piperidene;
307 63%
MeO MeO OH
I ( 1)B, M .O / --/--
MeO C HOAc MeO OMe
OMe 82% M308
OH MeO
combretastatin A-4 OMe
Scheme 2-39. Synthesis of combretastatin A-4 via Suzuki cross-coupling reaction

alkyne was then sequentially treated with dicyclohexylborane and acetic acid to give

combretastatin A-4 in 82% yield. This approach to combretastatin A-4 is much more

straight forward, as it requires no protecting group and the alkene moiety flanking the

aryl units is constructed with greater stereoselectivity.



Br
CHO Br Br B(OH)2
( PPh3; CBr4 BuSnH
S CH2CI2 Pd(PPh3)4
H 20% OH 85% OH MeO OMe
OMe OMe OMe OMe
309 310 311 312

(Ph3P)4Pd,
70'/ Na2CO3, DME

MeO

MeO p7"
OMe
combretastatin A-4 OMe
Scheme 2-40. Synthesis of combretastatin A-4








More recently, Lawrence et al. disclosed another short synthesis of combretastatin

A-4 ur, 7h ,r.,, Suzuki coupling reaction.314 In this method, the coupling of the cis-

vinylbromo styrene 311 with trimethoxyboronic acid 312 produced combretastatin A-4 in

three steps and eliminated the necessary reduction step required in the earlier syntheses

(Scheme 2-40).

Combretastatin D-1 and D-2

Combretastatin D-l and D-2 were isolated in trace amount from the South African

folk medicine tree Combretum caffrum. 315,316 Structurally they differ from the A and B

series in that they contain a 15-member biaryl ether macrolactone resembling that of the

.,nJ" ring system. These natural products and their derivatives, Figure 2-14, have

OMe OMe OMe

0 N" ^0 0


30 35
313 314 315
OMe OMe
N0 Br 0

0 0

3160 OH 317 OH
Figure 2-14. Combretastatin D derivatives

been shown to be active against the murine P-388 lymphocytic leukemia cell line.

Combretastatin D-l shows a PS cell line activity corresponding to ED5o 3.3 gg/mL and

D- 2 activity corresponding to ED50 5.2Ig/mL.316 Structure-activity-relationships for

combretastatin D-1 and D-2 and their derivatives on the tubulin

polymerization process at various concentration have been determined38 and the results

are summarized in Table 2-17.








Table 2-17. Effect of tested compounds on tubulin polymerization.'
Degree of tubulin polymerization
Compound
0.25mM 0.5 mM 1.0 mM 2.0 mM
Colchicine 4% 3% 4% 0%
313 16% 19% 27% 35%
314 0% 20% 27% 39%
315 5% 28% 35% 54%
316 27% 47% 62% 76%
317 3% 21% 39% 67%
Sassay with epothilone B39 (20pM) resulted in 100% polymerization.


In contrast to colchicines and combretastatins A, which inhibit tubulin

polymerization, combretastatin D derivatives, Figure 2-14, favor to various degrees the

formation of microtubules. Derivative 316 seems to be most active (Table 2-17),

capable of promoting 76% polymerization at concentration of 2.0 mM.

The structural simplicity of the combretastatin D series and their important

biological activity mentioned above make them attractive lead for the development of

new drugs. Couladouros and colleagues pioneer in this area, have developed efficient

methods for total synthesis of combretastatin D-1 (Scheme 2-41), 320 and D-2 (Scheme 2-

42).321

In an approach to combretastatin D-2, Wittig-type elongation of commercially

available para-bromobenzaldehyde 318 by means of Ph3P=CHCO2Et afforded conjugated

ester, which was subjected to diisobutyl aluminium hydride (DIBAL) reduction and the

resulting allylic alcohol was protected as benzyl ether 319. Epoxidation of 319 using m-

CPBA (3-chloroperoxybenzoic acid) and regioselective ring opening with DIBAL322

afforded, after silylation with TBSCI, the desired oxygenated synthon 320. Ullman-type








coupling of arylbromide 320 with phenol 321,317 followed by deprotection of the terminal

carboxylic and hydroxyl functionalities afforded the saturated acid 322.

Macrolactonization of 322 via a modified Mitsunobu-type condition323'324 afforded 323 in

91 % yield. Subsequent desilylation, promoted by fluoride anions, afforded the alcohol

324 in 94 % yield. Dehydration of the alcohol 324 was achieved using Sammuelson's

Br Br Br OMe
1) PhaP=CHCO2Et; OH
Benzene, rt I 1) m-CPBA, CH2Cl12
2) DIBAL (2eq), 2) DIBAL, toluene
CHO CH2CI, 3) TBSCI, imidazole OTBS
318 3) NaH, BnBr, THF DMF 30
318 319 320 32
OBn OBn CO2Et

1) CuBr-Me2S;
K2CO, Pyridine
2) LiOH:THF:MeOH
OMe 3) H2,Pd/C, EtOAc
OMe OMe OMe

TBAF 1 DEAD, PhP, OTBS
S0 0 toluene 0 OH

324 OH 230 OTBS322
1) Ph3P, 2, COH
imidazole, PhMe
2) KF, DMSO, 1150C
OMe
0 OH

O 1BI3, Benzene ,
S N,N-dimethylaniline O
325 0
combretastatin D-2
Scheme 2-41. Total synthesis of combretastatin D-2

conditions325 by first replacing the hydroxyl group with iodine and subsequent

dehydrohalogenation in refluxing DMF in the presence of excess KF afforded methyl

combretastatin D-2 325. Combretastatin D-2 was finally prepared after demethylation of

325 in 17 % yield following Boger's procedure.317










Br Br
1) Ph3P=CHC02Et;
Benzene, rt I
2) DIBAL,CH2CI2 -
CHO 3)PivCI,Pyr. DMF OBn
318 326
PivO CuBr-Me,S
OBn K2CO3, Pyridine
OH O1) DMFK2C03, BnCI OH 329
J 2) Ph3P=CHCO2Et; l
Y" Benzene, rt CO2Et
CHO 3) H2,Pd/C, PhH 1) K3[Fe(CN)6], K2C03
327 38 K2[OsO2(OH)4]
Et2C DHQD)2PHAL
IBuOH:H20:1:1
2) TBSCI, imidazole
OBn DMF


332 333 combretastatin D-1

Scheme 2-42. Total synthesis of combretastatin D-1

Similarly, the synthetic approach to combretastatin D-1 began with an Ullman-

coupling of the monobenzylated catechol derivative 328, which was prepared from

commercially available aldehyde 327 by selective benzylation, followed by Wittig-type

carbon elongation and reduction of the resulting double bond, with aryl bromide 318. The

biaryl ether 329 was subjected to asymmetric dihydroxylation by means of the well-

known Sharpless catalyst (DHQD)2 PHAL (hydroquinidine 1,4-phthalazinediyl

diether)326 followed by bis-silylation of the diol to arrive at the bis-silylether 330.





73

Subsequent hydrolysis of the ester functionality with LiOH and reductive cleavage of the

pivaloate ester with DIBAL afforded acid 331. Macrolactonization of this saturated

disubstituted acid under the same protocol previously used for the synthesis of

combretastatin D-1 furnished diol 332,after desilylation of the TBS-groups with TBAF.

The epoxide functionality of combretastatin D-I was installed via the well-known

phosphoranes and oxyphosphonium salts catalyzed diol cyclodehydration protocol,327.328

by heating diol 332 with excess diethyl azodicarboxylate (DEAD) and Ph3P at 1450C for

40 min followed by hydrogenolysis of the benzyl group to obtain combretastatin D- in

high overall chemical yield of 83 % after crystallization.













CHAPTER 3
RESULTS AND DISCUSSION

Pseudomonas putida F39D and E. coli JM109 (pDTG601) oxidation of 1-
phenyl-1-ethanol

The utility of cis-dihydroxydihydrobenzene metabolites obtained from TDO-

catalyzed cis-dihydroxylation of aromatic hydrocarbons in asymmetric synthesis is firmly

established. Several recent reviews highlight the growing activity in this field24.26.226 as

investigations continue to probe for the enzyme's properties and functions.329"332 To date,

over three hundred homochiral diols have been identified from the oxidations of simple,

fused, and biphenyl-type aromatics.2426,226 Despite this solid endeavor in the field, very

few studies have been designed to study the enzyme's capacity in resolving substrates

with remote chiral center of type 334 (Scheme 3-1).

Ri R R2 R R

& OH

334 335 336
HO


OH
337 338
Scheme 3-1. Biooxidation of substrates containing benzylic chiral centers


To date, there are only five instances of biooxidation of such compounds have been

reported. Gibson et al. reported the production of diol 337 in conjunction with their

studies of di- vs. monohydroxylation.333 The study of this substrate has been further

elaborated by Cripps and co-workers.334 Several studies were recently conducted by








Boyd et al. on -phenylethan-1 -ol and other substrates.335 Ribbons and Ahmed336 studied

the biooxidation of phenyl ethanols (R-, S-, and racemate) with blocked mutants and

reported a slight preference for the S-enantiomer. Racemic sec-butylbenzene 338 and

racemic 1-phenylpropan-l-ol also were oxidized, but the stereochemical outcome was not

reported. In view of these limited examples, several substrates with one or more remote

chiral centers were oxidized by means of parallel experiments with both blocked mutants

P. putida F39/D and recombinant organism, E. coli JM109 (pDTG601) to investigate the

kinetic resolution capability of TDO in resolving enantiomer pairs.29 200.337 The following

sections summarized the results obtain from this kinetic resolution study. The absolute

stereochemistry correlation of new metabolites are also presented.

At the onset of this study, Ribbons and Ahmed's experiments with phenethyl

alcohol were repeated as control experiments to ensure the viability of P. putida F39/D

and to verify the kinetic resolution they observed. When phenylethanol was inoculated

with toluene-induced P. putida F39/D, the S-enantiomer was observed to be consumed at

a faster rate than the R-antipode resulting in a 6:4 ratio of diastereomers 337a and 337b,

respectively, as confirmed by 'H-NMR spectroscopy and chiral HPLC analyses of the

metabolites formed (Table 3-1). This preliminary finding suggested that TDO is capable

of resolving racemic enantiomers and encouraged further investigation into the scope of

its kinetic resolution capacity.

Since the recombinant organism E. coli JM109 (pDTG601) is easier to handle and

grow, further investigation of TDO's kinetic resolution capability was conducted with the

E. coli strain. When racemic phenylethanol 339 was subjected to TDO dihydroxylation

with JM 109 (pDTG601), both enantiomers were converted to a 1:1 mixture of








diastereomers 337a and 337b, as revealed by both 'H and 3C NMR spectroscopy. In

experiments where the biooxidation process was terminated before completion, analyses

of the non-converted phenylethanol on a chiral support GLC revealed no difference in the

rate at which each enantiomer was metabolized. Moreover, in runs where the R- and the

S-enantiomers were oxidized separately, the same phenomenon was observed. These

results were unexpected, since the same dioxygenase enzyme operates in both P. putida

F39/D and JM109 (pDTG601). Table 3-1 summarized the biotransformation results of

phenylethanols by both organisms.330,331

Table 3-1. Biotransformation of phenylethanol by JMI09 (pDTG601) and Pseudomonas
putida F39/D
Substrates E. coli JM109 pDTG601) Pseudomonas putida F39/D
OH ,OH OH >OH OH


O339 H OH OH OH
339 337a H 337b H 337a H 337b
(1 : 1) (6 : 4)

SOH ,yOH ,>OH
.OH OH

339a 337a 337a

OOH OH

SOH H
339b 337b 337b


In light of the observed differences between the two organisms in oxidation of

phenylethanol, it was speculated that each organism might have different substrate

transport mechanisms. For example, in the P. putida F39/D, it is possible that the S-

enantiomer was transported to the enzyme active site at a faster rate than the R-antipode,

resulting in its consumption at a faster rate. By contrast, in the E. coli organism, the rate








of transport for both enantiomers maybe equal, which would result in both enantiomers

being metabolized at the same rate. This result also could be rationalized by slight

differences in the enzyme topology between the two organisms, perhaps resulting from

post-translational folding.

The absolute stereochemistry of metabolites 337a and 337b derived from

biooxidation of 1-phenyl-l-ethanols has been established333,334,336 and the diimide

reduction of the metabolites 337a and 337b via potassium azodicarboxylate (PAD) to

340a and 340b, was carried out to confirm a diastereomeric 1:1 ratio of the metabolites

(Scheme 3-2).

OH OH .OOH OH

JM109 H MeOH,
(pDTG601) AcOH, PAD +
339 337 OH 340OH 340b OH
Scheme 3-2. Biooxidation of racemic phenylethanol by JM109 (pDTG601)

The biooxidation of phenylethanol in both racemic and enantio-pure forms

provided us with a glimpse at the kinetic resolution power of TDO. During this

preliminary study, no evidence of enantiodiscrimination was found, except perhaps a

slight preference for the oxidation of the S-enantiomer 339a of I-phenyl-l-ethanol when

the blocked mutant P. putida 39D was used. On the other hand, no preference was

detected in the oxidations mediated by TDO expressed in the recombinant organism E.

coli JM109 (pDTG601). Nonetheless, it was envisioned that if the size of the substituents

at the benzylic stereogenic center of substrate were to increase, the enzyme would be

more discriminatory in its kinetic resolution of racemic enantiomers. We reasoned that

phenyl-cyclohexanone 341 and phenylcyclohexanol 342, would be ideal for this task as








they would, if successfully oxidized, furnish "resolved" synthons that would be useful in

construction of the dibenzofuran core of the morphine alkaloid (Scheme 3-3).

HO"
IA
B
W NMe
C

Morphine

R- R- R-

N --, P O


341 X=O 343 344
342 X=H, OH
Scheme 3- 3. Chemoenzymatic approach to morphine synthesis--introduction

Biotransformation and Absolute Stereochemistry of New Metabolites

Phenyleyclohexene

Before such undertaking was carried out, simpler substrates of similar structural

motif of 341 and 342 were screened to test for TDO's tolerance of such structural feature.

Phenylcyclohexene 345 was chosen for this experiment because of its having a structure

similar to the morphine model substrates 341 and 342. Biooxidation of




JM109 t
S(pDTG601) OH -OH

S OH
345 346 347
Scheme 3-4. Biooxidation of 1-phenylcyclohexene

phenylcyclohexene was carried out initially in a shake-flask experiment, as described in

the experimental section and metabolite 346 was detected by UV (Xma,=302 nm). Full-

scale fermentation of phenylcyclohexene was implemented in a 15-L fermentor to








generate 346 in a crude yield of 3 g/L (98% conversion) as an off-white solid.330'331 Diol

346 was observed to be acid and thermal unstable and re-aromatized readily to 347 at

room temperature (Scheme 3-4). For characterization of the new metabolite, it was

necessary to convert the triene diol 346 to a more stable derivative. This was achieved by

subjecting triene diol 346 to diimide reduction with PAD to furnish a much more stable

diol 352. Absolute stereochemistry of 346 therefore was established indirectly through

the chemical synthesis of 352. This was accomplished through coupling vinyl bromide

348, whose absolute stereochemistry is known,338 via its trimethylstannane derivative 349

with cyclohexanone enol triflate 350339 that was derived from cyclohexanone through a

palladium-catalyzed Stille coupling process340 (Scheme 3-5). The resulting coupled

product 351 was treated with THF:TFA:H20/4:1:1 mixture of reagents to furnished 352

whose physical data matched with that of the compound derived from fermentation of

phenylcyclohexene, and established the absolute stereochemistry of metabolite 346 as

depicted in Scheme 3-5.


Br SnMe3 TfO
S-BULi, 330
S MeSnCI L (CH3CN)2PdCI2 -
348 349 DMF


346 "'' 352 -
Scheme 3- 5. Absolute stereochemistry correlation of metabolite 346








Phenylcyclohexane



JM109 re-arnmatize.
(pDTG601) OH OH

/ \ OH
353 354 355
Scheme 3-6. Biooxidation of phenylcyclohexane

To explore further TDO's substrate specificity exploring effort, substrate 353 was

subjected to dihydroxylation with JM109 (pDTG601) to yield dienediol 354 in a good

yield of 1.8g/L (97% conversion) (Scheme 3-6). Because of the intrinsic tendency of

dienediol 354 to dehydrate to the more stable phenol form 355, metabolite 354 was

reduced with PAD to diol 356 for characterization and absolute stereochemistry

correlation (Scheme 3-7). The absolute stereochemistry of metabolite 354 was therefore

indirectly established through chemical synthesis of intermediate 356. This was

accomplished via coupling of acetonide-protected vinyl bromide 348 with cyclohexyl

triflate 358341 via the transformation of the bromide to the Grignard compound in the

presence of LiCuC14342 to arrive at acetonide protected ether 357. This latter compound

was matched in all respects to the one derived from metabolite 354 thus establishing the

absolute stereochemistry of the diol 354 as depicted in Scheme 3-7.

Br
U1. Mg, Et2O,
PAD, AcOH, DMP LiCuCI4
OH MeOH OHp-TsOH O 2. 348

S348
354 356 357
Scheme 3- 7. Chemical proof of the absolute stereochemistry of metabolite 354








2-Phenylcyclohexene

2-Phenylcyclohexene was another substrate of TDO successfully oxidized during

this kinetic resolution study. The biooxidation of substrate 359 furnished a 1:1 mixture

of diastereomers 360a and 360b, which were subjected to diimide reduction to reduce the

least sterically hindered olefins. However this process only provided an inseparable

mixture diol 361 and 362 (Scheme 3-8). To transform this diastereomer mixture of 361

and 362 into intermediate 357, the biochemically installed cis-diols of the mixture were

protected as acetonide and Wilkinson's catalyst was used to reduce the unconjugated

olefin to furnish 357 whose absolute stereochemistry has been established as described in

Scheme 3-7.



JM109 + "'H PAD, AcOH. +
)(pDTG601) OH OH MeOH OH OH

OH OH OH OH
359 360a 360b L 361 362
(1 : 1)
1. DMP, p-TsOH
2. PPh3)3RhCI, H2,
MeOH, 30PSI







357
Scheme 3-8. Biooxidation of 2-phenylcyclohexene and absolute stereochemistry of new
metabolite

2-phenylcyclohexanol

At this point it was clear that the cyclohexyl-substituted arenes are acceptable

substrates for TDO and that both enantiomers were processed by the enzyme to

corresponding diasteromers. Trans-2-phenylcyclohexanol 363, which has two








neighboring stereogenic centers that closely resemble the intended morphine models 341

and 342, was converted to the corresponding dienediol as a 1:1 mixture of diastereomers

364 without any observable preference of the enzyme for either enantiomers. The

diastereomers 364 were reduced to 365 for characterization (Scheme 3-9).



'OH 21OH JMI o "0OH 1" OHPAD.MeOH "OH OH
+ (.pDTGol) OH c OH AcOH OH I OH

OH OH OH OH
363a 363b 364a 364b 365a 365b

Scheme 3-9. Biooxidation of trans-2-phenylcyclohexanol 343

Attempts to separate 365a and 365b via fractional recrystalization, flash-

chromatography, and derivatization of the reduced triols were unsuccessful. Derivatives

367a and 367b were generated via protection of the biochemically installed cis-diol as

ketal followed by acylation of the free hydroxyl group. This produced a white solid



"'OH SIhOH "OH OH
SOHO OH DMP


365a 365b 366a 366b

Pyridine,
3,5-dinitrobenzoylchloride

O2N NO2 O2N NO2




0-0

367a 367b

Scheme 3-10. Derivatization of triols 365 for separation




Full Text
51
(Figure 2-8).24 Such primary synthons can be further manipulated into "secondary
synthons (Figure 2-9) for synthetic applications that have proved very fruitful.24 In this
R 191
R = C02Me, Cbz, Ts
192 OH
X
194
X XX
R = Cbz, 0O2Me r = THS, TBS
Figure 2-8. Examples of primary synthons
manner, cyclohexanoids such as (+)-pinitol228 and D-myo-inositol derivatives229 have
been prepared by making use of the possibility of functionalizing every carbon atom of
the glycol in a stereo-control approach. Cyclopentanoid synthons on the other hand have
been used for the synthesis of prostaglandins and terpenes via ring-opening/closure
sequence.33 Rare carbohydrates such as D- and L- erythrose230 and L-ribonolactone231
were obtained from chlorobenzene as were pyrolizidine alkaloids.232 In addition, other
complex natural products such as (-)-zeylena, (+)-lycoricidine, (-)-specionin, and (+)-
pancratistatin also have been synthesized efficiently using cis-dienediols.


107
toluene dioxygenase (TDO) [E. coli JM109 pDTG601A],200 Scheme 3-29. The
configuration of the central inositol unit can be readily synthesized to reflect all nine
inositols, as we have demonstrated previously.429 432 The possibility also exists to
approach the monomer synthesis from protected synthons in such cases the number of
isomeric possibilities in intermediates of this type would approach the theoretical limit of
64. As delineated in recent papers on combinatorial possibilities of oligoinositols 433,434
the number of possible isomers increases exponentially with higher oligomers once
secondary and tertiary structures are developed through hydrogen bonded forms and (3-
turns initiated by a 1,2-trans-connection in 447 430432'435
We were interested primarily in generating relating low molecular weigh polymers
and integrating physical and chemical properties of such compounds with an eye toward
for the cross-linking as well as chiral separation materials.
To test our approach to this problem, bis-ally ether vinylbromide 452, (Scheme 3-
30) was generated and subjected to polymerization with Grubbs First-generation
catalyst. The bis-ether was generated by first protecting the cis-diols of the
bromodienediol as acetonide, followed by epoxidation of the unhindered double bond to
afford epoxide 448. The BF3-Et20 catalyzed opening of epoxide 448 with ally alcohol
led to a low yield of ether 449 at the expense of generating the conduritol dimmer 450.
This problem was circumvented by treating epoxide 448 first with Amberlyst resin to
afford trans-diol 451. The diol was then allylated with allyl bromide to afford allyl ether
452 in 98% yield (Scheme 3-30).


220
(353) Frisch, M. J. TV, G.W., T.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
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D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.;
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(361) Boyd, D. R.; Hand, M. V.; Sharma, N. D.; Chima, J.; Dalton, H.; Sheldrake, G. N.
J. Chem. Soc., Chem. Commun. 1991, 1630.
(362) Yamamura, M.; Moritani, I.; Murahashi, S. I. J. Organomet. Chem. 1975, 91,
C39.
(363) Wender, P. A.; Takahashi, H.; Witulski, B. J. Am. Chem. Soc. 1995,117, 4720.
(364) Wender, P. A.; Nuss, J. M.; Smith, D. B.; Suarez-Sobrino, A.; Vagberg, J.;
Decosta, D.; Bordner, J. J. Org. Chem. 1997, 62, 4908.
(365) Wender, P. A.; Dyckman, A. J. Org. Lett. 1999,1, 2089.
(366) Wender, P. A.; Fuji, M.; Husfeld, C. O.; Love, J. A. Org. Lett. 1999,1, 137.
(367) Wender, P. A.; Dyckman, A. J.; Husfeld, C. O.; Scanio, M. J. Org. Lett. 2000, 2,
1609.
(368) Wender, P. A.; Bi, F. C; Brodney, M. A.; Gosselin, F. Org. Lett. 2001, 3, 2105.


73
Subsequent hydrolysis of the ester functionality with LiOH and reductive cleavage of the
pivaloate ester with DIBAL afforded acid 331. Macrolactonization of this saturated
disubstituted acid under the same protocol previously used for the synthesis of
combretastatin D-l furnished diol 332,after desilylation of the TBS-groups with TBAF.
The epoxide functionality of combretastatin D-l was installed via the well-known
phosphoranes and oxyphosphonium salts catalyzed diol cyclodehydration protocol,327'328
by heating diol 332 with excess diethyl azodicarboxylate (DEAD) and PhiP at 145C for
40 min followed by hydrogenolysis of the benzyl group to obtain combretastatin D-l in
high overall chemical yield of 83 % after crystallization.


13
industrial microbial transformation. Recombinant technology which is widely used
today, allows over-expression of desired proteins such as biocatalysts, thereby resulting
in higher yields and diminished biological waste. Almost all chemical and
pharmaceutical companies rely on one or more forms of this technology to meet the
challenges of their trade. Table 2-2 lists some representative industrial whole-cells
biotransformations for the production of specialty chemicals.60
Oxygenation Reactions
Molecular oxygen is a powerful oxidizing agent essential for lifes functions in
aerobic organisms. Oxygen interacts with a variety of biological systems including
oxidases, peroxidases and oxygenases that are important in cellular energy metabolism
and respiration.61 These enzymes utilize transition metals, most commonly Fe and Cu, or
heteroaromatic systems such as pteridium62 or flavin to activate dioxygen into highly
reactive singlet and hydroxyl radical species for electron transfer and oxidation of organic
molecules.
One of the most widely distributed enzyme systems among living organisms that
utilize oxygen is the iron-dependent cytochrome P-450 monooxygenases.63'66 These
enzymes are particularly abundant in detoxification organs such as the liver, but they are
also found in microorganisms. This class of enzymes was discovered in 194767'68 and has
been found to be involved in a variety of biochemical pathways including steroid
metabolism,69'71 assimilation of non-carbohydrate carbon sources72,73 and detoxification
of xenobiotics. Numerous studies have been devoted to this enzyme system and the
cyclic mechanism for P-450 catalyzed oxygenation reactions was first postulated in
1971,74 and the crystalline structure of the enzyme has been elucidated in the presence75




JUUUJ
3
2
1
-O
ppm


ee
EtOAc
Eu(hfc)3)
HPLC
HRMS
IPTG
KOH
LAH
LDA
L-DOPA
m-CPBA
MOMC1
MPB
MPLC
MsCl
MTPA
NDO
NMNO
NOEDS
OD
P-450
PAD
enantiomeric excess
ethyl acetate
tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorato]
europium (HI),
high-performance liquid chromatography
high resolution mass spectroscopy
iso-propyl-P-D-thiogalacto-pyranoside
potassium hydroxide
lithiumaluminum hydride
lithium diisopropylamide
3,4-dihydroxy-phenyl alanine
mera-chloroperbenzoic acid
methoxymethyl chloride
2-( 1 -methoxyethyl)benzeneboronic acid
medium-pressure liquid chromatography
methylsulfonyl chloride
R-or S-a-methoxy-a-(trifluoromethyl)phenylacetic acid
naphthalene dioxygenase
A-methylmorpholine-A-oxide
nuclear overhauser effect difference spectroscopy
optical density
cytochrome P-450
potassium azodicaboxylate
viii


OAc
11 10 987 6 5 4321


82
neighboring stereogenic centers that closely resemble the intended morphine models 341
and 342, was converted to the corresponding dienediol as a 1:1 mixture of diastereomers
364 without any observable preference of the enzyme for either enantiomers. The
diastereomers 364 were reduced to 365 for characterization (Scheme 3-9).
Scheme 3-9. Biooxidation of trans-2-phenylcyclohexanol 343
Attempts to separate 365a and 365b via fractional recrystalization, flash-
chromatography, and derivatization of the reduced triols were unsuccessful. Derivatives
367a and 367b were generated via protection of the biochemically installed cis-diol as
ketal followed by acylation of the free hydroxyl group. This produced a white solid
Pyridine,
3,5-dinitrobenzoylchloride
Scheme 3-10. Derivatization of triols 365 for separation


70
Compound
Degree of tubulin polymerization
0.25mM
0.5 mM
1.0 mM
2.0 mM
Colchicine
4%
3%
4%
0%
313
16%
19%
27%
35%
314
0%
20%
27%
39%
315
5%
28%
35%
54%
316
27%
47%
62%
76%
317
3%
21%
39%
67%
assay with epothilone B (20pM) resulted in 100% polymerization.
In contrast to colchicines and combretastatins A, which inhibit tubulin
polymerization, combretastatin D derivatives, Figure 2-14, favor to various degrees the
formation of microtubules. Derivative 316 seems to be most active (Table 2-17),
capable of promoting 76% polymerization at concentration of 2.0 mM.
The structural simplicity of the combretastatin D series and their important
biological activity mentioned above make them attractive lead for the development of
new drugs. Couladouros and colleagues pioneer in this area, have developed efficient
methods for total synthesis of combretastatin D-l (Scheme 2-41),320 and D-2 (Scheme 2-
42).32'
In an approach to combretastatin D-2, Wittig-type elongation of commercially
available para-bromobenzaldehyde 318 by means of Ph3P=CHC02Et afforded conjugated
ester, which was subjected to diisobutyl aluminium hydride (DIBAL) reduction and the
resulting allylic alcohol was protected as benzyl ether 319. Epoxidation of 319 using m-
CPBA (3-chloroperoxybenzoic acid) and regioselective ring opening with DIBAL322
afforded, after silylation with TBSC1, the desired oxygenated synthon 320. Ullman-type


Scheme 2-41. Total synthesis of combretastatin D-2 71
Scheme 2-42. Total synthesis of combretastatin D-l 72
Scheme 3-1. Biooxidation of substrates containing benzylic chiral centers 74
Scheme 3-2. Biooxidation of racemic phenylethanol by JM109 (pDTG601) 77
Scheme 3- 3. Chemoenzymatic approach to morphine synthesis-introduction 78
Scheme 3-4. Biooxidation of 1-phenylcyclohexene 78
Scheme 3- 5. Absolute stereochemistry correlation of metabolite 346 79
Scheme 3-6. Biooxidation of phenylcyclohexane 80
Scheme 3- 7. Chemical proof of the absolute stereochemistry of metabolite 354 80
Scheme 3-8. Biooxidation of 2-phenylcyclohexene and absolute stereochemistry of new
metabolite 81
Scheme 3-9. Biooxidation of trans-2-phenylcyclohexanol 343 82
Scheme 3-10. Derivatization of triols 365 for separation 82
Scheme 3-11. Absolute stereochemistry of new metabolites obtained from 83
Scheme 3-13. Biotransformation of enantiomerically pure 2-phenylcyclohexanone 84
Scheme 3-14. Absolute stereochemistry of metabolites obtained from biotransformation85
Scheme 3-15. Biooxidation of diphenylmethane 86
Scheme 3-16. Proposed reaction sequence for the formation of cis-dienediol 86
Scheme 3-17. Mechanistic possibilities for cis-dihydroxylation 87
Scheme 3- 18. Biotransformation of cyclopropylbenzene 88
Scheme 3-19. Absolute stereochemistry correlation of metabolite obtain from
biotransformation of cyclopropylbenzene 90
Scheme 3- 20. Synthetic potential of cyclopropyl epoxide 389 91
Scheme 3-21. An environmentally benign approach to 3,4-catechols through 97
Scheme 3- 22. Transformations of 3,4-bromocatechol 98
Scheme 3- 23. Retrosynthetic design of catechol (or arene-crs-diol) synthons 99
xv


84
(Scheme 3-12). Characterization of this product mixture was complicated by the
formation of the hemiketal 369a and 369c. In order to get a better picture of this
Scheme 3-12. Biotransformation of 2-phenylcyclohexanone
biotransformation process, pure enantiomers of 368a and 368b, obtained from pyridinium
chlorochromate (PCC) oxidation of commerically available alcohols 363a and 363b,
were each oxidized separately with JM109 (pDTG601) (Scheme 3-13). Ketone 368a,
upon oxidation, formed hemiketal 369, while ketone 368b gave a mixture of ketone 369b
and hemiketal 369c.
Scheme 3-13. Biotransformation of enantiomerically pure 2-phenylcyclohexanone
The absolute stereochemistry of the newly formed metabolites 369a, 369b, and
369c were established by first reducing the diastereomer mixtures with NaBH4 in MeOH
to afford a mixture of diastereomers of alcohols 370a and 370b. The least hindered
olefins in 370a and 370b was reduced with PAD and the cts-diol moiety was protected as


29
Above-mentioned Baeyer-Villiger oxidations are not trivial to perform on a
preparative scale. Whole-cell transformations are difficult to perform, as some strains
such as Acinetobacter calcoaceticus are pathogenic and suffer from low yields due to
over-metabolism and/or side reactions catalyzed by other active enzymes in the
microorganisms. On the other hand, the use of isolated mono-oxygenases for Baeyer-
Villiger oxidations is not trivial, as these enzymes are linked to NADPH-recycling, which
is a notoriously difficult process.
The difficulties posed by BVMOs have led investigators to seek other looked
elsewhere for alternatives. In this context, using yeast genetically engineered to contain
the cyclohexanone monooxygenase of Acinetobacter calcoaceticus, has been an excellent
and safe substitute for preparative applications.172175
Dioxygenases Catalyze Biotransformation
The benzene nucleus originating from both natural and anthropogenic sources is
ones of the most abundant chemical structures in the biosphere. This class of compounds
has attracted a lot of attention from the scientific communities due to its adverse health
affects. The relationship between cancer and exposure to this class of chemical was first
suggested in 1761176 and further supported by evidence presented in 1775,177 showing a
high incidence of scrotal skin cancer in chimney sweeps through exposure to coal-tar.
The link between exposure to aromatic hydrocarbons and cancer was unequivocally
proven in 1930.178'179 Within the last few decades there has been a virtual explosion of
scientific reports on aromatic hydrocarbons as carcinogenic agents that interfere with a
multitude of biological processes.180 Such investigations have contributed to the
realization that aromatic hydrocarbons are among the main causes of human cancers.181
The impetus drive for a better understanding of the biodegradation aromatic


J\
11
10


93
chemical methods. As a result, this class of compounds continue to elude synthetic
chemists and their non-trivial preparation is reflected in the pricing (Figure 3-3).
396 397
$9/g (TCI) $38/g (Aldrich)
F
OH
OH
277 399
$60/5g (Aldrich) $240/25mg
$23.40/g (ACRU) (Sigma-Aldrich
rare chemical)
398
400
$???/250mg
(Sigma-Aldrich
rare chemical)
Figure 3-3. Some commercially available catechols
378
$66.30/25g (Acros)
401
$673.80/g (ION)
The mild enzymatic approaches to catechols, on the other hand, are limited and
under-explored with only three such methods having been reported in the
literature.29'384'283 Among these, Frosts enzymatic synthesis of the parent catechol from
glucose via an engineered aromatic amino acid pathway is one of the most noteworthy.
This example demonstrates the power of biocatalysis in the synthesis of commodity
chemicals.
With the above limited information, we set out to explore the practical implication
of whole cell fermentation with E. coli JM109 (pDTG602) for generation of substituted
catechols as an environmentally benign and straightforward preparative procedure. This
organism, which was developed by D.T. Gibson in 1989, allows for an approach to the
whole-cell biotransformation of aromatic substrates to corresponding catechols. The
genetic make up that is responsible for such transformation came from the Pseudomonas
species whose metabolic pathway of aromatic degradation was elucidated by Gibson and


213
(220) Boyd, D. R.; Sharma, N. D.; Boyle, R.; Malone, J. F.; Chima, J.; Dalton, H.
Tetrahedron: Asymmetry 1993, 4, 1307.
(221) Resnick, S. M.; Torok, D. S.; Gibson, D. T. J. Org. Chem. 1995, 60, 3546.
(222) Boyd, D. R.; Dorrity, M. R. J.; Hand, M. V.; Malone, J. F.; Sharma, N. D.;
Dalton, H.; Gray, D. J.; Sheldrake, G. N. J. Am. Chem.. Soc. 1991, 113, 666.
(223) Jenkins, N.; Ribbons, D. W.; Widdowson, D. A.; Slavin, A. M. Z.; Williams, D. J.
J. Chem Soc., Perkin Trans. 1 1995, 2647.
(224) Boyd, R.; Sharma, N. D.; Agarwal, R.; Resnick, S. M.; Schoken, M. J.; Gibson,
D. T.; Sayer, J. M.; Yagi, H.; Jerina, D. M. J. Chem. Soc., Perkin Trans. 1 1997,
1715.
(225) Kumar, A.; Ernst, R. R.; Wuthrich, K. Biochem. Biophys. Res. Commun. 1980,
95, 1.
(226) Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep., 1998, 15, 309.
(227) Bowers, N. Ph.D. Thesis, The Queen's University of Belfast, 1997.
(228) Ley, S. V.; Stemfeld, F. Tetrahedron 1989, 45, 3463.
(229) Ley, S. V.; Parra, M.; Redgrave, A. J.; Stemfeld, F. Tetrahedron 1990, 46, 4995.
(230) Hudlicky, T.; Luna, H.; Price, J. D.; Rulin, F. Tetrahedron Lett. 1989, 30,4053.
(231) Hudlicky, T.; Price, J. D. Synlett. 1990, 159.
(232) Hudlicky, T.; Luna, H.; Price, J. D.; Rulin, F. J. Org. Chem. 1990, 55, 4683.
(233) Hudlicky, T.; Rulin, F.; Tsunoda, T.; Luna, H.; Andersen, C.; Price, J. D. Isr. J.
Chem. 1991, 31, 229.
(234) Hudlicky, T.; Olivo, H. F. Tetrahedron Lett. 1991, 32, 6077.
(235) Johnson, C. R.; Pie, P. A.; Su, L.; Heeg, M. J.; Adams, J. P. Synlett 1992, 388.
(236) Mandel, M.; Hudlicky, T.; Kwart, L. D.; Whited, G. M. J. Org. Chem. 1993, 58,
2331.
(237) Hudlicky, T.; Price, J. D.; Rulin, F.; Tsunoda, T. J. Am. Chem. Soc. 1990, 112,
9439.
(238) Hudlicky, T.; Pitzer, K. K.; Stabile, M. R.; Thorpe, A. J.; Whited, G. M. J. Org.
Chem. 1996, 61, 4151.


