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Application of Saccharomyces carlsbergensis Old Yellow Enzyme in Synthesis of Chiral Ketones and Building Blocks for Bet...

Permanent Link: http://ufdc.ufl.edu/UFE0021109/00001

Material Information

Title: Application of Saccharomyces carlsbergensis Old Yellow Enzyme in Synthesis of Chiral Ketones and Building Blocks for Beta-Amino Acids
Physical Description: 1 online resource (129 p.)
Language: english
Creator: Swiderska, Magdalena A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: betaaminoacids, biotransformation, cyclohexanones, oldyellowenzyme
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This dissertation describes the application of S. carlsbergensis old yellow enzyme (an NADPH dependent yeast oxidoreductase) in the biotransformation of different types of activated alkenes. Two classes of compounds, substituted cyclohexenones and beta-nitroacrylates, were synthesized and tested as potential substrates for OYE. Both 2- and 3-substituted 2-cyclohexenones (5a-b and 9a-e) were shown to be reactive with the mentioned protein. Chemo- and stereoselective alkene reductions were observed and no alcohol products were detectable. In most of the cases, biotransformations proceeded with high optical purities, with the exception 2-exo-methylene cyclohexanones (13a-b), which were obtained as racemic mixtures. The enantioselectivities of the reactions were determined based on the chiral GC separation of the derivatized biotransformation products. Enzymatic reductions of 2-substituted-beta-nitroacrylates (20c-f) occurred with 87-96% of enantiomeric excess (e.e.), with larger substrates providing greater stereoselectivities. The products of the biotransformations were further chemically reduced to amino acid esters (22c-f) and derivatized with TFAA (trifluoroacetic anhydride) in order to assess the enantiomeric excess values. The acid hydrolysis of esters gave optically active beta-2-amino acids (23c-f), important drug intermediates and subjects of biological studies. In the case of 3-substituted-beta-nitroacrylates (20a-b), only racemic products were observed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Magdalena A Swiderska.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021109:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021109/00001

Material Information

Title: Application of Saccharomyces carlsbergensis Old Yellow Enzyme in Synthesis of Chiral Ketones and Building Blocks for Beta-Amino Acids
Physical Description: 1 online resource (129 p.)
Language: english
Creator: Swiderska, Magdalena A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: betaaminoacids, biotransformation, cyclohexanones, oldyellowenzyme
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This dissertation describes the application of S. carlsbergensis old yellow enzyme (an NADPH dependent yeast oxidoreductase) in the biotransformation of different types of activated alkenes. Two classes of compounds, substituted cyclohexenones and beta-nitroacrylates, were synthesized and tested as potential substrates for OYE. Both 2- and 3-substituted 2-cyclohexenones (5a-b and 9a-e) were shown to be reactive with the mentioned protein. Chemo- and stereoselective alkene reductions were observed and no alcohol products were detectable. In most of the cases, biotransformations proceeded with high optical purities, with the exception 2-exo-methylene cyclohexanones (13a-b), which were obtained as racemic mixtures. The enantioselectivities of the reactions were determined based on the chiral GC separation of the derivatized biotransformation products. Enzymatic reductions of 2-substituted-beta-nitroacrylates (20c-f) occurred with 87-96% of enantiomeric excess (e.e.), with larger substrates providing greater stereoselectivities. The products of the biotransformations were further chemically reduced to amino acid esters (22c-f) and derivatized with TFAA (trifluoroacetic anhydride) in order to assess the enantiomeric excess values. The acid hydrolysis of esters gave optically active beta-2-amino acids (23c-f), important drug intermediates and subjects of biological studies. In the case of 3-substituted-beta-nitroacrylates (20a-b), only racemic products were observed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Magdalena A Swiderska.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021109:00001


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APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS
OF CHIRAL KETONES AND BUILDING BLOCKS FOR P-AMINO ACIDS




















By

MAGDALENA ALICJA SWIDERSKA


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

2007

































2007 Magdalena A. Swiderska

































To my loving parents, Helenie and Janowi Swiderskim.









ACKNOWLEDGMENTS

First of all, I would like to acknowledge Prof Jon D. Stewart for giving me an opportunity

to carry out my study in his group and for introducing me to the field of biocatalysis. I am deeply

thankful for his help, advice and encouragement. I thank my committee members: Dr. Nicole

Horenstein, Dr. William Dolbier, Dr. Tom Lyons and Dr. Keelnatham Shanmugam for their time

and contributions.

I also thank the Stewart's group members: Dr. Santosh Kumar Padhi, Neil Stowe, James

Melotek. Special acknowledgment goes to Dr. Despina Bougioukou who introduced me to the

techniques of molecular biology and always served with great advice. I cannot forget about

Dimitri Dascier who once was a good friend and helped me in bad times, I really appreciate it.

I would like to thank my loving mezczyzna Dr. Daniel Serra for his love and

encouragement. He was there for me both in difficult and happy moments.

Lastly and most importantly, I would like to thank my parents for their belief in me and for

their constant support. I thank my mom for understanding me and listening to my complaints,

and my dad for being proud of me. I cannot forget about my "little" brother Maciej who I would

like to thank for making me laugh when I felt sad. My family is always the most important for

me and without you I would not be the person I am today.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

L IST O F T A B L E S ...................................................................................................... . 8

L IST O F FIG U RE S ............................................................................... 9

A B STR A C T ........ ... ........ ... .. ............................ ..................... ..... 14

CHAPTER

1 HISTORICAL BACKGROUND OF OLD YELLOW ENZYME .............................16

Discovery and Structure of an Old Flavoprotein ................................ ...................16
Occurrence and Physiological Importance of OYE Family Members ..................................18
Com pounds Bound by OYE ........... ................................. .... ....................... 20
Catalytic Properties of an Old Flavoprotein .............. ................ ............. .................. 20
M echanism of Old Y ellow Enzym e ............................................... ............................ 24
Purification of O ld Y ellow Enzym e ............................................... ............................ 28

2 CHIRAL CYCLOHEXANONES ................................................ .............................. 32

Introduction .......... ...... .. ................ .. .............................. .32
Chemical and Enzymatic Methods toward Chiral Cyclohexanones...........................33
Old Yellow Enzyme Family Approach to Formation of Chiral Cyclohexanones.................38

3 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN
SYNTHESIS OF CHIRAL CYCLOHEXANONES............................... ...............43

Synthesis of a,P-Unsaturated Cyclic Enones.......................................................................43
General Procedure for Preparation of 2-Alkyl-2-Cyclohexen-1-ones.............................43
General Procedure for Preparation of 3-Alkyl-2-Cyclohexen-1-ones...........................44
General Procedure for Preparation of 2-Alkylidenecyclohexan-1-ones ....................45
Biotransformation of a,P-Unsaturated Cyclic Enones Using Old Yellow Enzyme ...............46
B iotransform ation U sing Isolated O Y E ..........................................................................46
Biotransformation Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b) ...............49
Biotransformation Using Sodium Dithionite as Reducing Agent for Old Yellow
E n zy m e ...............................................................................5 1
Conclusions.........................................53

4 C H IR A L P-A M IN O A C ID S ......................................................................... ...................55

Introduction ................................ ............... .................... ...............55
Chemical and Enzymatic Routes to P-Amino Acids ................................... .................57
Old Yellow Enzyme Approach to Chiral 3-Amino Acids.................... ........................... 67









5 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN
SYNTHESIS OF CHIRAL P-AM INO ACIDS........................................... .....................70

Synthesis of M ono-Substituted P-Nitroacrylates...............................................................70
Biotransformation of P-Nitroacrylates and Synthesis of P-Amino Acids ...........................73
Biotransform action U sing Isolated Enzym e ........................................ .....................73
Biotransformation Using Cell-Free Extract............ .........................................79
Conclusions........................................80

6 EXPERIM ENTAL SECTION ................................................................... ...............82

G general M methods and Instrum entation......................................................... ............... 82
Purification of S. carlsbergensis Old Yellow Enzyme........... ..... .................83
Cell Growth and Extract Preparation for Protein Isolation ..........................................83
Isolation of Old Yellow Enzyme ........... .. ........ ............................ 84
R generation of A affinity M atrix................ ......... ............... .................. ............... 84
E nzym e A activity A ssay .......................................................................... ...............84
Synthesis of 2-A lkyl-cyclohexane- 1,3-diones................................. ....................... .......... 85
Synthesis of 3-Isobutoxy-2-alkylcyclohex-2-enone ............................................................85
Synthesis of 2-A lkylcyclohex-2-enone...................................................................... ....... 86
Synthesis of 1,3-Cyclohexanedione.............................................. .............................. 87
3-A lkyl-2-C yclohexen- -ones ...................... .... ......... ............................. ....................... 88
2-(1-H ydroxyalkyl)cyclohexanes ........................................................... .. ............... 89
2-A lkylidenecyclohexanones..................... ..... ...............................................................90
Biotransformation of a,P-Unsaturated Cyclic Enones Using Isolated Old Yellow
E nzym e ........................................... ......................... .. .............. 9 1
Derivatization of Cyclohexanones with (2R,3R)-(-)-2,3-Butanediol .................................92
Biotransformation of a,P-Unsaturated Cyclic Enones Using Whole Cells ofE. coli
B L 2 1(D E 3)(pO Y E -pE T 3b) .......... ................................................................... ....... 92
Synthesis of 2-A lkyloxobutanoates ........................................................................................93
Synthesis of N itro A lcohols ........................................................................... ............... 94
Synthesis of 3- and 2-Alkyl Substituted P-Nitroacrylates...................................................96
Biotransformation of P-Nitro Acrylates Using Isolated Old Yellow Enzyme .....................97
Synthesis of -A m ino A cids...................................................................................... 99
Derivatization of P-Amino Acid Esters with TFAA.............. ...................... ...................100
Biotransformation of P-Nitroacrylates Using Cell Extract.............................. ..............100
G lu co se A ssay .............................................................10 1
Incubation of 21a in D 20 ........................................................................... 102
Biotransformation of (Z)-20c Using NADPD. ................................... ............... 102

APPENDIX

A GC ANALYSIS OF SUBSTITUTED CYCLOHEXANONES............ .................103

B NMR SPECTRA OF P-NITROACRYLATES AND P-AMINO ACIDS............................107





6









L IST O F R E F E R E N C E S .............................................................................. ..........................12 1

B IO G R A PH IC A L SK E T C H ............................................................................... ............... ..... 129









LIST OF TABLES


Table page

2-1 List of OYEs from yeasts and corresponding substrate specificity .................................41

2-2 List of OYEs from bacteria and corresponding substrate specificity...............................41

2-3 List of OYEs from plants and corresponding substrate specificity .................................42

3-1 Substituted cyclohexenones ......... ................. ................. ..................... ............... 43

3-2 Reduction of substituted cyclic enones by isolated old yellow enzyme..........................50

3-3 Reduction of substituted cyclic enones by whole cells................... ........................... 51

3-4 Reduction of substituted cyclic enones using sodium dithionite as reducing agent for
O ld Y yellow Enzym e .................. ............... ............................................ ........ .. .... 53

5-1 Reduction of substituted nitroacrylates by isolated old yellow enzyme and
production of -am ino acids ........................................... ................. ............... 75

5-2 Reduction of substituted nitroacrylates using E. coli cell-free extract with over
expressed O Y E and G D H .............. ........................................................ ........... .......... 81









LIST OF FIGURES


Figure pe

1-1 Reaction system of W arburg and Christian ............................ ....................16

1-2 Ribbon diagram of the oxidized Oyel monomer (PDB# Q02899-10YB) ........................17

1-3 Exam ples of typical ligands of O Y E ................. ......... .................................................21

1-4 Examples of a,P-unsaturated aldehydes, ketones and nitro compounds as substrates
fo r O Y E ............... ........................... ................................................ 2 2

1-6 Dismutation reaction catalyzed by OYE..................................... ......................... 23

1-7 Nitrate reduction by OYE 1: Pathway a ........................................ ......................... 24

1-8 N itrate reduction by OYE 1: Pathw ay b ........................................ ......................... 25

1-9 D enitration reaction ....................................... .. .......... ....... .... 25

1-10 Interaction between Thr-37 and FM N ............................... .. ................................. 26

1-11 Interaction between the substrate and Asn 194 and His 191 from OYE1 active site........27

1-12 Reduction mechanism for ketones and aldehydes catalyzed by OYE............................27

1-13 Reduction mechanism for nitrocyclohexene catalyzed by OYE ....................................28

1-14 Catalytic cycle for old yellow enzym e........................................ ........................... 29

1-15 Active site of OYE in complex with p-hydroxybenzaldehyde............ ................30

1-16 Kinetic m mechanism of OYE .......................... ...................................... ............... 30

1-17 Affinity m atrix for OYE purification........................................... .......................... 30

2-1 Application of 3-substituted chiral cyclohexanones.............. ..... .... ............... 32

2-2 Application of 3- and 2-substituted chiral cyclohexanones...............................................33

2-3 Enantioselective conjugate addition of R2Zn compounds to cyclic enones ......................34

2-4 Enantioselective conjugate reduction of P-substituted cyclic enones............................. 34

2-5 Chemical reduction of double bond of P-substituted cyclohexenones ...........................35

2-6 Enzymatic hydrogenation of C-C double bond of enones..............................................35









2-7 Enzymatic hydrogenation of a,P-unsaturated ketones ..................................................36

2-8 Catalytic enantioselective decarboxylative protonation ......................................... 36

2-9 Enantioselective alkylation of ketones via chiral enamines ............................................37

2-10 Enantioselective hydrolysis of enol esters........................................................ .............. 37

2-11 Enantioselective protonation of prochiral enolates using chiral imides ............................38

2-12 Substituted cyclohexenones as substrates for OYE1 .................... ............................... 39

2-13 Two-step conversion of ketoisophorone to (4R,6R)-actinol using old yellow enzyme
hom ologs and LVR .................. ..... ... ........ .......... .. ........... ............ 40

3-1 Synthesis of 2-alkyl-2-cyclohexen- 1-ones................................... ...............44

3-2 General synthesis of the 2-alkyl-2-cyclohexen-1-ones 5.............................................45

3-3 General synthesis of 3-alkyl-2-cyclohexen- -ones 9a-e ...................................45

3-4 General synthesis of 2-alkylidenecyclohexane-1-ones 13a and 13b..............................46

3-5 Biotransformation of cyclic enones using isolated OYE.............................................47

3-6 D privatization of cyclic enones............................................................... .....................48

