Enzymatic oxidation of aromatic substrates via toluene Dioxygenase and catechol dehydrogenase

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Enzymatic oxidation of aromatic substrates via toluene Dioxygenase and catechol dehydrogenase application to total synthesis of combretastatins A-1 and B-1
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xviii, 225 leaves : ill. ; 29 cm.
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Bui, Vu Phong
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Thesis (Ph.D.)--University of Florida, 2003.
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Includes bibliographical references.
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by Vu Phong Bui.
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Printout.
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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




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