37
better the substrate it is for the enzyme. For example, in the cts-dihydrodiols of
alkylbenzenes, the rate of metabolism for substrate with a methyl substituent was
observed to be twice as fast as for a substrate with n-butyl-substituent. The catabolism
rate for r-butylbenzene was even slower and the ci's-dihydrodiol metabolites of higher
homologues are unknown.194 Both naphthalene and biphenyl are good substrates for
TDO enzymes, perhaps indicating that flat, rigid molecules fit better into the enzymes
active site than molecules with bulky, flexible, substituents.
Toluene Dioxygenase Regioselectivity
The TDO-catalyzed cts-dihydroxylation of monosubstituted aromatic substrates is
very regioselective. With few exceptions of monohydroxylation, sulfoxidation,
dealkylation and desaturation of substituents, the cis-dihydroxylation of monosubstituted
benzenes by TDO occurred primarily at the benzene 2,3-bond. The hydroxylation at the
1,2-bond is the sole example of benzoic acid oxidation catalyzed by BZDO and no
examples of dioxygenase-catalyzed ris-dihydroxylation at a 3,4-bond have been found
(Scheme 2-22).
102
Scheme 2-22. Regioselectivity of dioxygenases in hydroxylation of aromatic substrates


54
OH OH OH
pancratistatin245,246 7-deoxypancratistatin246'247 lycoricidine248249
amino-inositol dimmer251
OH
PGE2a3335 (-)patchoulenone254 e-taxoids255 morphinan256
AB-ring system
zeylena257 Diels-Alder adduct258 Diels-Alder adduct258 Diels-Alder adduct258




63
combretastatins share a common binding site on tubulin and are competitive inhibitors of
colchicine, as summarized in Table 2-16, Experiment II. Combretastatin A-l was
found to be capable of displacing 97.8% of colchicine from its binding site, which
suggests its has an affinity for the active site on tubulin. These natural products also were
found to be potent inhibitors of microtubule assembly at 2-3pM (IC50) concentrations,
Table 2-16, Experiment I.
These biological properties demonstrated for combretastatin A-1 and B-1
encouraged further exploration and biological evaluation of this class of natural products.
Since the disclosure of combretastatin A-l and B-l, a series of combretastatins have been
reported, Figure 2-12. The majority of the members of this class of compounds have
been shown to be potent against a series of cancer cells lines. They interact with and
disrupt the vascular life support system of tumors, and in the process, starve and prevent
cancerous cells from metastasizing. The results of preclinical studies in which tumor


68
Br
Scheme 2-39. Synthesis of combretastatin A-4 via Suzuki cross-coupling reaction
alkyne was then sequentially treated with dicyclohexylborane and acetic acid to give
combretastatin A-4 in 82% yield. This approach to combretastatin A-4 is much more
straight forward, as it requires no protecting group and the alkene moiety flanking the
aryl units is constructed with greater stereoselectivity.
Br
CHO r^Br
PPh3; CBr4| ¡i¡l Bu,SnH,
^ ^ ch2ci2
HO 'Y' 20%
OMe
309
Pd(PPh3)4
85%
B(OH)2
OH MeO
OMe
combretastatin A-4 OMe
Scheme 2-40. Synthesis of combretastatin A-4


CHAPTER 5
EXPERIMENTAL SECTION
General Methods
All non-hydrolytic reactions were carried out in an argon atmosphere with standard
techniques for the exclusion of air and moisture. Glassware used for moisture sensitive
reactions was flame dried under reduced pressure or dried overnight at 80 C.
Tetrahydrofuran, benzene, and diethyl ether were distilled from sodium benzophenone
kethyl. Dichloromethane was distilled from calcium hydride. Microbial oxidations were
carried out in a 15 L B. Braun Biostat E fermentar and B. Braun Biostat D. All H and
13C NMR spectra were recorded at 300 and 75 MHz, respectively, using a Varian 300
spectrometer, unless otherwise stated. Chemical shifts are reported relative to TMS,
CDCI3 or DMSO-d6. Coupling constants are measured in Hz. Multiplicities in 13C NMR
are based on APT or DEPT. IR spectra were recorded on a Perkin-Elmer FT-IR (KBr)
and are given as wavenumbers in cm'1. Mass spectra are given with relative abundance
in parentheses. High resolution mass spectroscopy (HRMS) and elemental analyses were
performed at the University of Florida and Atlantic Microlab, Inc, respectively. Optical
rotations were recorded on the Perkin-Elmer 241 digital polarimeter (101 deg. cm2 g'1).
Melting points were obtained on a Thomas-Hoover capillary melting point apparatus and
are uncorrected. Analysis of enantiomeric mixtures was performed on a cyclodextrin
column (Chirasil DexCB, 25m x 0.32 mm with helium as the carrier gas). Reversed
phase HPLC analyses were performed on a C18 column (Phenomenex 250x10 mm) using
112


146
Polyhydroxylated Chiral Polymer Project
5-(5-AlIyIoxy-7-bromo-2,2-dimethyl-3a,4,5,7a-tetrahydro-
benzo[l,3]dioxol-4-yloxy)-7-bromo-2,2-dimeth
yl-3a,4,5,7a-tetrahydro-benzo[l,3]dioxol-4-ol (450). Into a flame-
dried 50 mL RBF equipped with a stir bar under argon atmosphere,
allylalcohol (0.56 g, 9.66 mmol) was dissolved in methylene
chloride (5 mL). Bromoepoxide 448 was then added and the reaction flask was cooled to -
78C for 20 min. BF3-Et20 was then added and the reaction was allowed to proceed over
night. The reaction was quenched with water (10 mL) and extracted with methylene chloride
(lOmL, 3X). The organic layers were combined and washed with 5% NaHC03 (10 mL,
IX), dry (MgS04) and evaporate to afford a reddish residue. The product was purified by
flash-chromatography (4: l/Hexanes:EtOAc) to yield the title compound as a white solid (256
mg, 56%). Rf = 0.65 (3:2/Hexanes:EtOAc); mp = 176-177C; [a]D31 = +47.35 (c, 0.665,
CHC13); IR (KBr) 3498, 2981, 1645, 1457, 1381, 1214, 1046, 998, 870, 513; 'H NMR (300
MHz, CDClj) 6 6.33 (d, 7= 1.71, 1H),6.28 (d, 7=1.71, 1H), 6.18-5.85 (m, 1H), 5.39-5.24 (m,
2H), 4.72 (d, 7=6.84, 1H), 4.47 (d, 7=6.84, 1H), 4.46 (s, 1H), 4.28-4.12 (m, 3H), 4.45-4.00
(m, 1H), 3.90 (d, J=8.30, 1H), 3.86-3.80 (m, 1H), 3.67 (m, 1H), 3.53 (t, J= 8.7, 1H), 1.58 (s,
3H), 1.57 (s, 3H), 1.42 (s, 3H), 1.41 (s, 3H); 13C NMR (75 MHz, CDC13) 8 135.1, 133.5,
132.8, 118.8, 118.1, 117.9,111.5, 111.1,84.4,84,1,78.7, 77.6, 77.20, 77.1,74.1,74.0,71.2,
28.2, 27.7, 25.9, 25.7. HRMS (FAB) caled for C2iH2907Br2: 551.0280. Found: 551.0045.
Anal, caled for C2iH2g07Br2: C, 45.67; H, 5.11. Found: C, 45.92; H, 5.28.
Br


92
biological activities. Morphine, for instance, is structurally small but its architecture
complexity has been an intriguing challenge for many synthetic chemists.
Figure 3-2. Oxygenated natural products containing a catechol subunit
Syntheses of simple catechols have been reviewed.375 For the most part, the
available chemical methods involve the cleavage of mono- or dialkylated catechols by
strong protic or Lewis acids under relatively harsh conditions.376'378 Other methods use
strong oxidants379'381 for direct hydroxylation of aromatic nucleus or cleavage of mono-
and diacetates.382'383 The arduous, non-selective and low-yielding difficulties associated
with these methods have hindered their widespread usage. Despite the effort invested,
there exists no reliable method for direct generation of catechols through conventional


211
(184) Gibson, D. T.; Subramanian, V. Microbial degradation of Organic Molecules',
Marcel Dekker: New York, 1984.
(185) Lipscomb, J. D.; Orville, A. M. Metal Ions Biol. Syst. 1992, 28, 243.
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(188) Boldt, Y. R.; Sadowsky, M. J.; Ellis, L. B. M.; Que, L., Jr.; ; Wackett, L. P. J.
Bacteriol. 1995,177, 1225.
(189) Whiting, A. K.; Boldt, Y. R.; Hendrich, M. P.; Wackett, L. P.; Que, L., Jr.
Biochemistry 1996, 35, 160.
(190) Gibson, D. T.; Chapman, P. J. Critical Reviews in Microbiology 1971, 199.
(191) Gibson D. T.; Zylstra, G. J.; Chauhan, S. Pseudomonas: biotransformations,
Pathenogenesis and Evolving Biochemistry; American Society for Microbiology,
1990.
(192) Resnick, S. M.; Lee, K.; Gibson, D. T. J. Ind. Microbiol. 1996,17, 438.
(193) Butler, C. S.; Mason, J. R. Adv. Microb. Physiol. 1997, 38, 48.
(194) Gibson, D. T.; Koch, J. R.; Kallio, R. E. Biochemistry 1968, 7, 2653.
(195) Rossiter, J. T.; Williams, S. R.; Cass, A. E. G.; Ribbons, D. W. Tetrahedron Lett.
1987, 28, 5173.
(196) Reineke, W.; Knackmuss, H. J. Biochim, Biophys. Acta 1987, 542, 412.
(197) Whited, G. M.; McCombie, W. R.; Kwart, L. D.; Gibson, D. T. J. Bacteriol. 1986,
166, 1028.
(198) Khan, A. A.; Wang, R. F.; Cao, W. W.; Franklin, W.; Cerniglia, C. E. Int. J. Sys.
Bacteriol. 1996, 46, 466.
(199) Reiner, A. M.; Hegeman, G. D. Biochemistry 1971,10, 2530.
(200) Zylstra, G. J.; Gibson, D. T. J. Biol. Chem. 1989, 264, 14940.
(201) Allen, C. C. R.; Boyd, d. R.; Dalton, H.; Sharma, N. D.; Haughey, S. A.;
McMordie, R. A. S.; McMurray, B. T.; Sheldrake, G. N.; Sproule, K. Chem.
Commun. 1995, 119.
(202) Gibson, D. T.; Cardini, G. E.; Mseles, F. C.; Kallio, R. E. Biochemistry 1970, 9,
1631-5.


374
140
120 100 80
60 40 20 0 ppm


207
(102) Weiler, E. W.; Droste, M; Eberle, J.; Halfmann, H. J.; Weber, A. Appl.
Microbiol. Biotechnol. 1987, 27, 252.
(103) Perlman, D.; Titius, E.; Fried, J. J. Am. Chem. Soc. 1952, 74, 2126.
(104) Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M.; Academic Press:
London, 1989, p 175.
(105) Fried, J.; Thoma, R. W.; Gerke, J. R.; Herz, J. E.; Donin, M. N.; Perlman, D. J.
Am. Chem. Soc. 1952, 74, 3692.
(106) Sawada, S.; Kulprecha, S.; Nilubol, N.; Yoshida, T.; Knoshita, S.; Taguchi, H.
Appl. Environ. Microbiol. 1982, 44, 1249.
(107) Olah, G. A.; Ernst, T. D. J. Org. Chem. 1989, 54, 1204.
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(109) Zimmer, H.; Lankin, D. C.; Horgan, S. W. Chem. Rev. 1971, 71, 229.
(110) Adachi, K.; Takeda, Y.; Senoh, S.; Kita, H. Biochemica et Biophysica Acta 1964,
93, 483.
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Communications 1973, 55, 888.
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69.
(115) Wiseman, A.; King, D. J. Topic Enzymol. Ferment. Biotechnol. 1982, 6, 151.
(116) Daly, J. W.; Jerina, D. M.; Witkop, B. Experientia 1973, 1129.
(117) Boyd, D. R.; Campbell, R. M.; Craig, H. C.; Watson, C. G.; Daly, J. W.; Jerina,
D. M. J. Chem. Soc. Perkin Trans. 1 1976, 2438.
(118) Klibanov, A. M.; Berman, Z.; Alberti, B. N. J. Am. Chem. Soc. 1981,103, 6263.
(119) Agarwal, M.; Shukla, O. P. Journal of Microbial Biotechnology 1988, 3, 18.
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1987, 76, 177.


34
oxygenase system, designated toluene dioxygenase (TDO).~05 The individual components
of toluene dioxygenase have been purified and are organized as shown in Scheme 2-21.
The enzyme is believed to go through the following sequence of actions during the
catabolism of aromatic substrate. Electrons are initially accepted from NADH by a
flavoprotein, reductase tol 206 and transferred them to a small iron-sulfur protein,
ferredoxin-roL207 FerredoxinjoL then reduces the terminal dioxygenase, which is a large
iron-sulfur protein that has been designated ISPtol208 The reduced oxygenase catalyzes
the addition of both atoms of molecular oxygen to toluene to form ds-toluene
dihydrodiol. The further metabolism of ds-toluene dihydrodiol involves NAD+-
dependent dehydrogenation reaction mediated by cis-toluene dihydrodiol dehydrogenase
to form 3-methylcatechol.209 Extradiol cleavage at the 2,3 position by 3-methylcatechol
2,3dioxygenase yields 2-hydroxy-6-oxo-2,4 -hepatadienoate, which is further
metabolized to 2-hydroxypenta-2,4-dienoate and acetate.
The enzyme system of toluene dioxygenase has been extensively studied since its
first disclosure in 1968. Individual enzymes within the metabolic pathway depicted in
Scheme 2-21 have been purified and characterized.206 Mutant P. putida with defective in
each of the bacteria genes also have been identified and isolated.210,211 A mutation that
occurred at the todD gene resulted in a mutant strain P. putida 39/D. This mutant strain
would no longer grow on toluene or benzene as the sole source of carbon. When P.
putida 39/D was grown on glucose, in the presence of benzene, ds-benzenediol
accumulated in the culture medium suggesting that the organism no longer carried a fully
functional enzyme system as disclosed in Scheme 2-21. It appears that the toluene
dihydrodiol dehydrogenase (todD) gene was disabled so that the metabolic pathway of


17
substrate. These two processes are among the most common and important in aromatic
hydrocarbon oxidation.
Dehydrogenases
SubH2 + Donor *- Sub + DonorH2
| cofactor-recycling |
Oxygenases
SubH2 + Donor + 02 Monooxygenases Sub + DonorH2 + h20
cofactor-recycling
Sub + 02 Di-Oxygenases Sub02
Oxidases
SubH2 + 02 Sub + H202
02 + 2e' Oj2' +,2H H202
02 + 4e' 202' 2H20
Peroxidases
2 SubH + H2O2 2 Sub* + 2 H2O Sub-Sub
Sub + H202 SubO + H20
Scheme 2-10. Enzymatic oxidation reactions
Monooxygenases Catalyze Biotransformation
Monooxygenases are ubiquitous in nature and serve many important biological
functions including detoxification of xenobiotics. They have a broad range of substrates
and can tolerate a very wide range of functional groups (Scheme 2-11).
Although the reaction mechanisms of various monooxygenases differ greatly
depending upon the sub-type of enzyme, their mode of oxygen-activation is the same in
term of their requirement of NAD(P)H and oxygen for function. In the following
sections, a series of monooxygenases that catalyze the mono-hydroxylation of alkanes,
arenes, and alkenes as well as enzymes that performed Baeyer-Villiger reactions are
described.


80
Phenylcydohexane
(pDTG601)
JM109
OH
re-aromatize.
OH
-OH
353
354
355
Scheme 3-6. Biooxidation of phenylcydohexane
To explore further TDOs substrate specificity exploring effort, substrate 353 was
subjected to dihydroxylation with JM109 (pDTG601) to yield dienediol 354 in a good
yield of 1.8g/L (97% conversion) (Scheme 3-6). Because of the intrinsic tendency of
dienediol 354 to dehydrate to the more stable phenol form 355, metabolite 354 was
reduced with PAD to diol 356 for characterization and absolute stereochemistry
correlation (Scheme 3-7). The absolute stereochemistry of metabolite 354 was therefore
indirectly established through chemical synthesis of intermediate 356. This was
accomplished via coupling of acetonide-protected vinyl bromide 348 with cyclohexyl
triflate 358341 via the transformation of the bromide to the Grignard compound in the
presence of LiCuCU342 to arrive at acetonide protected ether 357. This latter compound
was matched in all respects to the one derived from metabolite 354 thus establishing the
absolute stereochemistry of the diol 354 as depicted in Scheme 3-7.
354
356
357
Scheme 3- 7. Chemical proof of the absolute stereochemistry of metabolite 354


223
(407) Ray, W. H. Canadian Journal of Chemical Engineering 1991, 69, 626-9.
(408) Wulff, G. Angew. Chem., Int. Ed. Engl. 1989, 28, 21.
(409) Wulff, G.; Krieger, S. Macro mol, Chem. Phys. 1994, 195, 3665.
(410) Okamoto, Y.; Nakano, T. Chem. Rev. 1995, 94, 349.
(411) Crabtree, R. H. The Organometallic Chemistry of the
Transition Metals', 3rd ed.; Wiley and Sons: New York, 2001.
(412) Eleuterio, H. S. In US Patent 3,074,918,. USA, 1963.
(413) Rousch, W. R.; Hawkins, J. M.; Grubbs, R. H. Chemtract: Org. Chem. 1988, 1,
21.
(414) Okamoto, Y.; Hatada, K. Chromatographic Chiral Separations; Marcel Dekker:
New York, 1988.
(415) Williams, D. J. Angew. Chem., Int. Ed. Engl. 1984, 23, 690.
(416) Coates, G. W.; Waymouth, R. M. J. Am. Chem. Soc. 1993, 115, 91.
(417) Ito, Y.; Ihara, E.; Murakami, M. Angew. Chem., Int. Ed. Engl. 1992, 1509.
(418) Yokota, K.; Haba, O.; Satoh, T. Macromol. Chem. Phys. 1995,196, 2383.
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Commun. 1987, 7, 663.
(420) Kakuchi, T.; Harada, Y.; Hashimoto, H.; Satoh, T.; Yokota, K. J. Macromol. Sci.,
Pure Appl. Chem 1994, A31, 751.
(421) Kakuchi, T.; Kawai, H.; Katoh, S.; Haba, O.; Yokota, K. Macromolecules 1992,
25, 5545.
(422) Kakuchi, T.; Haba, O.; Fukui, N.; Yokota, K. Macromolecules 1995, 28, 5941.
(423) Haba, O.; Morimoto, Y.; Uesaka, T.; Yokota, K. Macromolecules 1995, 28, 6378.
(424) Nakano, T.; Okamoto, Y.; Sogah, D. Y.; Zheng, S. Macromolecules 1995, 28,
8705.
(425) Nakano, T.; Sogah, D. Y. J. Am. Chem. Soc. 1995, 117, 534.
(426) Satoh, T.; Yokota, K.; Kakuchi, T. Macromolecules 1995, 28, 4762.
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222
(388) Mason, H. S. J. Am. Chem. Soc. 1947, 69, 2241.
(389) Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K.
J. Med. Chem. 2000, 43, 2731.
(390) Brown, M. L.; Rieger, J. M.; Macdonald, T. L. Bioorg. Med.
Chem. Ber. 2000, 8, 1433.
(391) Pettit, G. R.; Lippert, J. W. I; Herald, D. L. J. Org. Chem. 2000, 65, 7438.
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(393) Cassar, L. J. Organomet. Chem. 1975, 93, 253.
(394) Sonogashira, K. In Metal-catalyzed Cross-coupling Reactions', Diederich, F.,
Stang, P. J., Eds.; Wiley-VCH: New York, 1998, p Chapter 5.
(395) Sonogashira, K. In Comprehensive Organic Synthesis', B. M. Trost, Ed.;
Pergamon: New York, 1991; Vol. 3, p Chapter 2.4.
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1730.
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J., Eds.; Wiley-VCH: Weinheim, Germany, 1998, p 49-89.
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2002, 40,2116-2133.




LIST OF SCHEMES
Scheme page
Scheme 1 -1. Aromatic degradation pathway of Pseudomonas putida F39D 3
Scheme 2-1. Vinegar production 6
Scheme 2-2. Reaction catalyzed by Bacterium xylinum 8
Scheme 2-3. Saccharomyces cerevisiae catalyzes acyloin condensation as the key step in
the production of L-Ephedrine 9
Scheme 2-4. Microbial dehydrogenation of D-sorbitol to L-sorbose as the key step in the
Reichstein-Grussner synthesis of L-ascorbic acid 9
Scheme 2-5. Two-stage fermentation for the production of 2-keto-L-gulonic acid from
D-glucose 10
Scheme 2-6. Microbial 11-a-hydroxylation of progesteron 11
Scheme 2-7. Chemical synthesis of cortisone 11
Scheme 2-8. Monooxygenase cytochrome P450s catalytic cycle 14
Scheme 2-9. Catalytic cycle of flavin-dependent mono-oxygenases 16
Scheme 2-10. Enzymatic oxidation reactions 17
Scheme 2-11. Monooxygenase catalyzed reactions 18
Scheme 2-12. Microbial hydroxylation of 1,4-cineole 19
Scheme 2-13. Possible mechanism in biodegradation of aromatics by eukaryotic cells .21
Scheme 2-14. Microbial hydroxylation of aromatics 21
Scheme 2-15. Mechanism of the Baeyer-Villiger reaction 25
Scheme 2-16. Progesterone metabolism by BVMOs in Cylindrocarpon radicicola 26
Scheme 2-17. Chemoenzymatic syntheses of various useful target molecules involving
the biotransformation by CHMO of bicyclo[3.2.0]-type ketones 27
xiii


40
are carried out primarily by dioxygenases such as NDO and BPDO215'216 that are capable
of accommodating larger substrates. A strong preference for dihydroxylation at the bay-
region bond is observed with a minor proportion of oxidation occurring at non-K
positions (Figure 2-3). It is noteworthy that little evidence has been obtained for attack at
the region of highest electron density in the angular arenes, Le. the K-region, with either
the NDO or BPDO enzymes.
Figure 2-3. Preferred regioselectivity of mono-and polycyclic arene cis-dihydrodiols
The presence of one or more heteroatoms in the arene ring has been found to exert
a strong influence on the regioselectivity of dioxygenase-catalyzed dihydroxylation. For
bicyclic heteroarenes quinoline,217 isoquinoline,217 quinoxaline,217 quinazoline,217
benzothiophene,218 2-methylbenzo-thiophene,219 benzofuran,220 and 2-
methylbenzofuran, the cis-dihydroxylation of the carbocyclic arene ring is generally
preferred over the heterocyclic ring. In some cases, mixture of metabolites have been
observed as a result of oxidation at both carbocyclic and heterocylic rings as in the case
of the benzothiophene and benzofuran, Table 2-7.218 219 However, where no carbocyclic


36
Several key features of TDO-catalyzed cis-dihydroxylation processes have been
elucidated, which have helped shed light into the nature of the active sites of toluene
dioxygenase enzyme system.
For example, substrates with charged or very polar substituents, such as, hydroxy,
amino, carboxy, nitro, sulfonic acid and sulfone, directly linked to the benzene ring have
been found to be resistant to TDO dihydroxylation. In spite of these limitations, the
dioxygenase enzymes are capable of accepting arene substrates with a wide range of
substituents.
The degree of substitution and types of substituents on the aromatic rings also are
key components in the TDOs catabolism processes of aromatic substrates. The
likelihood a substrate is accepted by TDO decreases as the degree of substitution
increases. When multiple electron-withdrawing substituents such as halogens are
present, they reduce electron density from the aromatic ring thus hindering the activity of
the aromatic moiety. On the other hand, when electron-donating groups are present, the
reactivity of the substrates are increased; however, electron rich substituents tend to
destabilize the rir-dihydrodiol metabolite and in many cases metabolites containing such
substituents have been found to decompose spontaneously during the biotransformation
or purification procedure. For the disubstituted substrates, a 1,3-arrangement of
substituents suffer the so-called meta effect that would result in an increased inhibitory
effect on the ring dihydroxylation rate in comparison with the substrates with substituents
at the 1,2 or 1,4 positions.
The size of substituents on the aromatic ring also plays a role in determining the
ability of TDO to accept the substrates. The smaller the substituent on the arene, the


Scheme 2-18. Degradation of aromatics by microbial dioxygenases 30
Scheme 2-19. Dihydroxylation of aromatic hydrocarbon by TDO 30
Scheme 2-20. Modes of catechol cleavage 31
Scheme 2-21. Catabolic pathway for toluene used by Pseudomonas putida FI. Genes
designations for individual proteins are shown in parentheses 33
Scheme 2-22. Regioselectivity of dioxygenases in hydroxylation of aromatic substrates37
Scheme 2-23. Regioselectivity of TDO in biooxidation of 1,4-disubstituted aromatic
substrates 38
Scheme 2-24. Regioselectivity of TDO in biooxidation of 1,2-disubstituted substrates. 38
Scheme 2-25. Chemoenzymatic approach to 3,4-cis-dihydrodiols 39
Scheme 2-26. Regioselectivity of 1,3-disubstituted aromatic substrates 39
Scheme 2-27. Biotransformation of 2-bromonaphthalene 43
Scheme 2-28. Preparation of Mosher ester 153 43
Scheme 2-29. Preparation of di-MTPA esters 158 for determination of c-dienediol 2
absolute stereochemistry 45
Scheme 2-30. Preparation of boronic ester 160 for determination of cA-dienediol
absolute stereochemistry 45
Scheme 2-31. Historically important application of arene cis-diols in synthesis 50
Scheme 2-32. Synthesis of methylcatechol from furfural 57
Scheme 2-33. Synthesis of bromocatechol 57
Scheme 2-34. Synthesis of bromocatechol from quaiacol 58
Scheme 2- 35. Synthesis of 3-fluorocatechol 58
Scheme 2-36. Synthesis of 3-methoxyatechol 59
Scheme 2-37. Total synthesis of combretastatins A-l and B-l 66
Scheme 2-38. Suzuki approach to combretastatin A-4 67
Scheme 2-39. Synthesis of combretastatin A-4 via Suzuki cross-coupling reaction 68
Scheme 2-40. Synthesis of combretastatin A-4 68
xiv


Br


LIST OF TABLES
Table Bags
Table 2-1. Examples of commercial steroid processes 11
Table 2-2. Some representative industrial whole-cells biotransformations 12
Table 2-3. Microbial epoxidation of alkenes 23
Table 2-4. Epoxidation of branched alkenes by Nocardia corallina 24
Table 2-5. Enzymatic Baeyer-Villiger oxidation of prochiral ketones 27
Table 2-6. Microbial Baeyer-Villiger oxidation of monocyclic ketones 28
Table 2-7. TDO-catalyzed biotransformation of substrate containing heterocyclic ring
system 41
Table 2-8. Preferred absolute configuration of cis-dihydrodiol metabolites of
monosubstituted benzenes 46
Table 2-9. cis-Dihydrodiols from ortho-substituted benzenes 47
Table 2-10. cis-Dihydrodiols from meta-substituted benzenes 47
Table 2-11. cis-Dihydrodiols from para-substituted benzenes 48
Table 2-12. cis-dihydrodiols from polycyclic aromatic hydrocarbons 49
Table 2-13. The use of cis-dienediols in synthesis 53
Table 2-14. Combretastatin A-1 andB-1 'H-NMR* 61
Table 2-15. Combretastatin A-1 andB-1 13C-NMR* 62
Table 2-16. Inhibition of microtubule assembly and biding of colchicine to tubulin by
combretastatin A-l and B-l 64
Table 2-17. Effect of tested compounds on tubulin polymerization.2 70
Table 3-1. Biotransformation of phenylethanol by JM109 (pDTG601) and Pseudomonas
putida F39/D 76
x


128
1-phenyl-l-ethanol. This afforded the acetals 369a and 369b together with the ketone
369c as a 2:1:1 mixture, respectively.
The proof of structures and the correlation of absolute stereochemistry was
provided by transforming the two enantiomers 368a and 368b separately as for racemic
368.
(4S,4aR,9aft)-4,6,7,8,9,9a-Hexahydro-4a//-dibenzofuran-4,5a-
diol (369a). XmK= 266 nm; Rf = 0.58 (1:1 hexane:EtOAc); [a]D28 +130.3
(c 0.7, MeOH); IR (film) 3378, 1449 cm1; 'H NMR (300 MHz, CDC13) 8
6.09 (dd, J= 4.8, 9.4, 1H), 5.98 (dd, J= 4.3, 9.4, 1H), 5.84-5.78 (m, 1H),
369a
4.76- 4.70 (m, 1H),4.25 (bs, lH),4.12(bs, 1H),3.97 (t, 7=5.5, 1H), 2.47- 2.38 (m, 1H),2.18-
2.06 (m, 1H), 1.83 -1.74 (m, 1H), 1.62-1.56 (m, 3H), 1.48 1.08 (m, 3H); 13CNMR(75
MHz, CDCb) 8 144.3, 127.8, 124.2, 114.9, 106.9, 79.7,61.6,49.1,33.9, 30.2,24.0, 22.8;
MS (Cl): m/z 192 (12), 191 (40), 132 (32), 128 (35), 119 (32), 107 (31), 105 (29), 91 (100),
83 (21), 81 (37); HRMS (Cl) m/z caled for Ci2H1302 (M-OH): 191.1072. Found: 191.1090.
(lS,5S,6R)-5,6-Dihydroxybicyclohexyl-l,3-dien-2-one
(369b). Rr = 0.42 (1:1 hexane:EtOAc); [cx]D28 +58.1 (c 0.6, MeOH); IR
(film) 3352, 1702, 1444 cm1; 'H NMR (300 MHz, CDC13) 8 6.06 (dd, J=
4.7, 9.5, 1H), 5.95 (dd, /= 5.6, 9.5, 1H), 5.81 5.77 (m, 1H), 4.74-4.69
(m, 1H), 4.20 (bs, 2H),3.95 (t, J= 5.6, 1H), 2.45 2.38 (m, 1H), 2.14-2.04 (m, 1H), 1.80 -
1.72 (m, 1H), 1.68-1.58 (m, 3H), 1.46-1.12 (m, 3H); 13CNMR (75 MHz, CDC13) 8213.2,
139.2, 129.2, 124.0, 114.6, 68.7, 68.5, 42.1, 30.1, 27.3, 25.9, 22.8; HRMS (Cl) m/z caled for
C,2H1703 (M+H): 209.2675. Found: 209.2669.
369b




145
solution stirred at 0 C for 2 h. H2O (30 mL) was added and the mixture extracted with
CH2CI2 (3 x 25 mL). The organic extracts were washed with saturated NaHCC>3 (3X, 25
mL), H20 (30 mL), dried (MgSCL) and concentrate in vacuo. The product was purified by
flash-chromatography affording the title compound as a red oil (80 mg, 80%). 1H NMR (300
MHz, CDCI3) 8 6.91 (d, J= 8.79,1H), 6.65 and 6.51 (AB system, J=12 Hz, 2H), 6.56 (d,
J=8.79 Hz, 1H), 6.48 (s, 2H), 5.19 (s, 2H), 5.12 (s, 2H), 3.82 (s, 6H), 3.65 (s, 6H), 3.60 (s,
3H), 3.57 (s, 3H); 13C NMR (75 MHz, CDC13) 8 153.2, 153.0, 149.3, 147.4, 139.0, 137.3,
132.7, 130.2, 126.1, 125.6, 125.3, 107.8, 106.4, 99.4, 98.8, 61.0, 57.7, 57.5, 56.2, 56.0.
Combretastatin A-l. Compound 442 (50 mg) was
dissolved in a 1:1 mixture of 3N HCLTHF solvent (5 mL)
and allowed to stir at ambient temperature for 3 hr. The
HO
OMe
MeO MeO OMe
combretastatin A-1
reaction mixture was diluted with H20 (10 mL) and extracted with CH2C12 (3X, 15 mL).
Organic layers were combined, dried (Na2S04), concentrated in vacuo, and the desired
product was purified by flash chromatography to afford the title compound as a white solid
(30.6 mg, 77% yield), mp = 140-142 C; Rf = 0.42 (l:l/hexane:EtOAc), H NMR (300 MHz,
CDCL) 8 6.76 (d, 7= 8.64, 1H), 6.59 and 6.53 (AB system, 7=12 Hz, 2H), 6.52 (s, 2H), 6.38
(d, 7=8.4, 1H), 5.41 (s, D20 exchangeable, 2H), 3.86 (s, 3H), 3.83 (s, 3H), 3.67 (s, 6H); ,3C
NMR (75 MHz, CDCI3) 8 153.0, 146.5, 141.8, 137.4, 132.8, 132.7, 130.5, 124.3, 120.6,
118.0, 106.1, 103.1,61.1,56.4, 56.1.


" 11111" 111 11111
200 180
160
T-p-T-T-T-TT
120
100




144
Combretastatin B-l: Compound 441 (49 1
mg) was dissolved in a 1:1 mixture of 6N HC1:THF
HO OH
solvent (2 mL) and allowed to stir at ambient
OMe
OMe
combretastatin B-l
temperature for lh. The reaction mixture was diluted with H20 (10 mL) and extracted
with CH2CI2 (3X, 15 mL). Organic layers were combined, concentrated and the desired
product was purified by flash chromatography to afford colorless oil (42 mg). Rf = 0.73
(7:3/hexane:EtOAc), IR (film) 3428, 2937, 1629, 1590, 1509, 1487, 1420, 1288, 1239, 1126,
1093, 1006; H NMR (300 MHz, CDC13) 8 6.57 (d, J= 8.28, 1H), 6.42 (s, 1H), 6.38 (d, 7=
8.47, 1H), 5.41 (s, D20 exchangeable, 1H), 5.39 (s, D2O exchangeable, 1H), 3.85, (s, 3H),
3.83 (s, 6H), 3.82 (s, 3H), 2.93-2.79 (m, 4H); 13C NMR (75 MHz, CDC13) 8 153.1, 145.5,
142.2, 138.4, 136.1, 132.4, 121.6, 120.3, 105.5, 102.5, 61.1, 56.3, 56.2, 36.9, 32.1;
MS(FAB): mJz 335 (M+, 71), 334 (100), 307 (24), 289 (15), 181 (58), 167 (30), 155 (45),
154 (130), 138 (53), 128 (30), 120 (21), 111 (37), 109 (41), 107 (36), 105 (30); HRMS
(FAB) mJz caled for C18H23O6: 335.1494. Found: 335.1490.
MOM-protected combretastatin A-l (442). MOf"'
Borane THF (5 mL, of a 1 M solution in THF) was MOMO 4 h 4 A OMe
slowly added to distilled cyclohexne (1.02 mL, 10 ^eO ^eO OMe
442
mmol) in THF (5 mL) at 0 C. This mixture was stirred for lh at 0 C, affording a dense
white precipitate after 10 min. The alkyne 426 (100 mg, 0.24 mmol) in THF (5 mL) was
added and the resulting yellow mixture was stirred for 1 h at 0C after which time the
precipitate had dissolved giving a clear yellow solution. The solution was allowed to warm
to ambient temperature and stirred for 30 min. HO Ac (1.72 mL, 30 mmol) was added and the


140
J=8.9, 1H), 6.45 (d, J=8.9, 1H), 3.76 (s, 3H). I3C NMR (75 MHz, CDC13) 8 149.0, 144.4,
136.2, 123.3, 105.8, 102.7, 57.1; MS(CI): m/z 218 (7), 209 (7), 185 (100), 93 (122); Anal,
caled for C7H7Br03: C, 38.38; H, 3.22. Found: C 38.49; H, 3.23.
3-Methoxy-6-methylbenzene-l,2-diol (407) was obtained through
biooxidation of 4-methyllanisole with E. coli JM109 (pDTG602) as described
in the general procedure for preparation of catechols. The title metabolite was
purified by flash chromatography (1:1 hexane:EtOAC) to yield 5.2 g (0.65g/L) of white
crystalline compound; mp 92-93.5 C (lit. 92.5-93.5 C); H NMR (300 MHz, CDC13)8 6.61
(d, 7= 8.3, 1H), 6.38 (d, 7=8.3, 1H), 5.49 (bs, 2H), 3.84 (s, 3H), 2.20 (s, 3H); l3C NMR (75
MHz, CDC13) 8 145.2, 142.4, 132.3, 120.7, 117.9, 102.5, 56.3, 15.2.
OMe
407
3-Cyclohexyl-benzene-l,2-diol (408) was obtained through
biooxidation of cyclohexylbenzene with E. coli JM109 (pDTG602) as
described in the general procedure for preparation of catechols. The catechol |
was purified by flash chromatography (1:1 Hexane:EtOAc) and recrystalized 408
from toluene:octane to afford 0.55g (0.07g/L) of a white solid. Rf = 0.68 (3:2
/hexane:EtOAc); mp 139-140 C; H NMR (300 MHz, DMSO-d6) 8 9.13 (s, OH, 1H), 7.95
(s, OH, 1H), 6.63-6.51, (m, 3H), 2.96-2.72 (m, 1H), 1.88-1.62 (m, 5H), 1.44-1.12 (m, 5H);
I3C NMR (75 MHz, CDC13) 8 143.0, 141.4, 134.5, 120.4, 119.2, 112.8, 37.6, 33.2, 27.1, 26.4.