3-7 Schematic diagram of the S. carlsbergensis old yellow enzyme active site...................49

3-8 Biotransformation of substituted cyclic enones using isolated old yellow enzyme ..........50

3-9 Biotransformation of substituted cyclic enones using whole cells........................ 51

3-10 Biotransformation of substituted cyclic enones using sodium dithionite as reducing
agent for old yellow enzym e......... .................................... ..................... ............... 52

3-11 Reduction of a,3-unsaturated ketones by Na2S204 ........................................................53

4-1 L near P-am ino acids ....................... .. ............................ .... ......... .. ............. 55

4-2 Nomenclature proposed by Seebach and co-workers ................................................55

4-3 Enantiopure P2-phenylalanine and P2-homovaline .................................... ............... 56

4-4 C ryptophycin and its precursor............................................................................ .... ... 56

4-5 Carbocyclic and heterocyclic -amino acids .......................................... ............... 57

4-6 (R)-(+)-3-Amino-3-phenyl-2,2-dimethylpropanoic acid and its derivative.....................57









4-7 A rndt-E istert hom ologation ....................................................................... ..................58

4-8 Arndt-Eistert homologation in 32-amino acids synthesis ...............................................58

4-9 Stereoselective acylation of one enantiomer of the racemic P-amino esters .................59

4-10 Hydrolysis of N-phenylacetyl derivatives of P-amino esters................ ..................59

4-11 Chem ical resolution of P-am ino acids ........................................ ......................... 60

4-12 Synthesis of a-hydroxy P-am ino acids........................................ ........................... 60

4-13 Asymmetric synthesis of P-amino acids derivatives.....................................61

4-14 Stereoselective preparation ofiturinic acid and 2-methyl-3-aminopropanoic acid .........61

4-15 Addition of a "chiral ammonia" equivalent to an acceptor .............. ............................62

4-16 Addition of a nitrogen nucleophile to a chiral acceptor ............... ............. .............62

4-17 Conjugate addition of amine nucleophile by asymmetric catalysis...............................62

4-18 Asymmetric synthesis of P-amino acids via conjugate addition of chiral metallated
a m in e s ............................................................................................ 6 3

4-19 Addition of a nitrogen nucleophile to a chiral acceptor..............................................63

4-20 Asymmetric catalysis in conjugate addition ........................................... ............... 63

4-21 Rhodium catalyzed hydrogenation of 3-aminoacrylates................ ..... ..........65

4-22 Rhodium-catalyzed hydrogenation .......................................................................65

4-23 Hydrogenation of p-enamino esters catalyzed by Pearlman's catalyst............................66

4-24 Direct reductive amination of P-keto esters with (R)-L-Ru catalyst..............................66

4-25 Synthesis of p-lactams via modified Staudinger reaction......................................68

4-26 Reduction of nitro-olefins by S. carlsbergensis old yellow enzyme .............................69

5-1 Synthesis of methyl 3-nitroacrylate using N204 .......................................................70

5-2 Synthesis of 0-nitroacrylates using N02C1 or NOC .................................. ............... 71

5-3 Synthesis of 0-nitroacrylates using nitrous acid ..................................... .................71

5-4 NaNO2-Ceric ammonium nitrate mediated conversion of acrylic esters into
B-nitroacrylates ............................................................................ 72









5-5 Proposed radical mechanism of 2-hydroxy-3-nitroacrylates formation ..........................72

5-6 Amberlyst A-21 mediated conversion of acrylic esters into P-nitroacrylates ..................73

5-7 Biotransformation of nitroacrylates by OYE towards P-amino acids.............................75

5-8 Proposed mechanism of reduction of substituted-2-cyclohexenones and
nitroacrylates by old yellow enzym e ........................................... .......................... 76

5-9 Biotransformation of nitroacrylates in D20 .......................... ... .............. ................... 77

5-10 Incorporation of deuterium into 21a....................................................... ...................77

5-11 Fragment of the 1H NMR analysis of the product of incubation................. ................78

5-12 Incubation of compounds 21b in D20 .............................. ... .................................. 78

5-13 Reaction of (E)-20c with NADPD ........... ......... ........................... ............... 79

5-14 Biotransformation of(E)-20d by isolated OYE1........................ ..............79

5-15 Incubation of 20d in K Pi buffer ........... .. ........................ .......... ....... ............... 79

5-16 Biotransformation of nitroacrylates using E. coli cell-free extract with overexpressed
OYE and GDH ......... ........... .............................................. ... ....... ........ 81

A-i GC chrom atogram of 14a, 15b and 15c..................................... ......................... 103

A -2 G C chrom atogram of 14b, 15a and 15d ........................... .................. ..................... 104

A-3 GC chromatogram of TFA derivatives of 22c and 22d.............................105

A-4 GC chromatogram of TFA derivatives of 22e and 22f ...........................106

B -l 13C N M R of spectrum of 20c ................................................................ ..................... 107

B-3 13C NM R spectrum of 20d ......................................................................... 109

B -4 1H N M R spectrum of 20d ..................................................................... .....................110

B -5 13C N M R spectrum of 20e ................................................... ..... ......................... ..

B -6 1H N M R spectrum of 20e .......................................................................... .............112

B-7 13C NM R spectrum of 20f......................................................................... 113

B -8 1H N M R spectrum of 20f........................................................................... .............114

B -9 13C N M R spectra of 23a and b .............................................. .................................... 115









B -10 13C N M R spectra of 23c and d ................................................................................116

B -11 13C N M R spectra of 23e and f............................................................................... ..... .. .117

B -16 H N M R spectrum of 21a ................................................................... ................ ..... .119

B-17 H NM R spectrum of deuterated 21c............................................................................. 120









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

APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS
OF CHIRAL KETONES AND BUILDING BLOCKS FOR P-AMINO ACIDS

By

Magdalena Alicja Swiderska

August 2007

Chair: Jon D. Stewart
Major: Chemistry

This dissertation describes the application of S. carlsbergensis Old Yellow Enzyme (an

NADPH dependent yeast oxidoreductase) in the biotransformation of different types of activated

alkenes. Two classes of compounds, substituted cyclohexenones and P-nitroacrylates, were

synthesized and tested as potential substrates for OYE.

Both 2- and 3-substituted 2-cyclohexenones (5a-b and 9a-e) were shown to be reactive

with the mentioned protein. Chemo- and stereoselective alkene reductions were observed and no

alcohol products were detectable. In most of the cases, biotransformations proceeded with high

optical purities, with the exception 2-exo-methylene cyclohexanones (13a-b), which were

obtained as racemic mixtures. The enantioselectivities of the reactions were determined based on

the chiral GC separation of the derivatized biotransformation products.

Enzymatic reductions of 2-substituted-P-nitroacrylates (20c-f) occurred with 87-96% of

enantiomeric excess (e.e.), with larger substrates providing greater stereoselectivities. The

products of the biotransformations were further chemically reduced to amino acid esters (22c-f)

and derivatized with TFAA (trifluoroacetic anhydride) in order to assess the enantiomeric excess

values. The acid hydrolysis of esters gave optically active p2-amino acids (23c-f), important drug









intermediates and subjects of biological studies. In the case of 3-substituted-P-nitroacrylates

(20a-b), only racemic products were observed.









CHAPTER 1
HISTORICAL BACKGROUND OF OLD YELLOW ENZYME

Discovery and Structure of an Old Flavoprotein

Saccharomyces carlsbergensis old yellow enzyme (OYE) is known as the first discovered

and characterized flavoprotein.1 It was isolated from brewers' bottom yeast by Warburg and

Christian2 in 1932 during their studies on the oxidation of glucose-6-phosphate by methylene

blue. They discovered that the reaction takes place only when oxygen is reduced to hydrogen

peroxide in the presence of additional elements called at that time "Zwischenferment"-identified

later as glucose-6-phosphate dehydrogenase, "Gelbe Ferment"-yellow protein, and a heat-stable

"Coferment"-NADP+ ( 1-1).


Zwischenferment
glucose-6-phosphate dehydrogenase




Cferment NADPH
NADP+


6-phospho
gluconolactone


H202 02
Gelbe Ferment
Old Yellow Enzyme

Figure 1-1. Reaction system of Warburg and Christian.

A few years after this discovery, another yeast flavoenzyme (D-amino acid oxidase) was

isolated and named "das neue gelbe Ferment".3 This led to the final name given to the Warburg's

protein: old yellow enzyme. In 1935, Hugo Theorell purified OYE and showed that it consists of

two components: a colorless apoprotein and a yellow dye, both essential for the enzyme activity.

In 1955, he identified the yellow element as flavin mononucleotide (FMN), which was later

found to bind non-covalently to the enzyme active site (Figure 1-2).4


glucose-6-
phosphate
































Figure 1-2. Ribbon diagram of the oxidized Oyel monomer (PDB# Q02899-10YB).

Despite the fact that OYE had been known since 1932, its first crystallization was not

achieved for nearly another 20 years by Hugo Theorell in 1955. Unfortunately, the quality of the

crystals was not sufficient for X-ray studies, which were later explained as a probable result of

the heterogeneity of natural OYE arising from the two genes present in S. carlsbergensis.1 Since

then, several X-ray data were published mainly by Fox and Karplus, who solved the crystal

structure at a resolution of 2.0 A for the oxidized and reduced forms of recombinant intact Oyel

and for its complex with p-hydroxybenzaldehyde.4

Based on Fox's results, the enzyme was found to exist as an a/f barrel in the form of a

dimer, with a monomer of- 45 kDa. Each single domain binds non-covalently the molecule of

flavin mononucleotide (FMN) that interacts by hydrogen bonds with surrounding it amino acids.5

Fox and Karplus used also spectroscopic techniques to obtain some additional information

on solvent accessibility to the active site of the OYE. 13C and 15N NMR together with the X-ray









experiments revealed that it is the si face of the FMN that is exposed to solvent, when the re face

is buried by interactions with the protein.

Since that time, OYE has been characterized in great detail, especially by Vincent

Massey's laboratory, and it has served as a model for studying other flavoproteins.1,6

Unfortunately, despite the extensive knowledge on the structure and reactivity of the old

flavoprotein, the physiological role of OYE has remained unknown.

Occurrence and Physiological Importance of OYE Family Members

Several proteins with amino acid sequence homologous to OYE have been found in yeasts,

plants and bacteria and their postulated physiological function gives some idea on the importance

of old yellow enzyme in Nature.7 Based on those reports a number of possible functions for

Saccharomyces carlsbergensis OYE were suggested, from assembly of the yeast cytoskeleton6 to

oxidative stress response in yeast.8

In Saccharomyces cerevisiae, two enzymes have been discovered, OYE2 and OYE3, with

sequence closely related to OYE1. The most recent studies on acrolein toxicity in this strain of

yeast (Saccharomyces cerevisiae) point to the role of OYE2 as a main agent mediating resistance

to small a,P-unsaturated carbonyl compounds, such as acrolein.9 However, based on the previous

reports on the role of OYE in the control of the redox state of the actin cytoskeleton, the authors

suggested that this group of enzymes is likely to have physiological functions beyond the simple

detoxification of harmful metabolites.

There have been found some other strains of yeasts containing enzymes with genes related

to OYE, such as

Candida albicans: contains estrogen binding protein (EBP1), which possesses desaturase
activity and reduces 19-nor-testosterone (OYE1 can only aromatize it).









Hansenulapolymorpha: contains hansenula yellow enzymes (HYE1, HYE2 and HYE3),
which have been found to increase the resistance towards high concentrations of allyl
alcohol in the presence of alcohol oxidase (AO).

Kluyveromyces lactis: contains kluveromyces yellow enzyme (KYE1), whose
physiological role is unknown.

Yarrowia lipolytica: contains N-ethylmaleimide reductase.

Candida macedoniensis: contains old yellow enzyme (oye).

A number of enzymes related to OYE have been identified in bacteria in the following strains

Gluconobacter suboxydans: contains old yellow enzyme, physiological role unknown.

Pseudomonasputida M10: contains morphinone reductase (morB), which reduces
double bonds of both morphinone and codeinone, producing important pharmaceutical
drugs like hydromorphone and hydrocodone, respectively.

Pseudomonasputida II-B and Pseudomonasfluorescenc I-C: contain nitroester
reductases (XenA and XenB, respectively), responsible for nitrate ester degradation.

Agrobacterium radiobacter: contains glycerol trinitrate reductase (Ner), responsible for
nitrate ester degradation.

Enterobacter cloacae Pb2: contains pentaerythritol tetranitrate reductase (Orn), which is
known for degradation of explosives, such as nitroglycerine (GTN) and 2,4,6-
trinitrotoluene (TNT).

Escherichia coli JM109: contains N-ethylmaleimide reductase (NemA)

Bacillus subtilis: contains YqjM (B]4911 08447), involved in detoxification.

Shewanella oneidensis: contains SYE1, SYE3 and SYE3 (AAN55488.1, AAN57126.1,
AAN56390.1, respectively), physiological role unknown.

Plant homologues of OYE were first identified during studies on elucidation of

octadecanoid biosynthesis by Vick and Zimmerman, who isolated 12-oxophytodienoic acid

reductase (OPR) from Zea mays, strain found in corn.10 The enzyme was found to catalyze one

of the steps in jasmonic acid biosynthesis, the reduction of the double bond of

12-oxo-10,15(Z)-hytodienoic acid (OPDA).11 After this discovery several other plant strains,

which contain proteins related to OYE, were identified, such as









Corydalis sempervirens: contains 12-oxophytodienoic acid reductase (OPRI), which
reduces (9R,13R)-cis and (9S, 13R)-trans diastereoisomers of OPDA.

Arabidopsis thaliana: contains 12-oxophytodienoate reductase 1 (OPRI), which reduces
(9R, 13R)-cis and (9S, 13S)-cis diastereoisomers of OPDA.

Arabidopsis thaliana: contains 12-oxophytodienoate reductase 2 (OPR2), which reduces
(9R, 13R)-cis diastereoisomer of OPDA.

Arabidopsis thaliana: contains 12-oxophytodienoate reductase 3 (OPR3), which reduces
(9R, 13R)-cis, (9S, 13S)-cis, (9R, 13S)-trans and (9S, 13R)-trans diastereoisomer of OPDA.

Oryza sativa L. (rice): contains 12-oxophytodecanoic acid reductase (opda).

Compounds Bound by OYE

In the process of searching for the physiological role of old yellow enzyme, researchers

identified a number of compounds capable of inhibiting this protein.12-14 The OYE ligands bind

with milimolar to micromolar affinities to the oxidized form of the enzyme.

Aromatic compounds with an ionizable hydroxyl substituent are one of the most studied

group of inhibitors of OYE (Figure 1-3). Upon binding, the phenolic ligand OYE forms deeply

colored long wavelength charge-transfer complexes, which can be detected by their green

color. 15

Two other classes of binding ligands are simple monovalent anions (e.g., acetate, chloride

or azide) and pyridine nucleotide derivatives such as the acid hydrolysis products of reduced

form of nicotinamide adenine dinucleotide phosphate (NADPH). All three types of inhibitors, as

well as substrates, share a common active site on the si-face of the flavin.

Catalytic Properties of an Old Flavoprotein

The first reports on the reactivity of old yellow enzyme can be found in the same studies

that helped Warburg discover this protein.1 During the attempts to elucidate the nature of

glucose6-phosphate oxidation process, he identified OYE as the component oxidizing NADPH

and reducing oxygen to hydrogen peroxide. Since then, several experiments proved that NADPH









0


HOe H HO C
p-hydroxybenzaldehyde p-chlorophenol



OH OH

H


0 : HO f!
testosterone P-estradiol

Figure 1-3. Examples of typical ligands of OYE

could be the physiological reductant for the old flavoprotein. There are some other compounds

capable of reducing OYE, like reduced form of nicotinamide adenine dinucleotide (NADH) or

sodium dithionite, but the efficiencies of the reactions are significantly lowered compared to

NADPH.16,17

Substrates capable of oxidizing OYE include methylene blue, Fe3+, quinones, cytochrome

c and ferricyanide.18 The reoxidation of the enzyme can also be effected by molecular oxygen,

producing H202.19 Recently, it has been found that a number of a,P-unsaturated aldehydes,

ketones,20,21 and nitro compounds22 could serve as more efficient substrates for this enzyme

(Figure 1-4).

These enzymatic reactions are chemoselective with exclusive reduction of the double bond

of the olefin, but not the carbonyl or nitro groups (Figure 1-5). The presence of a stoichiometric

amount of NADPH or another reductant for OYE is required during the reaction. It has been

found by Massey and co-workers that in the absence of an agent capable of reducing OYE, the

oxidative aromatization of the cyclic enones occurs.21 In this dismutation reaction, the substrate














3-oxodecalin-
4-ene


0





mesityl oxide


CHO



3-oxodecalin-
4-ene-1 0-carboxald ehyd e


cinnamaldehyd e


0




2-cyclohexenone


O


1,2-cyclohexanedione


0




3-methyl-
2-cyclohexenone


O


duroquinone


NO2


Q


NO2


SNO2


menadione nitrocyclohexene nitrostyrene nitrovinylthiophene

Figure 1-4. Examples of a,P-unsaturated aldehydes, ketones and nitro compounds that serve as
substrates for OYE

is first dehydrogenated, and subsequently the olefinic bond of a second substrate molecule is

reduced (Figure 1-6).

Another interesting class of reactions catalyzed by old yellow enzyme is reduction of

nitrate esters.23 The mechanism of the process is still not completely known but two pathways

were proposed

Pathway a: the reaction involves a hydride transfer from the reduced flavin to the
nitrogen of the nitrate residue (Figure 1-7). This step could be followed by or concerted
with electron rearrangement, resulting in liberation of nitrite and formation of the alcohol
product.

Pathway b: the reduction involves sequential electron and proton transfers as described
in Figure 1-8.








0


OYE. NADPH


2-cyclohexenone


NO2

\


OYE, NADPH


nitrocyclohexene


0




cyclohexanone



NO2




nitrocyclohexane


Figure 1-5. Example of reduction reaction catalyzed by OYE


OYE


0O
3-oxodecalin-
4-ene


Ho~\


3-hydroxy-
6,7,8,9-tetrahydronaphthalene


H


H
3-oxodecalin


0


OYE
-ON-


2-cyclohexenone


OH



phenol


0


6


cyclohexanone


Figure 1-6. Dismutation reaction catalyzed by OYE










"T ?I ii \.N N

+O Y
NO H O



I I ,H




02N O N





N0NH
'JrNN
N02 + OH 0

02N O

Figure 1-7. Nitrate reduction by OYE1: Pathway a
Two of the compounds were particularly studied: glycerin trinitrate (GTN) and propylene

dinitrate. The reactions resulted in product mixtures as the rate of reduction for primary and

secondary nitrate was different (Figure 1-9).

Mechanism of Old Yellow Enzyme
There are several residues that are assumed to have a major role in the catalytic function of

OYE. At least one of them, Thr-37, was found to strongly affect the reactivity of the enzyme by

direct interaction with the flavin molecule.19 It was suggested that this residue plays an important









role in controlling the redox potential of the enzyme by stabilizing the negative charge of the

reduced flavin by hydrogen bonding with the C-4 oxygen of the FMN (Figure 1-10).


02NO N
O" O+ O




O2NO /N
.0 OH





O2NO N
HO'" (OH



NO2+ +OH

02NO


H 0
I


,N N O
o NH

N
0O


Figure 1-8. Nitrate reduction by OYE1: Pathway b
ONO2 ONO OH
02NO ONO2 OYE, NADPH HO 0NO2 OH
02NO ON02 0-N02- + 02NO ON02
glycerine trinitrate
GTN


ON02
02N0,


OYE, NADPH 2
HO ,


OH
+ 02NO.


propylene dinitrate

Figure 1-9. Denitration reaction









The determination of the crystal structure4 of the complex between Oyel and

p-hydroxybenzaldehyde was critical in identifying two important residues of the enzyme:

histidine 191 and asparagine 194, which direct the positioning of the reactants (substrate or

inhibitor) in the active site of OYE by formation of hydrogen bonds (Figure 1-11). These

interactions are particularly important in the catalytic process, since they stabilize the anionic

form of the reactant, which acts as an electron acceptor and is involved in charge-transfer

interaction with the FMN group.

T37
T37
0.
\ ;H




N
H i
\ l 2.77





NN N


FMN

Figure 1-10. Interaction between Thr-37 and FMN. In this projection the view is on the si-face
of the flavin.

Further studies on the mechanistic role of the amino acid residues from the active site of

OYE provided some important information about the catalytic cycle of this enzyme.24 The

investigations were especially focused on tyrosine 196, which was suggested to be a proton

donor in the substrate double bond reduction. The experiments were performed with different

enones, including 2-cyclohexenone and 1-nitrocyclohexene. The results confirmed the

importance of Tyr-196 only in the case of the ketones, where single mutation of the tyrosine

residue to phenylalanine (Y196F) inhibited the process of reduction. Completely different








situation was observed for the nitro compound, where the same mutation (Y196F) almost did not

affect the reaction and it was possible to obtain a saturated product without a visible inhibition.

8+
Asn 194
S2.79

8-O""'""His 191
2.74
-J

8+

Figure 1-11. Interaction between the substrate and Asn 194 and His 191 from OYE1 active site.

These results suggest different mechanisms for those two types of compounds

Mechanism a: utilized by a,P-unsaturated ketones and aldehydes. This reaction may be
best described as a concerted mechanism in which the transfer of a hydride is possible by
the presence of Tyr-196 primed for protonation (Figure 1-12).

Mechanism b: utilized by 1-nitrocyclohexenones, may involve the formation of an aci-
nitro intermediate by transfer of hydride to the substrate followed by its protonation
either by Tyr-196 or water. In this case, the process does not depend on the presence of
the tyrosine residue (Figure 1-13).

6+
Asn 194
6- : 8+
0 OYE O'..His 191 C


5+ -H *- H
Hp-Tyrl96 H
FMNNg-H

Figure 1-12. Reduction mechanism for ketones and aldehydes catalyzed by OYE

In order to complete the model of the catalytic cycle for old yellow enzyme, some

additional studies were performed to establish the mechanism of the hydride transfer to the flavin

from NADPH, which is assumed to be the physiological reductant for OYE.21 According to the









results of this investigation, the pro-R-hydrogen of NADPH is transferred as a hydride to the

flavin Ns, then this is followed by the transfer of the same hydride to the 0-carbon of the

substrate (Figure 1-14). Base on the studies on the interaction between p-hydroxybenzaldehyde

ligand and the active site of OYE, the flavin should be always positioned in the re face of the

bound substrate that means that the hydride uptake is possible only from this direction, which

would make the reduction highly stereospecific (Figure 1-15).

8+ 8+
Asnl94 0 Asnl94



8+ 8+



-0
N-O-
C +

nitronate

Figure 1-13. Reduction mechanism for nitrocyclohexene catalyzed by OYE

It was suggested that NADPH and the substrate bind to the same site of the enzyme,

requiring the protein to act by a ping-pong mechanism, which is consistent with the steady state

kinetics of all forms of the enzyme studied (Figure 1-16).15'19'24'25

Purification of Old Yellow Enzyme

Old yellow enzyme was first isolated in the form of a homogeneous, crystalline protein by

Theorell and Akerson in 1956 as a result of long studies started by Warburg. However, the

original purification method was time-consuming with a very low efficiency. Since then, several

attempts were made in order to improve this process. During the development of the procedure,

it was discovered that the oxidized enzyme forms green colored charge-transfer complex with a










Oxidative
half-reaction


N ,NHO

5 NH


OYEox0




\, N N O
5I
NHNH

HR O

OYERed


Reductive
alf-reaction


HR Hs


N


NADPH
NADPH


NH2


NADP+


Figure 1-14. Catalytic cycle for old yellow enzyme

low molecular weight compound.12,13 Later, the ligand was identified as any aromatic molecule

with an ionizable hydroxyl substituent, e.g. naturally occurring p-hydroxybenzaldehyde.14

Additionally, it was found that the green complex dissociates upon reduction of the

enzyme. This characteristic interaction between phenolic compounds and OYE helped

Abramovitz and Massey to develop a simple purification procedure based on affinity

chromatography.17 They used 4-hydroxy-N-n-butylbenzamide as an affinity matrix (Figure

1-17). In this method, protein isolation process consists of 3 steps. First, the solution of crude

extract of brewer's bottom yeast is loaded on the column and only OYE binds to the agarose

containing phenolic ligand. The formation of the complex can be easily noticed as the gel

changes color from white to green. In order to remove all the unbound proteins the column is

washed several times with a Tris-HCl buffer (pH 8.0) that contains (NH4)2SO4 and PMSF. The


0








presence of the last reagent methylsulfonyl fluoride proved to increase the efficiency of the


process by inhibiting proteolysis.


Tyr 196


His 191




H

Asn 19

Asn 194


Tyr 375


O
H


Figure 1-15. Active site of OYE in complex with p-hydroxybenzaldehyde.


NADP+ Substrate
t 1


Substratend
I


OYEFMN OYE FMN OYE FMN OYEFMNH2
NADPH NADP+


OYE FMNH2 OYE FMN
Substrate Substrate.,


Figure 1-16. Kinetic mechanism of OYE


HN V aga rose


N-(4-hydroxybenzoyl)aminohexyl agarose

Figure 1-17. Affinity matrix for OYE purification


NADPH
I


OYEMN


I I









The desired protein is eluted from the column by using the same washing buffer but with

addition of 3 mM sodium dithionite, which acts as a reducing agent for the enzyme and releases

it from the complex with the ligand from the matrix. Upon elution from the column, the protein

becomes oxidized and returns to its yellow color. This procedure delivers the enzyme in nearly

homogeneous form with high efficiency. Additionally, this way of purification is simple and

does not require too many steps in comparison to the previous methods.









CHAPTER 2
CHIRAL CYCLOHEXANONES

Introduction

Optically active cyclic ketones, especially those bearing a stereogenic center a or P to the

carbonyl group, are important reaction intermediates (synthons) for asymmetric synthesis.

Among this group, chiral cyclohexanones are one of the most interesting synthons due to their

broad applications in the production of biologically active substances. The synthesis of

(-)-agarospirol, (-)-a-acorenol or (+)-p-acorenol, a family of sesquiterpenes used as ethereal oils

in perfumery, can serve as an interesting example (Figure 2-1).25,26

0





R-(+)-3-methylcyclohexanone






OH OH


OH

OH

(-)-agarospirol (+)-o3-acorenol (-)-a-acorenol

Figure 2-1. Application of 3-substituted chiral cyclohexanones.

Another class of compounds that find their origin in chiral a- or 3-substituted

cyclohexanones are lactones (Figure 2-2), which are present in various forms in numerous

naturally occurring substances like antibiotics and essential oils (Figure 2-2).28,29









0 o 0 0
CHMO 0 0

R ,/ R s R
.R
R R
3-substituted cyclohexanones Y
lactones
90.5% 9.5%
94% (94% ee) 6% (>99% ee)



O O

sR m-CPBA/CH2Cl2 0


2-substituted cyclohexanone lactone


R = Me, Et, n-Pr

Figure 2-2. Application of 3- and 2-substituted chiral cyclohexanones.

Chemical and Enzymatic Methods toward Chiral Cyclohexanones

Considering the variety of applications for optically active substituted cyclohexanones,

many research groups concentrate their work on developing methodologies to obtain these

important chiral intermediates. To date, several procedures, both chemical and biochemical, have

been developed. The two most widely used strategies are: i) the conjugate addition of

organometallic reagents to a,P-unsaturated compounds (Figure 2-3),30-32 and ii) the asymmetric

conjugate reduction of cyclic enones (Figure 2-4).33,34 Unfortunately, these methods are limited

only to formation of P-alkylated ketones.

Another procedure that is commonly applied in the synthesis of optically enriched a- or

P-substituted ketones is enantioselective reduction of the C-C double bond of the corresponding

enone, which can be carried out using both chemical (Figure 2-5) and biochemical (Figure 2-6)

catalysts.33-37









0


b


Et2Zn, 0
toluene, -300C
Cu(OTf)2 (0.5 mol%)
catalyst (1.0 mol%)
95% yield
e.e. >98%


Figure 2-3. Enantioselective conjugate addition of R2Zn compounds to cyclic enones catalyzed
by copper phosphoramidite.28-30


0


1 mol% (Ph3P)CuH
0.1-0.5% catalyst
2eq PMHS, PhMe


PMHS polymethylhydrosiloxane


98% yield
e.e. 90%


Figure 2-4. Enantioselective conjugate reduction of P-substituted cyclic enones.31'32


phosphoramidite catalyst


(R)-DTBM-SEGPHOS catalyst










0
EtO2C CO2Et 5 mol% catalyst salt
I + BI Bu20,
R N 600C,48h
R = Me, Et, -Pr 1.2 eq
R = Me, Et, i-Pr 1.2 eq


O


R
94-99% yield
94-98% e.e.