90
Br Br
Reagents:
i) PAD; AcOH; MeOH;
¡i) DMP; p-TsOH;
Hi) Mg; l2; EtzO
¡v) Pd(PPh3)4; THF; reflux
v) THF:H20:TFA/ 4:1:1
vl) Ac20, pyridine; DMAP, CH2CI2;
vil) JM109 (pDTG601)
Scheme 3-19. Absolute stereochemistry correlation of metabolite obtain from
biotransformation of cyclopropylbenzene
Synthetic Application Potential of Cyclopropylbenzene Metabolite
From a synthetic viewpoint the cyclopropyl system offers a wide variety of
opportunities for further synthetic manipulations and can be viewed as a chiral
vinylcyclopropane synthon for incorporation into future synthetic endeavors (Figure 3-1).
vinylcyclopropane
cycloadditions and
rearrangements
I\
AA/WWWV
.r
O'"
Suzuki
coupling
OH -
nucleophilic \ electrophilic
tethers tethers
Figure 3-1. Exploration of reactivity of oxygenated cyclopropylhexanyl systems
Two obvious applications are the tethered [5+2] annulation to 7-membered ring
systems, discovered and developed by Wender,363'369 and the simple thermolysis to
cyclopentenes,370 or the cascade radical cyclizations with concomitant unraveling of the


108
Br
Conditions: i) DMP, p-TsOH; ii) mCPBA,CH2CI2; iii) BF3-Et20, Allylalcohol;
iv) Amberlyst Resin v) DMF; NaH, Allylbromide
Scheme 3-30. Synthesis of monomer bis-allyl ether 438
Allylether 452 was subjected to ADMET conditions with Grubbs First-generation
catalyst. When the reaction was performed in dilute methylene chloride, no
polymerization occurred and only eight-member ring ether 453 formed (Scheme 3-31).
When the reaction conducted as neat, again, no polymer formed but only a mixture of
deallylated products 449 and 454 plus the starting material were isolated.
Br
Scheme 3-31. Polymerization of 452 with Grubbs' First-generation catalyst


66
ester, which allowed for specific methylation of the 4-hydroxy group by dimethylsulfate.
The reaction mixture was quenched with concentrated hydrochloric acid to afford
dihydroxybenzadehyde 293,310,311 which was then treated with diisopropylethylamine and
i-butyldimethylsily chloride (TBDMSC1) in dimethylformamide (DMF) to form 2,3-f-
butyldimethylsilyl ether 294.312The synthesis of combretastatin A-l was then
accomplished by coupling TBDMS-protected aldehyde 294 with phosphonium salt 296,
(readily prepared via treating 3,4,5-trimethoxybenzyl bromide 295 with r-BuLi and
followed with triphenylphosphine in THF) to give a 92.5% yield of a 9:1 ratio of TIE
combretastatin A-l (Scheme 2-37). The products mixture was reduced to via
hydrogenation to arrive at combretastatin B-l. The disadvantage of this approach to
combretastatins is the resulting mixture of products requires separation.
Conditions: i) Borax, NaOH; ii) (Me0)2S02; iii) HCI; iv) DMF;EtN(iPr)2,TBDMS-CI; v) BuLi,
THF, -15C-rt; vi) TBAF, THF; vii) H^C/Pd, MeOH
Scheme 2-37. Total synthesis of combretastatins A-l and B-l


43
tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorato] europium (HI), Eu(hfc)3),
has been useful in the determination of optical purity and absolute configuration of new
metabolites. For instance, this approach has been used in determining the optical purity
of metabolites obtained from biooxidation of 1-bromonaphthalene and 2-
bromonaphthalene. In this effort, metabolites 147 and 148, obtained from the
biooxidation of corresponding 1-bromonaphthalene, were separated and converted into
bis-MPTA protected ester 150 via catalytic hydrogenation and acylation of the cis-
hydroxyl groups with MPTA (Scheme 2-27).
H-NMR analysis of 150 derived from 147 and 148 in presence of an equal molar
quantity of Eu(hfc)3 showed a single signal for benzylic methane at 8 = 6.70 ppm that
matched with the standard sample of (-)-cw-tetrahydronaphthalene diol obtained from
hydrogenation of the product of microbial dihydroxylation of naphthalene. By contrast,
analysis of the 1 H-NMR of the racemic Mosher esters 153 obtained from chemical
Os04, NMO
acetone/H20
OH
(+)-R-MTPA, DMAP
DCC, CH2CI2
152
Scheme 2-28. Preparation of Mosher ester 153
OMTPA
153


This dissertation was submitted to the Graduate Faculty of the College of Liberal
Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
May 2003
Dean, Graduate School


CHAPTER 4
CONCLUSIONS
Toluene dioxygenase (TDO) is a powerful and excellent enzyme system that has
great potential for synthetic application. Using a combination of enzymatic oxidation and
chemical manipulation of the metabolites from TDO have allowed us to construct a
variety of highly oxygenated natural products and their derivatives. A series of bicyclic
substrates with and without remote chiral centers was oxidized and the enzyme was
found to be nondiscriminatory toward enantiomers. Cyclopropylbenzene was oxidized to
probe for mechanistic details of the biotransformation process. The E. coli JM109
(pDTG602) was used successfully for the generation of substituted catechols that can be
used in synthetic applications. For example, combretastatin A1 and B1 were successfully
synthesized
111




11
Scheme 2-6. Microbial 11-a-hydroxylation of progesteron
chemical syntheses of similar steroids were available from deoxycholic acid 26, they
were deemed lengthy, complicated and uneconomical, as in the case of cortisone 27
synthesis, which involves 31 steps (Scheme 2-7).58 The microbial 11-a-hydroxylation
deoxycholic acid 26 cortisone 27
Scheme 2-7. Chemical synthesis of cortisone
of progesterone quickly made the commercialization of steroid derivatives such as
cortisone a reality by reducing the price of this commodity from $200 to $6 per gram.
Table 2-1. Examples of commercial steroid processes
Substrate
Product
Microorganism
Manufacturer
Progesterone
11 a -Hydroxy-
progesterone
Rhizopus nigricans
Upjohn company
Component S
Cortisol
Curvularia lunata
Pfizer, Inc.,
Gist-Brocades
9a-Fluoro-
9a-Fluoro-16a-
Streptomyces
E. R. Squibb & Sons
cortisol
hydroxycortisol
roseochromogens
Lederle Laboratories
Cortisol
Prednisolone
Arthrobacter simplex
Schering Corp.
Progesterone
1 -Dehydro-
testololactone
Cylindrocarpon
radicicola
E. R. Squibb & Sons
p -Sitosterol
Androstadiendione
Mycobacterium sp.,
M. fortuitum mutants
G. D. Searle & Co.
Upjohn company


132
of the broth with base-washed ethyl acetate. This process provides
essentially pure dienediol which can be further purified by flash-chromatography
(Hexanes:EtOAc / 4:1) to afford a white solid; Rt=0.4 (hexanes:EtOAc / 1:1); mp=45-46C
(decomposition); [a]D28 +1 1.39 (c 1.75, CD3OD); IR (film) 3274, 3008, 2911, 1453, 1090,
992, 804 cm'1; 'H NMR (300 MHz, Acetone-d6) 8 5.75-5.85 (m, 1H), 5.54-5.66 (m, 2H),
4.17-4.26 (m, 1H), 3.63-3.81 (m, 2H), 3.40-3.51 (d, 7=7 Hz, 1H), 1.48-1.62 (m,lH), 0.40-
0.79 (m, 4H); 13C NMR (75 MHz, Acetone-d6) 8 130.2, 127.4, 124.7, 118.7, 71.2, 69.0, 15.8,
7.6, 6.6.
2-CycIopropylphenol was isolated as byproduct from the
biotransformation of cyclopropyl benzene. The phenol was purified by
distillation to afford a clear oil; Rf = 0.42 (hexane:EtOAc / 8:1); IR (film)
3536,1609, 1580, 1491,752 cm'1; H NMR (300 MHz, DMSO-de) 8 9.26 (s, 1H), 6.86-
6.92 (m, 1H), 6.63-6.76 (m, 3H), 2.02-2.12 (m, 1H), 0.80-0.87 (m, 2H), 0.55-0.61 (m, 2H);
13C NMR (75 MHz, CDClj) 8 155.8, 129.4, 125.9, 124.5, 119.0, 114.6, 9.3, 7.7; MS(EI) mJz:
136 (23), 134 (100), 133 (24), 115 (27), 107 (39), 106 (21), 105 (28), 91 (34), 89 (41), 78
(24), 77 (96), 61 (43); HRMS (El) mJz calcd for C9H10O (M+): 134.0732. Found: 134.0732.
7-Cyclopropyl-2,2-dimethyl-3a,4,5,7a-tetrahydrobenzo[l,3]
dioxole (386). To a well-stirred solution of diol 387 (0.150 g, 1.0 mmol) in
2,2-dimethoxypropane (DMP) (20 mL), a catalytic amount of p-
toluenesulfonic acid was added. The reaction mixture was stirred for 30
minutes then was quenched with 5% NaHC03 (2 mL). Solvent was removed under reduced
pressure. The residue was diluted with brine (20 mL) and extracted with CH2CI2 (3 x 30




131
a solvent mixture 4:1:1 THF:H20:TFA and allowed to stir at room temperature for one hour.
Organic solvent was then removed under vacuum and the residue was diluted with ethyl
acetate (10 mL), washed with 5% NaHC03 (5 mL, 2X). The organic layers were combined,
dry with Na2SC>4, and concentrated. The product was purified by flash chromatography to
afford a white solid (84 mg, 82%). Physical and spectroscopic data for this compound
obtained from this route matched with the PAD reduced diol 374 obtained from the
biooxidation, and thus proved the absolute stereochemistry of 373.
(IS, 2R)-3-Benzyl-cyclohex-3-ene-l,2-diol (374). An
aliquot of the crude extract (2 g, 9.9 mmol) from biooxidation of
substrate 372 was subjected to PAD (5.76 g, 29.7 mmol) reduction 374
according to the general procedure. The product was purified by chromatography (1:1
hexane:EtOAc) to afford a white solid (0.98 g, 49%). mp = 73.5-75C; [a]o26-160.4 (c 1.0,
MeOH); IR (KBr) 3281, 3024, 1602, 1494, 1453, 1260, 1078 cm1; 'H NMR (300 MHz,
DMSO-d6) 8 7.32-7.23 (m, 2H), 7.21-7.12 (m, 3H), 5.36 (s, 1H), 4.47 (d, J = 5.7, 1H), 4.31
(dd, 7 = 5.7, 1H), 3.62 (t, J = 4.5, 1H), 3.44-3.31 (m, 3H), 2.11-2.08 (m, 2H), 1.70-1.52 (m,
1H), 1.50-1.38 (m, 1H); 13C NMR (75 MHz, CDCI3) 8 139.7, 137.4, 129.2, 128.6, 127.2,
126.4, 69.8, 68.2,41.0,25.5,24.1; MS(FAB) m/z: 188(12), 187(100), 185(26), 169(19), 93
(31), 91(38); HRMS (Cl) m/z caled for Ci3H1602:(M+-0H): 187.1123. Found: 187.1125.
Anal, caled for C13H1602: C, 76.44; H, 7.90. Found: C, 76.20; H, 7.93.
Cyclopropylbenzene project
3-Cyclopropylcyclohexa-3,5-diene-l,2-diol (383) was isolated from
the crude mixture collected from the biotransformation of cyclopropyl
benzene. This was accomplished by centrifugation of the cells and extraction


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
y' jY
Tomas Hudlicky, Chair
Professor of Chemistry
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Dennis L. Wright,!
Assistant Professor of Chemistry
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
William R. Dolbier, Jr. Y
Professor of Chemistry *
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Steven A. Benner
Distinguished Professor of
Chemistry
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Lonnie O. Ingram '
Distinguished Professor of
Microbiology and Cell Science


125
carried out in neat dimethoxypropane with catalytic amount of p-toluenesulfonic acid
yields the corresponding ketals 366a and 366b. The crude mixture (378 mg, 1.5 mmol)
was dissolved in dry CH2C12 (10 mL) at 0 C and CBr4 (746 mg, 2.25 mmol) was added
followed by PPh3 (393 mg, 2.25 mmol) portion wise over 10-15 min. The mixture was
left for another 2 h at ambient temperature. Chromatography (2.5% EtOAc in hexane)
yielded the two diastereomeric bromides (441 mg, 90%) which were not characterized
but were reduced in the general manner using BusSnH and AIBN in dry, refluxing THF
affording crude 357 (377 mg, 80%). Purification by chromatography (EtOAc:hexane
1:9) yielded an analytical sample with all spectroscopic and physical data in accord with
357 obtained from the synthetic approach described below. [a]c28 + 49.6 (c 0.5, CHCI3).
2-(2,2-Dimethyl-3a,6,7,7a-tetrahydro-
benzo[l,3]dioxol-4-yl)-cyclohexanol (366a) and 2-(2,2-
Dimethyl-3a,6,7,7a-tetrahydro-benzo[l,3]dioxol-4-yl)-
cyclohexanol (366b). The triol mixture of 365a and 365b 366a 366b
(117 mg, 0.550 mmol) was dissolved in dimethoxypropane (DMP) (5 mL). To this
mixture a catalytic amount of pTsOH was added and the reaction was monitored by TLC.
After 30 min, the DMP solvent was removed under reduced pressure and the products
were purified by flash chromatography (4:1 hexane:ethyl acetate) to afford the 366a and
366b alcohols mixute as a yellow oil (110 mg, yield 79%). Rf = 0.79 (2:3 hexane:ethyl
acetate). *H NMR (acetone) 6 5.74 (m, 2 H), 4.23 (m, 2H), 4.24 (m,2H), 2.20-2.03
(m,2H), 2.06-2.01 (h, 2 H) 2.0-1.79 (m, 6 H), 1.78-1.53 (m, 10 H), 1.56-1.36 (m, 2 H),
1.34-1.11 (m, 20 H); 13C NMR (Acetone d6) 5: 138.46, 137.7, 130.3, 127.3, 108.3, 108.1,


95
Recognizing the importance of substituted catechol in synthesis, we set out to
investigate the capability of E. coli JM109 (pDTG602) to generate substituted catechols.
The biotransformation of various substituted arenes with E. coli JM109 (pDTG602) is
summarized in Table 3-2. The results illustrate the broad substrates specificity of the
enzyme as well as its excellent regioselectivity.
The size of the substituents on the aromatic ring seems to play a role in the
metabolism of the substrate by TDDH. The smaller the substituents the better the
substrate it is for the enzyme, thus affording higher yields. The bulkier the substituents
on the ring, the lower the yield of the metabolites. Although the yields reported in Table
3-2 appear to be low, they are a lot better when compared with the yield from multi-step
syntheses of similar catechols through conventional chemical methods. Comparison of
efficiency of non-enzymatic and biocatalytic syntheses is shown in Table 3-3. The
overall chemical yields are indicated as reported in the literature. The E value385 is
calculated by dividing the weight of all materials used in synthesis by the weight of
product. The EMY386 is calculated by expressing the weight of product as a percentage
of all non-benign mass used in the manufacturing. Solvents used for extractions are not
taken into account in either calculation because they would be recycled during
manufacturing. It is obvious that the EMY values are more informative about the
efficiency of the process than the overall yields, because the latter do not provide any
information about the process itself. It is also evident that the single step biooxidation is
far more efficient than traditional chemical preparation, even though the yield is limited
by the concentration of product at 1-2 g/L, beyond which it is toxic to the organism.283


HO
140
VO
O


97
(pDTG602) have been found exclusively at the 1,2-positions for mono-substituted
aromatic substrates. For 1,2- or 1,3 disubstituted substrates, the hydroxylation sites can
be predicted using Boyds model in his approach 3,4-cir-diols (Figure 3-4).
Based on the predictions of the above-mentioned model, in which the bulkiest
substituent of the ring directs the hydroxylation sites, substrates can be designed to
generate 2,3- or 3,4-catechols. The versatility of this method could allow investigators to
gain access to a variety of important catechols that otherwise would be impossible to
obtain through chemical means. Such catechols would facilitate synthesis of a variety of
naturally occurring amino acids, aromatic antibiotics, and many isoquinoline and
quinoline alkaloids.
For instance, substrates such as 413 (Scheme 3-21) can be designed to provide 3,4-
functionalized catechols following the electrochemical reduction of the directing halogen.
Such regiochemistry is commonly found in naturally occurring amino acids, aromatic
antibiotics, and many isoquinoline and quinoline alkaloids.
X = Cl, CN, Br, F, Alkyl
Vinyl, Propagyl
Scheme 3-21. An environmentally benign approach to 3,4-catechols through
The 3,4-catechol class of compounds, such as l-bromo-3,4-catechol 400 as shown
in Scheme 68, created through this technique can be used in a variety of chemical
transformations.


64
Table 2-16. Inhibition of microtubule assembly and biding of colchicine to tubulin by
combretastatin A-l and B-l
MeO^
rr
MeO
MeO^
rT
,OH
MeO^
V
rT
.OH
OMe
L I
OMe
'OH
280 I
'OH
281
OMe
Combretastatin A-1
OMe
Combretastatin B-1
Podophyllotoxin
Steganacin
Colchicine
Experiment I
Experiment II
Drug
Microtubule Assembly
Colchicine Binding
IDso (pM)
% of control
Combretastatin A-l
2
2.2
Combretastatin B-l
3
14
Combretastatin
11
34
Podophyllotoxin
3
13
Steganacin
6
49
Colchicine
6
Defined as the drug concentration inhibiting the extent of microtubule assembly by
50% (adapted from published work by Pettit et al.)291,298
cells were treated with combretastatins showed that these agents produced tumor
shrinkage and ultimately tumor disappearance. Thus, this class of natural products may
offer a new approach to cancer treatment that is a radical departure from conventional
drugs.
Among the combretastatins known to date, (Figure 2-12), combretastatin A-4 has
been identified as the most active vascular targeting and anti-angiogenesis agent.299 In
1998, clinical trials with its water-soluble phosphate derivative300301 291, Figure 2-13,


48
Dioxygenase-catalyzed cis-dihydroxylation of para-disubstituted benzene
substrates, on the other hand, produces cis-dihydrodiols with a wide range of
enantiopurity.212 The relative sizes of the substituents (CF3>I>Br>Cl~Me>F>H) provide
an empirical method for both the prediction of regioselectivity and facial selectivity
during TDO-catalyzed cis-dihydroxylation of benzene substrates. The general structure
depicted in Table 2-1 1226 indicates that the preference for a particular enantiomer will be
related to the differential in size between the large R and small R groups.
Table 2-11. cis-Dihydrodiols from para-substituted benzenes
R'
103
TDOi
V
R'
104
If: R>R'
^OH
vOH
HO""
R'
105
R>R
Compounds
R
R
% ee
176
I
F
88
177
I
Me
80
178
Br
Me
37
179
Cl
Me
15
180
Br
F
56
181
I
F
88
182
cf3
I
>98
183
cf3
Me
>98
groups. The absolute configuration of the preferred ct's-dihydrodiol enantiomer from
TDO-catalyzed oxidation for di-substituted benzene substrates is summarized in Figure
R>R' R>R' R>R'
Figure 2-6. Preferred absolute configuration of cis-dihydrodiol metabolites of
disubstituted benzenes


42
regioselectivity of multi-substituted substrates are more difficult because of (1)
uncertainty about the effective size of substituents in different conformations, (2) product
instability and (3) further dioxygenase-catalyzed oxidation of the substituent.
Toluene Dioxygenase Stereoselectivity
The ability of toluene dioxygenase to oxidize a wide range of aromatic substrates
while maintaining a high degree of optical purity is a remarkable biological process. The
combination of such properties has no doubt made TDO an attractive catalyst for the
production of synthetically useful chiral synthons. Indeed, the practicality of TDO in
organic synthesis continues to fuel much of the interest within the chemoenzymatic
synthesis arena. Today, many research groups from across the globe continue to devote
their time and energy to gain better understanding of the enzymes properties as well as
its synthetic potential. Thanks to their efforts, more than three hundred c/i-dihydrodiol
metabolites of arenes have been reported and much information about the properties of
the enzyme are known.
As the use of c-dienediols for synthesis becoming more routine, the search for
general methods for determining the optical purity and absolute stereochemistry of new
metabolites is underway.221'222 Traditional methods employed for such purpose have
included the synthesis of these metabolites,220 their chemical transformation to known
compounds, conversion of the metabolites to diastereomers and derivatives,204 X-ray
crystallography,223 stereochemical correlation5,6,8 circular dichroism,224 and NMR
spectroscopy.221,222,225
For example, the use of Moshers chiral acids, R-or S-a-methoxy-a-
(trifluoromethyl)phenylacetic acid (MTPA), to form NMR-distinguishable
diastereoisomeric esters in the presence of homochiral lanthanide shift reagent such as,


CHAPTER 6
SELECTED SPECTRAL DATA
152


151
1.49 (s, 6H), 1.40 (s, 6H); 13C NMR (75 MHz, CDC13) 8 138.6, 114.8, 114.7, 112.3, 109.6,
80.3, 79.4, 77.3, 76.7, 75.6, 74.2, 71.7, 70.9, 30.4, 29.9, 29.5, 29.3, 28.0, 26.5, 25.6, 24.3.
ADMET polymerization of diene 460 to polymer 461.
A 10 mL round-bottomed flask equipped with a magnetic stir bar,
monomer 460 (240 mg, 0.61 mmol) was charged and degassed
through several freeze-pump-thaw cycles under high vacuum.
Grubbs first generation catalyst (5 mg) was then added under argon atmosphere. After the
addition of Grubbs catalyst, very slow to moderate bubbling of ethylene was observed. The
bubbling reaction was then exposed to intermittent vacuum until the viscosity increased,
followed by exposure to high vacuum to remove ethylene, which is continuously generated
during the course of the polymerization. The reaction was maintained at room temperature
until the increase in viscosity prevented stirring. At this point, the reaction temperature was
slowly raised to 45C over a period of 3-5 days until bubbling ceased. The reaction mixture
was then cooled to room temperature, and quenched by exposure to air. The viscous residue
was dissolved in ethyl acetate and passed through a short column of silica to furnish a brown
viscous oil. H NMR (300 MHz, CDCl,) 8 5.50-5.40 (b, 2H), 4.25-4.15 (b, 4H), 3.80-3.65
(m, 4H), 3.31 (s, 2H), 2.20-1.80 (b, 4H), 1.75-1.55 (m, 4H), 1.50 (s, 6H), 1.33 (s, 6H). The
polymer 461 was dissolved in a solvent mixture of 4:1:1/THF:TFA:H20 (5 mL) to arrive at
fully hydroxylated polymer 462.


114
and the solvent evaporated. The crude diols were purified by recrystallization
(hexanes:ethyl acetate) to yield the pure diols. Only minimal data ('H NMR and/or
optical rotation) were obtained for metabolites that are unstable at room temperature.
Screening for new substrates
One liter of induced cells (OD>45) from the fermentor was centrifuged (3000 rpm,
10 min) and re-suspended in 300 mL of 0.20 M KPO4 buffer (pH 7.0) supplemented with
2% glucose. The substrate was added in small portions while the pH was maintained at
7.0 with 10 M NaOH. The progress of the biotransformation was monitored by UV
absorbance of the broth in the region of 260-270 nm and by TLC. Successfully
metabolized substrates were subjected to large scale biotransformation in a 15-L
fermentor.
Preparation of catechols
Substituted catechols were made using the same biotransformation procedures
described for E. coli JM109 (pDTG601) except E. coli JM 109 (pDTG602) was used
instead. All catechols were isolated by extraction of the cells-freed fermentation broth
with ethyl acetate, followed by drying (Na2S04) and concentrating of the organic layers
in vacuo. Analytical samples of catechols were obtained by distillation, recrystallization,
and/or flash chromatography.
General procedure for diimide potassium azodicarboxylate (PAD) reduction of the
diene diols
Potassium azodicarboxylate (PAD) (2.0 eq.) was added in small portions with
vigorous stirring to an ice-cold solution of the diene diol (1.0 eq.) in methanol (10 mL/
mmol), followed by dropwise addition of a solution of acetic acid (7.0 eq.) in methanol.
The solution was allowed to slowly warm to room temperature. Saturated NaHCCb


LIST OF ABBREVIATIONS
AIBN
azoisobutyronitrile
aq.
aqueous
BDO
benzene dioxygenase
Bn
benzyl
borax
sodium borate-decahydrate
BPDO
biphenyl dioxygenase
BVMO
Baeyer-Villiger Monooxygenase
Bz
benzoyl
BZDO
benzoate dioxygenase
CHMO
cyclohexanone monooxygenase
CSP-HPLC
chiral stationary phase-high pressure liquid chromatography
DEAD
diethylazodicarboxylate
DIBAL
diisobutyl aluminium hydride
DMAP
A.A-dimethyl-d-amino pyridine
DMDP
2,5-dideoxy-2,5-diimino-D-mannitol
DMF
dimethylformamide
DMP
2,2-dimethoxypropane
DMS
dimethylsulfide
DMSO
dimethylsulfoxide
E. coli
Escherichia coli
Vll


129
(4S,4aff, 9351-4,6,758,9,9a-Hexahydro-4a/-d¡benzofuran-4,5a-
diol (369c). ^.max=266nm; Rf = 0.49 (1:1 hexane:EtOAc); [a]o2S 126.3
(c 0.5, MeOH); IR (film) 3371, 1454 cm'1; 'H NMR (300 MHz, CDCI3) 8
6.05 (dd, J= 4.8, 9.4, 1H), 5.92 (dd, J= 4.3, 9.4, 1H), 5.80-5.75 (m, 1H),
4.72-4.68 (m, lH),4.20(bs, lH),4.10(bs, 1H),3.98 (t, 7=5.5, 1H), 2.48-2.40 (m, 1H),
2.17-2.07 (m, 1H), 1.85-1.77 (m, 1H), 1.60-1.54 (m, 3H), 1.45- 1.10 (m, 3H); 13C NMR
(75 MHz,CDC13) 144.8, 127.8, 124.4, 114.4, 105.7, 79.7,61.7,49.0, 33.8, 30.1,24.2,22.7;
HRMS (CI) m/z caled for C12H,302 (M-OH): 191.1072. Found: 191.1081.
Correlation of absolute stereochemistry of 369a, 369b, and 369c. The crude
mixture from the biotransformation of ()-368 was reduced with NaBFLj in MeOH to afford a
mixture of diastereomers of alcohols 370a and 370b. This product mixture was reduced with
PAD according to the general procedure and the biochemically installed eij-diol was
protected as a ketal using dimethoxypropane and catalytic amount of p-toluenesulfonic acid.
The crude mixture (756 mg, 3 mmol) was dissolved in dry CH2CI2 (25 mL) at 0C and CBr4
(1.49 g, 4.5 mmol) was added, followed by portion wise addition of PPh3 (786 mg, 4.5
mmol) over 15 min. The mixture was left for another two hours at ambient temperature.
Chromatography (2.5% EtOAc in hexane) yielded 702 mg (85%) of the two diastereomeric
bromides, which were reduced in the general manner using Bu3SnH and AIBN in dry,
refluxing THF, affording crude 357 (589 mg, 72 %). Purification by chromatography
(EtOAc:hexane 1:9) yielded an analytical sample and all spectroscopic and physical data
were in accord with synthetic 357, ([a]D28 + 50.1 (c 0.6, CHCI3)).
369c


PAHs
polycyclic aromatic hydrocarbons
PCBs
polychlorinated biphenyls
PCC
pyridinium chlorochromate
P. putida
Pseudomonas putida
pyr.
pyridine
TBDPSC1
chloro-ie-butyldiphenylsilane
TBDMSC1
r-butyldimethylsily chloride
TBSC1
chloro-rert-butyldimethylsilane
TBTH
tributyl-tin hydride
TDDH
toluene cis-dihydrodiol dehydrogenase
TDI1
iVAT-thiocarbonyldiimidazole
TDO
toluene dioxygenase
TFA
trifluoroacetic acid
THF
tetrahydrofuran
TLC
thin-layer chromatography
TsCl
para-toluenesulfonyl chloride
p-TsOH
para-toluenesulfonic acid
UV
ultraviolet
VIS
visible
IX


123
J=3.42, 1H), 4.56 (d, J=4.88, 1H), 4.37 (d, J=5.86, 1H), 4.31 (d, J=6.1, 1H), 4.25 (d, J=4.15,
1H), 3.88 (m, 1H), 3.73 (t, J=3.90, 1H), 3.48-3.28 (m, 3H), 3.27-3.15 (m, 1H), 2.14-1.44 (m,
4H), 1.94-1.78 (m,3H), 1.78-1.40(m, 11H), 1.40-1.02 (m, 8H). I3C (CDC13 75MHz) 8:
139.9, 138.7, 129.1, 125.5, 75.0, 71.8, 70.5, 70.0, 69.5, 65.6, 53.9, 50.0, 35.3., 34.2, 32.3,
32.0, 25.9, 25.7, 25.0, 24.8, 24.4, 24.3. IR (CHClj/cm1) v: 3355.8, 3017, 2933, 2858, 1449.
MS(CI+): mJz 213 (29%), 195 (100%), 117 (46%), 99 (51%). HRMS: caled for Ci2H2i03,
213.1490. Found 213.1490.
Bicyclohexyl-4,6-diene-2,3,2'-triol (364a) was obtained from
biooxidation of (lS,2R)-(+)-rai.v-2-phenyl-l -cyclohexanol (363a) with
E. coli JM109 (pDTG601) as described for the small scale transformation
of racemic 1-phenyl-1-ethanol. The progress of the biotransformations
was monitored by UV absorbance Ckmax = 270-272 nm) and TLC (EtOAc). The metabolite
was isolated according to the method described for ()-l-phenyl-l-ethanol. The crude
metabolite was immediately subjected to PAD reduction. Purification by flash
chromatography (1:4 hexane-EtOAc) afforded 365a (100 mg, 49%).
(lS,2/f,rf?,2S)-3-(2-Hydroxycyclohexanyl)-3-cyclohexene-l,2-
diol (365a). Rf = 0.68 (EtOAc); mp = 89-91C; [oc]D28 -82.3 (c 0.6,
CH2C12); IR (film) 3329, 1447 cm1; 'H NMR (300 MHz, DMSO-d6) 8
5.50 (m, 1H), 4.83 (d,7 = 4.5, 1H),4.73 (d,7=3.4, 1H), 4.30 (d, 7=6.1,
1H), 3.73 (t, 7=3.9, 1H), 3.46-3.38 (m, 1 H), 3.26-3.15 (m, 1 H), 2.1-1.0 (m, 13 H); 13C
NMR (75 MHz, CDC13) 8 139.9, 125.5, 75.0, 69.9, 69.5,49.9, 35.3, 32.3, 25.9, 25.1, 24.8,
24.2; MS(CI+): m/z 213 (22), 195 (100), 177 (87), 159 (29), 147 (59), 105 (24); HRMS:


57
The synthesis of toluene catechols, for instance, involves 7 steps with an overall
yield of 5% starting from furfural274 (Scheme 3-32).
Preparation of bromocatechol 270 was achieved by first converting 2,3-
dimethoxybenzadehyde 265 to 2,3-dimethoxybenzoic acid 266 through permanganate
oxidation.275 The acid was then converted to 2,3-dimethoxybenzamide 267, which was
then subjected to a modified Hoffmann rearrangement process276 to furnish 2,3-
dimethoxyaniline 268. The resulting aniline 268 was converted to 2,3-
dimethoxybromobenzene 269, using a modified Sandmeyer procedure.277 Finally,
demethylation of dimethoxybromobenzene 269 with aluminum trichloride in
chlorobenzene278 produced 3-bromocatechol 270 in 5 steps with an overall yield of 49%
(Scheme 2-33).
Scheme 2-33. Synthesis of bromocatechol


52
Q;x
OH
OH
X
J^Y 0Me
^yOMe
co<
O'
198
199
200 Fe(CO)3
201
.XX*
Co<
9H
*v^*o/x
&*
NHTs
202
HR
203
NHR
204
OR R = H, TBS
205
206 207
X = Cl, Br, I, CN, alkyl, aryl
Figure 2-9. Example of secondary synthons
OTBS R R = C02Me, Cbz, Ts
208 209
Hudlicky and his research group have been pioneers in this effort. Most of the
work of the group to date has been conducted with a diverse range of diols derived from
the biotransformation of corresponding arenes by TDO.25 Table 2-13 lists some
representative synthetic products that are illustrative of the applicability and versatility of
TDO as a synthetic tool.
The synthetic potential of dioxygenase enzyme system, such as TDO, provides a
viable research tool that currently is used by a growing number of research groups across
the globe for a variety of synthetic applications, and the activity within the field continues
to grow.


219
(337) Simon, M. J.; Osslund, T. D.; Saunders, R.; Ensley, B. D.; Suggs, S,; Harcourt,
A.; Suen, W. C.; Cruden, D. L.; Gibson, D. T, Gene 1993, 127, 31.
(338) Paquette, L. A.; Kuo, L. H.; Doyon, J. Tetrahedron 1996, 52, 11625-11636.
(339) Garcia Martinez, A.; Herrera, A.; Martinez, R.; Teso, E.; Garcia, A.; Osio, J.;
Pargada, L.; Unanue, R.; Subramanian, L. R.; Hanack, M. Journal of Heterocyclic
Chemistry 1988, 25, 1237-41.
(340) Scott, W. J.; Crisp, G. T.; Stille, J. K. Org. Synth. 1990, 68, 116.
(341) Koch, S. S. C; Chamberlin, A. R. J. Org. Chem. 1993, 58, 2725-37.
(342) Tamura, M.; Kochi, J. Synthesis 1971, 303.
(343) Baldwin, J. E.; Adlington, R. M.; Coates, J. B.; Crabbe, M. J.; Crouch, N. P.;
Keeping, J. W.; Knight, G. C.; Schofield, C. J.; Ting, H. H.; Vallejo, C. A.
BIOCHEMICAL JOURNAL 1987, 245, 831.
(344) Palissa, H.; von Dohren, H.; Kleinkauf, H.; Ting, H. H.; Baldwin, J. E. JOURNAL
OF BACTERIOLOGY 1989,171, 5720.
(345) Pang, C. P.; Chakravarti, B.; Adlington, R. M.; Ting, H. H.; White, R. L.;
Jayatilake, G. S.; Baldwin, J. E.; Abraham, E. P. BIOCHEMICAL JOURNAL
1984, 222,789.
(346) Baldwin, J. E.; Adlington, R. M.; Domayne-Hayman, P. P.; Knight, G. C.; Ting,
H. H. J. Chem Soc., Chem. Commun. 1987, 1661.
(347) Carredano, E.; Karlsson, A.; Kauppi, B.; Choudhury, D.; Parales, R. E.; Parales, J.
V.; Lee, K.; Gibson, D. T.; Eklund, H.; Ramaswamy, S. J. Mol. Biol. 2000, 296,
701.
(348) Birladeanu, L.; Hanafusa, T.; Johnson, B.; Winstein, S. J. Am. Chem. Soc. 1966,
88, 2316.
(349) Poulter, C. D.; Winstein, S. J. Am. Chem. Soc. 1969, 91, 3649.
(350) Poulter, C. D.; Winstein, S. J. Am. Chem. Soc. 1969, 91, 3650.
(351) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(352) Smith, D. M.; Nicolaides, A.; Golding, B. T.; Radom, L. J. Am. Chem. Soc. 1998,
120, 10223.


15
electron to 31 yields a ferric peroxide adduct 32, which can be protonated to give a
hydroperoxide complex. A second protonation of the same oxygen then leads to
heterolytic 0-0 bond cleavage, releasing water and generating the proposed oxo-ferryl
(0=Felv) porphyring radical 33a intermediate that is sometimes represented as (FeO)3+
33b. One atom of oxygen is expelled as water while the other forms the reactive
intermediate (FeO)3+ which oxidizes the substrate to form the product. The expulsion of
the product, SubO, regenerates the initial ferric iron porphyrin speciesreand closes the
catalytic cycle. The mechanistic representation of Cytochrome P-450 above is illustrative
of how an enzyme system can utilize oxygen in the catabolism of organic matter.
In contrast, flavin-dependent mono-oxygenases use a different mechanism, which
involves a flavin cofactor, Scheme 2-9.80 82 First, NADPH reduces the Enz-FAD complex
thus destroying aromaticity. The FADH2 so formed is oxidized via Michael-type
addition of molecular oxygen yielding a hydroperoxide species (FAD-4a-00H).
Deprotonation of the latter affords a peroxide anion, which undergoes a nucleophilic
attack on the carbonyl group of the substrate, usually an aldehyde or a ketone. The
tetrahedral intermediate thus formed, collapses via rearrangement of the carbon-
framework forming the product ester or lactone. Finally, water is eliminated from the
FAD-4a-OH species to reform FAD.
The cytochrome P450 mechanism resembles the chemical oxidation by hypervalent
transition metal oxidants through an electrophilic attack, while the FAD-dependent
mechanism parallels the oxidation of organic compound by peroxides or peracids, which
act as nucleophiles.83


71
coupling of arylbromide 320 with phenol 321,317 followed by deprotection of the terminal
carboxylic and hydroxyl functionalities afforded the saturated acid 322.
Macrolactonization of 322 via a modified Mitsunobu-type condition323'324 afforded 323 in
91 % yield. Subsequent desilylation, promoted by fluoride anions, afforded the alcohol
324 in 94 % yield. Dehydration of the alcohol 324 was achieved using Sammuelsons
combretastatin D-2
Scheme 2-41. Total synthesis of combretastatin D-2
conditions325 by first replacing the hydroxyl group with iodine and subsequent
dehydrohalogenation in refluxing DMF in the presence of excess KF afforded methyl
combretastatin D-2 325. Combretastatin D-2 was finally prepared after demethylation of
325 in 17 % yield following Bogers procedure.317


150
with water and extracted with methylene chloride (25 mL, 3X). The organic layers were
combined, dried (Na2S04) and concentrated. The product was purified by flash-
chromatography (5:l/hexanes:EtOAc) to yield the title compound (0.794g, 59%) as a
colorless semi-solid material. Rf = 0.25 (5:l/hexane:EtOAc), [a]o27 -29.4 (c 1.1, CH3OH);
IR (film) 3459, 2986, 1380, 1260, 1067, 1025 cm'1; 'H NMR (300 MHz, CDCI3) 8 5.92-5.74
(m, 1H), 5.10-4.92 (m, 2H), 4.28-4.12 (m, 4H), 3.96-3.84 (m, 1H), 3.70-3.50 (m, 2H), 3.30-
3.20 (m, 1H), 2.93 (s, exchanged with D20, 1H), 2.22-2.10 (m, 2H), 1.80-1.65 (m, 2H), 1.51
(s, 6H), 1.36 (s, 3H), 1.34 (s, 3H); 13C NMR (75 MHz, CDC13) 8 138.42, 115.11, 110.3,
110.0, 80.0, 79.3, 78.5, 76.9, 76.8, 71.5, 70,9, 30.5, 29.2, 27.9, 27.8, 25.4, 25.2; HRMS (El)
m/z calcd for C16H2506 [M-CH3]+: 313.1651. Found: 313.1649.
2,2,7,7-Tetramethyl-4,5-bis-pent-4-enyloxy-
hexahydro-benzo[l,2-d;3,4-cT]bis[l,3](iioxole (460). A 10
mL round-bottomed flash was charged with NaH (169 mg, 4.2
mmol) and covered with anhydrous DMF (5 mL). Alcohol
459 (138 mg, 0.42 mmol) dissolved in DMF (5 mL) was then added dropwise to the reaction
flask and allowed to stir at room temperature for 45 min. 5-Bromopentene (0.62 mL, 4.2
mmol) was then added and the reaction was allowed to proceed for 5h. The reaction was
quenched with H2O (5 mL) and extracted with Et20 (10 mL, 3X). The ethereal portions were
combined, washed with saturated NaCl (10 mL), H2O (10 mL), dried (MgS4) and
concentrated. The product was purified by flash-chromatography (9: l/Hexanes:EtOAc) to
yield a thick red oil (96 mg, 93%). Rf = 0.68 (5: l/Hexane:EtOAc), 'H NMR (300 MHz,
CDC13) 8 5.90-5.72 (m, 2H), 5.08-5.02 (m, 1H), 5.02-4.90 (m, 3H), 4.50-4.32 (m, 1H), 4.24-
4.10 (m, 4H), 3.80-3.66 (m, 4H), 3.36-3.26 (m, 2H), 2.20-2.06 (m. 4H), 1.76-1.63 (m, 4H),
\ S
0'"\ )o
' dKr
460