Figure 2-5. Chemical reduction of double bond of P-substituted cyclohexenones.


0 p90, NADPH

S35-95% yield
95-99% e.e.

R = Me, Et, n-Pr

p44, NADPH ,R


37-80% yield
>99% e.e.


p90, p44 reductases from Nicotiana tabacum

Figure 2-6. Enzymatic hydrogenation of C-C double bond of enones.

Most of the enzymatic reductions of a,P-unsaturated cyclic ketones yield not only saturated

cyclohexanones but also the corresponding alcohols37-39 (Figure 2-7). One of the newest methods


H3N ,C 2t-Bu

i-Pr


valine ester phosphate salt catalyst









leading to chiral a-substituted cyclohexanones is enantioselective decarboxylative protonation

recently reported by Stoltz and co-workers40 (Figure 2-8).

Enantioselective alkylation of ketones via chiral enamines, first reported by Horeau,41

subsequently modified by others, such as Meyers et al.,42 delivers a highly enantioselective and

efficient way of obtaining chiral a-substituted cyclohexanones (Figure 2-9).

0 0 OH OH

Reductase O+' C+




Reductase = Synechococcus sp. PCC 7942

Figure 2-7. Enzymatic hydrogenation of a,P-unsaturated ketones.


N ,P
Pd O






70
0 0 O-Pd' 0
O-^^ Pd,(dba),+L E HCO2Hb "10
Et20, rt, 10 h



L=
Ph2P NJ
t-Bu

Figure 2-8. Catalytic enantioselective decarboxylative protonation.

Among other interesting approaches to a-alkylated cyclohexanones is enzymatic

hydrolysis of prochiral a-substituted enol esters catalyzed by a number of esterases. The

intermediates of the reaction, a-substituted enols, undergo enantioselective rearrangement in the

active site of the enzyme to yield optically active ketones.28'43









Ph
H, -
H2N
OMe


0=O


Ph


NR
[::::O e


Ph
H LDA / THF
N _-200C
SOMe


RX
-780C


RX = Mel, EtI, n-PrI, BzBr


H3O+


87-99% e.e.


Figure 2-9. Enantioselective alkylation of ketones via chiral enamines.
One of the most recent works characterizing this type of reaction describes esterases I and

II isolated from cultured plant cells ofMarchantia polymorpha (Figure 2-10).44 Another route to

optically active substituted cyclohexanones is enantioselective protonation of prochiral enolates

using chiral imides. An interesting example of this type of reaction was reported by Yamamoto

et al. (Figure 2-11).45


OAc
esterase I
R KPi buffer (pH 7.0)

350C, 0.5h

R = Me, Et, i-Pr, n-Pr


R = Me, Et
>99% yield
14-99% e.e.


O


R. -R

R = i-Pr, n-Pr
10-99% yield
17-99% e.e.


Figure 2-10. Enantioselective hydrolysis of enol esters.
Despite the variety of existing methods that lead to optically active cyclohexanones, there

is constant demand for easier, environmentally friendly and universal for both alkyl substitutions









(a and p) synthetic routes. This fact forces chemists to explore new chemical and biochemical

procedures.

OLi 0
R(S,S)-imide R



R = Me, n-Bu, Ph, Bz 4-67% e.e.


Ph Ph

HN O
ON




(S,S)-imide


Figure 2-11. Enantioselective protonation of prochiral enolates using chiral imides.

Old Yellow Enzyme Family Approach to Formation of Chiral Cyclohexanones

Asymmetric hydrogenation by chiral rhodium or ruthenium phosphines has resulted in an

impressive number of enantioselective alkene reductions during the last 20 years.46'47 Despite the

tremendous progress in this area, high stereoselectivities nearly always depend on olefin

proximity to highly polar groups such as amides, acids and alcohols.48 Attempts to generalize

these procedures to aprotic oxygen functionalities such as aldehydes, ketones, esters or nitro

groups have been much less successful; although some exceptions were reported (those examples

were mentioned in the previous paragraph of this chapter). Moreover, the fact that

organometallic approach requires preparation of complex chiral reducing agents and extreme

reaction conditions makes this methodology unattractive, especially with respect to its

environmental issues. Enzymatic alkene reductions might be one useful solution to problem.









While several isolated enzymes have been reported to reduce a,P-unsaturated cyclic

ketones producing optically active saturated ketones,49'50 those of the old yellow enzyme (OYE)

family have been characterized most thoroughly. The Stott et al. study reported several enones as

good substrates for NADPH-mediated reduction by OYE1, among those many a,P-unsaturated

cyclic ketones were found as quite reactive species (Figure 2-12).21

Based on preliminary results on the reactivity of OYE1, many research groups extended

their studies to the search for the other OYEs from different organisms. OYE2 and OYE3 were

isolated from S. cerevisiae and both of them showed activity towards a,P-unsaturated cyclic

ketones.51'52 Similar results were obtained with old yellow enzyme from Candida macedoniensis.

The protein was discovered during the screening of different fungal species for the ability to

reduce stereoselectively C-C double bond of ketoisophorone (KIP) to produce (6R)-levodione, a

biologically important chiral synthon (Table 2-1).53 Reduction of unsaturated cyclic ketones was

also reported among some of the bacterial relatives of old yellow enzyme.


b OH

> > > >
0 0 0
2-cyclohexenone duroquinone cyclohexane-1,2-dione 4-oxo-isophorone menadione





H O
3-oxo-decalin-4-ene-10- 3-methyl-2-cyclohexenone
carboxaldehyde

Figure 2-12. Substituted cyclohexenones as substrates for OYE1 ordered from the least to the
most reactive enone.









In 1994, Bruce's laboratory isolated morphinone reductase from Pseudomonasputida

M10, enzyme responsible for reduction of olefin double bonds of morphinone and codeinone.54'55

The same group demonstrated the ability of this enzyme to reduce 2-cyclohexenone in an

NADH-dependent manner.

OYEs have been also detected in other Pseudomonas species54 and other bacteria, like:

Enterobacter cloacae,56 Escherichia oli,57 Bacillus subtilis,58 or ,s\'hn iell// a oneidensis.59 All of

them showed some reactivity towards 2-cyclohexenone (Table 2-2). Several OYEs were also

isolated from plants. Among those, some demonstrated ability to reduce double bond of

2-cyclohexenone (Table 2-3).60-63

Despite the extensive studies on the crystal structure and substrate specificity of proteins

from the old yellow enzyme family, the stereoselectivities of reductions catalyzed by those

enzymes were never determined. There are only two reported examples that employed homologs

of the S. carlsbergensis old yellow enzyme, both of which described production of

(6R)-levodione used in the synthesis of (4R,6R)-actinol (Figure 2-13).52,64 The above results

suggested a new view on old yellow enzyme as a stereoselective catalyst that may be used in

multi step reaction.

0 S. cerevisiae, OYE2 O O
or ,s V
C. macedoniensis, OYE Levodione reductase


O O OH
ketoisophorone (6R)-levodione (4R,6R)-actinol

Figure 2-13. Two-step conversion of ketoisophorone to (4R,6R)-actinol using old yellow
enzyme homologs and LVR.

Combining all these results and properties, the protein appears to be an effective and

inexpensive catalyst. In this work, we examined the substrate specificity and stereoselectivity of









the S. carlsbergensis old yellow enzyme and point to ways in which it can be employed in chiral

building block production. Our approach was based on the chemical synthesis of the starting

materials for the OYE1, in this case we concentrated on a,P-unsaturated cyclohexenones,

followed by their biohydrogenation using isolated enzyme or whole cells overexpressing OYE1.

Table 2-1. List of OYEs from yeasts and corresponding substrate specificity.
Organism and Protein Substrate
O 0




Saccharomyces cerevisiae 0
Old Yellow Enzyme 2 2-cyclohexenone menadione
Old Yellow Enzyme 3
Candida macedoniensis 0 0
Old Yellow Enzyme


O O
0 0
duroquinone 4-oxo-isophorone


Table 2-2. List of OYEs from bacteria and corresponding substrate specificity.
Organism and Protein Substrate
Pseudomonas putida II-B
Nitroester reductase RO
Pseudomonasfluorescens I-C 0
Nitroester reductase
Enterobacter cloacae 0 NCH3
Pentaerythritol reductase
Escherichia coli
N-ethylmaleimide reductase 0 2-cyclohexenone
Bacillus subtilis
YqjM R = H, morphinone
,\ll'n it ll// oneidensis R = Me, codeinone
SYE1, SYE3, SYE4









Table 2-3. List of OYEs from plants and corresponding substrate specificity.
Organism and Protein Substrate
Corydalis sempervirens 0
OPRI
Arabidopsis thaliana
12-oxophytodienoate reductase 2 and 3
Lecopersicon esculentum
12-oxophytodienoate reductase 1, LeOPR1 2-cyclohexenone
Pisum sativum
PsOPR1-6









CHAPTER 3
APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS
OF CHIRAL CYCLOHEXANONES

Synthesis of a,P-Unsaturated Cyclic Enones

A number of compounds were synthesized in order to serve as starting materials for the

enzymatic reductions with S. carlsbergensis old yellow enzyme. Early experiments on ligand

binding to OYE suggested that the enzyme should be reactive with some a,P-unsaturated

cyclohexenones.21'25 Moreover, the studies on the mechanism of ligand binding and interaction

with the active site of OYE1 suggested that the process may be highly stereoselective. Based on

already published data, we proposed three groups of substituted cyclohexenones that were

examined as substrates for OYE, as described in Table 3-1.

Table 3-1. Substituted cyclohexenones.
Cyclohexenones Substituents
0

2-alkyl-2-cyclohexen- 1-ones R6 R = Me, Et


0

3-alkyl-2-cyclohexen-l-ones 6 R = Me, Et, n-Pr, i-Pr, n-Bu

R
0

2-alkylidenecyclohexan-l-ones R R = Me, Et



General Procedure for Preparation of 2-Alkyl-2-Cyclohexen-l-ones

The first effort to obtain this class of compounds was concentrated on the procedure

reported by Ohta and coworkers,65 which consisted of two steps: a) bromination of

2-alkyl-cyclohexan-1-one with N-bromosuccinimide and b) subsequent dehydrobromination with

aniline to give the corresponding 2-alkyl-2-cyclohexen-1-one (Figure 3-1). Unfortunately, after

several attempts no positive result was obtained.











S a) N-bromosuccinimide, CC4 K

b) PhNH2


R = Me, Et

Figure 3-1. Synthesis of 2-alkyl-2-cyclohexen-1-ones based on method by Ohta et al.

We turned our attention to another procedure, which consisted of three-steps resulting in

the desired product. Although the first step of this method proceeded with very low yield, all the

reagents were readily available and inexpensive. The synthesis of 2-alkyl-2-cyclohexen- -ones

began with a Friedel-Crafts acylation of the appropriate carboxylic acid with glutaryl chloride 1

in the presence of aluminum chloride to yield the 2-alkylcyclohexane-1,3-diones 2a and 2b,66'67

which were converted to the corresponding 2-alkyl-3-isobutoxy-2-cyclohexenones 4a and 4b by

reaction with i-BuOH andp-TsOH.68'69 The resulting vinylogous esters were reduced by lithium

aluminum hydride (LAH) to afford the desired 2-alkyl-2-cyclohexen-1-ones 5 (Figure 3-2).70'71'72

General Procedure for Preparation of 3-Alkyl-2-Cyclohexen-l-ones

Our approach to 3-alkyl-2-cyclohexen-1-ones was based on a widely applied Grignard

reaction.73 The reason why we chose this route was practical. First of all, the reagents used were

readily available; second, the intermediates for all the 3-alkyl-2-cyclohexen-1-ones were the

same (cyclohexane-l,2-dione 6 and 3-isobutoxy-2-cyclohexenone 7).74,75 The synthesis began by

reacting 6 with 1-isobutanol to yield 7. This reaction was followed by addition of the appropriate

Grignard reagent 8a-e to 7, and then the crude product was hydrolyzed in aqueous acid. The final

enones 9a-e were purified by silica gel column chromatography (Figure 3-3).76-79









O 0
0C MACI R i-BuOH /p-TsOH
C1+ HOMeN28
C1 HO R 15-20% 80-95%

0
1 2aR=Me 3 a, b
bR= Et

0 0
R LAH
Et20 R
O[ 84-90%

4 a, b 5 a, b

Figure 3-2. General synthesis of the 2-alkyl-2-cyclohexen- -ones 5.

0 0 0
RMgBr (8 a-e)
i-BuOH/p-TsOH Et2O
o 85% O 70-83% R
6 7 9 a-e

RMgBr
8 aR = Me
bR= Et
c R= n-Pr
d R = i-Pr
e R= n-Bu

Figure 3-3. General synthesis of 3-alkyl-2-cyclohexen-1-ones 9a-e.

General Procedure for Preparation of 2-Alkylidenecyclohexan-l-ones

The synthesis was based on procedure developed by Huang et al.80 which gave acceptable

chemical yields. Although the authors reported that the method results in a mixture of cis- and

trans-isomers, always one of them is obtained in high excess. The procedure began with aldol

condensation of cyclohexanone with readily available aldehydes and gave expected aldols which

were dehydrated by mesylation, followed by treatment with DBU.81'82 Enones were obtained as a

mixture of isomers (80% of E; 20% of Z) (Figure 3-4).83,84 Unfortunately, attempts to separate

the isomers did not give positive results.









Biotransformation of a,P-Unsaturated Cyclic Enones Using Old Yellow Enzyme

There are two major approaches in biotechnology when it comes to the physical state of

biocatalyst, which can be applied either as isolated enzyme or in the form of whole

microorganism. The final decision as to which of them should be used depends on many factors,

such as: (i) the type of reaction, (ii) if there are cofactors to be recycled and (iii) the scale in

which the biotransformation has to be performed.85

0 O OH 0

1. LDA2.RCHO(lla,b) R 1.MeSO2CI / Et3N R
60-75% 2. DBU/ THF
10 12 a, b 64-71% 13 a, b

0

H'R
11 aR =Me
b R = n-Pr

Figure 3-4. General synthesis of 2-alkylidenecyclohexane-1-ones 13a and 13b.

In our studies, we first needed to examine the reactivity and stereoselectivity of the

enzyme. The most secure way to accomplish this task was by using purified protein. In this way,

we ensured that OYE is the source of chirality in the bioreduction products. The next step was to

scale up (up to 1 gram) the characterized reactions by using whole cells and determine their

efficiency.

Biotransformation Using Isolated OYE

The old yellow enzyme plasmid (pOYE-pET3b) (the plasmid was a gift from Professor

Massey's Laboratory) was transformed into BL21 (DE3) cells. The overexpressed protein was

purified based on the procedure developed by Massey and coworkers.17

Each of the ketones was tested as a substrate for the OYE using NADPH, which was

supplied by a cofactor regeneration system. This was based on the conversion of NADP+ and









glucose-6-phosphate into NADPH and 6-phosphoglucono-6-lactone, respectively, catalyzed by

glucose-6-phosphate dehydrogenase (Figure 3-5). The presence of NADPH was required in order

to avoid the dismutation reaction, catalyzed by OYE in the absence of this cofactor, which

results in formation of phenol compounds.21

O 0
R *R
Old Yellow Enzyme e
R' R'

a R alkyll, R' = H
b R = H, R' = alkyl NADPH NADP+
6-phosphogluconolactone glucose-6-phosphate

O R O R
^Glucose-6-phosphate Dehydrogenase
c

Figure 3-5. Biotransformation of cyclic enones using isolated OYE and NADPH regeneration
system.

The first step in the biotransformation of enones was to test them as potential substrates for

the enzyme. This procedure was performed on small scales, with substrate concentration of

3 mM, and the ketones were introduced to the reaction in the form of stock solutions with

ethanol, which increased the solubility of organic compounds in aqueous buffer. The reductions

were carried out at room temperature. The progress of the biotransformations was monitored by

taking 30 [tL samples and extracting them with 30 [tL of EtOAc. The organic phase was

analyzed by non-chiral-phase gas chromatography and mass spectrometry. Each of the reactions

went to completion, except 9e and 13b, which were not reduced.

The next step was to define the stereoselectivity of the enzyme. This was achieved by

scaling up the reactions to 20 mg of substrate, an amount sufficient for further analysis. A

stoichiometric quantity of P-cyclodextrin was added to promote the substrate solubility in the









reaction solution. The enantiomeric excess of the products obtained was assessed by chiral-phase

gas chromatography. The complete separation of the chiral ketones was possible only after their

derivatization with (2R,3R)-(-)-2,3-butanediol.86 The same procedure was applied to the

corresponding racemic cyclohexanones to demonstrate baseline resolution of enantiomers

(Figure 3-6). Product absolute configurations were revealed by comparison with authentic

standards available from earlier studies.87



O 0
R p -TsOH R
S+ CH2C12 ,
R H O OH R
substituted (2R,3R)-bu tane-2,3-diol cyclohexanone
cyclohexanone derivative

Figure 3-6. Derivatization of cyclic enones.

According to the results of the scaled up biotransformation with isolated enzyme, after 24

hours, only the least substituted substrates (5a and 9a) were completely reduced. Larger ketones

were not fully reduced, even after 24 h and the conversion did not proceed further after this time

(Table 3-2). Based on GC analysis, the conversion decreased with the length of the substituent,

with two examples of no conversion (9e and 13b).

The enzyme displayed high enantioselectivity towards 2- and 3-substituted cyclohexenones

(series of 5 and 9) with enantiomeric excess values raging from 90 to 97% (Table 3-2).

Moreover, the results of the absolute configuration assessment support the OYE reduction

mechanism proposed by Massey and coworkers. According to this model, reduction of

3-substituted cyclohexenones should deliver the S enantiomers, if we assume that the double

bond is at the right hand side as illustrated in Figure 3-7. This is a result of hydride donation by

reduced FMN from the re-face. With simultaneous a-face protonation by the phenol of Tyr 196,








which can be seen as formation of 2-substituted cyclohexanone with excess of its R enantiomer

(Figure 3-7). Attempted reductions of 2-alkylidenecyclohexanones resulted in very low extents

of conversions and almost racemic mixtures of products (Figure 3-8).

Biotransformation Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b)

The application of isolated enzymes in the biotransformation of organic compounds has its

pros and cons. One of the major disadvantages of this approach is the requirement for cofactor

recycling. In the case of old yellow enzyme, the regeneration of NADPH becomes very costly,

especially when applied to bigger scale reactions. One useful solution to this problem may be

biocatalysis mediated by whole cells.

Our strategy was to increase the scale of the enone reductions using whole cells of E. coli

BL21(DE3)(pOYE-pET3b) that overproduced the old yellow enzyme from S. carlsbergensis.

Flavoprotein expression was induced by adding isopropylthio-P-D-galactoside (IPTG) when the

cultures reached the early logarithmic phase of growth. After 30 min, the appropriate ketone and

stoichiometric amount of P-cyclodextrin (to increase substrate solubility) were added. The

reactions were allowed to proceed at room temperature until the bioconversions ceased.

/ Tyr196


Asn 194
As 19 His 191



HR' H


R' R
H
H:

Figure 3-7. Schematic diagram of the S. carlsbergensis old yellow enzyme active site.









0 0 0
R OYE -R R

NADPH NADP+
5a, b (R)-14 a,b (S)-14 a, b







R NADPH NADP+ R
9 a-e (S)-15 a-e (R)-15 a-e





R OYE $ j R R

NADPH NADP+
13 a, b (R)-16 a, b (S)-16 a,b

Figure 3-8. Biotransformation of substituted cyclic enones using isolated old yellow enzyme.

Table 3-2. Reduction of substituted cyclic enones by isolated old yellow enzyme.
Ketone R Conversion (%) ee (%) Configuration
5a Me 100 97 R
5b Et 40 92 R
9a Me 100 96 S
9b Et 81 95 S
9c n-Pr 30 90 S
9d i-Pr 23 92 S
9e n-Bu NRa
13a Me 40
13b n-Pr NRa
a No reaction

Experiments suggested that the enones were ultimately toxic to one or more of the reaction

components after extended periods. The biotransformation with whole cells delivered similar

results to those obtained with isolated enzyme (Table 3-3). The decrease in substrate conversion

was observed as the size of the substituents increased. 2-Exo-methylene cyclohexanones gave

either very low or no conversion. This was also the case for pure enzyme reactions. Additionally,










the enantioselectivity of the biotransformations mediated by whole cells was just slightly lower

than with isolated protein. One of the reasons for this may be the presence of other reductases in

the cells E. coli that could to small extent affect the reduction by OYE (Figure 3-9), (Table 3-3).

O O 0
EngineeredI R
S E. coli cells R R


5 a, b (R)-14 a, b (S)-14 a, b



o 0 0
Engineered
E. coli cells

R R R
9 a-e (S)-15 a-e (R)-15 a-e



0 0 0
Engineered
R E. coli cells R R

13 a, b (R)-16 a, b (S)-16 a, b

Figure 3-9. Biotransformation of substituted cyclic enones using whole cells.

Table 3-3. Reduction of substituted cyclic enones by whole cells
Ketone R Conversion (%) ee (%) Configuration
5a Me 100 96 R
5b Et 16 90 R
9a Me 100 94 S
9b Et 76 95 S
9c n-Pr 25 89 S
9d i-Pr 18 90 S
9e n-Bu NRa
13a Me 40
13b n-Pr NRa
a No reaction

Biotransformation Using Sodium Dithionite as Reducing Agent for Old Yellow Enzyme

The concept of using sodium dithionite as a reductant for the enzyme, instead of NADPH,

was based on the fact that the hydride that reduces the substrate does not come directly from









nicotinamide cofactor but from the flavin and so any chemical reagent (in this case Na2S204) that

can reduce the FMN can substitute for NADPH (Figure 3-10).

0 0 0

R OYE/Na2S2O4 R R

5 a (R)-14 a (S)-14 a



o 0 0

6 R OYE/Na2S204 R R

9 a-d (S)-15 a-d (R)-15 a-d

Figure 3-10. Biotransformation of substituted cyclic enones using sodium dithionite as reducing
agent for old yellow enzyme.

Since reactions with sodium dithionite require basic conditions and exclusion of oxygen,

which causes autooxidation of Na2S204, the buffer used for the biotransformations was degassed

and its pH increased from 7.0 to 8.0. Substrates (5a, 5b and 9a-e) and P-cyclodextrin were added

in a 1:1 ratio in the presence of the excess Na2S204. After 24 hours, although product formation

was detected, none of the reactions proceeded to completion (Table 3-4). Two major issues that

might have contributed to the decreased conversion are:

Oxidation of sodium dithionite by oxygen from the air that was not completely
eliminated.

Reaction between the substrates and sodium dithionite.

It has been reported in the literature that Na2S204 can serve as a strong reducing agent for

cyclohexenones (Figure 3-11).88 In this case, dithionite may reduce both enzyme and starting

material. The GC/MS spectra suggested that this may be the reason for the loss of substrate

without product conversion. On the other hand in the reaction analysis data there was no sign of









phenol compounds formation which could be the result of the dismutation reaction between OYE

and the cyclohexenones.

S2042- + H20 HSO2- + HSO3-


HSO2- + S2042- HSO3- + S2032-


R1 R, SO2H R, O
R2 0 HSO2- R2 2 or SO2H
0 or 2

R3 R4 R3 R4 R3 R4


H S=O

OH
RI
R2 O


Figure 3-11. Reduction of ca,3-unsaturated ketones by Na2S204.

Table 3-4. Reduction of substituted cyclic enones using sodium dithionite as reducing agent for
Old Yellow Enzyme.
Ketone R Conversion (%) ee (%) Configuration
5a Me 60 97 R
9a Me 60 96 S
9b Et 20 95 S
9c n-Pr 5 90 S
9d i-Pr NRa 92 S
a No reaction

Conclusions

Sketching the rough outlines of old yellow enzyme's substrate- and stereoselecivities was

the goal of this study, which employed a homologous series of simple alkyl-substituted enones.48

The enzyme displayed gratifying enantioselectivity. Moreover, the absolute configurations of the

products could be predicted reliably from a simple model derived from X-ray crystallography


R3',I- R4









data (Figure 3-7). Because of hydrogen bonding with the carbonyl oxygen and the requirement

that the P-carbon lie above N5 of the flavin, reduced FMN must deliver hydride to the re face of

the bound cyclohexenones while the protonation must occur from the si face.

Increasing the extents of conversion, particularly for larger substrates, is a key challenge

the must be overcome before the enzyme can be considered synthetically useful. Nonetheless,

the present results underscore the high potential of S. carlsbergensis old yellow enzyme and

probably related proteins in stereoselective organic synthesis.









CHAPTER 4
CHIRAL P-AMINO ACIDS

Introduction

P-Amino acids occur in nature both in free and bound form, and even though they are less

abundant than their a-analogues, they have become one of the most investigated subjects in

chemistry and biology. Especially interesting for scientists are the properties of oligomers

composed exclusively of p-amino acids (so called P-peptides), which are stable to metabolism,

exhibit slow microbial degradation and are stable to proteases and peptidases.89

There are three general types of open-chain chiral P-amino acids, depending on whether

the substitution takes place at the carbon bearing the carboxyl group (a-position), the carbon

bearing the amino group (P-position), or at both positions (a,P-disubstitution) (Figure 4-1).90

Recently, Seebach and co-workers91'92 proposed the terms P2- and 33-amino acid, where the

numbers indicate the position of the side chains, in order to distinguish positional isomers

(Figure 4-2).

O R O R' O

H2N OH H2N OH H2N OH
R R
a-substituted 0-substituted a,P-substituted

Figure 4-1. Linear P-amino acids

R3 0

H2N ; iOH
R,
R2

p2-amino acid, R3 = H
P3-amino acid, R2 = H

Figure 4-2. Nomenclature proposed by Seebach and co-workers.









Enantiopure P2-phenylalanine and P2-homovaline were recently synthesized by Gellman

and co-workers93 to provide access to new P-peptides with specific conformations and particular

functions (Figure 4-3). Besides their importance for peptidomimetic studies, P-amino acids are

also gaining attention as potential precursors for natural products and pharmaceuticals. Several

examples of this kind of amino acid structure can be found as an essential component of

biologically active compounds.94 One of the simplest p-amino acids,

(R)-2-methyl-3-aminopropionic acid, is a residue in cryptophycin, a potent antitumor

depsipeptide (Figure 4-4).95


0 0

H2N OH H2N OH




(S)-P2-phenylalanine (R)-32-homovaline


Figure 4-3. Enantiopure P2-phenylalanine and P2-homovaline.


0



H2N OH 0
H_ o 0 O OMe
(R)-2-methyl-3-aminopropionic acid L --------- J

cryptophycin


Figure 4-4. Cryptophycin and its precursor.

Carbocyclic P-amino acids, like cispentacin, and their heterocyclic analogues, such as

methylphenidate, are useful intermediates for the enantioselective synthesis of antifungal

antibiotics96 and mental disorder medications, respectively (Figure 4-5).97









(R)-(+)-3-Amino-3-phenyl-2,2-dimethylpropionyl derivative NSL-95301 is a novel trisubstituted

p-amino acid exhibiting potent inhibition of platelet aggregation, which makes it promising

antithrombotic agent (Figure 4-6).98



CO2CH3
NH
O .CO2H 'NH


cispentacin methylphenidate

Figure 4-5. Carbocyclic and heterocyclic P-amino acids.



0O

o H N
H2N OH H
NH

(R)-(+)-3-amino-3-phenyl- (R)-(+)-NSL-95301
2,2-dimethylpropanoic acid

Figure 4-6. (R)-(+)-3-Amino-3-phenyl-2,2-dimethylpropanoic acid and its derivative.

Chemical and Enzymatic Routes to p-Amino Acids

The importance of P-amino acids and their derivatives in the field of pharmacology and in

peptide chemistry is well represented by the multitude of reports that have been published in the

past decades. Additionally, since the far-reaching discovery that P-peptides form much more

stable structures than their a-peptidic natural counterparts, there has been an ever-growing

interest in synthesizing p-amino acids with various substitution patterns. In particular, the

preparation of enatiomerically pure P-amino acids has become an important and challenging

endeavor for organic chemists.89 There are eight main approaches available till date for









stereoselective synthesis of p-amino acids: homologation of a-amino acids, enzymatic resolution,

addition of enolates to imines, Curtius rearrangement, conjugate addition of a nitrogen

nucleophile to a,P-unsaturated esters or imides, hydrogenation, amino hydroxylation and

p-lactam synthesis.99

A number of homologation reaction examples in the synthesis of p-amino acids have been

reported,100-102 since this method is considered to be the best for one carbon chain elongation of

carboxylic acid. Seebach and co-workers have utilized the Arndt-Eistert procedure in P-peptide

synthesis to produce P-amino acid derivatives in enantiomerically pure form (Figure 4-7).103

Unfortunately, this method is limited to the synthesis of 33-amino acids, with only few

exceptions to their a-substituted equivalents. One of the examples of the application of the

Arndt-Eistert homologation in the synthesis of 32-amino acids is proposed by Yang and

co-workers104 synthesis of a-substituted-P-amino esters (Figure 8).