47
stereochemistry of the metabolites. With the exception of orf/to-difluorobenzene
substrate,227 the cts-dihydrodio! metabolites obtained from biotransformation of ortho-
disubstituted benzenes appear to be enantiopure. The lower ee values obtained for the
cis-dihydrodiol metabolites of orrfto-difluoro-benezene (64%) again may be a result of
the reduced steric requirements of the fluorine atoms (Table 2-9).226
Table 2-9. cis-Dihydrodiols from ortho-substituted benzenes
R'v.
s/y*
o> \ /
TDO R''
R
t
107
^OH
^OH
Metabolites
R
R
% ee
166
F
F
64
167
Me
F
>98
168
Br
F
>98
169
ch=ch2
Cl
>98
170
1
Cl
>98
For meta-disubstituted benzene substrates, similar phenomenon of regio- and
stereoselectivity was observed. Again, with the exception of ort/io-difluorobenzene,227
which afforded an ee of 64%, the enantiopurity of c/s-dihydrodiol metabolites obtained
from TDO-catalyzed oxidation processes are almost always greater than 98%. Table 2-
10, lists a few examples of TDO-catalyzed biooxidation of meta-substituted benzene.226
Table 2-10. cis-Dihydrodiols from meta-substituted benzenes
R
R
A
TDO
rVH
R1''
ra^aoh
Metabolites
R
R
% ee
171
F
F
56
172
1
Br
>98
173
CF3
F
>98
174
I
F
>98
175
Me
F
>98


174


o u


ACKNOWLEDGMENTS
I would like to take this opportunity to express my gratitude to everyone who
helped me arrive where I am today. First I sincerely thank my advisor, Professor Tomas
Hudlicky, for his guidance, support, and encouragement throughout my graduate studies
at the University of Florida. I am forever indebted to him for giving me the opportunity
and assistance to develop into the chemist that I am today. Special thanks go to Dr. Josie
Reed and John Patterson for their help and kindness. I am grateful to Dr. Louise
Greenberg and Dr. Cyrus Greenberg for their kind friendship and helpful advice
throughout my undergraduate and graduate school years and for their help in
proofreading this document. I also would like to thank Professors William Dolbier,
Steven Benner, Dennis Wright, and Lonnie Ingram for their helpful advice, and for
serving on my committee. I am grateful to Dr. Dennis Wright for his fruitful suggestions
and advice (on countless occasions) for solving many of my research-related difficulties.
I am grateful to the University of Florida and the Department of Chemistry for the
opportunity to further my education. Special thanks go to professor James Deyrup and
Lori Clark for their help and advice throughout my time at the University of Florida.
Throughout my career in the Hudlicky research group, I have had the opportunity
to work with a great group of people. I would like to take this moment to thank all past
and present group members for their contribution to my educational career. Special
thanks go to Drs. Mary Ann Endoma, David Gonzales, and Bennet Novak for passing on
their expertise and knowledge of the whole-cell biotransformation processes used in our


A series of substituted catechols were made using E. coli JM109 (pDTG602),
which carries the toluene cis-dihydrodiol dehydrogenase (TDDH), as the source of
biocatalyst. 4-Bromoanisole catechol obtained through biotransformation of
corresponding arene, 4-bromoanisole, with JM109 (pDTG602) was used in the total
syntheses of Combretastatins A-l and B-l. (IS, 2S)-3-bromocyclohexa-3,5-diene-1,2-
diol was also used as chiral synthon for synthesis of polyhydroxylated chiral polymer.
xviii


75
Boyd et al. on 1-phenylethan-l-ol and other substrates.335 Ribbons and Ahmed336 studied
the biooxidation of phenyl ethanols (R-, S-, and racemate) with blocked mutants and
reported a slight preference for the S-enantiomer. Racemic sec-butylbenzene 338 and
racemic 1-phenylpropan-l-ol also were oxidized, but the stereochemical outcome was not
reported. In view of these limited examples, several substrates with one or more remote
chiral centers were oxidized by means of parallel experiments with both blocked mutants
P. putida F39/D and recombinant organism, E. coli JM109 (pDTG601) to investigate the
kinetic resolution capability of TDO in resolving enantiomer pairs.29'200'337 The following
sections summarized the results obtain from this kinetic resolution study. The absolute
stereochemistry correlation of new metabolites are also presented.
At the onset of this study, Ribbons and Ahmeds experiments with phenethyl
alcohol were repeated as control experiments to ensure the viability of P. putida F39/D
and to verify the kinetic resolution they observed. When phenylethanol was inoculated
with toluene-induced P. putida F39/D, the S-enantiomer was observed to be consumed at
a faster rate than the R-antipode resulting in a 6:4 ratio of diastereomers 337a and 337b,
respectively, as confirmed by *H-NMR spectroscopy and chiral HPLC analyses of the
metabolites formed (Table 3-1). This preliminary finding suggested that TDO is capable
of resolving racemic enantiomers and encouraged further investigation into the scope of
its kinetic resolution capacity.
Since the recombinant organism E. coli JM109 (pDTG601) is easier to handle and
grow, further investigation of TDOs kinetic resolution capability was conducted with the
E. coli strain. When racemic phenylethanol 339 was subjected to TDO dihydroxylation
with JM109 (pDTG601), both enantiomers were converted to a 1:1 mixture of


113
mixtures of water and acetonitrile as eluents. For chromatographic purification, silica gel
(grade 60, 230^400 mesh Merck) was used.
Large Scale Biotransformation E. coli JM109 (pDTG601)
A 600-mL of mineral salts broth (MSB) containing 9.6 g K2HPO4, 8.4 g KH2PO4, 3
g (NH4)2S04, 9 g yeast extract, 18 g glucose, 1.2 g MgS04 7H20 was prepared and
divided into two 2.8 L fembachs for sterilization. Upon cooling to ambient temperature,
ampicillin (100 mg/L) was added, and the media were inoculated with single colonies of
E. coli JM109 (pDTG 601) from an agar plate. The cells were grown overnight on an
orbital shaker (35 C, 150 rpm). The cultures were then transferred into a 15-L fermentor
containing 8 L of a similar sterilized medium: 60 g KH2P04, 16 g citric acid, 40 g
MgS04'7H20, 16 mL trace metal solution (Na2S04 (lg/L), MnS04 (2g/L) ZnCl2 (2g/L),
C0CI2 6H:0 (2g/L), CuSO 5H20 (0.3g/L), FeS04 7H20 (lOg/L), pH =1.0), 9.6 mL
concentrated H2S04, 9.6 mL ferric ammonium citrate (270 g/L), neutralized with
concentrated ammonia and supplemented with 800 mg of ampicillin and 2.69 g thiamine
hydrochloride). The cells were grown for 24 h with concentrated glucose solution (720
g/L) as the carbon source and induced with 80 mg isopropyl p-D-thiogalactopyranoside
(IPTG) when the optical density (OD) of the medium is 15 or higher. When cells were
grown to an OD of 45 or higher, the cultures were used for screening of new substrates or
to carry out large scale biotransformation of aromatic substrates. The substrates were
added to the culture and their metabolic transformation was monitored UV ((A.max -260-
276 nm) or by TLC. After all metabolic activity ceases, the fermentation was terminated
and the pH of the culture was adjusted to 7.5 with ammonium hydroxide. Cells were
separated from the broth by centrifugation and supernatant was extracted with base-
washed ethyl acetate. The organic layer was dried with anhydrous magnesium sulfate


143
yellowish powder compound (0.256 g, 73%). mp = 84-85 C; Rf =0.48 (3:2/hexane:EtOAc):
IR (film); H NMR (300 MHz, CDC13) 8 7.26 (s, IH), 6.74 (s, 2H), 6.69 (d, J=8.4, 1H), 5.31
(s, 2H), 5.14 (s, 2H), 3.87 (s, 3H), 3.86 (s, 9H), 3.66 (s, 3H), 3.61 (s, 3H); 13C NMR (75
MHz, CDC13) 8 154.5, 153.3, 151.9, 139.2, 139.1, 138.9, 128.8, 118.7, 111.0, 108.8, 108.0,
106.8, 99.4, 98.9, 92.2, 85.1, 68.5, 68.4, 61.1, 57.8, 57.6, 56.3, 56.2; FABMS (%); m/z 418
(100) [M+], 387 (21), 343 (17), 342 (13), 154 (13), 137 (10), 95 (13), 83 (17), 81 (14);
HRMS (FAB) m/z caled for C^^iOg: 418.1627. Found; 418.1620. Anal, caled for
C22H2608: C, 63.15; H, 6.26. Found: C, 63.14; H, 6.17.
3-Methoxy-6-[2-(3,4,5-trimethoxy-
phenyl)ethyl]benzene-l,2-bis-methoxymethoxy-
benzene (441). Acetylene 440 (72 mg) in MeOH
MOMO, OMOM
MeO
441
(10 mL) and 10% Pd/C (10 mg) was treated with a positive pressure of H2 (30 PSI) at
ambient temperature overnight. The catalyst was removed by filtering through a bed of
silica. The product was purified by flash chromatography to afford the title compound 441
(65 mg, 93%) as colorless oil. Rf =0.46 (3:2/hexane:EtOAc); IR (film) 2939, 2839, 1589,
1495, 1458, 1277, 1126, 1065, 971, 804; 'H NMR (300 MHz, CDC13) 8 6.83 (d, J=
8.54,1H), 6.63 (d, 7=8.54, 1H), 6.40 (s, 2H), 5.15 (s, 2H), 5.12 (s, 2H), 3.83 (s, 3H), 3.82 (s,
9H), 3.60 (s, 3H), 3.58 (s, 3H), 2.88 (m, 4H); 13C NMR (75 MHz, CDC13) 8 153.1, 152.1,
149.6, 138.6, 138.0, 136.1, 128.1, 124.6, 107.7, 105.4, 99.8, 98.7,61.0, 57.8, 57.5, 56.1,56.1,
37.4, 32.1; FABMS(%): m/z 422 (80) [M+], 391 (28), 359 (51), 345 (68), 287 (19), 219 (15),
194 (21), 191 (26), 181 (91), 179 (22), 167 (27), 165 (100), 155 (21), 154 (57), 137 (38), 128
(38), 109 (38), 97 (48), 95 (45), 91 (28) 83 (68), 81 (61); HRMS (FAB) m/z caled for
C22H30O8: 422.1940. Found: 422.1951.


MeO
OMe
OMe
Combretastatin B-1 OMe
14
n-
12
TT"r
10
8
6
JlL
xJL
4
2
0


76
diastereomers 337a and 337b, as revealed by both 'H and l3C NMR spectroscopy. In
experiments where the biooxidation process was terminated before completion, analyses
of the non-converted phenylethanol on a chiral support GLC revealed no difference in the
rate at which each enantiomer was metabolized. Moreover, in runs where the R- and the
5-enantiomers were oxidized separately, the same phenomenon was observed. These
results were unexpected, since the same dioxygenase enzyme operates in both P. putida
F39/D and JM109 (pDTG601). Table 3-1 summarized the biotransformation results of
phenylethanols by both organisms.330-331
Table 3-1. Biotransformation of phenylethanol by JM109 (pDTG601) and Pseudomonas
putida F39/D
Substrates E. coli JM109 pDTG601) Pseudomonas putida F39/D
In light of the observed differences between the two organisms in oxidation of
phenylethanol, it was speculated that each organism might have different substrate
transport mechanisms. For example, in the P. putida F39/D, it is possible that the S-
enantiomer was transported to the enzyme active site at a faster rate than the R-antipode,
resulting in its consumption at a faster rate. By constrast, in the E. coli organism, the rate


103
Conditions: i) JM109 (pDTG602); ii) MOMCI; CH2CI2; EtN(iPr)2; iii) CBr4; PPh3; CH2CI2; 0C;
iv) nBuLi; THF; v) nBuLi, B(OiPr)3, DME:THF/10:1; -78C: vi) Pd(PPh3); 90C;
vii) 10% Pd/C/H2; (30 PSI); MeOH; viii) (CsHnlBH, 0C, 1h; then 20C, 30 min; HOAc; OC, 4h;
ix) 6N HCI:THF/1:1
Scheme 3-27. Enzymatic generation of catechol for approach to combretastatin A-1 and
B-l
In the preparation of combretastatin A-l, alkyne 440 was initially subjected to
Linlards reduction condition to selectively reduce the alkyne to alkene. However, this
method was not successful in generating cir-alkene as the sole product but rather a
complex mixture of cA-alkene 442 and its saturated analog 441 were produced that were


23
Besides Pseudomonas oleovorans, numerous bacteria have been shown to
epoxidize alkenes.136 As shown in Table 2-3, the optical purity of epoxides depends upon
the strain used, although the absolute configuration is usually R.137 This concept has been
recently applied to the synthesis of chiral alkyl and aryl gycidyl ethers.138,139 The latter
are of interest for the preparation of 3-substituted 1 -alkylamino-2-propanols, which are
widely used as adrenergic receptor blocking agents.140
Table 2-3. Microbial epoxidation of alkenes
R1^
54
P microorqanism _
02 'h-.o
55
Microorganism
Ri
R?
Config.
e.e.JWl
Pseudomonas
n-C5H||
H
R
70-80
oleovorans
n-CyHis
H
R
60
H
H
R
86
NH2C0-CH2-C6H4-0
H
S
97
CH,0(CH2)2-C6H4-0
H
S
98
Corynebacterium equi
ch3
H
R
70
n-CnH27
H
R
-100
Mycobacterium sp.
H
H
R
98
ch3
CH,
R,R
74
Ph-0
H
S
80
Xanthobacter Py2
Cl
H
S
98
ch3
ch3
R.R
78
Nocardia sp. IP 1
Cl
H
S
98
ch3
H
R
98
More recently, other microorganisms also have been found to perform epoxidation
of alkenes with excellent yield and optical purity. As can be seen from Table 2-4, non
terminal alkenes can be epoxidized by a Mycobaterium or Xanthobacter sp.,141 while
Nocardia coralline has been reported to convert branched alkenes into the corresponding
(R)-epoxides with good optical purities.


9
22,48 of benzaldehyde 11 and 2-oxo-propionic acid 12 could be achieved to furnish
levorotatory 1-hydroxy-l-phenylproan-2-one 13, when the substrates are added to a
carbohydrate solution undergoing active yeast fermentation. This process was later
modified to use acetaldehyde directly instead of 2-oxo-propionic acid to afford a much
better yield of a-hydoxy ketone 13 which is used industrially in the production of L-
ephedrinel449'50 (Scheme 2-3).
co2
Scheme 2-3. Saccharomyces cerevisiae catalyzes acyloin condensation as the key step in
the production of L-Ephedrine
The ability of microorganisms to carry out biotransformations such as acyloin
condensation mentioned above, was soon recognized as essential in the manufacture of
CH2OH
HO-
HO-
HO
CH2OH
Catalytic
reduction
HO-
HO-
-OH
T
ch2oh
CHO
D-glucose 15
Acetobacter 0=
suboxydans HO
OH f ^ |OH
NAD NADH2 HO
ch2oh
O.
HO-
HO-
HO-H
O

HO-
C
D-sorbitol 16
C02Na
Add Na0 |
treatment NaO
HO-
-OH
H2
ch2oh
L-sorbose 17
C02H
0=J
hoH
HO'
/
' Chemical
oxidation
V h2o
-OH
CH2OH
L-ascorbic acid 20
CH2OH
2-keto-L-gulonic acid 18
CH2OH
2-keto-L-gulonic acid/
sodium salt 19
Scheme 2-4. Microbial dehydrogenation of D-sorbitol to L-sorbose as the key step in the
Reichstein-Grussner synthesis of L-ascorbic acid


141
3-Phenoxy-pyrocatechol was obtained through biooxidation of
phenylether with E. coli JM109 (pDTG602), as described in the general
procedure for preparation of catechols. The metabolite was purified by
flash chromatograph. lH (DMSO) 8 9.35 (s, 1 H), 8.64 (s, 1 H), 7.37- 409
7.24 (m, 2 H), 7.06-6.94 (m, 1 H),6.88-6.78 (m, 2 H), 6.70-6.56 (m, 2H), 6.46-6.36 (m, 1 H)
13C (CDC13) 8: 156.9, 145.3, 144.0, 135.0, 130.0, 123.8, 120.5, 118.2,111.3, 110.9.
Chemoenzymatic approach to combetastatin A-l and B-l project
2-Methoxy-5-bromophenol this metabolite was obtained from
biotransformation of 4-bromoanisole. The product was purified by
chromatography and the red residue was triturated with pentane to afford a
OMe
Br
white precipitate: MP 64-65 (lit. 60-62 C), IR (KBr) 3400, 1592, 1262, H NMR (300 MHz,
DMSO-d6) 8 9.50 (s, OH, 1H), 6.93-6.83 (m, 3H), 3.73 (s, 3H); l3C NMR (75 MHz, CDClj)
8 146.7, 146.0, 123.0, 118.0, 113.4, 112.0, 56.3; MS (Cl); nt/z 202 [M+] (100), 194(42), 189
(40), 124 (41), 108 (64) 106 (142), 95 (22), 91 (174), 83 (120), 82 (35) 81 (197); HRMS (El)
m/z caled for C7H8Br02(M+): 201.9629. Found: 201.9627. Anal. Caled for C7H8Br02: C,
41.41; H, 3.48. Found: C, 41.67, H, 3.47.
l-Bromo-2,3-bis(methoxymethoxy)-4-methoxybenzene (436):
To a flame-dried round bottom flask fitted with a 3-way stopper, were
added bromoanisole catechol 406 (2.34 g, 10.6 mmol) and CH2C12
(15 mL) followed by EtN(iPr)2 (5.49 g 42.5 mmol) and then MOMC1 (3.42 g, 42.5
mmol). The reaction mixture was allowed to stir at room temperature under argon
atmosphere for 3 hours. The reaction mixture was diluted with CH2C12 (15 mL),washed with


7
they were a product of fermentation. During his investigation of the "Varner" condition
for the French wine industry, Pasteur examined specimens of wines under a microscope
and saw plump yeast cells in good wine and rod-like shapes in wine with too much acid.
Analysis of the wine showed that organic, or biological compounds were formed during
the fermentation, products that sugar alone could not produce. From his previous
research, Pasteur knew these compounds had to be formed by living cells. Pasteur
realized that the fermentation process was conducted by yeast and that other
microorganisms were responsible for turning the fermentation sour.
Pasteur eventually identified and isolated specific microorganisms responsible for
normal and abnormal fermentations of wine, beer and vinegar, and gave birth to the
science of bacteriology. He also showed that by heating each of the above liquids, the
microorganisms were killed and the wine was protected from further contamination.
With the identification of undesirable bacterial contamination of wines, volatile acidity
was eliminated and better quality wines were more routinely produced.
Pasteurs pioneer work in fermentation in 1862 conclusively disproved the theory
of spontaneous generation and led to the germ theory of infection. His work on wine,
vinegar, and beer resulted in the development of the pasteurization process.
In 1886, Brown confirmed Pasteurs findings and named Bacterium xylinum as the
agent responsible vinegar production.42 He also found that this organism could oxidize
propanol 7 to propionic acid 8 and mannitol 9 to fructose43 10 (Scheme 2-2).
In 1897, Eduard Buchner,44 a German chemist who also had been interested in the
problems of alcoholic fermentation since the 1880s, argued that enzymes were the


Br


98
RO
RO
R
Scheme 3- 22. Transformations of 3,4-bromocatechol
The availability of catechols and cis-diols produced through enzymatic means
would allow for an easy assembly of synthetic targets such as morphine, pancratistatin,
and combretastatins as shown in Scheme 3-23.
As demonstrated, functionalized catechols can be prepared directly from their
mono- or disubstituted aromatic precursors by biooxidation on a preparative scale. Such
compounds represent important starting materials for synthetic routes to many natural
products and are sometime unattainable by traditional methods (for instance
iodocatechol). The halogenated catechols are useful for the introduction of catechol
moiety into those synthons that rely on Heck cyclization or tethered radical for C-C bond
formation. Future direction of this research will clearly be concerned with the screening
of a broad range of substrates for the recombinant organism and establishment of the
substitution limits for recognition by the enzymes.


16
NADPH
y.pp*
Sub
XX
H H
NH
35 H o
[Enz-FADH2-Sub]
02"\
XX
nn^nY
NH
Xo o
[Enz-FAD-4a-OOH-Sub]
36 H .0 A
HO
Scheme 2-9. Catalytic cycle of flavin-dependent mono-oxygenases
Enzymatic oxygenation reactions are particularly intriguing since direct
oxyfunctionalization of unactivated organic compounds are difficult to attain through
conventional chemical methods. Thus transformations of cheap inert raw materials, such
as alkanes, alkenes and aromatics into oxidized products of greater value such as
aldehydes, ketones, alcohols, carboxylic acids, by means of biocatalysts are appealing
alternatives to industry. The highly regio- and enantiospecific properties of many
biocatalyst systems made them ideal for the precise manufacturing of specialty
chemicals. The enzymatic oxygenation of organic molecule may go through one of the
following processes depending upon the enzyme system involved, Scheme 2-10.
In reactions catalyzed by monooxygenases, one oxygen atom from molecular
oxygen is incorporated into the substrate and the other one is reduced to form water by
means of a cofactor usually NADH or NADPH. While in reactions catalyzed by
dioxygenases, the enzymes simultaneously incorporate both oxygen atoms into the


BIOGRAPHICAL SKETCH
Vu Phong Bui was bom in Nha Trang Viet Nam in 1974. He is the son of Mrs.
Hong Le. In 1990, he immigrated to America under the Orderly Departure Program
(ODP). Vu Bui graduated from Wakefield High School in Arlington Virginia, in 1994
and Dickinson College in 1998.
225


138
procedure for preparation of catechols. The catechol was purified by
Kugelrohr distillation (80-90 C/2mmHg) to yield 4.5 g (0.5 g/L) of white solid; mp 55-56 C
(lit.1 55-56); 'H NMR (300 MHz, DMSO-d6)5 9.74 (s, OH, 1H), 9.13 (s, OH, 1H), 7.08 (dd, J
= 1.5, 7.9, 1H), 6.76 (dd, J = 1.5, 7.9, 1H), 6.42 (t, J = 7.9, 1H); l3C NMR (75 MHz, CDC13)
8 143.6, 143.0, 129.5, 123.2, 116.0.84.5; MS(FAB) m/z: 236 (11), 209 (18), 185 (80), 93
(100); HRMS (Cl) m/z caled for C6H5I02 (M+): 235.9334. Found: 235.9250. Anal, caled for
C6H5I02: C, 30.53; H, 2.14. Found: C, 30.66; H, 2.19.
3-Ethynyl-benzene-l,2-diol (403) was obtained through biooxidation
of ethynylbenzene with E. coli JM109 (pDTG602) as described in the general
procedure for preparation of catechols. The metabolite was purified by flash-
chromatography (3:2 hexane:EtOAc) to yield white crystalline compound (0.75g/L). Rf
=0.35 (3:2 hexane:EtOAc); mp = 57.5-59.5 C; IR (film) 3490, 3284, 2101, 1617, 1586, 1469,
1332, 1266, 1200, 1064, 955, 831, 780, 673; 'H NMR (300 MHz, CDC13) 8 7.00-6.91 (m,
2H), 6.85-6.75 (m, 1H), 5.60 (bs, 2H), 3.45 (s, 1H);); 13C NMR (75 MHz, CDC13) 8 144.9,
143.9, 123.7, 121.2, 117.0, 108.6, 84.3, 78.3; MS (Cl); m/z 134 [M+] (100), 78 (88), 77 (87),
74 (23), 63 (18) 62 (33), 52 (47), 51 (97), 50 (73); Anal. Caled for C8H602: C, 71.64; H,
4.51. Found: C, 71.71, H, 4.56.
3-Cyclopropylbenzene-l,2-diol (404) was prepared by
biotransformation of the corresponding arene with E. coli JM109
(pDTG602). Rf = 0.52 (Hexane:EtOAc / 3:2); mp = 41 -42 C; IR (film)
3418, 1622, 1592, 1477, 775 cm'1; 'H NMR (300 MHz, DMSO-d6) 8 9.11 (s, 1H), 8.11
(s, 1H),6.50-6.58 (m, 1H), 6.49 (t, J = 7.82 Hz, 1H), 6.16-6.21 (m, 1H), 2.01-2.12 (m, 1H),
404
403


Kl


218
(318) Bollag, D. M.; McQuencey, P. A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, O.;
Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995,
55, 2325.
(319) Brossi, A.; Yeh, H. J. C.; Chrzanowska, M.; Wolff, J.; Hamel, E.; Lin, C. M.;
Quin, F.; Stuffness, M.; Silverton, J. Med. Res. Rev. 1988, 8, 77.
(320) Couladouros, E. A.; Soufli, L C. Tetrahedron Lett. 1994, 55, 4409.
(321) Couladouros, E. A.; Soufli, I. C.; Moutsos, V. I.; Chadha, R. K. Chem. Eur. J.
1998, 4, 33.
(322) Yoon, N. M.; Gyoung, Y. S. J. Org. Chem. 1985, 50, 2443.
(323) Mitsunobu, O. Synthesis 1981, 1.
(324) Justus, K.; Steglich, W. Tetrahedron Lett. 1991, 32, 5781.
(325) Garegg, P. J.; Samuelsson, B. J. Chem. Soc. Chem. Commun. 1979, 978.
(326) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.; Hartung, J.;
Jeong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.; Xu, D.; Zhang, X.-L. J.
Org. Chem. 1992, 57, 2768.
(327) Boigegrain, R.; Castro, B. Tetrahedron Lett. 1976, 1283.
(328) Robinson, P. L.; Barry, C. N.; Bass, S. W.; Jarvis, S. E.; Evans, S. A. J. J. Org.
Chem. 1983, 48, 5396.
(329) Hudlicky, T.; Novak, B. Tetrahedron Asymmetry 1999, 10, 2067.
(330) Bui, V.; Vidar Hansen, T.; Stenstrom, Y. R.; W.;, D.; Hudlicky, T. J. Chem. Soc.,
Perkin Trans. 1 2000, 11, 1669-1672.
(331) Bui, V. P.; Vidar Hansen, T.; Stenstrom, Y.; Hudlicky, T.; Ribbons, D. W. New J.
Chem. 2001,25, 116-124.
(332) Bui, V. P.; Nguyen, M.; Hansen, J.; Baker, J.; Hudlicky, T. Canadian Journal of
Chemistry 2002, 80, 708-713.
(333) Gibson, D. T.; Gschwendt, B.; Yeh, W. K.; Kobal, V. M. Biochemistry 1973, 8,
1520.
(334) Cripps, R. E.; Trudgill, P. W.; Whateley, G. Eur. J. Biochem. 1978, 86, 175.
(335) Boyd, D. R.; Sharma, N. D.; Bowers, N. I.; Duffy, J.; Harrison, J. S.; Dalton, H. J.
Chem Soc., Perkin Trans. 1 2000, 1345.
(336) Ahmed, S. PhD, Imperial College of Science, 1991.




69
More recently, Lawrence et al. disclosed another short synthesis of combretastatin
A-4 utilizing Suzuki coupling reaction.314 In this method, the coupling of the cis-
vinylbromo styrene 311 with trimethoxyboronic acid 312 produced combretastatin A-4 in
three steps and eliminated the necessary reduction step required in the earlier syntheses
(Scheme 2-40).
Combretastatin D-l and D-2
Combretastatin D-l and D-2 were isolated in trace amount from the South African
folk medicine tree Combretum caffrum.315 316 Structurally they differ from the A and B
series in that they contain a 15-member biaryl ether macrolactone resembling that of the
caffrane317 ring system. These natural products and their derivatives, Figure 2-14, have
OMe OMe OMe
Figure 2-14. Combretastatin D derivatives
been shown to be active against the murine P-388 lymphocytic leukemia cell line.
Combretastatin D-l shows a PS cell line activity corresponding to ED50 3.3 pg/mL and
D- 2 activity corresponding to ED50 5.2pg/mL.316 Structure-activity-relationships for
combretastatin D-l and D-2 and their derivatives on the tubulin
polymerization process at various concentration have been determined318 and the results
are summarized in Table 2-17.


130
(IS, 2R)-3-benzyl-3,5-cyclohexadien-l,2-diol (373) was
obtained from biotransformation of diphenylmethane (372) according
to the general procedure for large scale biotransformation. To 8L of
fully grown (OD = 65) cells, diphenylmethane (17 g, 101 mmol) was slowly added over a
period of 3 h, and the progress of the biotransformation was monitored by UV (Xmax= 266
nm). After the usual workup including extractions and concentration, the process gave (8.8
g, 1.1 g/L) metabolite 373 as an off-white solid. Rf= 0.47 (2:3 Hexane:EtOAc); mp = 69-71
C; [a]u33 + l 17 (c 1.0, CH2C12); IR (film) 3440, 3054, 2987, 1643, 1422, 1265, 896, 745,
705; *H NMR (300 MHz, DMSO-dc) S 7.34-7.26 (m, 2H), 7.23-7.16 (m, 3H), 5.85-5.75 (m,
1H), 5.68-5.60 (m, 1H), 5.54 (d, 7= 4.43, 1H), 4.64 (d, 7=6.36, 1H),4.52 (d, 7= 6.17, 1H),
3.70 (t, J=6.16, 1H), 3.46 (s, 2H); l3C NMR (75 MHz, CDC13) 8 141.3, 139.1, 129.2, 128.7,
128.2, 126.6, 124.9, 121.1, 69.9, 69.3, 40.3; MS(FAB) miz: 202 (12), 185 (56), 184 (14),
107 (14), 93 (31), 91(100); HRMS (Cl) mJz caled for C13H|402: 202.0994. Found: 202.0997.
Correlation of absolute stereochemistry of (IS, 2R)-3-benzyl-3,5-cyclohexadien-
1,2-diol (373). Into a flame-dried one-necked 25 mL round bottom flask equipped with a 3-
way stopper, vinyl bromide 348338 (172 mg, 0.746 mmol) was dissolved in freshly distilled
THF (10 mL). The reaction vessel was cooled to -78C, upon which r-BuLi (0.86 mL, 1.12
mmol) was added dropwised via syringe. The reaction was allowed to stir at -78C for 45
minutes. Benzyl bromide (0.191 g, 1.12 mmol) was then added and the reaction was allowed
to proceed for one hour at -78C and then allowed to slowly warm to room temperature. The
reaction was then quenched with 10% NH4CI (5 mL). The organic layer was diluted with
ethyl acetate (10 mL). The layers were separated and the organic layer was washed with DI
water (10 mL, 2X), dry with MgS04. The solvent was removed under vacuum to afford a
crude yield of 123 mg (68%) as light yellow oil. The crude product was dissolved in 6 mL of


8
^^OH Bacterium^ ^
y xylinum
H-
H-
HO-
HO-
CH2OH
OH
OH
H
H
CH2OH
9
ch2oh
H OH
Bacterium | H OH
xylinum HO H
o=
CH2OH
10
Scheme 2-2. Reaction catalyzed by Bacterium xylinum
causative agents responsible for conversion of alcohol to acid and not whole yeast cells as
was originally thought. This view was not in line with the conventional belief at that
time, namely, that without life there could be no fermentation. Despite of this, Buchner
and his brother Hans Buchner in 1897 succeeded in preparing a cell-free liquid extract
that was capable of converting sugar into ethanol and carbon dioxide, just as though
whole yeast cells had been present. He named the causative agent as zymase.45 Buchner
succeeded in the proving this concept where many others, including Liebig and Pasteur,
had failed. He did this by grinding yeast cells with sand and then filtering the crude
preparation for enzyme isolation. Eventually, it became clear that the conversion of
sugar into alcohol by means of yeast juice entails a series of stepwise reactions, and that
zymase really is a mixture of several enzymes. Buchners discovery of zymase then led
to establishment of the enzyme theory of fermentation. This gave birth to the science of
biochemistry, in which where much effort over the years has been invested in gaining a
thorough understanding of biological systems.
As biochemical knowledge of microorganisms and their environmental function
become more evident, microorganisms are being used more frequently for the production
of fine chemicals. Neuberg and Hirsch46'47 in 1921 showed that acyloin condensation20


41
alternative exists, dihydroxylation occurs at the heteroarene ring itself, as in the case of
thiophene and 3-methylthiophene.219
Table 2-7. TDO-catalyzed biotransformation of substrate containing heterocyclic ring
system.
Substrates Metabolites
OH
144 145
Electron rich functional groups such as cyano-, vinyl-, alkoxy- and alkylthio-,
appear to have a relatively strong directing effect. However, predictions of


31
dioxygenases,184 are known to be involved in the ring fission process of transforming
catechol precursors into aliphatic products, as illustrated in Scheme 2-20. Generally, the
catechol dioxygenases require no cofactor and can cleave the catechol moiety via either
an intradiol or extradiol fashion, as depicted in Scheme 2-20. The intradiol-cleaving
enzymes utilize Fe(HI) to give metabolite 96, while the extradiol-cleaving enzymes
utilize Fe(II)185'186 and in a few cases, Mn(II)187189 to give metabolite 97.
Scheme 2-20. Modes of catechol cleavage
intradiol
cleavage
extradiol
cleavage
There are diverse classes of dioxygenases existing in nature that are capable of
oxidizing a variety of organic substrates. However, the breakdown of the aromatic
nucleus has received the most attention. Numerous studies have been launched to probe
for a better understanding of this important metabolic process. As a result, there is a
great deal of knowledge known about dioxygenases. They are multicomponent enzyme
systems involving several non-heme iron proteins and require NADH for function.184'190
192 To date there are a number of microorganisms that have been found to be capable of
utilizing a wide range of carbon sources for energy and growth and these enzymes have
been classified according to the type of substrate they oxidize. The following are some of
the most common dioxygenases found to date: benzene dioxygenase (BDO), toluene
dioxygenase (TDO), naphthalene dioxygenase (NDO), benzoate dioxygenase (BZDO),
and biphenyl dioxygenase (BPDO). The overlapping in substrate specificity among the


28
substituent in the 4-position, to give predominantly the (S)-configuration products, A
switch to the R-Iactone was observed with 4-n-butylcyclohexanone. Simple models were
recently developed which allow the prediction of the stereochemical outcome of Baeyer-
Villiger oxidation catalyzed by cyclohexanone mono-oxygenase of Acinetobacter and
Pseudomonas sp by determining which group within the Criegee-intermediate is prone to
167,168
migration.
Table 2-6. Microbial Baeyer-Villiger oxidation of monocyclic ketones
0
J-is d Acinetobacter sp.
u 0
Ck* (V
84
85 86
R
e.e. of lactone [%]
n-C<;Hi i
97
/¡-C7H15
95
-C9H19
85
n-CnH23
73
Racemic ketones can be resolved by BVMOs, in which only one enantiomer gets
oxidized and its counterpart remains unchanged,169 Table 2-6. For example, a-substituted
cyclopentanones were stereospecifically oxidized by an Acinetobacter sp. to form the
corresponding (S)-configurated 8-lactones,170 substances which constitute valuable
components of various fruit flavors.
Bicyclic halo-ketones, which are used for the synthesis of anti-viral 6-fluoro-
carbocyclic nucleoside analogues, were resolved by using the same technique.171 Both
enantiomers were obtained with >95% optical purity. The exquisite enantioselectivity of
the microbial oxidation is due to the presence of the halogen atoms, since the de-
halogenated bicycle[2.2.1 ]hepan-2-one was transformed with low selectivity.


148
7-Bromo-2,2-dimethyl-4,5-bis-pent-4-enyloxy-3a,4,5,7a-
tetrahydro-benzo[l,3]dioxole (455). A 50 mL oven-dried round-
bottomed flask was charged with NaH (0.66 g, 22 mmol) under
argon atmosphere at room temperature. Anhydrous DMF (10 mL)
was then added, followed by bromodiol 451 (0.73 g, 2.75 mmol)
and the reaction mixture was allowed to stir for lh at room temperature. 5-Bromopentene
(3.28 g, 22 mmol) dissolved in DMF (10 mL) was then added drop-wise to the reaction
mixture, which was allowed to stir for another 6h. The reaction was quenched with water (5
mL) and extracted with diethyl ether (25 mL, 3X). The ethereal fractions were combined,
dried (MgSCL), and concentrated under reduced pressure to afford a reddish oil. The
products were purified by flash-chromatography (9: l/Hexanes:EtOAc) and the fractions
containing the titled compound were combined and concentrated. The resulting yellowish oil
was distilled under reduced pressure (155C, 0.2mmHg) using Kulgerolh apparatus to furnish
the titled compound as a colorless oil (0.35g, 32%). 'H NMR (300 MFIz, CDC13) 8 6.2 (d,
7=3.05 Hz), 5.92-5.74 (m, 2H), 5.05 (J=2.05, 1.54, 1H), 5.03-4.92 (m, 3H), 4.63 (dd, 7=1.03,
6.41, 1H), 4.21-4.12 (dd, 7=6.41, 8.46 Hz, 1H), 3.86-3.67 (m, 3H), 3.65-3.51 (m, 7=2.56,
5.64, 6.41, 6.67 Hz, 2H), 3.39 (t, 7= 8.1 Hz), 2.20-2.08 (m, 4H), 1.77-1.63 (m, 4H), 1.54 (s,
3H), 1.41 (s, 3H); 13C NMR (75 MHz, CDC13) 8 138.5, 138.2, 133.8, 118.8, 115.2, 114.8,
110.7, 80.0, 79.0, 78.0, 77.5, 72.3, 70.0, 30.4, 30.8, 29.5, 29.3, 28.3, 26.3; HRMS (El) m/z
calcd for Ci9H2904Br: 400.1249 Found: 400.1256.
Br


2
1
-0 ppm


81
2-Phenylcyclohexene
2-Phenylcyclohexene was another substrate of TDO successfully oxidized during
this kinetic resolution study. The biooxidation of substrate 359 furnished a 1:1 mixture
of diastereomers 360a and 360b, which were subjected to diimide reduction to reduce the
least sterically hindered olefins. However this process only provided an inseparable
mixture diol 361 and 362 (Scheme 3-8). To transform this diastereomer mixture of 361
and 362 into intermediate 357, the biochemically installed cis-diols of the mixture were
protected as acetonide and Wilkinsons catalyst was used to reduce the unconjugated
olefin to furnish 357 whose absolute stereochemistry has been established as described in
Scheme 3-7.
357
Scheme 3-8. Biooxidation of 2-phenylcyclohexene and absolute stereochemistry of new
metabolite
2-phenyIcyclohexanol
At this point it was clear that the cyclohexyl-substituted arenes are acceptable
substrates for TDO and that both enantiomers were processed by the enzyme to
corresponding diasteromers. 7>an.s-2-phenylcyclohexanol 363, which has two


33
putida FI (PpFl)194 that could utilize benzene, toluene, and ethylbenzene as sources of
carbon for energy and growth. Subsequent studies showed that the organism would not
grow if the aromatic hydrocarbons were added directly to the growth media. However,
good growth was observed when the substrate was added as a vapor. Toluene appeared
to be the best substrate for cell growth. Cells that were induced with toluene were found
to be able to metabolize a variety of other substrates. Isopropylbenzene was oxidized at
approximately half the rate observed with toluene. Propylbenzene and butylbenzene
were metabolized even slower. This evidence led investigators to believe that the
bulkiness of the substituent on the aromatic ring has an inverse effect on the metabolic
rate of the substrates. Oxygen uptake experiments202 indicated that the organism
consumed 1.0 mole of oxygen per mole of substrate and the oxidation process was
initiated by incorporating both atoms of molecular oxygen into the aromatic nucleus to
form (+)-c¡x-(lS,2R)-dihydroxy-3-methylcyclohexa-3,5-diene.203'204 This metabolic
transformation catalyzed by a multicomponent mononuclear non-heme iron
Toluene dioxygenase
94
. . M.ntr TT+
(,todD)
Scheme 2-21. Catabolic pathway for toluene used by Pseudomonas putida FI. Genes
designations for individual proteins are shown in parentheses


206
(82) Ghisla, S.; Massey, V. Eur. J. Biochem. 1989, 181, 1.
(83) Mansuy, D.; Battioni, P.; Wiley: New York, 1989, p 195.
(84) Crabtree, R. H. Chem. Rev. 1985, 85, 245.
(85) Shilov, A. E. Activation of Saturated Hydrocarbons by Transition Metal
Complexes;; D. Reidel Publishing: Dordrecht, 1984.
(86) Sheldon, R. A.; Kochi, J. K. Metal-Catalyzed oxidation of organic Compounds',
Academic Press: New York, 1981.
(87) Breslow, R. Acc. Chem. Res. 1980, 13, 170.
(88) Fossey, J.; Lefort, D.; Massoudi, M.; Nedelec, J.-Y.; Sorba, J. Can. J. Chem.
1985, 63, 678.
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Soc. 1989, 111, 7144.
(90) Johnson, R. A.; Trahanovsky, W. S., Ed.; Academic Press: New York, 1978, p
131.
(91) Dalton, H. Adv. Appl. Microbiol. 1980, 26, 71.
(92) Kieslich, K. Bull. Soc. Chim. Fr. 1980, 11, 9.
(93) Sariaslani, F. S. Crit. Rev. Biotechnol. 1989, 9, 171.
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Biotechnol. 1995, 13, 200.
(95) Ribbons, D. T.; Michalover, J. L. FEBS Letters 1970, 11, 41.
(96) Colby, J.; Stirling, D.; Dalton, H. Biochemical journal 1977, 1965, 395-402.
(97) Colby, J.; Dalton, H. Biochemical Journal 1979, 177, 903-908.
(98) Tonge, G. M.; Harrison, D. E. F.; Higgins, I. J. Biochemical Journal 1977, 161,
333.
(99) Burrows, K., J.;; Cornish, A.; Scott, D.; Hoggins, I. J. J. Gen. Microbiol. 1984,
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(100) Rosazza, J. P. N.; Steffens, J. J.; Sariaslani, S.; Goswami, A.; Beale, J. M.; Reeg,
S.; Chapman, R. Appl. Environ. Microbiol. 1987, 53, 2482.
(101) Liu, W. G.; Goswami, A.; Steffek, R. P.; Chapman, R. L.; Sariaslani, F. S.;
Steffens, J. J.; Rosazza, J. P. N. J. Org. Chem. 1988, 53, 5700.