R R H R O
SOH 1. Et3N/CICO2Et P J UV light NOM
N 2. CH N N N OMe
H 2.CH2N2 H 2 or cat. PhCOAg H
O O in Et3N/MeOH

P = Boc or Cbz 88-98% yield
R= Me, i-Pr, i-PrCH2
Figure 4-7. Arndt-Eistert homologation.

O 1.i-BuOCOC / Et3N O R2
R, H 2. CH2N, R N2 h v, MeOH/ CH2Cl2 R1 OMe
NHBoc 3. KHMDS / HMPA, R2X BocHN R BocHN


R, = alkyl
R2 = H or alkyl

Figure 4-8. Arndt-Eistert homologation in p2-amino acids synthesis.

Enzymatic resolution of P-amino acids is a cheap and environmentally friendly approach to

their optically active derivatives. This method can be applied in several ways, but two commonly









used strategies are stereoselective acylation of one enantiomer of the racemic P-amino esters105

and hydrolysis of N-phenylacetyl derivatives, both of which are catalyzed by lipases106

(Figure 4-9 and 4-10).


NH2O

R-<>"O~t


R = Me, Et, n-Pr, i-Pr


lipase

PrCOOR1


0

Pr NH 0
S R OEt


NH2 O

R ~JOEt


R = CH2CF3 or Bu


lipase lipase A from Candida antarctica

Figure 4-9. Stereoselective acylation of one enantiomer of the racemic P-amino esters.


NH2 O

R-'K>KH


Et3N

Ph -4,
Co


0
Ph
Ph ,iNH 0 acylase

R OH H20


R= C6H, 4-F-C6H4
2-F-C6H4, 4-MeO-C6H4


0

Ph ,NHO0

RI6kAOH


acylase penicillin acylase from
E. coli ATCC 9637


NH2 O
R -OH


6N HCI NH2 0
500C R)OH

Figure 4-10. Hydrolysis of N-phenylacetyl derivatives of p-amino esters.

Similar chemical procedures may be achieved by formation of diastereomeric salts via

complexation with a chiral base, for example (-)-ephedrine (Figure 4-11).107 The diastereomeric

salts a and b can be separated by fractional crystallization due to their difference in solubility in










a suitable solvent (Figure 4-11). However, the process of multistep recrystallizations is long and

tedious.

Enzymatic resolution was also applied as one of the steps in the synthesis of a-hydroxy

p-amino acids, which are important class of compounds that possess interesting bioactivities.94

Cardillo and Gentilucci reported a two-step approach to the production of syn-a-hydroxy

p-amino acids (Figure 4-12).108 The two key steps in this process were PGA catalyzed kinetic

resolution of a racemic amino acid ester, followed by the highly diastereoselective formation of

trans-oxazoline. Escalante and Juaristi have demonstrated the utilization of pyrimidinones in the

synthesis of a-hydroxy P-amino acids (Figure 4-13).109 Another interesting way of obtaining

Ph OH
NH2 0

R O H2N
a I
NH20 OH +
+ROH + h Ph OH
S+ Ph NH2 0 Y
NHMe
(-)-ephedrine R O H2
b I

Figure 4-11. Chemical resolution of P-amino acids.

O O
NH 0 NH1. EtN, PhCOCI NH
Bn NH ONPGA O2 0 2. SOCI2/MeOH Ph O
Ph- OH Ph' OH Ph OMe



0 Ph 0

LiHMDS/I Ph NH 0 N'O 1M HCI Ph' NH 0
Ph OMe Ph' OMe
Ph CO Me O
I 2 OH


PGA penicillin G acylase

Figure 4-12. Synthesis of a-hydroxy P-amino acids.










chiral P-amino acids is transformation of the carboxy into an amino group by means of a Curtius

rearrangement (Figure 4-14). Sibi and Deshpande used this methodology in stereoselective

preparation of iturinic acid and 2-methyl-3-aminopropanoic acid, components of biologically

important peptides iturin and cryptophycin.110

Among various strategies available to date, conjugate addition of an amine nucleophile to

a,P-unsaturated carboxylic acid derivatives represents one of the most attractive methods for the

stereoselective synthesis of P-amino acids.94,111 There are basically three ways to achieve

asymmetric induction using this methodology


Bz'N N

Ph O

perhydropyri-
midinone


LDA


Bz.
BzN N/ 1.6N HCI NH O

h 2. TMSCI/ MeOH Ph l" OMe
Phe -O 3.BzC / Et3N OH
OHOH
(2R,3R)-methyl 3-benzamido-
2-hydroxy-3-phenylpropanoate


Figure 4-13. Asymmetric synthesis of P-amino acids derivatives.

0 0
A OtBu NaHMDS 0 OtBu
Oo_ RBr X T "
Ph R O
Ph 81-97% d.e.
X
O 1. Pd/C/H2
LiOH/H202 OtBu Curtius BocHN OtBuO 2. TFA BocHN ,OH
R 0 one-pol Y 3. Boc20/El3N H
R O R O R = undec-3-ene O


O 0 0
c__[ .OH Curtius LiOOH
TFA Xc one pot* Xc NHBoc LiOOH HO NHBoc
R 0 R R
R =Me
component of
cryptophycins

Figure 4-14. Stereoselective preparation of iturinic acid and 2-methyl-3-aminopropanoic acid
using Curtius rearrangement. 1) Et3N, CICO2Et, acetone, 0C, lh; 2) NaN3, H20,
acetone, 0C, lh; 3) toluene, heat, lh; 4) t-BuOH, heat, 12-24h








Method 1: addition of a "chiral ammonia" equivalent to an acceptor (Figure 4-15).

Method 2: addition of a nitrogen nucleophile to a chiral acceptor (Figure 4-16).

Method 3: asymmetric catalysis (Figure 4-17).99

The first case is well represented by the protocol developed by Davies and co-workers,112

who used lithium amides as synthetic equivalents of ammonia (Figure 4-18). The second case

can be described by the addition of diphenylmethanamine to chiral crotonates, reported by

Chiaroni and co-workers (Figure 4-19).113 The synthesis of P-aryl-P-amino acid derivatives using

catalytic amounts of a chiral Lewis acid can serve as an example for the third case of conjugate

addition (Figure 4-20).114

Chiral "NH3" NH

Z .^ R ZRC

Z = achiral template

Figure 4-15. Addition of a "chiral ammonia" equivalent to an acceptor.


0 Chiral "NH3"
Xc

Xc = chiral auxiliary


O NH2

Xc R


Figure 4-16. Addition of a nitrogen nucleophile to a chiral acceptor.

0 Nitrogen Nu: O NH2
Z ) R chiral Lewis acid Zl R

Z = achiral template

Figure 4-17. Conjugate addition of amine nucleophile by asymmetric catalysis.









S Ph

O ., Ph N O
0R O Ph" N Ph P ) 0
R1 / OR Li 1 RliC OR
R2 R2H,

R1 = Ph or Me >95% d.e. H H
R2 = H orMe N-H
2Li
NH2 0 Rj-
1. Pd(OH),/C/H Ot-Bu
2.TF R OH O
2. TFA
3. Dowex R2 Proposed model

Figure 4-18. Asymmetric synthesis of 3-amino acids via conjugate addition of chiral metallated
amines.


0 Ph2CHNH2 Ph Ph 0
14-15 bar HN 0 J

O ^^ -OR*
Ar HNCHPh,
R* chiral auxiliary HNCPh
Proposed model


Figure 4-19. Addition of a nitrogen nucleophile to a chiral acceptor.

O 0 2.2 eq BnNHOH, -600C
A N 0.3 eq Mg(CIO4) or Mgl, OzNz Ph H2 /Pd HO r

0 O 'Ar
N Ns,,




Figure 4-20. Asymmetric catalysis in conjugate addition.

Reductions of a,P-unsaturated esters or nitriles can serve as another interesting approach to

enantiopure P-amino acids, which can be achieved in two ways: 1) catalytic reduction and 2)

reductive amination. These two strategies are exemplified by









Synthesis of p-amino esters and synthesis of p2-amino acids: proceed through rhodium
catalyzed hydrogenation of 3-aminoacrylates (Figure 4-21)115and rhodium-catalyzed
hydrogenation of P-phthalimide acrylates (Figure 4-22).116

Synthesis of P-aryl-p-amino esters and synthesis of p-amino esters: proceed through
hydrogenation of p-enamino esters catalyzed by Pearlman's catalyst (Figure 4-23)117 and
direct reductive amination of P-keto esters with NH4OAc and H2 in the presence of
(R)-L-Ru catalyst (Figure 4-24).11s

One of the most important derivatives of P-amino acids are p-lactams with a broad

application in the production of biologically active substances. Despite their significance, there

are only few protocols for their construction. The development of the efficient methods for their

asymmetric synthesis is still a very active area of research.119 The Staudinger reaction, which

involves [2+2] ketene-imine cycloaddition, is one of the most reliable methods available for the

construction of p-lactam rings.120 Lecka et al. developed a modified Staudinger reaction, where

nucleophilic ketene was generated in the presence of 10 mol% ofbenzoylquinine (BQ) (Figure

4-25).121

Each of mentioned above approaches has advantages along with limitations.

The most important advantages of available approaches are:

Arndt-Eistert homologation uses ready available, inexpensive and high
enantiomerically pure a-amino acids as starting materials.

Asymmetric addition of enolates or silyl enolates to imines gives very high
e.e., around 98%.

Catalytic hydrogenation process mostly uses the systems that tolerate an E/Z
mixture of the substrates (derivatives of acrylic acid or nitrile), which
simplifies the starting material preparation process.

The major disadvantages of mentioned methods are:

Arndt-Eistert homologation is not suitable for large scale synthesis due to the
high cost of the silver catalyst and danger of working with the hazardous
reagent CH2N2.











AcHN

R CO2Me

E


AcHN CO2Me
or
R

Z



Ph2P
T4-


1 mol% Rh(I)+L
toluene, rt
H2 pressure:
40 psi, E
294 psi,Z


AcHN CO2Me

R
R = alkyl, 90-99% e.e
R = Ph, 65-66% e.e.


(R,R)-BICP


(R,R)-Me-DuPhos


L

Figure 4-21. Rhodium catalyzed hydrogenation of 3-aminoacrylates.


HCHO DABCO H CHCOCI oJ
H CO2Et THF/HO H0 CO2Et H CO2Et
eth acrlate hdxaclate acetoxacrat
ethyl acrylate hydoxyacrylate acetoxyacrylate


I N OEt

0 0
phthalimido-protected
0-amino acid


SN OEt [Rh(COD)2]BF4 +L
H2 (10 atm), CH2Cl2
O O rt
P-phthalimide acrylate
0 Ph O

HCI HO NH, O
resin-- HO NH2 0

(R)-(-)-a-methyl-
p-alanine Ph 0


Figure 4-22. Rhodium-catalyzed hydrogenation.


L = ManniPhos











0
Ar, CO2Me
Ar "


+ H2N

OMe


AcOH/toluene

650C, reduced
pressure


HN -
MeO2C Ar
Ar


HN

Ar OMe
CO2Me


I 2eq. BF3-Et2O


Pd(OH)2/C/H2


NH2
MeO2C~
MOCAr


Figure 4-23. Hydrogenation of 3-enamino esters catalyzed by Pearlman's catalyst.


SOt
R' kOEt


(R)-L-Ru (1 mol%)
NH40Ac, TFE
H2


NH2 0



p-amino ester


OH O


-hydroxy ester
P-hydroxy ester


R = alkyl, aryl C1



MeO PPh2
MeO PPh2


Cl
L = (R)-CIMeOBIPHEP


Figure 4-24. Direct reductive amination of P-keto esters with (R)-L-Ru catalyst


'OMe









Classical resolution of P-amino acids requires multistep fractional
recrystalization, and therefore the sequence is long and tedious.

The enzymatic resolution reactions need to be stopped at 50% of the
conversion and so yield is usually low; another problem is a narrow tolerance
of the enzymes toward racemic P-amino acids.

Oppolzer's sultam-chiral auxiliary is unstable at temperatures greater than -
450C.

Evans' chiral auxiliary is too expensive for the large-scale synthesis.

Catalytic hydrogenation requires the use of expensive rhodium catalysts.

All the mentioned methods (besides enzymatic resolution) are
environmentally unfriendly chemical processes.

Based on above information on the synthesis of chiral P-amino acids, the development of

an efficient, easy to operate, inexpensive and suitable for large-scale synthesis process, still

remains a significant issue.

Old Yellow Enzyme Approach to Chiral P-Amino Acids

Old yellow enzyme is well known for its reduction of the olefinic bond of a,P-unsaturated

carbonyl compounds by using NADPH as a cofactor. Moreover, we proved that these reactions

are highly stereospecific.122

Another class of similar compounds is that of unsaturated nitro compounds. Like a

carbonyl group, a nitro group exerts a strong electron attracting influence within the molecule,

enhancing the acidity of the hydrogen atoms attached to the carbon a to the substituent group.

Nitro compounds also exhibit a tautomerism analogous to keto-enol tautomerism. Massey and

co-workers found that old yellow enzyme is capable of reducing double bond conjugated with a

nitro group (Figure 4-26).18










Similar reactions can be catalyzed by other members of old yellow enzyme family, such as

old yellow enzyme from Candida macedoniensis, N-ethylmaleimide reductase from Escherichia

coli or YqjM from Bacillus subtilis.49'53'54

It was discovered that the reduction catalyzed by OYE1 proceeds in a stepwise manner,

with formation of a nitronate intermediate which is the result of hydride transfer to the P-carbon

of the olefin. This process is followed by protonation of the nitronate at a-carbon to form the

saturated nitroalkane. Both steps are catalyzed by the enzyme.

Meah and Massey suggested, based on results from theoretical model of interaction

between active site of the protein and nitrocyclohexenone, that reduction of nitro-olefins by old

yellow enzyme may proceed via a trans-addition across the double bond.18 If that is the case, it

seemed reasonable to conclude that the reaction may be stereoselective, like it was showed for

unsaturated cyclohexenones.

Based on this information, we extended our studies on the stereoselectivity and substrate

specificity of old yellow enzyme to the substituted nitro acrylates. The reduction products served

as intermediates for P-amino acids synthesis.

O Cl N/Ts proton sponge

) + H10 mol% BQ
R EtO2C H 10 mol% In(OTf)
OMe

N


OCOPh
BQ


S0 BQ Ts 0

R H R H EtO2C R

96-98% e.e.

Figure 4-25. Synthesis of p-lactams via modified Staudinger reaction.










S. carlsbergensis, OYE1
NADPH


nitrocyclohexene


N,


nitrocyclohexane


S. carlsbergensis, OYE1
NADPH


O ,O
011+,


nitrostyrene


nitrovinylthiophene


nitroethylbenzene


0+,,0

s N


nitroethylthiophenc


Figure 4-26. Reduction of nitro-olefins by S. carlsbergensis old yellow enzyme.


N


I


0+ ,0 S. carlsbergensis, OYE1
S N NADPH
CtJ --









CHAPTER 5
APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS
OF CHIRAL P-AMINO ACIDS

Synthesis of Mono-Substituted P-Nitroacrylates

The preparation of p-nitro acrylic esters has been reported by several groups. Shechter et

al. proposed their method based on nitration of corresponding acrylic esters with dinitrogen

tetroxide (Figure 5-1).123 The reaction usually yielded a mixture of products in the form of

methyl 3-nitrocrylate, methyl 2-hydroxy-3-nitropropionate, oxalic acid dihydrate and

nitrogen-containing polymers of methyl acrylate.

ONO

0 1. H20
O + N204 02 NO 0 2. CO(NH2)2
O 3. Dist.
ONO2
methyl acrylate 0O

NO0 0




OH

Y O + 0 + HO2C-CO2H H20
NO0 0 N0, 0

methyl 2-hydroxy- oxalic acid
3-nitroacrylate 3-nitropropionate dihydrate
13% 27% 80%

Figure 5-1. Synthesis of methyl 3-nitroacrylate using N204

Besides this, nitryl chloride (Figure 5-2),123 nitrosyl chloride (Figure 5-2)124 and NaNO2 in

aqueous solution of CH3CO2H (Figure 5-3) have also been employed to synthesize










P-nitroacrylates from the corresponding acrylic esters.125 Unfortunately, the low boiling point of

some of these reagents makes them inconvenient, especially if the reactions are carried out on

small scales.


NO2CI
0 or
R NOCI

ethyl a,P3-unsaturated
carboxylate


NO2 0 H20 NO, O Ac 20 NO2 O
RO RO R
ONO OH OCOCH3
a-nitrito-p- ethyl a-hydroxy- ethyl a-acetoxy-P-
nitrocarboxylate P-nitrocarboxylate nitro carb oxylate

+ /base


NO2 0

ci
Cl
a-chloro-p-
nitrocarboxylate


NO2 0



ethyl a,P-un saturated
p-nitro acrylate


Cl 0

R@O
Cl
cp-dichloro-
carboxylate

Figure 5-2. Synthesis of 3-nitroacrylates using N02C1 or NOC1.


0


NaNO2-aqueous AcOH
or N203


methyl methacrylate


R CH3, C2H5, n-C3H7, i-C3H7, CAH


-Y0-
NO20

methyl 2-methyl-3-
nitroacrylate


Figure 5-3. Synthesis of P-nitroacrylates using nitrous acid.

Recently, Vankar et al. reported their approach using NaNO2-ceric ammonium nitrate

(CAN) in CH3CN in order to convert acrylic esters into p-nitro alcohols that were dehydrated via

their mesylates by following modified McMurry's method (Figure 5-4).126 The reaction is

believed to proceed via radical intermediates, which may be the reason for formation of side









products and low yield of reaction (13-25%) (Figure 5-5). Despite our attempts to improve the

efficiency (10-13% average yield) of the Vankar's procedure, we were not able to obtain

sufficient amount of products for further application.


R1 CO2R2
RI CO^


R H

R = H, Me, Ph
R1 = H, Me
R2 = Me, Et


AN-NaNO2
CH3CN
OoC-r.t.


OH
R1 CO2R2 R1 C02R2
CH3SO2CI, Et3N R

R H -200C 2N R
NO2


Figure 5-4. NaNO2-Ceric ammonium nitrate mediated conversion of acrylic esters into P-
nitroacrylates.


MeO2C NaN
SCNaNO
CAN


MeO2C ONO2


NO2


MeO2C



NO2


MeO2C


NO2


MeO2C


Hydrolysis


CAN
ONO2
NO2




MeO2C OH


NO2


Figure 5-5. Proposed radical mechanism of 2-hydroxy-3-nitroacrylates formation.

After considering several synthetic methods to p-nitro acrylic esters, we decided to follow

the protocol developed by Palmieri.127 The first step of this approach was a nitroaldol (Henry)

reaction carried out under heterogeneous catalysis using a solid-phase base (Amberlyst A-21)

along with the appropriate a-keto-ester (either commercially available or prepared by the method

of Macritchie et al.128) and nitroalkane. The NMR spectral data for nitroaldol adducts 19a-e, g

matched those reported previously. 127,129-131 These were converted to the corresponding









nitroacrylates via mesylate derivatives (Figure 5-6). Both 3-alkyl-substituted nitroacrylates 19a

and b were obtained predominantly in the (E)-form (~90%)127 whereas the (Z)-isomers

predominated for 2-substituted alkenes (-80%) 19c-g, possibly as a result of E1cB reaction on

the corresponding mesylates. Because olefin geometry may directly impact the stereoselectivity

of enzymatic reactions, the major alkene isomers were chromatographically enriched (>95%

geometric purity).

R1
O R2 R1CH2NO, (18) R1 MsCI, Et3N CO2Et
S Amberyst A-21 02N R2 2N
CO Et HO CO2Et R
E-20a, b
17 19a R = Me, R2 = H -
b R = Et, R = H
cR1 = H, R2 =Me Ri
dR = H, R2 Et 02 R2
eR = H, R2 = n-Pr 2
fR = H, R2 = i-Pr CO2Et
g R = H, R = Ph Z-20c-g


Figure 5-6. Amberlyst A-21 mediated conversion of acrylic esters into P-nitroacrylates.

Biotransformation of P-Nitroacrylates and Synthesis of p-Amino Acids

We studied two approaches towards the bioreduction of P-nitroacrylic esters: i)

biotransformation using isolated enzyme and ii) in the form of enzyme extract. The first method

served for determination of the stereo- and enantioselectivity of the protein towards

nitroacrylates. The reactions with extract were used to scale up the process.

Biotransformation Using Isolated Enzyme

Biocatalytic reductions utilized old yellow enzyme that had been purified by affinity

chromatography. NADPH was supplied by a cofactor regeneration system (glucose-6-phosphate

/ bakers' yeast glucose-6-phosphate dehydrogenase). Preliminary studies had revealed that olefin

isomerization was more rapid under alkaline conditions, and pH 6.93 was selected to minimize

this side-reaction while maintaining acceptable enzyme efficiency. A two-fold molar excess of









P-cyclodextrin (relative to the nitroacrylate) was also included to enhance substrate solubility

under aqueous conditions. Unfortunately, the solubility of the P-cyclodextrin / 20g complex was

still too low for efficient reduction, and the observed conversion was too low for further analysis

of the product. Both the substrates (20a-f and glucose-6-phosphate) and the two enzymes were

added portionwise to enhance the longevity of the processes, which were monitored by GC/MS

and complete substrate consumption was observed after ca. 8 hr in all cases except for (E)-20b.

The NMR and GC analysis of the crude products verified that only the double bond of the olefins

had been reduced and the nitro groups remained intact. No significant levels of side products

were observed and yields after purification ranged from 74-98%. Because it was not possible to

determine the optical purities of the obtained products by chiral-phase chromatography, the

crude materials were hydrogenated in the presence of Raney-Ni to the corresponding amines

(23a-g) (75-85% yield) (Figure 5-7). Enantiomer separations were then possible by chiral-phase

GC following derivatization with trifluoroacetic anhydride.

The racemic standards, required for enantioselectivity assignment, were obtained by

hydrogenation of corresponding P-nitroacrylates catalyzed by Raney-Ni. Good optical purities

were obtained from 2-alkyl-substituted nitroalkenes 20c-f; by contrast, 3-alkyl-substituted

products were obtained in essentially racemic form.105,132-135 The absolute configurations were

assigned by the direction of optical rotations of the free p2-amino acids as their hydrochloride

salts (obtained by acid hydrolysis in 88-95% yields). Overall yields of 12-amino acids from

P-nitroalkenes ranged from 57-73% (Table 5-1).

The results of our previous studies of alkyl-substituted-2-cyclohexenone reductions by old

yellow enzyme122 were consistent with the net trans-hydrogenation mechanism elucidated by

Massey and Karplus.4'15'24 Hydride P-addition (from reduced FMN) occurs from the re-face









while a-protonation likely involves the phenol side-chain of Tyr-196. Carbonyl activation is

achieved via hydrogen bonding by the side-chains of His-191 and Asn-194. For the acyclic

P-nitroacrylates investigated here, analogous binding could occur in which one nitro-oxygen

occupies the same location as the carbonyl oxygen and the alkene is positioned similarly (Figure

5-8).

R1

02N CO2Et
R2
E-20a,b 1. OYE, NADP+, cof actor R1
regeneration system H2N CO2H

RI 2. H Ra-Ni R
N R2 3. HCI, A 2
02N 2 23a-g
CO2Et
Z-20c-g

Figure 5-7. Biotransformation of nitroacrylates by OYE towards B-amino acids.

Table 5-1. Reduction of substituted nitroacrylates by isolated old yellow enzyme and production
of p-amino acids.
Nitroolefin Conversion (%) ee (%) [a]Da1
(E)-20a >98 8(R) -1.1 c 1.0 (-39.50 c 0.56)136
(E)-20b 50 ---
(Z)-20c >98 87 (R) -13.0 c 0.94 (-12.60 c 1)137
(Z)-20d >98 91 (R) -2.70 c 1.0 (-2.90 c 1)137
(Z)-20e >98 94 (R) +1.20 c 1.0 (3.50 c 1)137
(Z)-20f >98 96 (R) -1.60 c 1.0 (-14.40 c 1)138
a Measured in aqueous solution at the indicated concentrations from hydrochloride salts at room
temperature.

This substrate binding orientation was verified by carrying out the enzymatic reductions of

(E)-20b and (Z)-20c in D20 (Figure 4-9) All MS and NMR data were consistent with deuterium

incorporation only on nitro-bearing carbon in both cases, although they did not eliminate the

possibility of the hydrogen exchange with water after the biotransformation, which could explain

the racemization at the 3-carbon.










In order to determine whether the mentioned hydrogen incorporation occurs we examined

the deuterium exchange between the enzymatic reduction product 21a and deuterated phosphate

buffer. After only five hours the deuterium incorporation could be detected in high levels in MS

chromatogram by measuring the ratio of peak 117 to 116 as observed in Figures 5-10 and Figure

5-11.

Based on the above results, the formation of the racemic center at the P-position was

attributed to the process of the epimerization related to the presence of the acidic hydrogen at the

asymmetric P-carbon (Figure 5-12). Unfortunately, this data was not sufficient to confirm

proposed mechanism of the biotransformation and additional tests using deuterated NADPH

were required.

H+
0-f OYE, O
-R NADPH

H
H:


H+
oOYE, o o 0
+ / OYE, +/, + +1 H
-O-N H NADPH -N H -O-N H O-N R
EO2C R X 2 HR2
EtO R EtO2tC ~E C EtO2C 2 H
2 [2 H H
H:


H+
O OYE, O O + i
O-N H NADPH H O-N H O -N --H ORN CHO2Et
2 C R" -/CO2EtH
R2 CO2Et 2 CO2Et R2 H H

H:

Figure 5-8. Proposed mechanism of reduction of substituted-2-cyclohexenones and
nitroacrylates by old yellow enzyme.












2N CO2Et
ON


20 b


N CO2Et
O2N 2


OYE
NADPH
D2O


20 c


Figure 5-9. Biotransformation of nitroacrylates in D20.


3.0


2.5 -


2.0


1.5

1.0 -


0.5


0 5 10 15 20 25


time hourlyl

Figure 5-10. Incorporation of deuterium into 21a.


OYE
NADPH
D20


CO2Et


21b


SH
D COEt
02N H
TH


21 c






















I I T I I II I I
5 4 3

O,18 0.96
2.00 0.95


Figure 5-11. Fragment of the 1H NMR analysis of the product of incubation.


HN C02Et D20 O CO2Et
2N H H
H H
21 b 21 b

Figure 5-12. Incubation of compounds 21b in D20.

There are two different approaches to the reactions with isotope labeled nicotinamide:

either using isolated NADPD or applying NADPD regeneration system directly to the analyzed

reaction. The first method seems to be rather unattractive considering the high cost of the

reagents and the time consuming purification process. Instead, we turned our attention to the

second method by using Thermoanaerobium brockii alcohol dehydrogenase (TBADH) and

isopropanol-d8 in OYE reduction of (Z)-20c (Figure 5-13).139

The MS and NMR data supported proposed net trans-addition of hydrogen to 20c-f that

leads to the observed (R)-products. Additionally, in a preliminary experiment, (E)-20d was

reduced by the OYE with largely (S)-stereoselectivity, as would be expected from the model









described in Figure 5-14. Within 7 hours, initially pure (Z)-20d incubated in buffer alone

afforded three by-products in a combined conversion of ca. 50%. They were identified by

GC/MS as (Z)-20d, the alternate acrylate alkene regioisomer of 20d and the water addition

product from 20d (in racemic form) (Figure 5-15). These observations underscore the need to

reduce P-nitroacrylates rapidly and minimize their exposure to the aqueous medium conditions.

H O Old Yellow Enzyme H H O

02N OC2H 02N OC2H
D
NADPD NADP+
acetone-d6 isopropanol-d8



Alcohol Dehydrogenase

Figure 5-13. Reaction of (E)-20c with NADPD.