49
TDO also is capable of oxidizing a wide range of polycyclic aromatic hydrocarbons
(PAHs) and heterocyclic aromatic hydrocarbons such quinoline, isoquinoline,
quinoxaline, with good yields and excellent enantiomeric excess,217 Table 2-12226 lists
some of these metabolites to illustrate the structural diversity obtained from this
transformation.
Table 2-12. cis-dihydrodiols from polycyclic aromatic hydrocarbons
Substrates Products ee%
Synthetic Applications of Dihydrodiols
The synthetic utility of the new chiral centers derived from the cij-dihydroxylation
of arene via TDO has been extensively reviewed. Despite the important original


119
(lS,2R)-3-(l-Cyclohexyl)-3,5-cyclohexadien-l,2-d¡ol (354) was
generated via biooxidation of phenylcyclohexane 353 according to the
general procedure for the large scale biotransformation. Addition of
phenylcyclohexane (26.5 g; 165 mmol) over three hours during the
fermentation process afforded 14.0 g (1.8 g/L) of 354 as an off-white solid. Rf = 0.32 (1:1
hexane:EtOAc); mp = 63-65C; Xmax= 268 nm; [a]D30 -73.8 (c 1.0, CH2C12); IR (KBr) 3312,
3048, 1646, 1588, 1447; *H NMR (DMSO-d6) 8 5.80 (ddd, 7= 2.3, 5.4, 9.5, 1H), 5.62-5.56
(m, 2H), 4.67 (d, 7= 6.4, OH), 4.30 (d, 7= 6.3, OH), 4.08 4.00 (m, 1H), 3.75 (bt, 7= 5.4, 1
H), 2.10-1.98 (m, 1 H), 1.86- 1.60(m, 5 H), 1.34-0.96 (m, 5 H); 13C NMR (DMSO-d6) 8
146.9, 129.5, 123.0, 116.7, 69.7, 67.0,41.4, 32.8,31.2,26.3,26.2,25.9.
Correlation of absolute stereochemistry of metabolite 354. The cis-diene diol
354 was reduced with PAD according to the general procedure. Purification by flash
chromatography (hexane-EtOAc 1:4) afforded 0.98 g (86%) of 356 as a crystalline
compound.
(l.S',2/i)-3-(l-cyclohexyl)-3-cyclohexene-l,2-diol (356). Rf =
0.35 (1:1 hexane:EtOAc); mp = 68-69 C; [a]D28 -75.2 (c 1.1, CH2C12);
IR (KBr) 3330, 1448 cm1; H NMR (300 MHz, DMSO-d6) 6 5.34 (t, 7
= 3.5,1 H), 4.31 (d, 7 = 5.9, 1H, OH), 4.27 (d, 7 = 5.6, 1H. OH), 3.80
(t, 7 = 4.5, 1H),3.45-3.33 (m, 1H),2.12-1.85 (m, 3H), 1.82-1.52 (m, 6H), 1.49-1.39 (m,
1H), 1.32 0.90 (m, 5H); 13C NMR (75 MHz, DMSO-ds) 8 143.8, 120.9, 69.4, 67.0,41.5,
38.0, 31.6, 26.6, 26.4, 26.0, 24.9, 24.2; MS (Cl) m/z 195 (3, M-l), 179 (100), 161 (18), 93
356


109
Diol 451 was alkylated with 5-bromopentene to furnish bis-allylated ether 455.
The ether was subjected to polymerization reaction to furnish 456, with an estimated
molecular weight of 4657 (Scheme 3-32). Monomer 455 also has been polymerized
using Grubbs second-generation catalyst.
Br Br Br
Conditions: i) DMF, NaH, 5-Bromopentene; ii) Grubbs' catalyst 1st generation
Scheme 3-32. Generation and polymerization of pentenylether 451
The synthesis of the fully hydroxylated polymer was carried out as outline in
Scheme 3-33.
Conditions: i) DMP, p-TsOH; ii) Aceton, H20, NMO, 0s04; iii) Bu3SnH, AIBN, Benzene;
iv) mCPBA, CH2CI2; v) BF3-Et20, CH2CI2, 4-penten-1-ol; vi) DMF, NaH,
5-bromo-pentene; vii) Grubbs catalyst 1st Generation; viii) H+
Scheme 3-33. Chemoenzymatic approach to fully hydroxylated chiral polymer
In our approach to polyhydroxylated polymer 462, the di-diol 221 was first
protected and the more electron rich double bond was converted to dj-diol 457.
Debromination of 457 was achieved with tributyltinhydride and subsequent epoxidation


kOH
352


121
(lS,2f?)-3-(2-Cyclohexenyl)-3,5-cyclohexadien-l,2-diol (360) was
made according to the general procedure for the small scale
biotransformation. Addition of 3-phenylcyclohexene438 359 (500 mg)
afforded a crude yield of 120 mg of 360 as a reddish oil. X,max= 262;
Rf= 0.53 (2:3/hexane:EtOAc); 'H NMR (DMSO-d6) 5 5.86-5.75 (m, 4H), 5.66-5.51 (m,
6H), 4.64 (d, J= 5.97, 2H), 4.41 (d, J= 6.36, 2H), 4.05 (bs, 2H), 3.78 (t, J= 5.97, 2H), 2.95
(bs, 2H), 1.98-1.91 (m, 4H), 1.86-1.70 (m, 2H), 1.60-1.40 (m, 6H).
Correlation of absolute stereochemistry of (360). The crude metabolites (7.47 g,
38.9 mmol) from a large scale biotransformation of 359 were subjected to PAD reduction
according to the general procedure to give rise to a mixture of inseparable diols 361 and
362. The crude mixture of products 361 and 362 were protected as acetonides and
subjected to catalytic hydrogenation in MeOH at 30 PSI with Wilkinsons catalyst. The
product was isolated and purified by flash chromatography to afford a colorless oil
(2.12g) whose spectroscopic data and physical data ([ajo28 +52.8 (c 0.6, CHCI3)) were in
accord with synthetic 357 and thus proved the absolute stereochemistry of 360.
360
3-(2-hydroxycycIohexanyl)-3,5-cyclohexadien-l,2-dioI
(364a and 364b) were obtained from biooxidation of (+)-
ran-2-phenyl-l-cyclohexanol (363) according to the general
procedure for large scale fermentation. Into a 15-L fermentor 364a 364b
of JM109 (pDTG601)cells (ODMonm = 45), ()-trani-2-phenyl-! -cyclohcxanol 363 (10.29
g) was added (2 g every 30 min). The progress of the biotransformation was monitored by
UV absorbance (X=270 nm). Three hours after the first addition, the absorbance at 270 nm


3
2
T
1
-O
ppm


I L
u
' '-''-'-I 1 ' r |''i' I-1-'
160 140
120 100 80
60 40
20 0


221
(369) Wender, P. A.; Barzilay, C. M.; Dyckman, A. J. J. Am. Chem. Soc. 2001, 123,
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Green Chemistry 1999,1, 57.
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M. G. Bioorg.&Med. Chem. Lett. 2001, 11, 627.


203
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Greenwich, CT, 1995; Vol. 1.
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8,3.
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3795.
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61
0.7g of combretastatin A-l (9.Ixl04 %) and 39.6mg (5.1x10 s %) of combretastatin B-
l.290 Structural analyses of combretastatin A-l and B-l via IR and UV suggested the
presence of aromatic systems. High resolution EIMS indicated molecular formula
C18H20O6 and C18H22O6for combretastatin A-l and B-l, respectively. 'H-NMR
spectroscopic data of the natural products exhibited signals for four methoxy group
protons and indicated that combretastatin B-l is a dihydro derivative of A-1. 1 H-NMR
data of combretastatin A-1 showed four aromatic protons, two of which are magnetically
identical and relatively shielded at 8 6.460. The other two are doublets of an AB spin
system appearing at 8 6.310 and 8 6.691 with a coupling constant (7=8.64 Hz) typical of
two ortho coupled aromatic protons. Another set of AB spin system appears as doublets
at 8 6.453 and 6.523 with coupling constant (7=12.2 Hz) and two exchangeable protons at
8 5.438, suggesting the presence of dihydroxy functionality. The 'H-NMR data for
combretastatin A-l and B-l is summarized in Table 2-14.
Table 2-14. Combretastatin A-1 and B-l 'H-NMR*
Combretastatin A-1
Combretastatin B
-1
Coupling
Coupling
protons
PPm
Constant (Hz)
protons
PPm
(Hz)
3,5-OCHj
3.597 (6H, s)
CH2-CH2-
2.851(4H,m)
4-OCH3
3.760 (3H, s)
4-OCH3
3.827(3H,s)
4-OCH,
3.770 (3H, s)
3,5-OCHj
3.831 (6H,s)
2,3-OH
5.438 (2H, s)
4-OCHj
3.856(3H,s)
5-H
6.310 (1H, d)
Jab =8.64
2,3-OH
5.382(lH,s)
CH=CH
6.453 (1H, d)
Ja-b-12.2
5.398(lH,s)
2,6-H
6.460 (2H, s)
5-H
6.390(1 H,d)
Jab =8.36
CH=CH
6.523 (1H, d)
Jba=12.2
2,6-H
6.420(2H,s)
6-H
6.691 (1H, d)
Jba =8.6
6-H
6.577(1 H,d)
Jba =8.36
chemical shift assignments relative to TMS in CDCI3 solution. For proton-numbering -
see Figure 2-11
Further NMR studies, including l3C-NMR and nuclear overhauser effect difference
spectroscopy (NOEDS), of combretastatin A-l and B-l, suggested structural features as




MOMO


4
illustrate the versatility as well as the practicality of TDO as a biocatalyst for synthesis.
The syntheses of a few substituted catechols by conventional chemical methods as well
as by enzymatic means are included. The last part of the historical chapter is saved for
reviewing a class of natural products, combretastatins, including their discovery,
biological activities, and syntheses because combretastatins A-l and B-l are among the
synthetic targets of this work.
The results and discussion chapter of this document is devoted to explore the
synthetic utility of toluene dioxygenase (TDO) and the second enzyme, toluene cis-
dihydrodiol dehydrogenase (TDDH), of Pseudomonas putida F39D through gaining
better understanding of the enzymes properties and functions. The kinetic resolution
capacity of TDO in resolving aromatic substrates with proximal and distal stereogenic
centers and the possibility of the enzyme going through a radical intermediate during the
catabolism of aromatic substrates are among the main topics of this chapter. The utility
of TDDH to generate substituted catechols also will be discussed as an environmental
benign method to generate synthons for approaches to combretastatins A-l and B-l. The
synthesis of polyhydroxylated chiral polymer is the last part of this chapter.


122
ceased to increase. The pH of the medium was adjusted to 7.8 with concentrated NH4OH,
cells were removed by centrifugation and the supernatant was extracted with base-washed
ethyl acetate to afford 10.25g (1.3 g/L) of a gray solidas a 1:1 mixture of inseparable
diastereomers of 3-(2-hydroxycyclohexanyl)-3,5-cyclohexadien-l,2-diol (364a and 364b).
H (300MHz, DMSO-ds) 8 5.87-5.75 (m, 2H), 5.75-5.68 (m, 2H), 5.65-5.55 (m, 2H), 4.77 (d,
7=6.59, 1H), 4.74-4.65 (m, 2H), 4.59 (d, 7=5.43, 1H), 4.53-4.47 (m, 1H), 4.29 (d, 7=4.1,
1H), 4.18-4.08 (m, 2H), 3.84 (t, 7=5.42, 1H), 3.72 (t, 1H), 3.40-3.23 (m, 1H), 2.04-1.80 (m,
4H), 1.77-1.54 (m, 6H), 1.42-1.06 (m, 9H). I3C (75MHz, CDC13) 8 143.8, 141.7, 129.7,
128.1, 124.4, 123.6, 123.5, 119.4, 75.6, 71.2, 71.1,70.7, 69.2, 66.1, 52.9, 48.6, 35.2, 34.1,
32.2, 31.5, 25.7, 25.5, 25.0, 24.8.
Bicyclohexyl-6-ene-2,3,2'-triol (365a and 365b).
Crude mixture products 364a and 364b (5.034 g, 23.94 mmol)
obtained from biooxidation of ()-iratu-2-phenyl-l-
cyclohexanol (363) was dissolved in 20 mL of cold MeOH.
The reaction flask was placed in an ice bath. With stirring, potassium axodicarboxylate
(6.933 g, 35.67 mmol) was added. The solution of AcOH (9.53 mL, 166.46 mmol) in MeOH
(10 mL) was dropwise added to the reaction mixture. The reaction was allowed to proceed
overnight. Saturated NaHC03 was added until effervescence ceased and the mixture was
evaporated to remove MeOH and the residue was diluted with water (10 mL). The aqueous
solution was extracted with EtOAc (4x30 mL) and dried with MgSOi. The concentrated
EtOAc residue was purified by flash-chromatography (4:l/EtOAc:Hexanes). The eluent was
concentrated to afford a yellow oil (1.2284g, 24%). Rf= 0.68 (EtOAc), [a]n28 =-67.8 (c=1.0
CHCI3). H (300MHz, CDC13) 8 5.51 (m, 1H), 5.45 (m, 1H), 4.84 (d, J=4.39, 1H), 4.75 (d,
365a 365b


139
0.79-0.86 (m, 2H), 0.50-0.57 (m, 2H); 13C NMR (75 MHz, CDC13) 5 143.4, 142.9, 128.8,
120.6, 119.7, 113.5, 9.4, 6.1; MS(EI) m/z\ 150 (100), 149 (6), 107 (26), 77 (44); HRMS (El)
m/zcalcdforC9H10O2(M+): 150.0681. Found: 150.0675. Anal, calcd for C9Hl0O2: C,
71.98, H, 6.71. Found: C, 72.07, H, 6.69
6-Chloro-3-methoxycatechol (405) was obtained through biooxidation
of 4-chloroanisole with E. coli JM109 (pDTG602) as described in the general
procedure for preparation of catechols, the metabolite was purified by flash-
chromatography (4:1 Hexane:EtOAc). The fractions were combined and
concentrated under reduced pressure. Pentane was added to afford 6-chloro-3-
methoxycatechol as white precipitate (4.0g, 0.5g/L). Rf = 0.37 (3:2 hexane:EtOAc); mp =
115-116 C; IR (KBr) 3379, 3081,3059, 3025, 1600, 1492, 1451,756,700; H NMR (300
MHz, DMSO-dj) 8 9.14 (s, 1H), 8.94 (s, 1H), 6.56-6.53 (m, 1H), 6.51-6.48 (m, 1H), 3.75 (s,
3H); 13C NMR (75 MHz, CDClj) 8 146.2, 140.2, 133.8, 119.6, 113.3, 103.7, 56.5; MS(CI):
m/z 176 (33),174 (100), 155 (27), 154 (88), 138 (38) 137 (56), 136 (53)107 (21), 91 (13), 83
(13), 81 (12); Anal, caled for C7H7CIO3: C, 48.16; H, 4.04. Found: C, 48.13; H, 4.01.
l-Bromo-2,3-dihydroxy-4-methoxybenzene (406) was obtained
through biooxidation of 4-bromoanisole with E. coli JM109 (pDTG602) as
described in the general procedure for preparation of catechols, the metabolite
was purified by flash-chromatography (1:1 Hexane:EtOAc) and recrystallized
from octanes:pentane solvent mixture to afford white crystalline l-bromo-2,3-dihydroxy-4-
methoxybenzene: Rf = 0.58 (1:1 hexane:EtOAc); mp = 121-122 C; IR (KBr) 3442, 3054,
1486, 1434, 754, 688; H NMR (300 MHz, DMSO-d6) 8 9.15 (s, 1H), 8.98 (s, 1H), 6.87 (d,
OMe
Br
406
OMe
Cl
405


58
Bromination and demethylation of quaiacol is another approach to 3-
bromocatechol. Bromination of quaiacol in a solution of f-BuNH2 and toluene provided
2-bromo-6-methoxyphenol 272 as the major product plus a small amount of 3-bromo-6-
methoxyphenol 273 as well as trace amount of polybrominated products 274. The side-
products were minimized when two or more molar equivalents of quaiacol were used to
give the desired product in a 60% yield. Demethylation of 2-bromo-6-methoxyphenol
272 with boron tribromide in methylene chloride resulted in 3-bromocatechol,275 as
illustrated in Scheme 2-34.
Scheme 2-34. Synthesis of bromocatechol from quaiacol.
In the synthesis of 3-fluorocatechol 277, 2,3-dimethoxyaniline 268 was treated with
nitric acid and quenched with floroboric acid to give diazonium salt 275.280 The salt was
pyrolyzed to give 2,3-dimethoxyfluorobenzene 276, which was subjected to
demethylated by aluminum chloride to give 3-fluorocatechol 277 in a 29% overall yield
(Scheme 2-35).281
AICI
PhCI
^ I
OH
OH
Scheme 2- 35. Synthesis of 3-fluorocatechol


Combretastatin D-l and D-2 69
3 RESULTS AND DISCUSSION 74
Pseudomonas putida F39D and E. coli JM109 (pDTG601) oxidation of 1 -phenyl-1-
ethanol 74
Biotransformation and Absolute Stereochemistry of New Metabolites 78
Phenylcyclohexene 78
Phenylcyclohexane 80
2-Phenylcyclohexene 81
2-phenylcyclohexanol 81
2-Phenylcyclohexanone 83
Diphenylmethane 85
Biooxidation of Cyclopropylbenzene 86
Mechanistic Investigation 87
Absolute Stereochemistry Correlation of Cyclopropyldiol 383 89
Synthetic Application Potential of Cyclopropylbenzene Metabolite 90
Substituted Catechols 91
Chemoenzymatic approach to combretastatins A-l, and B-l 99
Polyhydroxylated Chiral Polymer 105
4 CONCLUSIONS Ill
5 EXPERIMENTAL SECTION 112
General Methods 112
Large Scale Biotransformation E. coli JM109 (pDTG601) 113
Screening for new substrates 114
Preparation of catechols 114
General procedure for diimide potassium azodicarboxylate (PAD) reduction of
the diene diols 114
Experimentis 115
TDO-catalyzed dihydroxylation of arenes 115
Cyclopropylbenzene project 131
Substituted Catechol Project 135
Chemoenzymatic approach to combetastatin A-l and B-l project 141
Polyhydroxylated Chiral Polymer Project 146
6 SELECTED SPECTRAL DATA 152
LIST OF REFERENCES 202
BIOGRAPHICAL SKETCH 225
vi


co-workers in the late 1960s (Scheme 1-1). The genes responsible for the production of
TDDH in P. putida F39D were cloned and expressed in the E. coli JM109 (pDTG602)
host. Although the organism has been in existent for more than a decade, its use has been
Table 3-2. Catechols From Fermentation of Corresponding Arenes with
JM109 (PDTG602)
[2g/L]
[1.5 g/L] [1.2 g/L]
[0.5 g/L]
[2.2 g/L]
Notes: Numbers in brackets denote the not optimized isolated yield of
metabolite in g/L.
almost non-existent, despite its great potential as a synthetic tool. This is surprising in
view of the importance of simple catechols as synthetic building blocks, the common
occurrence of these functionalities in natural products, and the difficulty associated with
their synthesis in polyfunctional molecules.


26
A number of bacterial enzymes have been purified and characterized,155 however,
the best characterized Baeyer-Villiger Monooxygenase (BVMO) to date is
cyclohexanone monooxygenase (CHMO) from Acinelobacter calcoaceticus. The
complete sequence of the enzyme is available,156 and the enzyme is believed to be
monomeric with only one active site.157
The importance of BVMOs was first recognized over 40 years ago during the initial
era of steroid biotransformation when the biotransformation of progesterone 42 by
66 '2 'z 65
testololactone Baeyer-Villiger
Scheme 2-16. Progesterone metabolism by BVMOs in Cylindrocarpon radicicola
Cylindrocarpon radicicola158 was used in the synthesis of testololactone 66 (Scheme 2-
16). Over the intervening years, the scope of the reaction was vigorously studied. The
enzyme is found to be capable of oxidizing a variety of substrates with excellent regio-
and stereoselectivity,159161 making it an ideal synthetic tool for chemoenzymatic
approaches to synthesis of a wide range of useful compounds.162
Biotransformations of various unsubstituted bicyclic ketones containing a furan or
pyran ring have been used to yield chiral synthons for chemoenzymatic syntheses of


10
many biologically active compounds. The ability of Acetobacter suboxydans5' to
oxidize D-sorbitol 16 to L-sorbose 17 was discovered in 1928 and used as the key step in
the Reichstein-Grussner synthesis of L-ascorbic acid 20 in 193 452 (Scheme 2-4). The
importance of L-ascorbic acid in the production of vitamins and its use as an antioxidant
in the manufacture of food accounts for its worldwide production of about 40,000 tons
each year.
Today, in addition to the Reichstein-Grussner synthesis (Scheme 2-4), the
commercial production of L-ascorbic acid is carried out using Erwinia sp. and Coryne
bacterium sp. in two fermentation steps to provide approximately 1 kg of L-ascorbic acid
for every 2 kg of glucose used (Scheme 2-5).53
H-
CHO
-OH
HO H Erwinia sp. HO H
C02H
0=1
co2h
0=1
Coryne-
HO-
-OH
-OH
CH2OH
D-glucose
H OH bacteriur SP- H OH
o=
CH2OH
2,5-diketo-D-gluconic acid
21
HO-
-H
L-ascorbic acid
-H
CH2OH
2-Keto-L-gulonic acid
22
Scheme 2-5. Two-stage fermentation for the production of 2-keto-L-gulonic acid from
D-glucose
Another industrially important microbial transformation was disclosed in 19525'
for the conversion of that progesterone 23 to 11-a-hydroxyprogesterone 24 by the molds
Rhizopus arrhius.56 The significance of this microbial transformation was soon realized
when the hydroxylated metabolite 24 was used in the synthesis of therapeutic steroids
and their derivatives.57 The microbial hydroxylation step simplified and improved the
efficiency of many approaches to synthesis of corticosteroid hormones and their
derivatives. An example of such an application is illustrated in the production of
prednisone 25, from 11-hydroxyprogesterone 24 (Scheme 2-6). Although the


38
Regioselectivity of TDO-catalyzed cis-dihydroxylation of disubstituted benzene
substrates is restricted to unsubstituted arene bonds. For para-disubstituted benzene
substrates of type 102, the oxidation of the aromatic moiety will occur at bond b to afford
ct.v-dienediol 103 if R>R and give diol 104 if R>R, (Scheme 2-23).
R R R
R' R' R'
102 103 104
If: R>R' R'>R
Scheme 2-23. Regioselectivity of TDO in biooxidation of 1,4-disubstituted aromatic
substrates
For ortho-disubstituted benzene substrates, Scheme 2-24, TDO-catalyzed oxidation
will occur at either bonds b or d depending upon the size of the substituents on the ring.
With a preference being shown for b hydroxylation if substituent R was larger than R
the oxidation will occur primary at bond b giving rise to metabolite a-hydroxylated
metabolite. On the other hand, if R was larger than R, oxidation at bond d will be
preferred.212 213 When both substituents are of similar size, a mixture of ris-dihydrodiols
formed due to oxidation at both bonds b and d.
R R R
105 106 1075h
If: R>R' R>R
Scheme 2-24. Regioselectivity of TDO in biooxidation of 1,2-disubstituted substrates
The directing effect of the substituents has practical implications and has been
exploited for the preparation of crs-dienediols of type 110 and 113 (Scheme 2-25) that are
not obtainable through conventional biotransformation. Ortho- or meta-substituted


100
combretastatins as angiogenesis inhibitors offers new hope and approach to cancer
391
treatment.
Because of their valuable biological activity, much effort has been invested in the
synthesis of these natural products, especially combretastatin A-l and A-4, and their
derivatives. Early approaches to the combretastatins utilize the Wittig coupling reaction
of 294 and 295 as the key step to install the olefin flanking the two aromatic portions of
the natural products.291 This approach gives rise to a mixture of cis- and frans-isomers of
combretastatin A-1 which are difficult to separate (Scheme 2-37).
In our approach to this problem, it was envisioned that the catechol moiety of the
natural products could be constructed by the TDDH carrying organism, E. coli JM109
(pDTG602).200 Oxidation of the para-substituted anisles such as 430, and 434 using E.
coli JM109 (pDTG602) would, in one operation, generate the functionalized catechol unit
of combretastatin A-l and B-l. We envisioned that palladium catalyzed Sonogashira or
Suzuki coupling of the catechol moiety with the appropriate 3,4,5-trimethoxy unit as
illustrated in Scheme 3-24 would provide the natural products main skeleton as an
internal alkyne 429. Selective hydrogenation of 429 via Lindlars catalyst would provide
cis-alkene, combretastatin A-l, or by Pd/C/H2 would provide the fully saturated alkane,
combretastatin B-l (Scheme 2-24). Of the potential substrates for this task, 430, and 434,
only 4-bromoanisole was successfully oxidized to the corresponding catechol with a yield
of 1,2g of catechol per L of fermentation broth. Bromocatechol was then used to
assemble combretastatin A-l and B-l.


Br


364a
*OH
'OH
a:
364b
OH
OH
m,
200
180
140
,L h i
HI
L c 0
100 80
30


MeO
MeO
OMe
MeO 440 MOMO OMOM
: S J
wkw^wwwrttwM
150 140 13f
120 lit
100 90 80 70


85
a ketal using dimethoxypropane and a catalytic amount of p-toluene sulfonic acid to give
alcohol 371a and 371b (Scheme 3-14). The alcohols were treated with CBr4 and PPhi to
generate corresponding bromides, which were reduced with BusSnH to give 357 whose
spectroscopic and physical data were in accord with its synthetic version as disclosed in
Scheme 3-7.
Scheme 3-14. Absolute stereochemistry of metabolites obtained from biotransformation
Diphenylmethane
The biotransformation of diphenylmethane 372 was carried out according to the
general procedure for large scale biotransformation in an 8 L fermentor to furnish 1. lg/L
(89% conversion) of metabolite 373 as an off-white solid. Absolute stereochemistry of
373 was established by first reducing the electron rich olefin with PAD to diol 374, which
was chemically synthesized by coupling vinyl bromide 348 with benzylchloride using s-
BuLi as the metal halogen exchange reagent, followed by deprotection of the acetonide
under acidic condition to arrive at compound 374 (Scheme 3-15).


53
Table 2-13. The use of cis-dienediols in synthesis
Diols Product of asymmetric syntheses
211 212
conduritol C233 conduramineA-1234'235
OAc
D>Ln>D
D D'7] P'OMe
AcO iv OAC
D OAc
215
deuterated hexose234,235,238
D-chiro-inositol2
(-)-pinitol237
OR
OH
x = ch2, nh, o
Y = NH, OH
L-ascorbic acid hexoses;2 u aminohexoses,
azahexoes;240 carbasugar240


357
I'1 1" ' '1 11 '
80 160 140
' 1 1 1 i
IZO
II 1 | 1 1 | IT
100 80
60 <
0
ZO 0 ppm


88
When cyclopropylbenzene was subjected to dihydroxylation with JM109
(pDTG601), only ci's-dienediol 383 metabolite was isolated and no trace of the
cyclopropyl-ring opened product was observed (Scheme 3-18).
JM109 (pDTG601)
377
Scheme 3- 18. Biotransformation of cyclopropylbenzene
This intriguing result prompted us to investigate and compare the reactive tendency
of a conjugated cyclopropylcarbinyl radical such as 378 to the well-known
cyclopropylcarbinyl 375 rearrangement348'350 through computational analysis using the
unrestricted Beckes hybrid three-parameter functional (UB3LYP).351 This method
provides a reliable level of theory for examining the cyclopropylcarbinyl radical
system,352 and the density functional theory methodology was used to examine the
kinetics of the ring opening of the cyclopropylcarbinyl radical system 381 to the
conjugated butenyl system 382 (Scheme 3-17). Calculations were performed using the
Gaussian 98 program package.353 Structures were optimized and vibrational frequency
calculations were performed using UB3LYP level of theory and the 6-31G(d) basis set.354
The transition structure was characterized by a single imaginary frequency, and
thermochemical information was obtained using unsealed frequencies. Single-point
energies were calculated using UB3LYP level of theory using the 6-311+G(2df,2p) basis
set.355
At 35 C the ring opening of the cyclopropylcarbinyl radical, 375, (kr = 1.4 X 108 s
1^356-359 js iq8 tjmes faster tj,an that 0f radical 381 (krcalc =1.1 s'1). Radical stabilizing


89
substituents at the carbinyl position are known to retard the rates of ring opening.360 Even
if a radical is formed at C3, ring opening may not occur if the radical has a short lifetime.
From the computational calculations it appeared that a radical of type 381 would be
very unlikely to participate in a ring opening reaction and this is supported by
experimental results as evidenced by the fact that no open-ring product formed during the
biotransformation process.
The calculation described here for species, such as 378, however, does not
eliminate the possibility of intermediates of this type in the oxidation of cyclopropyl
benzene even though products derived from the ring opening of the cyclopropane have
not been observed in the reaction mixtures. While the nature of the cis-dihydroxylation
remains a mechanistic enigma, the recent evidence by Gibson points to a possible
stepwise mechanism for this unique reaction. Whether the mechanism involves radical or
anionic iron-bound species is at the moment unknown.
Absolute Stereochemistry Correlation of Cyclopropyldiol 383
The absolute stereochemistry of diol 383 was confirmed by an independent
synthesis of vinylcyclopropane 387 from bromodienediol whose absolute stereochemistry
is known (Scheme 3-19). Compound 387 was prepared by the selective reduction of diol
383 with potassium azadicarboxylate (PAD) in 73% yield. Bromodienediol also was
reduced with PAD (86%) and protected to provide acetonide 348 (78%).361 The Grignard
reagent, 385, derived from cyclopropyl bromide 384 was coupled with 348 via a
modified Corey-House synthesis362 to give vinylcyclopropane 386 in a low yield (16%).
Deprotection of this material with THF:H20:TFA/4:1:1 afforded diol 387 whose aD (-
126) matched that of the material obtained via fermentation of cyclopropylbenzene.




180
120


96
Table 3-3. Comparison of efficiency of the enzymatic vs. non-enzymatic preparation of
3-bromo-, 3-methyl-, 3-iodocatechols
Catechols
Chemical
Overall yield = 35 %a; 52 %
Overall yield = 63 %
OH
E value = 47.50a; 36.25 b
E value
= 1.25
"OH
EMY =2.1 % a; 2.8 % b
EMY = 75.0 %
OH
Overall yield = 1 %c
Overall yield = 45 %
Evalu = 63.50c
E value
= 2.25
OH
EMY = 1.6 % c
EMY = 60.0 %
Overall yield = 34 %
OH
OH
No synthesis
E value = 2.0
EMY = 38 %
Enzymatic
Br
5
270
Note: Superscripts denote previous synthesis of corresponding catechols.a Ref.
b From procedure in Ref.388.c From procedure in Ref.274.
Since TDO is the enzyme that precedes catechols dehydrogenase in the aromatic
degradation pathway of P. pulida, the regioselectivity of TDO presets the regiochemistry
of catechols produced in the process. As with TDO, the hydroxylation sites for JM109
small
Figure 3-4. Chemoenzymatic approach to 3,4-cis-dihydrodiols via directing


135
g, 2.0 mmol) was followed by dimethylaminopyridine catalyst (0.2 mg, 0.20 mol%). The
reaction mixture was allowed to stir for 20 hours and quenched with saturated citric acid (2
mL). The reaction mixture was diluted with brine (20 mL) extracted with CH2CI2 (3 x 30
mL). The organic layers were combined, dried (Na2SC>4), and concentrated under reduced
pressure. The crude residue was purified by flash silica gel chromatography (3:2
Hexane:EtOAc) to afford 388 (0.156 g, 96%) as a white solid: Rf = 0.79 (Hexane:EtOAc /
3:2); mp = 80.5-81.5 C; [cx]D29-196.9 (c 1.0, CH,OH); IR (film) 1739, 1460, 1248 cm1; 'H
NMR (300 MHz, CDCI3) 8 5.82 (t, J = 3.2 Hz, 1H), 5.52 (d,/= 3.2 Hz, 1H), 4.92-4.99 (m,
1H), 2.14-2.22 (m, 2H), 2.10 (s, 3H), 2.01 (s, 3H), 1.84-1.91 (m, 1H), 1.70-1.74 (m, 1H),
1.22-1.28 (m, 1H), 0.50-0.64 (m, 2H), 0.30-0.47 (m, 2H); l3C NMR (75 MHz, CDC13) 8
170.9, 170.6, 135.6, 125.5, 70.8, 68.4, 24.3, 22.6, 21.3, 21.2, 14.8, 5.6, 5.0; MS (El) m/z: 179
(100), 119 (92), 91 (24); HRMS (El) m/z calcd for C13H1904 (MH+): 239.1283. Found:
239.1289. Anal, calcd for C,3H1804: C, 65.63; H, 7.61. Found C, 65.43; H, 7.60.
Substituted Catechol Project
3-methylcatechol (98) was obtained through biooxidation of toluene
with E. coli JM109 (pDTG602) as described in the general procedure for
preparation of catechols. The catechol was purified by Kugelrohr distillation
(90-95C/2- 3mm Hg) yielding 19g (2.0g/L) of a white solid; mp 45-46 C (lit.41 55-56); IR
(KBr) 3422, 1624 cm1; H NMR (300 MHz, DMSO-d6) 8 9.04 (s, OH, 1H), 8.06 (s, OH,
1H), 6.64-6.55, (m, 1H), 6.53-6.44 (m, 2H), 2.10 (s, 3H); 13C NMR (75 MHz, CDC13) 8
143.2, 142.2, 124.8, 123.2, 120.4, 113.2, 15.6; MS (FAB) m/z: 93 (85), 124 (100); HRMS
(Cl) m/z caled for C7H802(M+): 124.0524. Found: 124.0590.


55
Br
rf
narciclasine2
Diols Products of non-asymraetric syntheses


65
OH
combretastatin combretastatin A-1 combretastatin A-2 combretastatin A-4
combretastatin A-5 combretastatin B-1 combretastatin D-1 combretastatin D-2
Figure 2-12. Structural variation in Combretastatins
began302 and are continuing into phase n. Today studies continue to investigate the
structure-activity relationship of substituted stilbene derivatives of this type.303306
Figure 2-13. Water soluble phospate derivative of combretastatin A-4
From these studies it has been shown that the 3,4,5-trimethoxy aryl unit, a small
group on the 4-position, and separation of two aryl rings by a two carbon unit, are
important for antimitotic activity, while substitution with a hydroxyl group on the 3-
position is not essential.303,307'309
Combretastatins Syntheses
Combretastatin A-1 and B-1
Early syntheses of combretastatins and their analogues used the Wittig reaction as
the key step for installing the biaryl moieties flanked by a methylene bridge. In the
synthesis of combretastatins A-1 and B-1, 2,3,4-trihydroxybenzaldehyde 292 was treated
with sodium borate-decahydrate (borax) in water for selective formation of the 2,3-borate


216
(280) Balz and Schiemann Ber. Dtsch. Chem. Ges. 1927, 60, 1186.
(281) Corse, J.; Ingraham, L. L. J. Org. Chem. 1951, 16, 1345.
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J. Chem. 1982, 60, 1374.
(287) Malcolm, S. A.; Sofowora, E. A. Lloydia 1969, 32, 512.
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Pflanzen 1975, 168, 15.
(289) Watt, J. M.; Breyer-Brandwijk, M. G. The Medicinal and Poisonous Plants of
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1987,50, 119.
(292) Hamel, E.; Lin, C. M. Biochem. Pharmacol. 1983, 32, 3864.
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(294) Lelleher, J. K. Mol. Pharmacol. 1977, 13, 232.
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(296) Loike, J. D.; Brewer, C. F.; Stemlicht, H.; Gensler, W. J.; Horwitz, S. B. Cancer
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(297) Dixon, M.; Webb, E. C.; Thorne, C. J. R.; Tipton, K. F. Enzymes', Academic
Press: New York, 1979.
(298) Furstner, A.; Nikolakis, K. Liebigs Ann. Chem. 1996, 2107.
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Cancer Res. 1997, 57, 1829.


,7?6
UNIVERSITY OF FLORIDA
3 1262 08556 6197


25
ago.146 Fifty years later, the mechanism of this reaction was deciphered by Criegee
and is still accepted in its original formulation (Scheme 2-15). The reaction proceeds via
A + HSO5
R 58 R
O
X-COOH
59
O
1
rA0RI
R
A.
61
OR'
HSO4
O
x-ccf
Scheme 2-15. Mechanism of the Baeyer-Villiger reaction
the formation of an intermediate resulting from addition of the peracid to the carbonyl
group to form the tetrahedral Criegee-intermediate 60 followed by migration of one of
the substituents on the peroxy oxygen and formation of the product. The migration step
is concerted, rate determining and product determining. The regiochemistry of oxygen-
insertion of the chemical and the enzymatic Baeyer-Villiger reaction can usually be
predicted by assuming that the carbon atom best able to support a positive charge will
migrate preferentially.148 The applications of the Baeyer-Villiger reaction in organic
synthesis are widespread and have been reviewed.91,133'148*152
In biological systems, the Baeyer-Villiger reaction is carried out by
monooxygenases. Enzymatic Baeyer-Villiger oxidation is a flavin-dependent process
that catalyzes the breakdown of carbon structures containing a ketone moiety.
Mechanistically, the process is closely related to the purely chemical process80,153
proposed by Griegee. Of the enzymes studied to date, almost without exception all have
been found in bacteria and fungi.154


217
(300) Pettit, G, R.; Temple, C.; Narayanan, V. L.; Varma, R.; Simpson, M. J.; Boyd, M.
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(304) Sackett, D. L. Pharmac. Ther. 1993, 59, 163.
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229.
(306) Olszewski, J. D.; Marshalla, M.; Sabat, M.; Sundberg, R. J. J. Org. Chem. 1994,
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126
74.2,73.9, 73.86,773.7, 72.2,71.7,54, 50.6, 35.2, 34.0, 32.77, 32.0, 28.0, 27.9,26.67,
26.28, 26.25, 26.0, 26.0, 25.98, 25.25, 24.88, 21.28, 21.0; IR (CDC13) v: 3462.1, 2983,
2856, 1448, 1377; Anal, caled, for C12H24O3: C 71.39, H 9.59; Found C 71.37, H 9.76.
-0>\ no2 kAA N2
3,5-Dinitro-benzoic acid 2-
(2,2-dimethyl-3a,6,7,7a-tetrahydro-
benzo[l,3]dioxol-4-yl)-cyclohexyl
ester (367a) and 3,5-Dinitro- 367a 367b
benzoic acid 2-(2,2-dimethyl-3a,6,7,7a-tetrahydro-benzo[l,3]d¡oxol-4-yl)-cyclohexyl
ester (367b). Into a round-bottomed flask, alcohol mixture 366a and 366b (160 mg,
0.634 mmol) was dissolved in pyridine (5 mL) at ambient temperature. 3,5-
Dinitrobenzylchloride (95 mg, 0.413 mmol) was then added and the progress of the reaction
was monitored via TLC. After 1 h of stirring at room temperature, the reaction mixture was
diluted with EtOAc (20 mL) and washed with 5%, NaHCC>3 (10 mL, 2X), 5% CuSCL (10
mL, 3X). The organic layer was dried with Na2S04 and concentrated under reduced pressure.
The 3,5-dinitrobenzoate derivatives 367a and 367b were purified via recrystallization (EtOH)
to give (190 mg, 67%) white solid, mp= 125-130 C; Rf = 0.54 (4:1 hexane-EtOAc), [a]D27 =
+17.6 (c 0.5, CH2CI2); 'H (CDCI3) 8 9.20 (q, 7= 2.2, 1 H), 9.11 (d, 7= 2.2, 1 H), 9.08 (d, 7=
2.2, 1 H), 5.93 (t, 7= 4.1, 1 H), 5.71 (t, 7= 3.5, 1 H), 5.25 (ddd, 7= 4.2, 10.5, 14.9, 1 H), 5.05
(ddd,7=4.5, 10.8, 15.2, 1 H),4.45 (t,7= 6.1, 2 H), 4.28-4.16 (m, 2H), 2.48-1.22 (m, 24
H), 1.37 (s, 3 H), 1.33 (s, 3 H), 1.26 (s, 3 H), 1.10 (s, 3 H); l3C (CDCl,) 8 162.2, 162.1,
148.7, 136.9, 136.5, 135.3, 134.6, 129.6, 129.4, 128.7, 126.8, 122.4, 122.1, 108.3, 108.2,
79.6, 77.4, 76.6, 73.8, 73.6, 72.9, 72.3, 49.3, 48.1, 32.5, 32.4, 32.2. 31.8, 28.1, 28.1, 26.5,
26.4, 26.1, 26.0, 25.7, 25.7, 24.8, 24.7, 21.5, 20.9; IR (CDCI3) 3102, 1726, 1628, 1600, 1547;


224
(428) Nakno, T. J. Chromatogr. 2001, 906, 205.
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Journal of the American Chemical Society 2002, 124, 10416-10426.
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(433) Dolhaine, H.; Honig, H. MATCH 2002, 46, 71-89.
(434) Dolhaine, H.; Honig, H. MATCH 2002, 46, 91-119.
(435) Hudlicky, T.; Abboud, K. A.; Bolonick, J.; Maurya, R.; Stanton, M. L.; Thorpe,
A. J. Chemical Communications 1996, 15, 1717-1718.
(436) Hudlicky, T.; Stabile, M. R.; Gibson, D. T.; Whited, G. M. Organic Syntheses
1999, 76, 77-85.
(437) Hudlicky, T.; Endoma, M. A. A.; Butora, G. J. Chem. Soc., Perkin Trans. 1 1996,
77,2187-2192.
(438) Murray, R. W.; Singh, M.; Williams, B. L.; Moncrieff, H. M. J. Org. Chem. 1996,
61, 1830.
(439) Berti, G.; Macchia, B.; Macchia, F.; Monti, L. J. Chem. Soc. C, 1971, 3371.


212
(203) Gibson, D. T.; Hensley, M.; Yoshioka, H.; Mabry, T. J. Biochemistry 1970, 9,
1626.
(204) Kobal, V. M.; Gibson, D. T.; Davis, R. E.; Garza, A. J. Am. Chem.. Soc. 1973, 95,
4420.
(205) Yeh, W. K.; Gibson, D. T.; Liu, T. N. Biochem. Biophys. Res. Commun. 1977, 78,
401.
(206) Subramanian, V.; Liu, T. N.; Yeh, W. K.; Narro, M.; Gibson, D. T. J. Biol. Chem.
1981,256, 2723.
(207) Subramanian, V.; Liu, T. N.; Yeh, W. K.; Serdar, C. M.; Wackett, L. P.; Gibson,
D. T. J. Biol. Chem. 1985, 260, 2355.
(208) Subramanian, V.; Liu, T. N.; Yeh, W. K.; Gibson, D. T. Biochem. Biophys. Res.
Commun. 1979, 91, 1131.
(209) Rogers, J. E.; Gibson, D. T. J. Bacteriol. 1977,130, 1117.
(210) Finette, B. A.; Subramanian, V.; Gibson, D. T. J. Bacteriol. 1984,160, 1003.
(211) Finette, B. A.; Gibson, D. T. Biocatalysis 1988, 2, 29.
(212) Boyd, D. R.; Sharma, N. D.; Hand, M. V.; Groocock, M. R.; Kerley, N. A.;
Dalton, H.; Chima, J.; Sheldrake, G. N. Chem. Commun. 1993, 974.
(213) Boyd, D. R.; Sharma, N. D.; Barr, S. A.; Dalton, H.; Chima, J.; Whited, G.;
Seemayer, R. J. Am. Chem. Soc. 1994, 116, 1147.
(214) Boyd, D. R.; McMordie, R. A. S.; Porter, H. P.; Dalton, H.; Jenkins, R. O.;
Howarth, O. W. J. Chem. Soc.,Chem. Commun. 1987, 1722.
(215) Jerina, D. M.; Selander, H.; Yagi, H.; Wells, M. C; Davey, J.; Mahadevan, V.;
Gibson, D. T. J. Am. Chem. Soc. 1976, 98, 5988.
(216) Koreeda, M.; Akhtar, M. N.; Boyd, D. R.; Neill, J. D.; Gibson, D. T.; Jerina, D.
M. J. Org. Chem. 1978, 43, 1023.
(217) Boyd, D. R.; Sharma, N. D.; Dorrity, M. R. J.; Hand, M. V.; McMordie, R. A. S.;
Malone, J. F.; Dalton, H.; Chima, J.; Sheldrake, G. N. J. Chem. Soc., Perkin
Trans. 1 1993, 1065.
(218) Boyd, D. R.; Sharma, N. D.; Boyle, R.; McMurray, B. T.; Evans, T. A.; Malone,
J. F.; Chima, J.; Dalton, H.; Sheldrake, G. N. J. Chem. Soc., Chem. Commun.
1993, 49.
(219) Boyd, D. R.; Sharma, N. D.; Brannigan, I. N.; Clarke, D. A.; Dalton, H.;
Haughey, S. A.; Malone, J. F. Chem. Commun. 1996, 2361.


39
iodobenzenes can be oxidized to the corresponding enantiopure c/s-dihydrodiols and the
metabolites was then subjected to catalytic hydrogenation would give rise to a new range
of 3,4-ct's-dihydrodiols unattainable by direct enzyme-catalyzed asymmetric
dihydroxylation, (Scheme 2-25).213
Scheme 2-25. Chemoenzymatic approach to 3,4-cis-dihydrodiols
Regioselectivity for meta-disubstituted benzenes in TDO-catalyzed
biotransformation is similar to that for ortho-disubstituted benzene substrates. There is a
preference for attack at bonds b if R>R to afford cir-dienediol type 115. On the other
hands, dihydroxylation of bond c will be predominant if R>R to afford metabolite 116 as
the major product (Scheme 2-26).
If: R>R' R>R
Scheme 2-26. Regioselectivity of 1,3-disubstituted aromatic substrates
Dihydroxylation of tri-substituted benzene substrates by TDO is rare and follows a
pattern of regioselectivity similar to that observed in the oxidation of di-substituted
benzene substrates by TDO. The broad substrate specificity of TDO allows for
hydroxylation of bicyclic arenes, such as naphthalene and heteroarenes.183,214
Dihydroxylation of polycyclic arene of higher homologues of naphthalene also occur but


Dedicated to my grandmother, Nguyen Thi Sau


3
The Hudlicky research program is heavily involved in using the toluene
dioxygenase-carrying microorganism, Pseudomonas putida F39D, as the source of
biocatalyst for generation of chiral synthons for application toward numerous synthetic
targets. The P. putida F39D was discovered by Gibson and colleagues in the late 1960s,
and is noteworthy for its ability to metabolize aromatic hydrocarbons to afford
synthetically useful chiral synthons of type 2 (Scheme l-l).29 With the disclosure of
racemic pinitol synthesis by Ley in 1987,30 the practical utility of TDO in synthesis
toluene
dioxygenase
1
wild strain of P. putida
c/s-dihydrodiol
dehydrogenase
.OH
further
metabolisms
acetate
3
OH
mutant strain I R=H, alkyl, aryl, halogen ...
P. putida 39/D
Scheme 1-1. Aromatic degradation pathway of Pseudomonas putida F39D
was realized. Since then, metabolites obtain through biooxidation of aromatic substrates
with P. putida F39D have been used in various enantioselective syntheses, including
polyphenylene,31,32 prostaglandin E2a,33-35 and indivir36,37 that are of industrial and
medical significant.
The historical chapter of this dissertation gives some important developments in the
realm of biotransformation. The use of molecular oxygen as oxidizing agent by
monooxygenases and dioxygenases-carrying microorganisms for the conversion of
alkanes, alkenes, and aromatic to more polar identities is also discussed. A section of this
chapter is designated for a brief review of TDO properties, including the enzymes
substrate specificity, regioselectivity, and stereoselectivity. Selected syntheses of highly
oxygenated natural products and targets of industrial significant are highlighted to


60
established, the use of the second enzyme, TDDH, has been limited to explorations in
cloning.29 This is surprising in view of the potential of simple catechols to serve as
synthetic building blocks for various synthetic applications.
In 1989 the gene todClC2BAD encodes for TDDH, which is responsible for the
transformation of aromatics to corresponding catechols was cloned and expressed into an
E. coli host JM109 (pDTG602).200 Although the organism has been in existence for more
than a decade, there are few reports of the use of the organism for production of
catechols.
Combretastatins
Isolation and Biological Activity
The importance of natural products in biomedical sciences is well established, as
illustrated by the number of scientific publications devoted to this subject. New natural
products are being discovered everyday and their biological affects are routinely being
evaluated. Today, natural products and their derivatives are integral parts of every drug
discovery program. As part of the effort in combating chronic diseases such as cancer,
AIDS, and TB, the worldwide exploratory survey of plants for natural products with
desirable biological activity is an active and promising field, offering hope for combating
chronic diseases.
In this effort, George Pettit and co-workers have traveled to all comers of the world
to explore new sources of natural products from medicinal native plants. In 1981, their
efforts led them to investigate the medicinal potential of an African bush willow tree,
Combretum caffrum, of the Combretaceae family.286 The tree has been used by the
natives for centuries for treatment of leprosy, cancer, and mental illness.287-289 ln 1985, a
large-scale extraction of the bark of the African bush willow tree was initiated, yielding


LIST OF REFERENCES
(1) Keith, L. H.; Telliard, W. A. Environ. Sci. Technol. 1979, 13, 416.
(2) Gibson, D. T.; Subramanian, V. Microbial Degradation of Organic Compounds',
Marcel Dekker Inc.: New York, 1984.
(3) Reineke, W. Annu. Rev. Microbiol. 1988, 42, 263.
(4) Abraham, W. R.; Stumpf, B.; Arfmann, H. A. J. Essent. Oil. Res. 1990, 2, 251.
(5) Boyle, A. W.; Silvin, C. J.; Hasset, J. P.; Nakas, J. P.; Tanenbaum, S. W.
Biodegradation 1992, 3, 285.
(6) Daly, J. W.; Jerina, D. M.; Witkop, B. Experientia 1972, 28, 1129.
(7) Cemiglia, C. E. Biodegradation 1992, 3,351.
(8) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem. lnt. Ed.
Engl. 1995, 34, 412.
(9) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry,
Pergamon: Oxford, 1994.
(10) Drauz, K.; Waldmann, H. Enzyme Catalysis in Organic Synthesis', VCH
publishers: New York,, 1995.
(11) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. Rev. 1996, 96, 443.
(12) Fessner, W. D.; Sinerius, G. Angew. Chem. Int. Ed. Engl. 1994, 33, 209.
(13) Nilsson, K. G. I. Trends in Biotechnol. 1988, 6, 256.
(14) Okamoto, K.; Goto, T. Tetrahedron 1990, 46, 5835.
(15) Roberts, S. M.; Turner, N. J. Journ. of Biotech. 1992, 22, 227.
(16) Kadyrov, R.: Selke, R. Organic Synthesis Highlights IV, 2000.
(17) Taschner, M. J.; Black, D. J. J. Am. Chem. Soc. 1988, 110, 6892.
(18) Martinez, C. A.; Stewart, J. D. Current Organic Chemistry, 2000, 4, 263.
202


99
pancratistatin R = H, OMe, CHO
7-deoxypancrastistatin X = H, CN
R = H, Br, C = X = Cl, Br, I, C =
Scheme 3- 23. Retrosynthetic design of catechol (or arene-ci's-diol) synthons
Chemoenzymatic approach to combretastatins A-l, and B-l
Oxygenated natural products containing a catechol subunit are ubiquitous in nature.
Combretastatins isolated from the bark of an African willow tree Combretum caffrum291
have such moiety embedded in their structures (Figure 2-12). Biological studies of these
compounds have revealed that they are among the most cytotoxic agents tested so far
against a series of cancer cell lines.302,313,389'390 They share a common binding site on
tubulin with the well-known antimitotic agents colchicine, podophyllotoxin, and
steganancin and are capable of inhibiting microtubule assembly at relatively low
concentrations (Table 2-16).291 The fact that combretastatin A-l is capable of displacing
97.8% of colchicine from its binding site at 2 pM concentration shows that it is by far the
best antimitotic agent compared with previously known drugs. The high potency of


35
the P. pulida 39/D is terminated at the cis-dienediol stage, Scheme 1-1. The early
termination in this metabolic pathway makes this organism a valuable tool for the
generation of synthetically useful chiral synthons.
Escherichia coli JM109 (pDTG601)
In order to study the individual enzymes, namely toluene dioxygenase, toluene
dihydrodiol dehydrogenase, and 3-methylcatechol 2,3-dioxygenase, of the P. putida FIs
metabolic pathway in detail, Gibson and colleagues constructed three clones that over
expressed the first three enzymes of the metabolic pathway shown in Scheme 2-21. The
genes todCl C2BA that encode for toluene dioxygenase (reductase-rot., ferredoxin-roL, and
ISPtol) was cloned into a plasmid designated as pDTG601 and the expression of the
genes was placed under control by a lac promoter which can be chemically induced by
isopropyl-P-D-thiogalactopyranoside (IPTG). The plasmid was then placed in an E. coli
host named JM109 (pDTGOl).200 The development of this recombinant microorganism
has many advantageous over the P. putida F39D strain in generating chiral metabolites
for synthetic applications. Since handling and growing JM109 (pDTGOl) is much
easier than handling and growing P. putida F39D, JM109 (pDTGOl) has enabled
toluene dioxygenase to become much more accessible for scientific research. Large
amount of JM109 (pDTGOl) can be grown easily through fermentation processes and
induced to over-express TDO for used in manufacturing of chiral synthons for various
synthetic applications. Today the use of JM109 (pDTGOl) has become routine and is an
integral part of many synthetic ventures.
Toluene Dioxygenase Substrate Specificity
The broad substrate specificity of toluene dioxygenase has allowed for the
generation of a diverse group of metabolites with varying degrees of substitutions.


10
UL
i > > 1 i 1 1 1 i 1 1 "r 1 i 1 1 1 i ''
5 4 3 2 1


205
(59) Sebek, O. K.; Perlman, D. In Microb. Technol.; 2nd ed. 1979; Vol. 1, p 483-488.
(60) Liese, A.; Seelbach, K.; Wandrey, C.; VCH-Wiley: New York, 2000, p 10.
(61) Nunn, J. F. In Oxygen: in applied respiratory physiology, 2nd ed. Boston,
Betterworths, 1977, p 375.
(62) Dix, T. A.; Benkovic, S. S. Acc. Chem. Res. 1988, 21, 101.
(63) Dawson, J. H.; Sono, M. Chem. Rev. 1987, 87, 1255.
(64) Sato, R.; Omura, T. cytochrome P-450; Academic Press: New York., 1978.
(65) cytochrome P-450; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York,
1986.
(66) Omura, T.; Ishimura, Y.; Fujii-Kuriyama, Y. Cytochrome P-450; Second ed.;
VCH: Tokyo, 1992.
(67) Garfinkel, D. Arch. Biochem. Biophys. 1957, 71, 111.
(68) Klingenberg, M. Arch. Biochem. Biophys. 1958, 75, 376.
(69) Estabrook, R. W.; Cooper, D. Y.; Rosenthal, O. Biochem. Z. 1963, 338, 741.
(70) Tan, L.; Muto, N. Eur. J. Biochem. 1986, 156, 243.
(71) Shikita, M; Hall, P. F. J. Biol. Chem. 1973, 248, 5598.
(72) Katagiri, M.; Ganguli, B. M.; Gunsalus, I. C. J. Biol. Chem. 1968, 243, 3543.
(73) Guengerich, F. P.; Kim, D.; Iwasaka, M. Chem. Res. Toxicol. 1991, 4, 168.
(74) Estabrook, R. W.; Hildebrandt, A. G.; Baron, J.; Netter, K. J.; Leibman, K.
Biochem. Biophys. Res. Commun. 1971, 42, 132.
(75) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987,195, 687.
(76) Poulos, T. L.; Finzel, B. C.; Howard, A. J. Biochemistry 1986, 25, 53140.
(77) Sligar, S. G. Biochemistry 1976, 15, 5399.
(78) Kassner, R. J. J. Am. Chem. Soc. 1973, 2674.
(79) Mueller, E. J.; Loida, P. J.; Sligar, S. G.; 2nd ed.; Plenum: New York, 1995, p 83.
(80) Ryerson, C. C.; Ballou, D. P.; Walsh, C. Biochemistry 1982, 21, 2644.
(81) Visser, C. M. Eur. J. Biochem. 1983, 135, 543.


78
they would, if successfully oxidized, furnish resolved synthons that would be useful in
construction of the dibenzofuran core of the morphine alkaloid (Scheme 3-3).
344
OH
Scheme 3- 3. Chemoenzymatic approach to motphine synthesis-introduction
Biotransformation and Absolute Stereochemistry of New Metabolites
Phenylcyclohexene
Before such undertaking was carried out, simpler substrates of similar structural
motif of 341 and 342 were screened to test for TDOs tolerance of such structural feature.
Phenylcyclohexene 345 was chosen for this experiment because of its having a structure
similar to the morphine model substrates 341 and 342. Biooxidation of
JM109
(pDTG601)
345
346
347
Scheme 3-4. Biooxidation of 1 -phenylcyclohexene
phenylcyclohexene was carried out initially in a shake-flask experiment, as described in
the experimental section and metabolite 346 was detected by UV (kmax=302 nm). Full-
scale fermentation of phenylcyclohexene was implemented in a 15-L fermentor to


24
Table 2-4. Epoxidation of branched alkenes by Nocardia corallina
A
56
Nocardia corallina.
02 H20
57 H
R
e.e. [%]
n-C3H7-
76
II-C4H9-
90
11-C5H11-
88
However, the disadvantages associated with mono-oxygenase catalyzed
epoxidation reactions include the enzymes instability, its dependency upon cofactors and
the precise coordination requirement of its subunits for proper function. These factors
discourage the exploitation of isolated enzymes as biocatalysts for epoxidation of
alkenes. Instead, whole-cell fermentations are generally employed. The drawback of this
approach is often linked to the epoxides tendency to react with cellular proteins and
enzymes leading to cell death. If the problem of product toxicity is surmounted, the
microbial epoxidation of alkenes also will be feasible on an industrial scale.142'143 The
toxic effects of the epoxide formed and its further (undesired) metabolism by epoxide
hydrolases can be reduced by employing biphasic media144 for the production of chiral
epoxides. Despite such difficulties, enzymatic epoxidation of alkenes continues to be a
reliable alternative for numerous synthetic applications, an example of which is the
generation of the epoxy-phosphonic acid derivative, fosfomycin,145 whose
enantiospecific synthesis by classical methods would have been extremely difficult. This
was accomplished by a microbial epoxidation reaction using Pemcillim spinulosum.
Baeyer-Villiger Reactions
The Baeyer-Villiger oxidation of ketone with a peroxy acid to produce
corresponding lactone/ester was first reported by Baeyer and Villiger over a century


27
useful compounds, such as multifiden 70 and viridiene 71,163 the antibiotic sarkomycin A
75,164 and the insect antifeedant clerodin 80 (Scheme 2-17).165
cr v
crfsms- c:>+c -
67 68 69
h^b:> o-oS
73 'X
72 O
70
Multifidene
O
(V
71
Viridiene
O
O
Scheme 2-17. Chemoenzymatic syntheses of various useful target molecules involving
the biotransformation by CHMO of bicyclo[3.2.0]-type ketones
BVMOs are also an excellent source of catalyst for desymmetrization of prochiral
ketones to yield the corresponding lactones.17,166 As depicted in Table 2-5, CHMO
catalyzed oxygen insertion occurred on both sides of the ketone, depending upon the
Table 2-5. Enzymatic Baeyer-Villiger oxidation of prochiral ketones
R
Configuration
e.e. [%]
CH3-O-
S
75
Et-
S
>98
n-Pr-
S
>98
f-Bu-
S
>98
n-Bu-
R
52


105
synthetic exercise as a follow up project once the synthesis of the parent compounds has
been completed successfully.
R, = Halogen, Alkyl, OAc
Scheme 3-28. Chemoenzymatic approach to combretastatin analogs
Polyhydroxylated Chiral Polymer
Polymer chemistry is a fascinating and growing field. This branch of chemistry has
attracted a great deal of attention within the last few decades as polymer usefulness
becoming more and more widespread. Polymers such as plastics, fibers, elastomers,
coatings adhesives, rubber, protein, and cellulose have become essential commodities
within modern societies. Thanks to the seemingly endless possibilities, research
activities within this field remain relentless, as the search for better materials continues.
Radical, anionic, or cationic methods for the synthesis of polymeric materials
usually do not impart stereo- or enantio-selectivity to the process.405'407 Chiral polymers
have been of considerable interest for many years.408'410 Methods for the preparation of
chiral polymers remain a challenging field with many difficulties. The introduction of
metal catalysts by Ziegler and Natta in the late 1940s411,412 offered new approaches and


110
with m-CPBA followed by protection of the cis-diol provided epoxide 458. BFj-EtiO
catalyzed epoxide opening with 4-penten-l-ol gave alcohol 459, which was alkylated
with 5-bromopentene to furnish the desired monomer 460. Exposure of this monomer to
Grubbs catalyst (neat) gave polymer 461, which upon deprotection under acidic
condition gave rise to the fully hydroxylated polymer 462, with MW estimated at
-10,000 via GPC.


32
enzymes is common and therefore it is difficult to provide definitive information on the
most appropriate type of dioxygenase for a particular type of biotransformation. In
general terms, however, the TDO system is most suitable for ds-dihydroxylation of
substituted benzene substrates and bicyclic arenes, NDO is particularly useful for bi- and
tri-cyclic arenes, BPDO is more appropriate for larger polycyclic aromatic hydrocarbons
and BZDO is required for benzoic acid substrates. More than twelve types of
multicomponent ring-hydroxylating dioxygneases have been identified and purified.193
A number of mutant dioxygenases also have been identified and characterized and
these have been instrumental in providing ds-dihydrodiols chiral synthons for numerous
synthetic applications. For example, Pseudomonas putida mutant strain F39D190'194 and
UV431 provide a source of TDO, while JT103,195 B13,196 and BG1197 are a source of
BZDO and 9816/11192 has been identified as the mutant strain for NDO. A mutant strain
of a Beijerinckia species, Spingomonas yanoikuyae B8/36198 has been identified as the
source of BPDO and Alcaligenes eutrophus 335/B9 for BZDO.199 Since, these mutant
strains carry an ineffective c-dihydrodiol dehydrogenase gene, the aromatic
hydrocarbon metabolic pathways is terminated after the initial cis-dihydrodioxylation
process. As a result, the ds-dihydrodiol metabolite accumulates. The genes encoding the
TDO components in both F39D and UV4 strains have been cloned and expressed in
Escherichia coli (pDTG601)2and (pKST 11),201 respectively. The expression of these
dioxygenases in E. coli hosts has been extremely useful in facilitating the production of
chiral synthons for synthetic applications.
Pseudomonas putida F39D
In 1968 after a series of studies on microbial oxidation of aromatic hydrocarbons
by soil bacteria, Gibson and colleagues discovered a strain of the bacteria, Pseudomonas


120
(33); HRMS (Cl) m/z caled for Ci2H190 (M-OH): 179.1436. Found: 179.1436. Anal, caled
for C12H20O2: C, 73.43; H. 10.27. Found: C, 73.46; H, 10.38.
This compound upon protection as an acetonide furnished a sample whose
spectroscopic and physical data were in accord with synthetic 357.
(2,2-dimethyl-3a,7a-dihydrobenzo[l,3]dioxol-4-yl)cyclohexane
(357) was synthesized from a procedure adapted from a report by Tamura
and Kochi.342 In a 100 mL 3-neck round bottom flask, magnesium metal
(98 mg, 4 mmol) was covered with dry Et20 (5 mL) and the solvent
degassed with ultrasound under argon atmosphere for 30 min. Then 7-bromo-2,2-dimethyl-
3a,4,5,7a-tetrahydrobenzo[l,3]-dioxole 348,338 (0.699 g, 3.0 mmol) was added together with
dry Et20 (5 mL) and left in the ultrasound bath for 1 h. The reaction mixture was then cooled
down to -78C and with rigorous stirring Li2CuCLi (30 mL, 0.1 M) in Et20 was added over
15 min. Stirring was continued for another 30 min and then cyclohexyltriflate341 (390 mg,
2.0 mmol) in dry Et20 (10 mL) was added. The reaction mixture was stirred for 3 hours.
Work up in the usual manner followed by chromatography (5% EtOAc in hexane) afforded
18 mg (38%) of the product. [ct]D27 = + 49.2 (c 0.9, CHC13); H NMR (300 MHz, CDC13) 8
5.52 (t, 7 = 3.0, 1 H), 4.39 (d, 7 = 5.6, 1H), 4.22-4.17 (m, 1H), 2.18-2.04 (m, 1H), 2.02-1.93
(bt, 7 = 11.3, 1H), 1.87-1.54 (m, 7H), 1.35 (s,6H), 1.34-0.93 (m, 6H); 13C NMR (75 MHz,
CDCI3) 8 141.2, 122.9, 107.9, 73.6, 72.9,40.8, 33.3, 31.5, 28.0, 26.8, 26.6, 26.5, 26.4, 25.6,
20.7; MS (FAB) m/z 236 (0.6), 207 (40), 191 (21), 177 (12), 161 (18), 153 (42), 147 (100),
133 (42), 105 (21), 95 (35), 91 (63), 83 (70); HRMS (FAB) m/z caled for C1SH2402:
236.1776. Found: 236.1774.


79
generate 346 in a crude yield of 3 g/L (98% conversion) as an off-white solid.330-331 Diol
346 was observed to be acid and thermal unstable and re-aromatized readily to 347 at
room temperature (Scheme 3-4). For characterization of the new metabolite, it was
necessary to convert the triene diol 346 to a more stable derivative. This was achieved by
subjecting triene diol 346 to diimide reduction with PAD to furnish a much more stable
diol 352. Absolute stereochemistry of 346 therefore was established indirectly through
the chemical synthesis of 352. This was accomplished through coupling vinyl bromide
348, whose absolute stereochemistry is known,338 via its trimethylstannane derivative 349
with cyclohexanone enol triflate 350339 that was derived from cyclohexanone through a
palladium-catalyzed Stille coupling process^40 (Scheme 3-5). The resulting coupled
product 351 was treated with THF:TFA:H20/4:1:1 mixture of reagents to furnished 352
whose physical data matched with that of the compound derived from fermentation of
phenylcyclohexene, and established the absolute stereochemistry of metabolite 346 as
depicted in Scheme 3-5.
351
THF:H20:TFA
(4:1:1)
r^T
^OH
352
'OH
Scheme 3- 5. Absolute stereochemistry correlation of metabolite 346


45
Ph
0^Vs6
n-n15
Ph'NA.
J ft R
.OMTPA
/Lx^Lomtpa
158
Reagents: i) (R) or (S)-x-methoxy-x-(trifluoromethyl)phenylacetyl chloride.
Scheme 2-29. Preparation of di-MTPA esters 158 for determination of di-dienediol 2
absolute stereochemistry
More recently enantiopurity of ds-dihydrodiols can be determined using R- and S-
2-(l-methoxyethyl)benzeneboronic acid (MPB) 159 followed by 1 H NMR analysis
(Scheme 2-30).221
160
Scheme 2-30. Preparation of boronic ester 160 for determination of ci's-dienediol
absolute stereochemistry
The attractive features of this method include the smaller quantity of ds-
dihydrodiols required and the rapid rate of reaction, which allows less stable metabolites
to be analyzed under milder conditions. The most sensitive and direct method for
enantiopurity determination of ds-dihydrodiols involves the use of chiral stationary
phase-high pressure liquid chromatography (CSP-HPLC). However, this method
requires both enantiomers to be available in order to demonstrate their separation. Since
the majority of ds-dihydrodiol metabolites are rather difficult to obtain in racemic form
and appear to be enantiomerically homogeneous, the CSP-HPLC analysis method212 is
generally used in a complementary manner with the other methods.221,222
In term of stereoselectivity, it is now commonly accepted that biotransformation of
arenes using TDO produces a single enantiomer. The oxidation of monosubstituted


46
benzenes by TDO, with the exception of fluorobenzene, gives almost exclusively a single
enantiomer of the absolute configuration and with an enantiomeric excess of greater than
R
Figure 2-5. The preferred absolute stereochemistry of metabolites obtained from
biotransformation of mono-substituted aromatic substrates
98% (Figure 2-5). The size of substituents on the aromatic ring plays a key role in
defining the absolute stereochemistry of the metabolites. The bulkier the substituent, the
greater directing effect it has on the stereochemical outcome of the oxidation process.
When the size of the substituent is too small, as in the case of fluorobenzene, low
stereocontrol was observed with enantiomeric excess ranging from 60-80%. This
phenomenon is probably due to the relatively small size of the fluorine atom, which
would allow more flexibility of the substrate within the active site of the TDO enzyme.
Table 2-8226 provides a few examples of TDO-catalyzed biotransformations of mono-
substituted benzene with excellent enantiomeric excess.
Table 2-8. Preferred absolute configuration of cis-dihydrodiol metabolites of
monosubstituted benzenes
R
R
rii TDOr
Yoh
u
"k>H
Metabolites
R
% ee
98
ch3
>98
161
Br
>98
162
CH2CH(CH3)2
>98
164
ch=ch2
>98
165
ch2och3
>98
For ori/io-disubstituted benzene substrates, the bulkiest substituent on the ring
exerts the strongest influence on the hydroxylation site and therefore controls the


19
alkanes, alkenes, aromatic, and heterocyclic compounds," sometimes with little
regioselectivity control. Complex substrates have been converted into more than one
oxidation product as in the case of hydroxylation of 1,4-cineole 38 by Streptomyces
griseusm and Bacillus cereus10' (Scheme 2-12). When Streptomyces griseus was used,
8-hydroxycineole 41 was observed to be the major product along with trace amounts of
exo- and endo-2-hydroxy derivatives with low optical purity. On the other hand, when
Bacillus cereus was used, (2R)-exo- and (2R)-emfo-hydroxycineoles were obtained,
exclusively in a ratio of 7:1, both in excellent enantiomeric excess.
1,4-cineole
38
2R-exo
39
2R-endo
40
41
Microorganism
e.e. [%] exo
e.e. [%] endo
exo/endo ratio
Streptomyces griseus
46
74
1:1.7
Bacillus cereus
94
94
7:1
8-hydroxycineole was the major product
Scheme 2-12. Microbial hydroxylation of 1,4-cineole
However there are monooxygenase systems that are extremely precise in regio
selectivity and give rise to only one oxidation product. The importance of such
biocatalyst systems have been realized and used industrially for hydroxylating of the 9a-
and 16a-carbons the steroid framework in the production of therapeutic agents.102'103
Today, almost all carbon centers of the steroid skeleton can be selectively hydroxylated
by using an appropriate microorganism.104 For example, the 1 la-hydroxylation of
progesterone 42 can be achieved with Rhizopus arrhizus55 or Aspergillus niger105 and is
used industrially in manufacturing of 1 la-hydroxyprogesterone (Figure 2-2). The 7p-
hydroxylation of lithiocholic acid 43 via Fusarium equiseti106 is another good example of



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133
mL). The organic layers were combined, dried (MgS04), and concentrated under reduced
pressure. The crude residue was purified by flash chromatography (8:1 HexaneiEtOAc) to
provide 386 (0.151 g, 80%). Rf =0.57 (HexaneiEtOAc / 8:1); [a]D20 +58.99 (c 1.0, CH3OH):
IR (film) 3082, 2985, 1456, 1367, 870 cm1; 'H NMR (300 MHz, CDC13) 8 5.51 (t, 7=3.91
Hz, 1H), 4.37 (d, J = 4 Hz, 1H), 4.25-4.31 (m, 1H), 2.09-2.22 (m, 1H), 1.75-1.96 (m, 2H),
1.60-1.72 (m, 2H), 1.42 (d, J = 5 Hz, 6H), 0.32-0.71 (m,4H); 13C NMR (75 MHz, CDC13) 8
137.3, 123.4, 108.5, 74.5, 73.8, 28.2, 26.8, 25.9, 20.9, 14.6, 5.9, 5.1; MS(EI) m/z: 179 (57),
137 (33), 136 (66), 121 (29), 108 (25), 107 (28), 95 (21), 91 (100), 79 (45), 77 (23)55 (26);
HRMS (El) m/z calcd for Ci2Hi802 (M+): 194.1307. Found: 194.1317.
3-cyclopropylcyclohex-3-ene-l,2-diol (387):
A) From diol 383: To a well-stirred and ice-cooled solution of diol
383 (17 g, 0.11 mol) in methanol (50 mL), potassium azadicarboxylate
(PAD) (76 g, 0.35 mol), was added. A solution of acetic acid (64 mL, 1 mol) in methanol
(50 mL) was added dropwise to the reaction mixture at such a rate that produced 1-2 bubbles
per second. The mixture was allowed to warm to ambient temperature, with the color
changing from yellow to milky white. The reaction mixture was stirred overnight and then
quenched with saturated NaHC03 until effervescence ceased. Methanol was removed under
reduced pressure. The residue was diluted with brine (20 mL), and extracted with ethyl
acetate (4 x 40 mL). The combined organic phases were dried (MgS04) and concentrated to
yield a reddish oil. The crude product was purified by flash silica gel chromatography (3:2
Hexane:Ethylacetate) to afford 387 (12.41 lg, 73%); as a white solid: Rf = 0.20
(Hexane:EtOAc / 3:2); mp = 96-98 C; [a]D29 -126.0 (c 1.0, CH3OH); IR (film) 3272, 3012,
1461, 989 cm1; 'H NMR (300 MHz, DMSO-d6) 8 5.32 (s, 1H), 4.32-4.36 (m, 1H), 3.65 (t, J


6
The story of microbial biotransformations is closely connected with vinegar
OH
biocatalyst
O
A,
5
OH
h2o
Scheme 2-1. Vinegar production
production, dating back to 2000 years B.C. Vinegar production, perhaps the oldest
example of microbial oxidation, shows important developments in the field of
biotransformation by living cells (Scheme 2-1). However, much of the scientific
evidence for this important microbial transformation did not emerge until the mid-1800s.
The ability of a microbe to carry out kinetic resolution of racemic compounds was
first recognized by Pasteur in 1858 when ()-tartaric acid was resolved with Penicilium
co2h
HOjH
Hj OH
co2h
(-)-(S,S)-tartaric acid 6
Figure 2-1. Pasteurs product of the first resolution reaction.
glaucum.40 He showed that the organism resolved racemic tartaric acid by consuming
only the (+)-tartaric acid, while leaving the (-)-enantiomer 6 untouched (Figure 2-1).41
From his work in this area, Pasteur became interested in fermentation and the concept of
spontaneous generation, a widely held belief that living organisms can somehow arise
spontaneously from nonliving matter.
Over the next few years, Pasteur became deeply involved with the French wine
industry investigating the Iamer condition, which turned wine sour, and destroyed
large quantities of the best Burgundy every year. At the time, fermentation was thought
to be a straightforward chemical breakdown of sugar into alcohol, carbon dioxide and
water (as a result of inherent instability of sugar). Scientists had found yeast cells in
fermenting vats of wines and recognized them as being living organisms, but believed


group. I am indebted to Dr. Kofi Oppong, Dr. Stefan Schilling, Dr. Jerremy Willis, Stan
Freeman, and Minh Nguyen for their help and friendship throughout my career here. I
also thank Drs. Duan Caiming, Dean Frey, Pierce Kerry, and Dr. Martin Mandel for
lending their vast chemical knowledge and laboratory techniques to my research
endeavors. I thank the current group members Uwe Rinner, Josef Zezula, Kevin Finn,
David Adams, and Fleather Hillebrenner for their friendship and for sharing with me the
daily duties of keeping the lab functioning smoothly throughout our time together in the
group.
Lastly and most importantly, I would like to acknowledge my family for supporting
me throughout my time at the University of Florida.
tv


116
3-(l(S)-Hydroxyethyl)cyclohexa-3,5-diene-l(S),2(/?)-diol (337a)
was obtained from biooxidation of (S)-l- phenyl-l-ethanol (339a)
according to the procedure described for racemic 1-phenyl-l-ethanol with
P. putida F39/D to provide standard metabolites for monitoring the biotransformation of
racemic phenylethan-l-ol. The conversion was monitored by UV spectroscopy (A, =266
nm). After the usual work up, the process provided an isolated yield of lg/L of metabolite
337a.
3-(l(/f)-Hydroxyethyl)-cyclohexa-3,5-diene-l(S),2(/?)-diol (337b)
was obtained from biooxidation of (5)-l- phenyl-l-ethanol (339b)
according to the procedure described for racemic 1-phenyl-1 ethanol with -
P. putida F39/D to provide a standard metabolite for monitoring the biotransformation of
racemic phenylethan-l-ol. The conversion was monitored by UV spectroscopy (A.max
=266 nm) and after the usual work-up, the process provided an isolated yield of lg/L of
metabolite 337b.
337b
337a
Biooxidation of ()-l-phenyl-l-ethanol (339) with E.
coli JM109 (pDTG601). The cultures were prepared by
using the procedure of Hudlicky et a/.436,437 The progress of
/2 jj/b
(1 : 1)
the biotransformation was monitored by UV (A.raax = 266 nm). GLC analysis of the
metabolites formed showed that the process gave rise to a 1:1 mixture of diastereomers
337a and 337b. 'H NMR spectra of the metabolites were in agreement with the literature


115
solution was added until the solution become slightly basic (pH=8). The methanol was
removed under reduced pressure and the remaining aqueous solution was extracted with
ethyl acetate (4X, 30 mL). The combined organic layers were dried (MgSCL) and the
solution was filtered through a bed of silica gel. The solvent was removed under reduced
pressure to yield the reduced product (85-90%). The reduced diols can be further
purified by recrystallization from hexanes:ethyl acetate for analytical purposes.
Experimentis
TDO-catalyzed dihydroxylation of arenes
Biooxidation of ()-l-phenyl-l-ethanol (339) with P.
putida F39/D. The culture was prepared according to the
procedure of Hudlicky et al.,436 The culture was induced by
adding 150-200 mg of toluene to the media and incubated
in a shaker for 2 hours. Cells were centrifuged (3000 rpm, 10 min) and re-suspended in
the same nutrient broth (250 mL). The racemic phenylethanol (300 mg) was then added
to the culture and the progress of the biotransformation was monitored by UV (266 nm)
and TLC. After 48 h, cells were removed by centrifugation and the supernatant was
extracted with EtOAc (5x 250 mL). The organic layers were combined, dried (NaiSCL)
and concentrated in vacuo to give (138 mg) a 3:2 mixture of diastereomers 337a and
337b respectively according to GLC analysis on an optically active cyclodextrine column
and the 'H NMR spectrum of the resulting metabolites were in accord with the reported
value in literature.




387
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209
(142) Furuhashi, K. Biological Methods to Optically active epoxides. In Chirality in
Industry:', Wiley: New York, 1992.
(143) Takahashi, O.; Umezawa, J.; Furuhashi, K.; Takagi, M. Tetrahedron Lett. 1989,
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(144) Furuhashi, K. Chem. Econ. Eng. Rev. 1986, 18, 7.
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(148) Lee, J. B.; Uff, B. C. Quart. Rev. 1967, 21, 429.
(149) Leffler, J. E. Chem. Rev. 1949, 45, 385.
(150) Hassall, C. H. Org. React. 1957, 9, 73.
(151) House, H. O. In Modem Synthetic Reactions', Benjamin: New York, 1972, p 327.
(152) Plesnicar, B. Oxidation in Organic Chemistry, Part C New York, 1978.
(153) Schwab, J. M.; B.;, L. W.; Thomas, L. P. J. Am. Chem.. Soc. 1983,105,4800.
(154) Willetts, A. Trends in Biotechnology 1997,15, 55.
(155) Nealson, K. H.; Hastings, J. W. Microbiol. Rev. 1979, 43, 496.
(156) Chen, Y.-C. J.; Peoples, O. P.; Walsh, C. T. J. Bacteriol. 1988, 170, 781.
(157) Donoghue, N. A.; Norris, D. B.; Trugill, P. W. Eur. J. Biochem. 1976, 63, 175.
(158) Fried, J.; Toma, R. W.; Klingsberg, A. J. Am. Chem. Soc. 1953, 75, 5764.
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York, 1995, p 745-772.
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1991, 2385.


149
ADMET polymerization of vinylbromo ether
455 to polymer 456. A 10 mL round-bottomed flask
equipped with a magnetic stir bar, monomer 455 (1.68
g, 4.19 mmol) was charged and degassed through
several freeze- pump-thaw cycles under high vacuum.
Grubbs second-generation catalyst (35 mg, 1%) was then added under argon
atmosphere. A few drops of dry CDC13 were added on occasion in order to facilitate
reaction initiation. After the addition of Grubbs catalyst, very slow to moderate
bubbling of ethylene was observed. The bubbling reaction was then exposed to
intermittent vacuum until the viscosity increased, followed by exposure to high vacuum
to remove ethylene, which is continuously generated during the course of the
polymerization. The reaction was started and maintained at room temperature until the
increase in viscosity prevented stirring. At this point, the reaction temperature was
slowly raised to 55C over a period of 3-5 days until a very high viscosity was obtained
or bubbling ceased. The reaction mixture was then cooled to room temperature, and
quenched by exposure to air. The viscous residue was dissolved in ethyl acetate and
passed through a short column of silica to furnish a brown viscous oil (1.58 g).
2,2,7,7-Tetramethyl-5-pent-4-enyIoxy-hexahydro-
benzo[l,2-d;3,4-d']bis[l,3]dioxol-4-ol (459). The di-acetonide
protected epoxide 458 (1.0 g, 4.13 mmol) was dissolved in freshly
distilled methylene chloride (15 mL) along with the 4-penten-l-ol
(0.64 mL, 6.19 mmol) and cooled in an ice-bath for 15 minutes. BF3-Et20 (10 mol %) was
then added and the reaction was allowed to proceed overnight. The reaction was quenched
Br
456


204
(38) Sheldon, R. A. Chirotechnology: Industrial Synthesis of Optically Active
Compounds; Marcel Dekker,: New York, NY 1993.
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Vol. 8.
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(51) Kluyver, A. J.; de Leeuw, F. J. Tijdschr. Verg. Geneesk. 1924, 10, 170.
(52) Reichstein, T.; Grussner, H. Helv. Chim. Acta 1934,17, 311.
(53) Crueger, W.; Crueger, A.; 2nd ed.; Brock, T. D., Ed.; Sinauer Associates, Inc.:
Sunderland, MA, 1989, p 298.
(54) Peterson, D. H.; Murray, H. C. J. Am. Chem. Soc. 1952, 74, 1871.
(55) Peterson, D. H.; Murray, H. C.; Epstein, S. H.; Reineke, L. M.; Weintraub, A.;
Meister, P. D.; Leigh, H. M. J. Am. Chem. Soc. 1952, 74, 5933.
(56) Charney, W.; Herzog, H. L. Microbial transformations of Steroids', Academic
Press: New York, 1967.
(57) Crueger, W.; Crueger, A.; 2nd ed.; Brock, T. D., Ed.; Sinauer Associates, Inc.:
Sunderland, MA, 1989, p 294.
(58) Sarett, L. H. J. Biol. Chem. 1946,162, 601.


Table 3-2. Catechols From Fermentation of Corresponding Arenes with JM109
(pDTG602) 94
Table 3-3. Comparison of efficiency of the enzymatic vs. non-enzymatic preparation of
3-bromo-, 3-methyl-, 3-iodocatechols 96
xi


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
LIST OF ABBREVIATIONS viii
LIST OF TABLES xi
LIST OF FIGURES xiii
LIST OF SCHEMES xiv
ABSTRACT xviii
CHAPTERS
1 INTRODUCTION 1
2 HISTORICAL 5
Biotransformations 5
Oxygenation Reactions 13
Monooxygenases Catalyze Biotransformation 17
Hydroxylation of Alkanes IS
Hydroxylation of Arenes 20
Epoxidation of Alkenes 22
Baeyer-Villiger Reactions 24
Dioxygenases Catalyze Biotransformation 29
Pseudomonas pulida F39D 32
Escherichia coli JM109 (pDTG601) 35
Toluene Dioxygenase Substrate Specificity 35
Toluene Dioxygenase Regioselectivity 37
Toluene Dioxygenase Stereoselectivity 42
Synthetic Applications of Dihydrodiols 49
Substituted Catechols 56
Catechol Dehydrogenases Escherichia coli JM109 (pDTG602) 59
Combretastatins 60
Isolation and Biological Activity 60
Combretastatins Syntheses 65
Combretastatin A-l and B-l 65
Combretastatin A-4 67
v


215
(260) Butora, G.; Hudlicky, T.; Feamley, S. P.; Gum, A. G.; Stabile, M. R.; Abboud, K.
Tetrahedron Lett. 1996, 37, 8155.
(261) Butora, G.; Hudlicky, T.; Feamley, S. P.; Stabile, M. R.; Gum, A. G.; Gonzales,
D. Synthesis 1998, 665.
(262) Tran, C. H.; Crout, D. H. G.; Errington, W.; Whited, G. M. Tetrahedron:
Asymmetry 1996, 7, 691.
(263) Stabile, M. R.; Hudlicky, T.; Meisels, M. L.; Butora, G.; Gum, A. G.; Feamley, S.
P.; Thorpe, A. J.; Ellis, M. R. Chirality 1995, 7, 556.
(264) Johnson, C. R.; Pie, P. A.; Adams, J. P. J. Chem. Soc., Chem. Commun. 1991,
1006.
(265) Johnson, C. R.; Adams, J. P.; Collins, M. A. J. Chem. Soc., Perkin Trans.1 1993,
1.
(266) Mandel, M.; Hudlicky, T. J. Chem. Soc., Perkin Trans. 1 1993,13, 1537.
(267) Hudlicky, T.; Restrepo-Sanchez, N.; Kary, P. D.; Jaramillo-Gomez, L. M.
Carbohydr. Res. 2000, 324, 200.
(268) Ensley, B. D.; Ratzkin, B. J.; Osslund, T. D.; Simon, M. J.; Wackett, L. P.; T., G.
D. Science 1983, 222, 167.
(269) Cavalca, L.; Gilardi, F. Atti Soc. Lomb. Sci. Med. Biol. 1955, 154.
(270) Winterhoff, H.; Gumbinger, H. G.; Sourgens, H. Planta Med. 1988, 54, 101.
(271) Siddiqui, B. S.; Adil, Q.; Siddiqui, S. Pak. J. Pharm. Sci. 1993, 6, 53.
(272) Ladd, D. L.; Weinstock, J. J. J. Org. Chem. 1981, 46, 203.
(273) Ladd, D. L.; Gaitanopoulos, D.; Weinstock, J. J. Synth. Commun. 1985, 15, 61.
(274) Murakami, M.; Chen, J. C. Bull. Chem. Soc. Jpn. 1963, 36, 263.
(275) Perkin and Robinson J. Chem. Soc. Perkin Trans. 1 1914,105, 2383.
(276) Ide, B. a.; Blatt, A. H Ed.; John Wiley &Sons, Inc.: London, 1943; Vol. U, p 44.
(277) Bigelow, A. organic Syntheses', John Wiley and Sons, Inc.: New York, N. Y.,
1941; Vol. 2.
(278) Dawson; Wasserman; Keil J. Am. Chem. Soc. 1946, 68, 534.
(279) Klix, R. C.; Chain, M. H.; Bhatia, A. V. Tetrahedron Lett. 1995, 36, 6413.


30
hydrocarbons biodegradation is therefore essential, since such understanding may allow
development of bioremediation technology and production of chiral precursors for
synthetic applications.
The degradation of aromatic hydrocarbons is carried out almost exclusively by
dioxygenase-carrying microorganisms.182 The initial step of the metabolic process is
often the cycloaddition of oxygen to the aromatic moiety forming an unstable
dioxetane183 which is then enzymatically reduced to yield synthetically useful cis-glycols.
This process constitutes the key step in the aromatic hydrocarbon degradation pathway
and is a prerequisite for further fission and catabolism of the aromatic compounds,
Scheme 2-18.
dioxetane
c/s-glycol
further
metabolism
NAD(P)+ NAD(P)H
Scheme 2-18. Degradation of aromatics by microbial dioxygenases
Dioxygenases are involved in several stages of the aromatic hydrocarbon
degradation pathway. Some diooxygenases, such as TDO, Scheme 2-19, are responsible
for hydroxylation of the aromatic ring, while other dioxygenases, such as catechol
02 + NADH
94
95
Scheme 2-19. Dihydroxylation of aromatic hydrocarbon by TDO




127
MS(CI+) mJz 446(2), 445 (9), 389 (16), 195(20), 177(100), 159 (81), 91 (97); HRMS:
caled, for C22H27N2O8 (M+H) 447.1767, found 447.1764; Anal. Caled, for C22H26N2Og: C
59.19, H 5.87, N 6.27; Found C 59.30, H 5.92, N 6.27.
([{)- and (S)-2-Phenylcyclohexanone (368a and 368b) were made by oxidizing
the corresponding optically active trans-2-phenylcyclohexanol with pyridinium
chlorochromate (PCC) in CH2CI2 at 5 C with the usual work up.
(i?)-2-Phenylcyclohexanone (368a): Rf = 0.30 (Hexane:Et20 4:1);
mp 48-51 C; [a]D27 +108.7 (c 0.20, benzene), lit.439 [a]D26 +114.7 (c 0.45,
benzene); IR (KBr) 3090, 1699 cm1; H NMR (300 MHz, CDCI3) 8 7.34 -
7.16 (m, 5H), 3.62 (dd, J = 12.0, 5.5 Hz, 2H), 2.55-2.38 (m, 2H), 2.27-2.22
(m, 1H), 2.14-2.08 (m, IH) 2.02-1.95 (m, 2H), 1.85-1.77 (m, 2H); 13C NMR (300 MHz,
368a
CDCb) 8 210.2, 138.8, 128.8, 128.5, 126.7, 57.7,42.0, 35.3,27.6, 25.0.
(5)-2-Phenylcyclohexanone (368b): [a]D27 -109.8 (c 0.30, benzene),
lit.439 [o]d24-1 13.5 (c 0.60, benzene); IR (KBr) 3092, 1695 cm'1; H NMR
(300 MHz, CDCI3) 8 7.36-7.15 (m,5H), 3.62 (dd,7= 12.0, 5.5 Hz, 2H),
a
6
368b
2.56-2.39 (m, 2H), 2.28-2.24 (m, 1H), 2.13-2.08 (m, 1H) 2.02-1.95 (m, 2H),
1.85-1.78 (m, 2H);13C NMR (75 MHz, CDClj) 8 210.2, 138.8, 128.8, 128.5, 126.7,57.7,
42.0, 35.3, 27.6, 25.0.
Biooxidations of ()-2-phenylcyclohexanone (368) with E. coli JM109
(pDTG601) were carried out as described for the small scale transformation for racemic


102
Scheme 3-25. Sonogashira coupling approach to combretastatins
A variation of Suzuki coupling reaction between 3,4,5-trimethoxyacetylene 306
and dimethoxybromocatechol 438 was attempted as a model study (Scheme 3-26). The
coupling reaction proceeded successfully to afford 439, which offers a direct avenue for
construction of combretastatin A-l and B-l 397 401
Conditions: i) nBuLi, B(OiPr)3, DME:THF/10:1, -78 C; ii) Pd(PPh3)4; DME:THF/10:1, Reflux
Scheme 3- 26. Suzuki approach to combretastatins
In our approach to combretastatin A-l and B-l, p-methoxybromocatechol 406
prepared through bio-oxidation of the corresponding arene, was first functionalized as
MOM-protected derivative 436 and then subjected to Suzuki-Miyaura coupling
conditions with alkynylboronic ester of 438 to form internal alkyne 440. Hydrogenation
of 440 using 10% Pd/C at 30 PSI for 30 minutes afforded the MOM-protected
combretastatin 441 in 93-95% yield. Acid hydrolysis of 441 in a 1:1 mixture of 3N
HCkTHF solution provided combretastatin B-l, in an overall yield of 52% over4 steps,
excluding the biotransformation process. The spectroscopic data for combretastatin B-
lwere in accord with the literature values402 (Scheme 3-27).


124
caled for Ci2H2i03, 213.1490. Found 213.1456. Anal, caled for C|2H20O3: C, 67.89; H, 9.50.
Found: C, 67.60; H, 9.55.
Bicyclohexyl-4,6-diene-2,3,2'-tr¡ol (364b) was obtained from
biooxidation of (lR,2S)-(+)-frans-2-phenyl-l-cyclohexanol (363b) with
E. coli JM109 (pDTG601) as described for the small scale transformation
of racemic 1-phenyl-l-ethanol. The progress of the biotransformations of
racemic 1-phenyl-l-ethanol. The progress of the biotransformations was monitored by
UV absorbance (Lraax =270 nm) and TLC (EtOAc). The crude metabolite was reduced
with PAD and purified by flash chromatography (1:4 hexane-EtOAc) to afford 102 mg
(50%) of 365b.
(IS, 2R, lS, 2R)-3-(2-hydroxycyclohexanyl)-3-cyclohexene-l,2-
diol (365b). Rf = 0.68 (EtOAc); mp = 103-106C; [a]D31 -148.0 (c 1,
CH2C12); IR (film) 3364, 1448 cm1; 'H NMR (300 MHz, CDC13) 5 5.70
(dd, J = 2.6, 4.5 Hz, 1H), 4.16 (d, J = 3.3, 1H), 3.70-3.60 (m, 2H), 3.10
(bs, 1H), 2.85 (bs, 1H),2.30-2.00 (m, 5H), 1.90-1.62 (m, 4H), 1.58-1.50 (m, 1H), 1.41-1.20
(m, 4H); l3C NMR (75 MHz, CDC13) 6 138.9, 129.5, 72.1, 70.7, 65.8, 54.1, 34.5, 32.2, 25.9,
25.3, 25.2, 24.7; MS(CI+): m/z 213 (29), 195 (78), 177 (100), 159 (28), 147 (16), 105 (14);
HRMS: caled for C12H2i03, 213.1490. Found: 213.1511. Anal, caled for Ci2H20O3: C,
67.89; H, 9.50. Found: C, 67.78; H, 9.62.
Correlation of absolute stereochemistry of 364a and 364b. The crude mixture
from the biotransformation of ()-363 was reduced with PAD to afford 365a and 365b.
Protection of the mixture of the vi'c-cis-diols 365a and 365b (392 mg, 2.0 mmol) were
365b
364b


CHAPTER 2
HISTORICAL
Biotransformations
Throughout the history of mankind, microorganisms have been of tremendous
social and economic importance. Without us even being aware of their existence,
microorganisms were used in food and beverage production very early in history.
Sumerians and Babylonians practiced beer brewing before 6000 B.C. Egyptians,
Hebrews, Greeks, and later Gauls and Iberians, began using yeast to produce fermented
food products such as bread, wine, and beer as early as 4000 B.C. It was only in the 19th
century that scientific progress revealed the secrets behind yeast fermentation. The
pioneering work of French chemist, Louis Pasteur, in the mid-1800s, led to a scientific
understanding of yeast fermentation that eventually became industrially important in
producing chemical commodities such as alcohols and organic material by fermentation.
Lactic acid was probably the first optically active compound to be produced industrially
by fermentation, in 1880.38
In the course of time, it was discovered that microorganisms could modify certain
compounds by simple, chemically well-defined reactions catalyzed by enzymes.39 Today,
these processes are termed biotransformations. The essential difference between
fermentation and biotransformation is that there are several catalytic steps between
substrate and product in fermentation; while there are only one or two steps in
biotransformation. Another distinction is that the chemical structures of the substrate and
the product are similar in biotransformation, but not necessarily in fermentation.
5


62
MeO
MeO^
MeCr
V
rT
-OH
MeO^
^rOH
OMe
'OH
OMe
280
OMe
281
OMe
combretastatin A-1 combretastatin B-1
Figure 2-10. combretastatin A-1 andBl
shown in Figure 10. 13C-NMR data of combretastatin A-1 and B-1 are summarized in
Table 2-15.
Table 2-15. Combretastatin A-1 and B-1 l3C-NMR*
Carbon
Combretastatin A-1
Combretastatin B-1
l
132.49
138.18
2
106.13
105.67
3
152.80
153.05
4
132.67
132.35
5
152.80
153.05
6
106.13
105.67
la
130.21
36.49
la
124.06
31.82
r
117.91
121.55
2
141.72
142.19
3
137.42
136.21
4
146.37
145.40
5
102.98
102.52
6
120.17
120.32
3,5-OCH3
55.85
56.12
4-OCH3
60.79
60.18
4-OCH3
56.16
56.18
*see Figure 2-11 for carbon-numbering
Combretastatin A-ls structure was subsequently and unequivocally established by
X-ray crystallography, Figure 2-11.291
In numerous studies of the biological properties of combretastatins A-1 and B-1,
the compounds were shown to cause mitotic arrest292 in cultured cells. Competitive
inhibition studies between combretastatins A-1 and B-1 and the well-known antimitotic
agents podophyllotoxin 282, steganancin 283, colchicines 284, suggested that


20
methane oxygenase transformation to generate drugs capable of dissolving cholesterol
and which also can be used for therapeutic treatment of gallstones (Figure 2-2).
Rhizopus arrhizus Fusarium equiseti
Aspergillus niger
Figure 2- 2. Microbial hydroxylation of steroids
Hydroxylation of Arenes
Aromatic hydrocarbons are stable identities and are notoriously inert to most
chemical manipulations. Harsh conditions, which are often associated with low
selectivity and uncontrollable formation of byproducts, are often required for
functionalization of the aromatic moiety. The difficulties associated with such methods
make them unappealing and impractical for synthetic applications.107'109
By contrast, selective hydroxylation of aromatic compounds can be achieved
through enzymatic means. Monohydroxylation of aromatic substrates by
monooxygenases has been the subject of many studies. Most monooxygenases require
NAD(P)H to reduce the flavin to a 4a-hydroxyperoxyflavin required for hydroxylation of
the aromatic ring. The transformation is generally very regioselective and the
hydroxylating sites tend to be either ortho-"0'"1 or para"2'"3- to the existing substituent
of the aromatic ring. A/eio-hydroxylation on the other hand is rarely observed.114
Mechanistic studies of monooxygenases in eukaryotic cells such as fungi, yeasts
and higher organisms suggested that the process proceeds predominantly via epoxidation
of the aromatic species to generate unstable arene-oxides 45 as the first step in the


210
(164) Konisgsberger, K.; Griengl, H. Bioorg. Med. Chem. 1994, 2, 595.
(165) Petit, F.; Furstoss, R. Synthesis 1995, 1517.
(166) Taschner, M. J.; J.;, B. D.; Chen, Q.-Z. Tetrahedron: Asymmetry 1993, 4, 1387.
(167) Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.; Wright, M. A. J. Chem.
Soc., Chem. Commun. 1995, 729.
(168) Ottolina, G.; Carrea, G.; Colonna, S.; Ruckemann, A. Tetrahedron: Asymmetry
1996, 7, 1123.
(169) Ouazzani-Chahdi, J.; Buisson, D.; Azerad, R. Tetrahedron Lett. 1987, 28, 1109.
(170) Alphand, V.; Archelas, A.; Furstoss, R. Biocatalysis 1990, 3, 73.
(171) Levitt, M. S.; Newton, R. F.; Roberts, S. M.; Willetts, A. J. J. Chem.. Soc., chem..
Commun. 1990, 619.
(172) Stewart, J. D.; Reed, K. W.; Kayser, M. M. J. Chem. Soc., Perkin Trans. 1 1996,
755.
(173) Stewart, J. D.; Reed, K. W.; Martinez, C. A.; Zhu, J.; Chen, G.; Kayser, M. M. J.
Am. Chem. Soc. 1998,120, 3541.
(174) Kayser, M. M.; Chen, G.; Stewart, J. D. J. Org. Chem. 1998, 63, 7103.
(175) Kayser, M.; Chem, G.; Stewart, J. D. Synlett. 1999, 153.
(176) Redmond, D. E. J. N. Engl. J. Med. 1970, 282, 18.
(177) Pott, P. Natl. Cancer Inst. Monogr. 1963, 10, 7.
(178) Kennaway, E. L.; Heiger, I. Br. Med. J. 1930, 1, 1044.
(179) Cook, J. W.; Hewett, L.; Heiger, I. J. Chem. Soc. Perkin Trans. 1 1933, 395.
(180) Freudenthal, R. I.; Jones, P. W. Carcinogenesis: A comprehensive Survey, Raven
Press: New York, 1978; Vol. 1.
(181) Higginson, J.; Muir, C. S. Cancer Medicine', Lea and Febiger: Philadelphia, 1973.
(182) Dagley, S. In the Bacteria', Sokatch, J. R., Ed.; Academic: London, 1986; Vol. 10,
p 527-55.
(183) Jeffrey, A. M.; Yeh, H. J. C.; Jerina, D. M.; Patel, T. R.; Davey, J. F.; Gibson, D.
T. Biochemistry 1975, 14, 575.




86
348
OH
372
373
374
Scheme 3-15. Biooxidation of diphenylmethane
Biooxidation of Cyclopropylbenzene
The remarkable ability of TDO to process various aromatic substrates has for years
been a subject of study involving scientists from many disciplines. Today much progress
has been made in gaining better understanding of the enzymes properties, including
substrate specificity, regioselectivity, and kinetic resolution capacity. However, many
mechanistic aspects of the enzyme functions remain obscured and unresolved.
R
R
R
dioxetane
Scheme 3-16. Proposed reaction sequence for the formation of cis-dienediol
Early studies by Gibson and colleagues suggested that a hypothetical dioxetane was
an intermediate, which would account for the cis-configuration of diol 2 found in
metabolites from biotransformation by prokaryotic organisms (Scheme 3-16). This
proposal was supported by lsO incorporation experiments where both atoms of oxygen in
the metabolite were shown to come from molecular oxygen.202 The pathway proposed
for the oxidation of benzene by P. putida is given in (Scheme 2-21). However, the
minute details surrounding the oxygen incorporation step, i.e. whether or not the process
goes through a radical intermediate, has not been deciphered.


118
Synthesis of (lS,2R)-3-(l-Cyclohexenyl)-3-cyclohexene-l,2-diol
(352). Into a flame-dried 50 mL round bottom flask equipped with a 3-
way stopper, (CHiCN^PdCL (5 mg, 0.019 mmol) was added together
with 5 mL of anhydrous DMF. A solution of cyclohexanone enol
triflate339(2 1 6 mg, 0.94 mmol)in dry DMF (5 mL) was added to the reaction flask via a
syringe under argon. Trimethyl tin derivative of the vinyl bromide 348338 (296 mg, 0.94
mmol) was dissolved in dry DMF (5 mL) and the solution was added drop wise to the
reaction flask via a syringe. The starting material was consumed after 4 h of stirring at room
temperature according to TLC. The reaction mixture was washed with 5% NaHC03 (2 x 10
mL), and the aqueous layer was extracted several times with pentane. The organic layers
were combined, dried with NaiSCL, and the solvent was removed under vacuum to afford a
red oil. The residue was dissolved in a solvent mixture of THFtFLOtTFA (4:1:1; 10 mL),
and stirred for 1 h. THF and TFA were removed under vacuum and the residue was washed
with 5% NaHCC>3 (2x 5 mL) and water (2x 5mL). The organic layer was dried NaiSCL and
the product was purified by flash chromatography (1:4 hexane-EtOAc) to afford 352 as an
off-white solid 43 mg (18%). Rf = 0.54; mp = 143-144 C; [a]D28 -88.8 (c 1.0, CHC13); IR
(KBr) 3222, 3060, 1451 cm1; 'H NMR (300 MHz, DMSO-ds) 8 5.99 (m, 1H), 5.64 (m, 1H),
4.41 (d,7 = 6.1, 1H), 4.24 (d, 7= 5.2, lH),4.12(m, 1H), 3.44-3.33 (m, 1H), 2.19-1.98 (m,
6H), 1.75-1.38 (m, 6H); 13C NMR (75 MHz, CDC13) 8 137.6, 134.3, 124.3, 123.5,70.0, 65.9,
25.84, 25.77, 24.94, 24.85, 22.87, 22.21; MS(FAB) m/z: 194 (5), 177(22), 93(100); HRMS
(Cl) m/z caled for C12Hi802 (M+): 194.1307. Found: 194.1253.
The title compound was also made via diimide reduction of the metabolite 346
obtained from biotransformation of 1-phenylcyclohexene 345.


147
4,5-Bis-allyloxy-7-bromo-2,2-dimethyl-3a,4,5,7a-tetrahydro-
Br
benzo[l,3]dioxole (452). Into a 50 mL oven-dried round-bottomed
O
flask, NaH (2.41 g, 45 mmol) was added under argon atmosphere and
O
\
covered with anhydrous DMF (20 mL). The mixture was allowed to
452
stir for lh at room temperature under argon atmosphere after the addition of bromodiol 451
(2.41 g, 9.1 mmol). Allylbromide (5.46 g, 45 mmol) dissolved in 10 mL of DMF was then
dropwise added to the reaction mixture and allowed to proceed for another 8h after the
completion of allylbromide addition. The reaction was quenched with water and extracted
with diethylether (25 mL, 3X). The product was purified by chromatograph
(9: l/hexanes:EtOAc) and the fractions containing the titled compound were combined and
concentrated to yield a yellowish oil, which was further purified by distillation at high
temperature and under reduced pressure (150 C, 0.2 mm Hg) using Kulgerolh apparatus to
afford bis-allylether 452 as a colorless oil (3.14 g, 98%). Rf = 0.44 (6:l/hexanes:EtOAc);
[a]D27 = 36.85 (c 1.1, CDC13); IR (film)3078, 2987, 1646, 1456, 1381, 1246, 1218, 1073,
869, 794 cm1; 'H NMR (300 MHz, CDC1,) 6 6.21 (d, 7=2.19, 1H), 6.02-5.84 (m, 2H), 5.33
(dt, 7=1.47, 1.71, 1H), 5.28 (dt, 7=1.47, 1.71 Hz, 1H), 5.24-5.14 (m, 2H), 4.65 (dd, 7=1.22,
6.35 Hz, 1H), 4.40-4.25 (m, 2H), 4.24-4.19 (dd, 7=6.35, 8.55 Hz, 1H), 4.16 (t, 7=1.46 Hz,
1H), 4.14 (t, 7=1.46 Hz, 1H), 3.90-3.84 (ddd, 7=1.22, 0.98, 7.82 Hz, 1H), 3.52 (t, 7=8.05 Hz,
1H), 1.55 (s, 3H), 1.41 (s, 3H); 13C NMR (75 MHz, CDC13) 5 135.2, 134.5, 133.6, 119.0,
117.4, 117.4, 110.8, 79.2, 78.0, 77.9, 77.65,73.5,71.7, 28.2, 26.3; HRMS (El) m/z calcd for
C14H180,,Br [M-CH3]+: 329.0388. Found: 329.0271. Anal, calcd for C^HjiCLBr: C, 52.19;
H, 6.13. Found C, 52.35; H, 6.23.


104
inseparable by standard chromatography techniques. Therefore an alternative approach
for the selective reduction of alkyne 440 to cis-alkene 442 was explored. It was found
that hydroboration was the method of choice for such transformation to give the required
cii-olefin 442 in 84% yield. Final acidic deprotection of the MOM-protective groups
provided for a concise preparation of Combretastatin A-l in an overall yield of 43-51%
over 5 steps in a convergent manner starting from bromoanisole, (Scheme 3-27). This
method is as good or better than previous literature syntheses.291,403'404
The versatility of bio-oxidation with TDO/TDDH, as demonstrated above has
allowed for the generation of functionalized catechols that can be incorporated into the
synthesis of natural products. Such methods also can be used to generate combretastatin
derivatives as in Scheme 3-28. Catechols of type 445 may contain either carbon
substituents or halogens para to the acetylene unit, allowing for analog synthesis through
Suzuki type coupling once the combretastatin nucleus is assembled. Because iodo-,
bromo-, and chloroarenes react at different rates under a variety of C-C bond forming
conditions,397'401,392"390 it is possible to assemble a complete combretastatin nucleus which
still retains a halogen atom para to the styrene unit. In such a case, the R| substituent
could be vary at a late stage of synthesis. On the other hand, such variations in R| may be
introduced early at the stage of 443 and prior to the enzymatic oxidation. One can
envision either p-bromoiodobenzene or p-bromochlorobenzene as convenient starting
points for the synthesis of combretastatin analogs. The hydroxyl groups in 445 or 446
may be differentiated based on the nature of Rj, and functionalized either before or after
the assembly of the combretastatin nucleus. The synthesis of combretastatin analogs for
optimization of their activity, as envisioned in Scheme 3-28, would be an appropriate




12
Further improvements of this biotransformation process have led to a current price of less
than $1 per gram of cortisone.59
The microbial reaction steps listed in Table 2-1 are of great economic significance
for the commercial production of steroids. For example, the transformation of
progesterone to a C 19-steroid is used industrially in testosterone and estrogen production
and the microbial dehydration of ring A is also used in estrogen production.57
In the 1950s, the DNA double helix structure the genetic code of heredity and
the chemical nature of RNA were discovered. This discovery can be regarded as a major
milestone among this centurys main scientific achievements. It led to the synthesis of
recombinant DNA and gave impetus to genetic engineering in the sixties and seventies.
This development quickly made recombinant DNA technology an integral part of
Table 2-2. Some representative industrial whole-cells biotransformations.
Products
Biocatalyst
Since
Company
Vinegar
bacteria
1823
Various
L-2-Methylamino-1-
pheny lpropan-1 -ol
yeast
1930
Knoll AG, Germany
L-Sorbose
Acetobacter suboxydans
1934
Various
Prednisolone
Arthrobacter simplex
1955
Schering AG, Germany
L-Aspartic acid
Escherichia coli
1958
Tanabe Seiyaku
Co., Japan
7-ADCA
Bacillus megaterium
1970
Asahi Chemical
Industry, Japan
L-Malic acid
Brevibacterium
ammoniagenes
1974
Tanabe Seiyaku
Co., Japan
D-p-Hydroxy-
phenylglycine
Pseudomonas striata
1983
Kanegafuchi, Chemical
Co., Japan
Acrylamide
Rhodococcus sp.
1985
Nitto Chemical
Ltd, Japan
D-Aspartic acid
Pseudomonas
1988
Tanabe Seiyaku
and L-Alanine
dacunhae
Co., Japan
L-Camitine
Agrobacterium sp.
1993
Lonza, Czech. Rep.
2-Keto-L-gluconic
acid
Acetobacter sp.
1999
BASF, Merck,
Cerestar, Germany


14
and absence of substrate.76 A summary of the catalytic cycle catalyzed by cytochrome
450 is depicted in Scheme 2-8.
P-
Sub
rh-
N |-N
Cys^33b
Sub
? H20 SubO
NIN
SubO HoO Sub
V } N N
ly^ l
N |-N
Cys/29
-e'
r
Sub
Nri!7N
¡to
N |N
Sub o
o-^
m
N -N
S
31b^Cys
Scheme 2-8. Monooxygenase cytochrome P450s catalytic cycle
In the resting stage, the iron species is coordinated equatorially by a heme moiety
with water or hydroxide as an exchangeable axial ligand trans to the thiolate of a cysteine
residue, 28. The binding of substrate is accompanied by the desolvation of the binding
pocket and expulsion of the quao axial ligand77 to give rise to species 29. This process
alters the enzymes configuration and increases its redox potential by 130 mV.78 These
changes allow the enzyme to accept an electron from NAD(P)H to give species 30 onto
which a molecular oxygen is bound to give a Cytochrome P-450 dioxygen complex that
can be represented by two resonances forms, 31a and 31b. The addition of a second


137
chromatography to yield white powder, mp 35-37C, 'H NMR (300 MHz, CDCI3) 8 6.75 (t,
7= 8.3, 1H), 6.60 (dd, 7 = 1.3,83, 1H), 6.46 (dd, 7= 13,8.3, 1H), 3.89 (s, 3H), 1.52 (bs,
2H); l3C NMR (75MHz, CDClj) 8 146.9, 144.0, 132.4, 119.7, 108.7, 103.0,56.1.
3-Chlorocatechol (398) was obtained through biooxidation of
chlorobenzene with E. coli JM109 (pDTG602) that was carried out as
described in the general procedure for preparation of catechols, yielding a 398
concentration of 0.5 g/L;mp 46-48 C; 'H NMR (300 MHz, DMSO-d6)8 9. 69 (s, OH, 1H),
9.05 (s, OH, 1H), 6.76 (dd,7= 1.7, 8.1, 1H), 6.71 (dd,7= 1.7, 8.1, 1H), 6.61 (t,7=8.1, 1H).
3-Isopropyl-6-methylcatechol (400) was obtained through
biooxidation of p-cymene with E. coli JM109 (pDTG602) as described in the
general procedure for preparation of catechols. The catechol was purified by
Kugelrohr (75-80 C /2 mmHg)distillation yielding 3.6g (0.4g/L) of the title
compound; mp 47-48 C (lit. 47.5-48); IR (KBr) 3447, 1629, 1578 cm'1; 'H NMR (300
MHz, DMSO-d6) 8 8.09 (s, OH, 1H), 7.97 (s, OH, 1H),6.51 (d,7=8.1, 1H), 6.49 (d, 7 = 8.1,
1H), 3.16 (heptet, 7 = 6.8, 1H), 2.09 (s, 3H), 1.11 (d. 7 = 6.8, 6H); l3C NMR (75 MHz,
CDC13) 8 141.6, 141.1, 132.7, 122.1, 121.4, 117.6, 27.2, 22.8 (x2), 15.5; MS(FAB) m/z: 166
(100), 151 (51), 125 (30), 93 (75); HRMS (Cl) m/z caled, for C10Hl4O2 (M+): 166.0993.
Found: 166.0985. Anal, caled for C|0H14O2; C, 72.26; H, 8.49. Found: C, 72.37; H, 8.61.
3-Iodocatechol (402) was obtained through biooxidation of
iodobenzene with E. coli JM109 (pDTG602) as described in the general
procedure for preparation of catechols. The catechol was purified by
402
400


LIST OF FIGURES
Figure page
Figure 2-1. Pasteurs product of the first resolution reaction 6
Figure 2- 2. Microbial hydroxylation of steroids 20
Figure 2-3. Preferred regioselectivity of mono-and polycyclic arene cis-dihydrodiols... 40
Figure 2-4. The used of Moshers acid, MTPA, to determine optical purity of 1-
bromonaphthalene diol 44
Figure 2-5. The preferred absolute stereochemistry of metabolites obtained from
biotransformation of mono-substituted aromatic substrates 46
Figure 2-6. Preferred absolute configuration of cis-dihydrodiol metabolites of
disubstituted benzenes 48
Figure 2-7. Local bond-forming sites in the cis-dienediols 50
Figure 2-8. Examples of primary synthons 51
Figure 2-9. Example of secondary synthons 52
Figure 2-10. combretastatin A-l and B1 62
Figure 2-11. Crystal structure of combretastatin A-l 63
Figure 2-12. Structural variation in Combretastatins 65
Figure 2-13. Water soluble phospate derivative of combretastatin A-4 65
Figure 2-14. Combretastatin D derivatives 69
Figure 3-1. Exploration of reactivity of oxygenated cyclopropylhexanyl systems 90
Figure 3-2. Oxygenated natural products containing a catechol subunit 92
Figure 3-3. Some commercially available catechols 93
Figure 3-4. Chemoenzymatic approach to 3,4-cis-dihydrodiols via directing 96
xii


ENZYMATIC OXIDATION OF AROMATIC SUBSTRATES VIA TOLUENE
DIOXYGENASE AND CATECHOL DEHYDROGENASE: APPLICATION TO
TOTAL SYNTHESIS OF COMBRETASTATINS A-1 AND B-1
By
VU PHONG BUI
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
2003


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
ENZYMATIC OXIDATION OF AROMATIC SUBSTRATES VIA TOLUENE
DIOXYGENASE AND CATECHOL DEHYDROGENASE: APPLICATION TO
TOTAL SYNTHESIS OF COMBRETASTATINS A-l AND B-l
By
VU PHONG BUI
May 2003
Chair: Tomas Hudlicky
Major Department: Chemistry
Toluene dioxygenase (TDO)-carrying microorganisms, the Pseudomonas putida
F39/D and the genetically engineered Escherichia coli JM109 (pDTG601), were used to
investigate the enzymes kinetic resolution power in resolving aromatic substrate with
remote chiral center. In the biotransformation of 1-phenyl-ethanol with P. putida F39/D,
the S-enantiomer was consumed at faster rate than its R-counter part. However, when the
same substrate was oxidized using E. coli JM109 (pDTG601), both enantiomers were
oxidized indiscriminately at the same rate. Similar substrates with larger substituents at
the stereogenic center were also oxidized without any preference for either enantiomer by
JM109 (pDTG601). Biotransformation of cyclopropylbenzene was carried out to
investigate whether or not the biotransformation process goes through a radical
intermediate. New metabolites obtain in these studies were isolated, characterized, and
their absolute stereochemistry were established through conventional chemical
manipulations.
XVII


106
insight into how chiral polymers can be made on an industrial scale. These polymers
have been found helpful as catalysts for asymmetric induction in organic
synthesis,408'409'413 chiral chromatographic separations,414 and ferroelectric and nonlinear
optical applications 415 The types of synthetic chiral polymers that have been reported
include the following: (1) polymers that adopt helical conformations, such as poly-
(chloral), poly(isocyanates), poly(isocyanides), and poly-(triarylmethyl methacrylates);
these polymers are obtained via the so-called helix-sense-selective polymerization from
achiral monomers using either a chiral initiator or catalyst;410 (2) polymers whose optical
activity is derived either from main chain or side chain chirality; they are obtained by
cyclopolymerization of diolefins 416 diisocyanides 417 divinyl ethers,418'421
bis(styrenes),408'409'420'421 dimethacrylates,422'425 diepoxides,426 and peptides427'428 that
mimic natural chiral polymers.
We became interested in polyhydroxylated polymers or high oligomers of type 447,
(Scheme 3-29) which would be derived from the monomers of established configuration
via acyclic diene metathesis (ADMET) of vicinally bis-alkylated olefinic side chains.
The corresponding monomers can be obtained easily from bromodienediol, the product of
the whole cell fermentation of bromobenzene with recombinant organism expressing
Scheme 3-29. Chemoenzymatic approach to polyhydroxylated chiral polymer


CHAPTER 1
INTRODUCTION
Aromatic compounds are ubiquitous in the environment and originate from both
natural and anthropogenic sources. The latter has become an issue of public concern for
human health and environment, as more and more of these compounds find their ways
into the ecosystem through misuse, accidental spillage, and improper disposal.'Although
many aromatic hydrocarbons are biodegradable by existing microorganisms,2 there are
many man-made xenobiotics, such as polychlorinated biphenyls (PCBs) that are
extremely stable and some are non-biodegradable.35 As a result, bioaccumulation of
these recalcitrant chemicals has occurred and has given rise to a whole host of biological
problems including mortality, edema, hepatotoxicity, teratogenicity, immunotoxicity,
reproductive toxicity, and promotion of cancer, affecting organisms at all levels.6,7
The toxic effect of aromatic hydrocarbons on living systems brought about
awareness of the need for prevention and cleanup of already polluted areas. In this effort,
bioremediation technologies have recently emerged as economical and practical viable
alternatives to conventional physicochemical methods. Because of microorganisms
ability to adapt quickly to a changing environment, a very diverse collection of
microorganisms now can be found in many kinds of extreme environments. These
microorganisms are capable of metabolizing a variety of environmental contaminants,
ranging from heavy metals to recalcitrant aromatic hydrocarbons. While a detailed
review on bioremediation technology is beyond the scope of this discussion, it is evident
1


91
cyclopropylcarbinyl system. These cascade reactions could offer a nice approach to
tricyclic ring system for construction of allocyathin B2 and scabronine E as depicted in
Scheme 3-20.
Scheme 3- 20. Synthetic potential of cyclopropyl epoxide 389
Substituted Catechols
The catechol unit, free or alkylated, is a ubiquitous component of countless natural
products. Morphine,261'371 narciclasine,372 pancratistatin,245,246,373 combretastatins A-
1291,374 and B-l291 are a few of such natural products that are of great interest within the
Hudlickys research program because of their extraordinary architectures and interesting


173
ppm


67
Combretastatin A-4
Since combretastatin A-4 is the most potent anticancer agent among the
combretastatins discovered to date, this drug and its derivatives have been the focus of
many syntheses. As shown in the Scheme 2-38, arylacetylene 302, which can be
fashioned in a few steps, was the key intermediate in an approach developed by Furstner
et al. to combretastatin A-4.298 The key step in this approach involved the coupling of
arylbromide 300 with the lithiated trimethoxyacetylene 301 via a 9-MeO-9-BBN-
mediated Suzuki-type cross coupling reaction to produce internal alkyne 302. Selective
hydrogenation of 302 with a Lindlars catalyst (5%Pd/C, ethylenediamine) in MeOH,
followed by deprotection of the TBDMS-group with aqueous TBAF provided a 86:14
mixture of combretastatin A-4 and its saturated analogue 303 (Scheme 2-38).
Scheme 2-38. Suzuki approach to combretastatin A-4
Using similar strategy, Lawrence and colleagues disclosed their approach to
combretastatin A-4 with an overall yield of 29% over 5 steps (Scheme 2-39).313 Again,
the key step in the synthesis involved the coupling of the readily available
trimethoxyacetylene 306298 with aryl iodine 307 to give diarylalkyne 308. The internal


CHAPTER 3
RESULTS AND DISCUSSION
Pseudomonas putida F39D and E. coli JM109 (pDTG601) oxidation of 1-
phenyl-l-ethanol
The utility of cis-dihydroxydihydrobenzene metabolites obtained from TDO-
catalyzed cis-dihydroxylation of aromatic hydrocarbons in asymmetric synthesis is firmly
established. Several recent reviews highlight the growing activity in this field24'26,226 as
investigations continue to probe for the enzymes properties and functions.329332 To date,
over three hundred homochiral diols have been identified from the oxidations of simple,
fused, and biphenyl-type aromatics.24,26'226 Despite this solid endeavor in the field, very
few studies have been designed to study the enzymes capacity in resolving substrates
with remote chiral center of type 334 (Scheme 3-1).
338
Scheme 3-1. Biooxidation of substrates containing benzylic chiral centers
To date, there are only five instances of biooxidation of such compounds have been
reported. Gibson et al. reported the production of diol 337 in conjunction with their
studies of di- vs. monohydroxylation.333 The study of this substrate has been further
elaborated by Cripps and co-workers.334 Several studies were recently conducted by
74


83
mixture of 367a and 367b that were inseparable by conventional chemical techniques,
(Scheme 3-10).
In order to characterize these metabolites individually, biotransformation of the
individual enantiomers was necessary. In two separate experiments, 363a and 363b were
individually oxidized and metabolites were separately reduced with PAD to triol 365a
and 365b, respectively (Scheme 3-11). The biochemically installed cts-diols were
protected as acetonide, and the resulting free alcohols were individually treated with CBr4
363b 365b
Scheme 3-11. Absolute stereochemistry of new metabolites obtained from
and PPI13 followed by BujSnH to furnish 357, whose absolute stereochemistry had been
established, as described in Scheme 3-7. Thus, the chemical manipulations shown in
Scheme 3-11 established the absolute stereochemistry of the metabolites obtained from
biooxidation of rra/ti-2-phenylcyclohexanol.
2-Phenylcyclohexanone
When biotransformation of racemic 2-phenylcyclhexanone was carried out, an
inseperable mixture of metabolites 369a, 369b, and 369c in a ratio of 2:1:1 was isolated




50
disclosure made by Gibson on aromatic degradation by P. pulida in 1968, it took almost
20 years for the synthetic application of this discovery to be realized with the syntheses
of racemic pinitol by Ley10 in 1987 and polyphenyene31,32 by ICI (Scheme 2-31).
?H
(*)-pinitol, 187
Scheme 2-31. Historically important application of arene cis-diols in synthesis
Research groups around the world soon followed these initiatives and demonstrated the
diversity of substrate range of the dioxygenase enzymes. To date, several hundred cis-
diols that have been isolated and identified, and new metabolites continue to be
discovered that could eventually lead synthetic chemists toward new applications and
more diverse targets. The rich functionality of the metabolites offers flexibility for
chemical manipulations including: asymmetric Diels-Alder reactions, epoxidation,
photochemical oxygenation, metallation, diol cleavage, diene cleavage, carbene additions
and ozonolysis that are synthetically useful (Figure 2-7).
Figure 2-7. Local bond-forming sites in the cis-dienediols
Such chemical manipulations have been extremely rewarding in the creation of a
whole new class of primary synthons that possess even greater versatility and
applicability than the original metabolites for synthesis of complex synthetic targets


77
of transport for both enantiomers maybe equal, which would result in both enantiomers
being metabolized at the same rate. This result also could be rationalized by slight
differences in the enzyme topology between the two organisms, perhaps resulting from
post-translational folding.
The absolute stereochemistry of metabolites 337a and 337b derived from
biooxidation of 1-phenyl-1-ethanols has been established333,334'336 and the diimide
reduction of the metabolites 337a and 337b via potassium azodicarboxylate (PAD) to
340a and 340b, was carried out to confirm a diastereomeric 1:1 ratio of the metabolites
(Scheme 3-2).
Scheme 3-2. Biooxidation of racemic phenylethanol by JM109 (pDTG601)
The biooxidation of phenylethanol in both racemic and enantio-pure forms
provided us with a glimpse at the kinetic resolution power of TDO. During this
preliminary study, no evidence of enantiodiscrimination was found, except perhaps a
slight preference for the oxidation of the S-enantiomer 339a of 1-phenyl-1-ethanol when
the blocked mutant P. pulida 39D was used. On the other hand, no preference was
detected in the oxidations mediated by TDO expressed in the recombinant organism E.
coli JM109 (pDTG601). Nonetheless, it was envisioned that if the size of the substituents
at the benzylic stereogenic center of substrate were to increase, the enzyme would be
more discriminatory in its kinetic resolution of racemic enantiomers. We reasoned that
phenyl-cyclohexanone 341 and phenylcyclohexanol 342, would be ideal for this task as


72
Scheme 2-42. Total synthesis of combretastatin D-l
Similarly, the synthetic approach to combretastatin D-1 began with an Ullman-
coupling of the monobenzylated catechol derivative 328, which was prepared from
commercially available aldehyde 327 by selective benzylation, followed by Wittig-type
carbon elongation and reduction of the resulting double bond, with aryl bromide 318. The
biaryl ether 329 was subjected to asymmetric dihydroxylation by means of the well-
known Sharpless catalyst (DHQD)2 PHAL (hydroquinidine 1,4-phthalazinediyl
diether)326 followed by bis-silylation of the diol to arrive at the bis-silylether 330.


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Scheme 3-24. Chemoenzymatic retrosynthetic possibilities to combretastatins 101
Scheme 3-25. Sonogashira coupling approach to combretastatins 102
Scheme 3- 26. Suzuki approach to combretastatins 102
Scheme 3-27. Enzymatic generation of catechol for approach to combretastatin A-l and
B-l 103
Scheme 3-28. Chemoenzymatic approach to combretastatin analogs 105
Scheme 3-29. Chemoenzymatic approach to polyhydroxylated chiral polymer 106
Scheme 3-30. Synthesis of monomer bis-allyl ether 438 108
Scheme 3-31. Polymerization of 452 with Grubbs' First-generation catalyst 108
Scheme 3-32. Generation and polymerization of pentenylether 451 109
Scheme 3-33. Chemoenzymatic approach to fully hydroxylated chiral polymer 109
xvi


2
that microorganisms possess great potential that remains to be exploited for scientific
endeavors.
In recent years, microbes have been recognized as indispensable biocatalysts for
manufacturing enantiomerically pure materials in the chemical and pharmaceutical
industries. Biocatalysts have been used to simplify and enhance the production process
of complex chemicals and drug intermediates. They can add stereospecificity to the
process, eliminating the need for complicated separation and purification steps.
In the field of organic synthesis, the application of enzymes has led to significant
developments in stereoselective approaches to well-known reactions, such as aldol-
condensations,8'14 ketone-reductions,15'16 Baeyer-Villiger oxidations,17,18 and acyloin
condensation.19'23 Although the introduction of chirality is one of the main aims, the use
of microbial enzymes as catalysts for the production of chemically useful compounds
such as alcohols, acids, and amides has been the focus of attention for many years. In
most cases, enzymes have been used for reactions difficult to achieve by purely chemical
methods, e.g., regioselective functionalization of a non-activated carbon center or
regioselective conversion of one out of several similar functional groups in a molecule.
These tasks are facilitated by the mild conditions under which enzymatic reactions take
place.
Recognizing the great potential of microorganisms as tools in synthesis, the last
two decades have witnessed a steady growth of activity within the field.24'28 Research
groups from across the globe continue to explore for useful applications of microbes in
their synthetic designs.


142
NaHC03 (2X, 10 mL), then with water (2X, 10 mL). The organic layer was dried with
MgS04 and the solvent was removed under reduced pressure. The resulting red oil was
purified by flash-chromatography (9:l/pentane:acetone) to afford a colorless oil (2.96 g,
89%). Rf = 0.33 (9:1 pentane:acetone); IR (film) 3087, 2961, 2835, 1577, 1477, 1390, 1292,
1221, 1159; H NMR (300 MHz, CDC13) 8 7.25 (d, /= 8.9, 1H), 6.61 (d, 7=8.9, 1H), 5.2 (s,
2H), 5.13 (s, 2H), 3.82 (s, 3H), 3.66 (s, 3H), 3.60 (s, 3H); l3C NMR (75 MHz, CDC13) 8
153.5, 148.4, 140.2, 127.7, 109.1, 108.9, 99.6, 98.9, 58.4,57.7, 56.3; MS(FAB): mJz 306
(M+, 35), 277 (61), 275 (66). 244 (55), 242 (57), 231 (77), 230 (100), 229 (82) 165 (26), 151
(33); HRMS (FAB) mJz caled for CiiHisBrOs: 306.0102. Found: 306.0157. Anal, caled for
CH,5Br05: C, 43.02; H, 4.92. Found: C, 43.48; H, 4.92.
3-Methoxy-6-[2-(3,4,5-trimethoxy-
phenyl)ethynyl]benzene-l,2-bis-
methoxymethoxy-benzene (440). 3,4,5-
trimethoxy-ethynylbenzene (306) (239 mg, 1.246 mmol) was dissolved in a 10:1 mixture of
DME:THF (5 mL) under Argon. The mixture was cooled to -78 C for 10 min. and nBuLi
(1.33 mmol) was then added and allowed to stir at -78 C for 45 min. Upon which time,
B(OiPr)3 (234 mg, 1.246 mmol) was added and the reaction was allowed to proceed for
another hour. The reaction mixture was then transferred into another reaction vessel
containing MOM-protected-bromoanisole catechol 436 (256 mg, 0.831 mmol) in
10:1/DME:THF (10 mL) along with Pd(PPh3)4 (29 mg, 0.025 mmol) under refluxing
conditions, with constant argon passing through. The reaction was allowed to proceed at
reflux for another 4h. The catalyst was removed by filtration through a silica bed. The
filtrate was concentrated and the product was purified by flash-chromatography to afford
MeO MOMO OMOM
MeO
440


22
industrially (Scheme 2-14). The production of the pharmacologically important (3-
blocker, racemic prenalterol 53, via selective p-hydroxylation of a simple aromatic
precursor, using Cunninghamella echinulata,'2'is another good example of whole-cell
biotransformation.122126
Epoxidation of Alkenes
Chiral epoxides are extensively employed as high-value intermediates in the
synthesis of chiral compounds. In recent years much research has been devoted to the
development of catalytic methods for their production.127 The Katsuki-Sharpless
methods for the asymmetric epoxidation of allylic alcohols'28 and the asymmetric
dihydroxylation of alkenes are now widely applied as reliable procedures. Catalysts for
the epoxidation of non-functionalized olefins have been developed more recently.129'130
Although high selectivity has been achieved for the epoxidation of cis-alkenes, the
selectivity achieved with trans- and terminal olefins were less selective and continue to
be a challenge within the synthetic community.
By contrast, monooxygenases-catalyzed epoxidation can be achieved with excellent
stereoselectivity from a wide range of alkenes that are not accomplishable via traditional
methods.131'132 Despite the wide distribution of mono-oxygenases within all types of
organisms, only alkane and alkene utilizing bacteria, and to a lesser extent fungi, have
been shown to be capable of epoxidizing alkenes.4'91'133
The most intensively studied microbial epoxidizing agent is the co-hydroxylase
system of Pseudomonas oleovorans,134 This consists of three protein components: (i)
rubredoxin, (ii) NADH-dependent rubredoxin reductase and (iii) an co-hydroxylase (a
sensitive non-heme iron protein), and catalyzes not only the hydroxylation of alphatic C-
H bonds, but also the epoxidation of alkenes.135


18
Sub + 02 + H+ + NAD(P)H Monooxygenases. Sub0 + NAD(P)+ + H20
OH
O
Q
EC
1
Cr
0
0
Rn-X=0
rA>-
X = N, S, Se, P
Substrate
Product
Type of reaction
Type of cofactor
alkane
alcohol
hydroxylation
metal-dependent
aromatic
phenol
hydroxylation
metal-dependent
alkene
epoxide
epoxidation
metal-dependent
ketone
ester/lactone
Baeyer-Villiger
flavin-dependent
heteroatom3
heteroatom-oxide
heteroatom oxidation
flavin-dependent
Scheme 2-11. Monooxygenase catalyzed reactions
Hydroxylation of Alkanes
Hydroxylation of unactivated hydrocarbon is a subject of great interest within the
synthetic community.84'86 However, despite the invested effort, functionalization of
unactivated hydrocarbon remains an unresolved challenge. Available approaches to the
problem are limited87'89 and problematic with little regio- and stereoselectivity control.
By contrast, biochemical hydroxylation of unactivated hydrocarbon is common and
is among the most useful processes90'94 with great synthetic utility. Biooxidation of the
hydrocarbon of steroids and terpenoids, for example, are industrially important processes.
In this aspect, methane oxygenases play many important roles. This class of enzyme
specializes in the conversion of alkane to alcohol in the presence of NADH.95 The
enzymes from two obligate methylotrophs, Melhylococcus capsulatus (Bath)96,97 and
Methylosinus Iricosporium OB3b98 have been isolated, purified, and studied. They have
been shown to have a very broad substrate range and are capable of oxidizing a variety of


134
= 4Hz, 1H), 3.35-3.42 (m, 2H), 1.92-1.99 (m, 2H), 1.52-1.63 (m, 1H), 1.38-1.46 (m, 2H),
0.33-0.56 (m, 4H); 13C NMR (75 MHz, CDCl,) 6 138.6, 123.4, 70.0, 68.9, 25.3, 24.2, 14.9,
5.6; MS(FAB) mJz\ 137(8), 93(100); HRMS (FAB) m/z calcd for C,Hl30, (MH+-H20):
137.0966. Found: 137.0967; Anal, calcd for C9H14O2: C, 70.1; H, 9.15. Found C, 70.17; H,
9.18.
B) From vinyl bromide 348: A 50 mL round bottom flask equipped with a condenser and an
addition funnel under argon atmosphere was charged with magnesium metal (0.76 g, 31.2
mmol) and a crystal of iodine. The addition funnel was charged with a solution of
cyclopropyl bromide (0.76 g, 6.24 mmol) in anhydrous diethyl ether (10 mL) and this
solution was added dropwise into the reaction flask. Once the reaction proceeded on its own,
the addition was continued to maintain a constant reflux. After the addition was completed
the reaction flask was heated to reflux for an additional 45 minutes. The Grignard reagent
384 was then transferred into another reaction flask containing bromoacetonide 348 (1.29 g,
5.61 mmol) and tetrakis(triphenylphosphine) palladium (0.194 g, 0.17 mmol) in TFLF (25
mL). The reaction was refluxed for 5 hours and the reaction mixture was filtered through a
thin layer of silica. The filtrate was concentrated under reduced pressure. Flash
chromatography provided the coupled product 386 (0.174 g, 16%). The acetonide 386 was
deprotected using a 4:1:1/THF:H20:TFA (10 mL) solvent mixture affording diol 387 (0.104
g, 75%), whose physical data matched the compound obtained via the biotransformation of
cyclopropylbenzene followed by PAD reduction.
Acetic acid 6-acetoxy-2-cyclopropyIcyclohex-2-enyl ester (388): To
a well-stirred solution of diol 387 (0.105 g, 0.68 mmol) in CH2Cl2(20 mL),
pyridine (0.16 g, 2.0 mmol) was added. Addition of acetic anhydride (0.21
388


160
140
120
100
80
60
40
20
0 ppm
200


44
manipulation of 1,2-dihydronaphthalene 151, Scheme 2-28, showed well-resolved signals
for benzylic methines (8 a-isomer = 6.85 ppm and p.¡SOmer = 6.70 ppm) of both ester isomers.
The combination of methods used above led to the establishment for enantiomeric excess
of diol 147 and 148 to be >98%.
Ll
w\ -l
ft
X
X
ILL,
(a)
(b)
(c)
(d)
6 (ppm)
I (B)
Reagents: i) Ha/Pd/C; i¡) MTPA; iii) OsO
Figure 2-4. The used of Moshers acid, MTPA, to determine optical purity of 1-
bromonaphthalene diol.
The formation of syn-cycloadducts between the cts-dihydrodiol metabolite of type
2 and 4-phenyl-l,2,4-triazoline-3,5-dione 156, followed by protection of the free diols to
form corresponding di-MTPA esters 158 provided another general method for
enantiopurity determination H-NMR analysis (Scheme 2-29). The advantages of this
method include: (1) the formation of crystalline products, which allow absolute
configurations to be confirmed by X-ray crystallography, (2) good resolution of signals
using 19F NMR analysis and (3) ready availability of both R and S-enantiomers of MTPA.


i Lju,
8
7
6
5
ill
VO
4
3
2
1
-0 ppm


C*


214
(239) Banwell, M.; Blakey, S.; Harfoot, G.; Longmore, R. J. Chem. Soc., Perkin Trans.
1 1998,3141.
(240) Hudlicky, T.; Entwistle, D. A.; Pitzer, K. K.; Thorpe, A. J. Chem. Rev. 1996, 96,
1195.
(241) Rouden, J.; Hudlicky, T. J. Chem. Soc., Perkin Trans. I 1993, 1095.
(242) Hudlicky, T.; Rouden, J.; Luna, H.; Allen, S. J. Am. Chem. Soc. 1994, 116, 5099.
(243) Hudlicky, T.; Natchus, M. J. Org. Chem. 1992, 57, 4740.
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(246) Hudlicky, T.; Tian, X.; Konigsberger, K.; Maurya, R.; Rouden, J.; Fan, B. J. Am.
Chem. Soc. 1996, 118, 10752.
(247) Tian, X.; Maurya, R.; Konigsberger, K.; Hudlicky, T. Synlett 1995, 1125.
(248) Hudlicky, T.; Olivo, H. F. J. Am. Chem. Soc. 1992, 114, 9694.
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1997,62, 1194.
(251) Hudlicky, T.; Abboud, K. A.; Entwistle, D. A.; Fan, R.; Maurya, R.; Thorpe, A.
J.; Bolonick, J.; Myers, B. Synthesis 1996, 897.
(252) Banwell, M. G.; McRae, K. J. J. Org. Chem. 2001, 66, 6768.
(253) Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J. Am. Chem. Soc. 1997,
119, 4856.
(254) Banwell, M.; McLeod, M. Chem. Commun. 1998, 1851.
(255) Banwell, M. G.; Darmos, P.; McLeod, M. D.; Hockless, D. C. R. Synlett 1998,
897.
(256) Butora, G.; Gum, A. G.; Hudlicky, T.; Abboud, K. A. Synthesis 1998, 275.
(257) Hudlicky, T.; Seoane, G.; Pettus, T. J. Org. Chem. 1989, 54, 4239.
(258) Hudlicky, T.; Boros, C. H. Tetrahedron Lett. 1993, 34, 2557.
(259) Frey, D. A.; Duan, C.; Hudlicky, T. Org. Lett. 1999, 1, 2085.


208
(122) Wigne, B.; Archelas, A.; Furstoss, R. Tetrahedron 1991, 47, 1447.
(123) Yoshioka, H.; Nagasawa, T.; Hasegawa, R.; Yamada, H. Biotechnol. Lett. 1990,
679.
(124) Theriault, R. J.; Longfield, T. H. Appl. Microbiol. Biotechnol. 1973, 25, 606.
(125) Glockler, R.; Roduit, J.-P. Chimia 1996, 50,413.
(126) Watson, G. K.; Houghton, C.; Cain, R. B. Biochem. J. 1974, 140, 265.
(127) Schurig, V.; Betschinger, F. Chem. Rev. 1992, 92, 873.
(128) Johnson, R. A.; Sharpless, K. B.; Ojima, I., Ed.; Verlag Chemie: New York, 1993,
p 103-158.
(129) Jacobsen, E. N.; Zhang, W.; Muci, A. R.; Ecker, J. R.; Deng, L. J. Am. Chem..
Soc. 1991,113, 7063.
(130) Konish, K.; Oda, K.; Nishida, K.; Aida, T.; Inoue, S. J. Am. Chem. Soc. 1992,
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(131) de Bont, J. A. M. Tetrahedron: Asymetry 1993, 4, 1331.
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(134) de Smet, M.-J.; Witholt, B.; Wynberg, H. enzyme Microb. Technol. 1983, 5, 352.
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1988, 10, 214.


87
Mechanistic Investigation
To probe for a better understanding of this mechanistic aspect of TDO,
cyclopropylbenzene was chosen as model substrate for tracking radical intermediates if
such species formed during the biotransformation process. If the biotransformation
process goes through a radical intermediate of type 378, there is a chance that it could
AH*= 18.1 kcal/mol
ASF = 1.27 cal/(mol K)
AGT= 17.7 kcal/mol
381 382
Scheme 3-17. Mechanistic possibilities for cis-dihydroxylation
lead to the fragmentation of the cyclopropyl ring to form the radical anion 380 (Scheme
3-17). Such fragmentation would be likely to occur, since cyclopropylcarbinyl type
rearrangements have been observed during the investigation of the mechanism involved
in the isopenicillin N synthase reaction343'346 and Iron IV-sulfur-bound species347 have
been proposed to account for the results where trapping of the butenyl radical by the
sulfur atom have been observed.


ZL\


59
The synthesis of anisle catechol 261 was accomplished via demethylation of aryl
methyl ether with thioethoxide ion in dimethyl formamide to give an excellent yield of
96% (Scheme 2-36).282
OMe OMe
Scheme 2-36. Synthesis of 3-methoxyatechol
The above examples are representative of chemical methods available for the
syntheses of 1,2-monosubstituted catechols. Most methods involve the use of harsh
conditions such as strong acids, or oxidants to cleave alkylated catechols with poor
selectivity and low yields.
Enzymatic approaches to catechol synthesis are available but under-explored. To
the best of our knowledge, only three or four methods of making catechols through
enzymatic means exist, in addition to the original report by Gibson. The process by
Frost283 involves the use of E. coli AB2834/pKD136/pKD9.069A for conversion of
glucose to the parent catechol. Another method involved the oxidation of substituted 2-
nitrophenols to various catechols with nitrophenol oxygenase via Pseudomonas Sp.
Strain T-12 to generate various substituted catechols.284 Recently, Yoshida and co
workers reported a new organism capable of oxidizing aromatic substrates to catechols in
one step.285
Catechol Dehydrogenases Escherichia coli JM109 (pDTG602)
Studies of soil bacteria degradation of aromatic compounds led David Gibson in
the late 1960s to elucidate the metabolic pathway as shown in Scheme 1-1. Although
the use of TDO as a means of generating chiral synthons for synthetic applications is well


117
values.333 The biooxidation of racemic 1-phenyl-1-ethanol was also carried out on a
large scale and gave the same 1:1 mixture of 337a and 337b.
3-(Hydroxyethyl)cyclohexa-3-ene-(lS, 2/)-diol (340) was
made by treating the crude dienetriol 337 (1.50 g, 9.6 mmol) obtained
from biooxidation of corresponding phenylethanol with PAD in the
usual manner as described in the general procedure to afford the title compound as a
colorless oil (1.4 g, 92%). H NMR (300 MHz, CDC13)6 6.00 (bt, J = 3.5, 1H), 5.89 (bt, J=
3.7, 1H), 4.47-4.42 (m, 1H), 4.40^1.26 (m, 4H), 4.17-3.90 (m, 5H), 3.76-3.52 (m, 2H), 2.30-
2.12 (m, 2H), 2.11-1.90 (m,2H), 1.85-1.56 (m, 4H), 1.36-1.20 (m, 6H); ,3C NMR (75MHz,
CDC13) S 140.3, 139.2, 126.5, 124.9,71.0, 69.6, 69.2, 69.1, 67.6, 65.8, 24.9, 24.7, 23.9, 23.4,
21.8,21.6.
(lS,2R)-3-(l-Cyclohexenyl)-3,5-cyclohexadien-l,2-diol (346)
was obtained from biotransformation of 1 -phenylcyclohexene (345)
with the E. coli JM109 (pDTG601) according to the general procedure
for the large scale fermentation. Addition of 1-phenylcyclohexene
(15.7 g, 99.2 mmol) afforded a crude yield of 18.4 g (3 g/L) of 346 as an off-white solid.
K= 302 nm; mp 90-93 C; [ 'H NMR (300 MHz, CDClj) 8 6.16 (bt, J= 4.2, 1H), 6.01-5.89 (m, 2H), 5.75 (bd, J= 9.5,
1H), 4.49-4.43 (m, 1H), 4.34 (bd, J= 5.5, 1H), 2.70 (bs, 2H), 2.27-2.14 (m, 4H), 1.79-1.55
(m, 4H); 13C NMR (75 MHz, CDCI3) 8 138.8, 133.8, 130.7, 125.7, 124.0, 117.9, 71.4, 66.7,
26.0, 25.3, 22.6, 22.0; MS(CI): tn/z 193 (1), 191 (27), 175 (100), 174 (79), 173 (22); HRMS
(Cl) m/z caled for C|2Hi702(M+H): 193.1228. Found: 193.1213.


169
- C-J
- fO
r
r ^
- m
- ID
-
r
I- CO
- CD
ppm




101
OMe OMe
combretastatin A-1 combretastatin B-1
Conditions: i) JM109 (pDTG602); ii) (a) PPh3, CBr4, CH2CI2; (b) BuLi, THF, -78C; Hi)
CH2I2, AIBN, PhCH3; iv) HCCNa, BOMe)3, Pd[CI2(dppf)]; v) Sonogashira coupling
vi. Lindlar's catalyst
Scheme 3-24. Chemoenzymatic retrosynthetic possibilities to combretastatins
Initially, Sonogashira coupling conditions392'396 were used in an attempt to couple
trimethoxyacetylene 306 and 436. This approach failed to give any desire coupled
product 437 (Scheme 3-24), and starting materials were recovered unchanged.


136
3-Bromocatechol (270) was obtained through biooxidation of
bromobenzene with E. coli JM109 (pDTG602), as described in the general
procedure for preparation of catechols. The catechol 3- bromocatechol was
purified by distillation using Kugeirohr apparatus (80-90C/2mmHg) to yield 9g (lg/L)
of white solid; mp 39-40C (lit.388 40.5-41.5); IR (KBr) 3484, 1592; H NMR (300 MHz,
DMSO-d6) 5 9.76 (s, OH, 1H), 9.07 (s, OH, 1H), 6.90(dd,7= 1.5,8.1, 1H), 6.76 (dd,/ =
1.5, 7.9, 1H), 6.57 (dd, 7 = 7.9, 8.1, 1H); l3C NMR (75MHz, CDC13) 8 146.4, 142.9,
122.7, 120.1, 114.5, 109.8; MS(FAB) m/z: 190 (6), 188 (6), 137 (60), 136 (70), 107 (33),
95 (50), 81 (63); HRMS (Cl) m/z caled for C6H5Br02 (M+): 187.9473. Found: 187.9469;
Anal, caled for C6H5Br02: C, 38.13; H, 2.67. Found: C, 38.14; H, 2.69.
3-Fluorocatechol (277) was obtained through biooxidation of
fluorobenzene with E. coli JM109 (pDTG602) that was carried out as
described in the general procedure for preparation of catechols. The
catechol 3-fluorocatechol was purified by distillation using Kugeirohr apparatus (80-
90C/0.2mmHg) to yield 12g (1.3g/L) of white solid; mp 69-70 C; IR (KBr); 'h NMR
(300 MHz, DMSO-d6) 8 9.44 (s, OH, 1H), 8.97 (s, OH, 1H), 6.62-6.52 (m, 3H); 13C
NMR (75MHz, CDC13) 8 ; HRMS (FAB) m/z caled for C6H502F (M+): 128.0273.
Found: 128.0275; Anal, caled for C6H502F: C, 56.26; H, 3.93. Found: C, 56.26; H, 4.05.
3-Methoxycatechol (279) was obtained through biooxidation anisle
with £ coli JM109 (pDTG602) as described in the general procedure for
preparation of catechols. The metabolite was purified by flash-
OMe
279
F
277


56
251 252 253 254
(+)-conduritol C264 (-)-conduritol C264 (-)-methyl shikimate265 (+)-methyl
shikimate265
OH QH OH
Substituted Catechols
Catechol and its derivatives are common structural features of many natural
products. Many of them are known to possess diverse biological activity, such as
antihyperglycemic,269 antimutagenic,270 antibiotic, and cytotoxic activities.271 For these
reasons, the role of catechol derivatives has become a topic of increased study in recent
years. However, despite their chemical and biological importance, only a limited number
of studies have been devoted to the synthesis of catechols derivatives. Available
synthetic methods for generation of substituted catechols tend to be cumbersome, non-
selective and involve harsh conditions.272,273 The following paragraphs provide a few
examples of how substituted catechols are made through the use of conventional
chemical methods. These examples give details that are illustrative of the difficulties
associated with using currently available methods for the synthesis of such compounds.


21
biotransformation process. The instability of arene-oxides has been shown to be
responsible for toxic and mutagenic effect by reacting with DNA, RNA and proteins.115
Arene-oxide can rearrange easily via hydride anion (NIH-shift)116 migration to form the
phenolic product117 49 or hydrolyzed to a irans-diol 46 by epoxide hydrolase (Scheme 2-
13). Phenolic components are often further oxidized by polyphenol oxidases to
corresponding catechols118 which can undergo further metabolism.
R R
Scheme 2-13. Possible mechanism in biodegradation of aromatics by eukaryotic cells
Enzymatic hydroxylation of aromatic substrates is a well-established industrial
process for the production of therapeutic agents. The production of L-DOPA (3,4-
dihydroxy-phenyl alanine)119 for treatment of Parkinsons Disease120 and 6-
hydroxynicotinic acid 51 from nicotinic acid by either Pseudomonas or Bacillus sp. are
just two examples of monooxygenase-catalyzed aromatic hydroxylation reactions used
CY
C02H Pseudomonas
or Bacillus sp.
C02H
'N'
50
HO N
51
6-hydroxynicotinic acid
Cunninghamella
echinulata
o2
Scheme 2-14. Microbial hydroxylation of aromatics