02N CO 2EO


E-20d


OYE
NADPH 2
NADPH 02NO2Et



(S)-21d


Figure 5-14. Biotransformation of (E)-20d by isolated OYE1.

KP. buffer
N pH7.0 O2N) + 02N
CO2Et 7CO^Et
Z-20d (Z)-20d (


*( CO2Et


E)-20d


H
+ O CO2Et
+2N 1dOH

19d


Figure 5-15. Incubation of 20d in KPi buffer.

Biotransformation Using Cell-Free Extract

Old yellow enzyme requires NADPH as cofactor which in purified form is very

expensive in big amounts. The need to scale up the bioreductions forced us to look for


O









regeneration of NADPH by other means. One solution to this problem is application of the

coupled-enzyme regeneration protocol.140 The procedure is based on combining cell extracts of

glucose dehydrogenase from B. subtilis responsible for cofactor regeneration and old yellow

enzyme S. carlsbergensis, both over expressed in E. coli. Cells over expressing those two

proteins were grown separately in 1 liter of LB medium and harvested just before they reached

the stationary phase. Next, they were lysed and centrifuged in order to remove cell debris. The

extracts were then mixed together in 100 ml KPi buffer (100 mM) and the biotransformation

performed by portionwise addition of the appropriate nitroacrylate (20a, c, e at final

concentrations of 13 mM), a two-fold molar excess of P-cyclodextrin (relative to the

nitroacrylate) and glucose (at a final concentration of 10 mM). The pH of the solution was

constantly controlled and kept at 6.93. The reactions were allowed to proceed in room

temperature with gentle stirring until the bioconversions ceased after 11 hours.

Although the chemical yields of the bioreductions were satisfactory (72-85%), the

enantioselectivity of the transformations of 20c and 20e was lower than in the case of isolated

enzyme reactions (Table 5-2). The reason for this could be the presence of small amounts of

other reductases in the extract (e.g. old yellow enzyme from E. coli: NemA) that to some extent

affected the final optical purity of the product.

Another problem may be insufficient amount of intercellular NADP+ and decreased

production of the NADPH required by OYE1 as a cofactor. Additionally, the MS and NMR

analysis identified by-products: (E)-20a, 20c, 20e and the water addition product from 20a, 20c

and 20e (in racemic form) (Figure 5-16).

Conclusions

Old yellow enzyme-mediated reductions of 3-alkylsubstituted P-nitroacrylates 20a and 20b

yielded essentially racemic products.141 Given the highly stereoselective a-protonation observed









in 2-cyclohexenone reductions, this result was surprising. The isotope wash-in following

reduction analysis revealed chiral instability of the P-carbon of the biotransformation product,

which undergoes spontaneous epimerization in aqueous media. The above explanation is further

supported by Ohta and co-workers who reported racemization of similar a-substituted nitro

compounds occurring both in basic and acidic water solutions.142 Further studies with deuterium

labeled NADPH confirmed proposed net trans-addition mechanism by incorporation of

deuterium at the a-carbon.

RI
02 CO2Et
2 1. E. coli cell extract
E-20a with over expressed R
OYE and GDH H2N_ CO2H
RI 2. H2, Ra-Ni R2

02N R2 3. 23ac,e
CO2Et
Z-20c,e

Figure 5-16. Biotransformation of nitroacrylates using E. coli cell-free extract with
overexpressed OYE and GDH.

Table 5-2. Reduction of substituted nitroacrylates using E. coli cell-free extract with over
expressed OYE and GDH.
Nitroolefin Conversion (%) e.e. (%) Configurationa
(E)-20a 96 --- ---
(Z)-20c 98 73 R
(Z)-20e 97 89 R
a Determined by chiral-phase GC following nitro group reduction and derivatization with
trifluoroacetic anhydride.

In conclusion, our results have uncovered a new application for S. carlsbergensis old

yellow enzyme in synthesizing optically active p2-amino acids. The synthetic route is concise

and utilizes inexpensive starting materials. The major difficulties lie in suppressing alkene

isomerization prior to reduction and ensuring active site protonation of the nitronate

intermediate.









CHAPTER 6
EXPERIMENTAL SECTION

General Methods and Instrumentation

Standard media and techniques for growth and maintenance ofE. coli were applied.

Luria-Bertani (LB) medium used for bacterial cultivation contained 1% Bacto-Tryptone, 0.5%

Bacto-Yeast Extract and 1% NaC1. Synthetic reactions were carried out under argon atmosphere,

with the exception of water containing reactions. Dichloromethane, diethyl ether and

tetrahydrofuran were dried on an MBRAUN solvent purification system using a double 4.8 L

activated alumina columns type A2. Triethylamine was dried by distillation in the atmosphere of

argon and stored over molecular sieves in -20C. An ion-exchange resin Amberlyst A-21 was used

in the water-moist free base form in nitroaldol reaction procedures. Reactions were monitored by

thin-layer chromatography (TLC), using precoated silica gel plates (EMD Chemicals), or by gas

chromatography using DB-17 column (0.25 mm x 25 m x 0.25 [m thickness) with a flame

ionization detector. Products were purified by flash chromatography on Purasil silica gel 230-

400 mesh (Whatman). For chiral separation, gas chromatography was applied which utilized a

Chirasil-Dex CB column (0.25 mm x 25 m x 0.25 [m thickness) with mass spectrometric

detection. NMR spectra were measured in CDC13, CDOD or D20 solutions and recorded at room

temperature on a Varian Mercury 300 spectrometer operating at 300 MHz for 1H and 75 MHz for

13C, respectively, with chemical shifts (6, ppm) reported relative to tetramethylsilane (1H NMR)

or residual solvent (13C NMR). IR spectra were obtained as neat films on NaCl plates using a

Perkin-Elmer Spectrum One FT-IR spectrophotometer. Racemic P-amino acid esters were

prepared from the corresponding nitroacrylates by hydrogenation at 500 psi in the presence of

Raney nickel. A solution of 6M HC1 was used in hydrolysis procedures of P-amino acid esters.









Purification ofS. carlsbergensis Old Yellow Enzyme

All enzyme purification steps were carried out in 4C. The plasmid encoding S.

carlsbergensis old yellow enzyme (pOYE-pET3b) was a gift from Professor V. Massey's

laboratory. Routine E. coli strain BL21(DE3) (Novagen) transformations with pOYE-pET3b

were performed by electroporation. The affinity matrix (N-(4-hydroxybenzoyl)aminohexyl

agarose) was synthesized based on reported procedure.14

Cell Growth and Extract Preparation for Protein Isolation

An overnight culture of E. coli (BL21(DE3)(pOYE-pET3b)) grown in LB medium

containing 100 [g/ml ampicillin was diluted 1:100 into 4 L of the same medium in a New

Brunswick M19 fermenter. The culture was stirred at 700 rpm with aeration at 4 L/min in 37C

until the optical density at 600 nm reached 0.8, then the enzyme overproduction was induced

with isopropylthio-p-D-galactoside (IPTG) at a final concentration of 400 [M and the culture

was stirred for an additional 2.5 hours at room temperature. The cells were harvested by

centrifugation (5,000 rpm for 15 min at 40C), washed twice with cold sterile water and then

resuspended in 15 mL of buffer (40 mM Tris-C1, 10 mM MgC12, 10 mM DTT, 200 mM KC1,

1 mM PMSF and 10% glycerol, pH 8.0). The cells were lysed using a French Press and debris

was removed by centrifugation at 20,000 rpm for 60 min at 40C. The pH of supernatant was

adjusted to 8.5 by adding concentrated ammonium hydroxide and the solution was made to 78%

saturation with ammonium sulfate added portionwise over an hour. Then it was left for another

30 min stirring and the mixture was centrifuged at 20,000 rpm for 60 min at 40C. The pellet was

resuspended in 20 mL of buffer (0.1 M Tris-C1, 0.1 M ammonium sulfate, 10 [M PMSF, 10 [M

sodium dithionite, pH 8.0). The resulting solution was dialyzed against Tris-buffer (0.1 M

Tris-C1, 0.1 M ammonium sulfate, 10 [M PMSF, 10 [M sodium dithionite, pH 8.0) which was









changed three times over 20 hours, the second and third time without sodium dithionite. After 20

hours of dialysis the crude material was centrifuged at 20,000 rpm for 10 min at 40C.

Isolation of Old Yellow Enzyme

A 10 mL affinity column was washed with 600 mL of the starting buffer (0.1 M Tris-C1,

0.1 M ammonium sulfate, 10 [M PMSF at pH 8.0) at a flow rate of 0.5 mL/min. The crude

extract was applied and the column was washed with 1 L of the same buffer until the absorbance

at 280 nm was less than 0.2. The enzyme was eluted with washing buffer (0.1 M Tris-C1, 0.1 M

ammonium sulfate, 10 [M PMSF, pH 8.0) which had been degassed and flashed with oxygen-

free argon in a 500 ml suction flask and then supplemented with 3 mM sodium dithionite. The

enzyme was collected in three or four 4 mL fractions in test tubes, concentrated by ultrafiltration

and stored at -200C.

Regeneration of Affinity Matrix

The N-(4-hydroxybenzoyl)aminohexyl agarose was regenerated by washing with 0.2 M

sodium acetate buffer, pH 5.0, containing 6 M guanidine HC1. Storage of the gel in Tris buffer,

pH 8.0, with 1 mM sodium azide prevented microbial damage.

Enzyme Activity Assay

Old yellow enzyme activity was assayed by measuring the rate of NADPH oxidation at

340 nm at 250C. The standard assay system contained 0.2 mM NADPH (10 iL of 20 mM stock

solution prepared immediately before use in 0.1 M KPi buffer, pH 7.0), 2.5 mM 2-cyclohexenone

(100 iL of 25 mM stock solution in EtOH) and 1 [L of enzyme in a total volume of 1 mL.

Glucose Dehydrogenase activity was assayed by measuring the rate of NADP reduction at 340

nm at 250C. The standard assay system contained 0.5 mM of glucose (0.5 iL of 1 M stock

solution in water), 0.1 mM NADP+ (5 [L of 20 mM stock solution in KPi buffer) and 1 [L of

GDH. The slope was calculated and used to find the specific activity. The background NADPH









oxidation was measured using an identical to above mentioned solution in which the amount of

substrate was replaced by phosphate buffer. A unit of enzyme activity was defined as the

quantity sufficient to oxidize 1 imol ofNADPH per minute in the mixture described above. Unit

per ml of enzyme preparation were calculated based on the following equation:

Units / mL = [(dA / dt x 1000) / (e340 1)] x (Vassay / Venzyme) x Dilution, where dA / dt is the

slope in AU / min, e340 = 6270 L / mol cm, 1 = 1 cm, Vassay = 1 mL, Venzyme = volume of enzyme

added in mL, Dilution = dilution factor.

Synthesis of 2-Alkyl-cyclohexane-1,3-diones

General procedure: Glutaryl chloride (6.7 g, 40 mmol) and 80 mmol of the appropriate

acid were added to a suspension of 13.58 g (103 mM) of AlC13 in 13 mL nitromethane with

cooling in the atmosphere of argon. The mixture was heated for 3 hours at 800C, then cooled to

10C and poured onto 20 g of ice. After cooling to about 0C, the crude product that had

separated was filtered off, washed with 5 ml of cold water, and recrystalized from water (with

charcoal). The aqueous phase of the filtrate was boiled with charcoal, filtered and extracted with

ether. A second fraction crystallized after concentration of the ether extract.

2-Methylcyclohexane-1,3-dione (3a). White solid (1.0 g, 20%). 1H NMR (CDC13): 6 0.75

(s, 3H,), 1.80 (m, 2H), 2.29 (t, 2H), 3.29 (t, 1H) ppm. 13C NMR (CDC13): 7.7, 21.0, 33.0, 110.0

ppm (C2 signal not observed).

2-Ethylcyclohexane-l,3-dione (3b). Beige solid (0.84 g, 15%). 1H NMR (CDC13): 6 0.75

(s, 3H,), 1.76 (m, 2H), 2.08 (q, 2H), 2.24 (t, 2H), 3.18 (t, 1H) ppm. 13C NMR (CDC13): 12, 17.5,

21.5, 32.5, 108.0 ppm (C2 signal not observed).

Synthesis of 3-Isobutoxy-2-alkylcyclohex-2-enone

3-Isobutoxy-2-methylcyclohex-2-enone (4a). To a stirred solution of

2-methyl-1,3-cyclohexanedione (3a) (1.08 g, 8.57 mmol) and p-toluenosulfonic acid (108 mg) in









9 ml of benzene was added i-BuOH (2.6 mL). The mixture was heated at reflux under a Dean-

Stark trap for 3 hours. The reaction mixture was cooled down to room temperature and poured

into 6.5 mL of saturated aqueous NaHCO3 solution and extracted with ether (3 x 5 mL). The

combined extracts were washed with 5 mL of brine and dried over MgSO4. The solvent was

removed under reduced pressure, solution purified by column chromatography (silica gel,

hexane/ethyl acetate = 5/1) to give 3-isobutoxy-2-methylcyclohex-2-enone as yellow oil (1.48 g,

95%). H NMR (CDC13): 6 0.97 (d, 6H,), 1.71 (s, 3H), 1.93 (m, 1H), 2.31 (t, 2H), 2.51 (t, 2H),

3.39 (t, 2H), 3.73 (d, 2H) ppm. 13C NMR (CDC13): 7.0, 18.7, 21.3, 27.3, 32.2, 72.3, 105.8, 167.4,

185.9 ppm.

3-Isobutoxy-2-ethylcyclohex-2-enone (4b). To a stirred solution of 2-methyl-

1,3-cyclohexanedione (3a) (0.90 g, 6.45 mmol) andp-toluenosulfonic acid (80 mg) in 7 mL of

benzene was added i-BuOH (1.95 mL). The mixture was heated at reflux under a Dean-Stark

trap for 3 hours. The reaction mixture was cooled down to room temperature and poured into 5

mL of saturated aqueous NaHCO3 solution and extracted with 3 x 5mL of ether. The combined

extracts were washed with 4 mL of brine and dried over MgSO4. The solvent was removed under

reduced pressure and the residue was purified by column chromatography (silica gel,

hexane/ethyl acetate = 5/1) to give 3-isobutoxy-2-ethylcyclohex-2-enone as yellow oil (1.01 g,

80% yield. H NMR (CDC13): 6 0.96 (d, 6H,), 1.1 (t, 3H), 1.67 (m, 3H), 2.25 (t, 2H), 2.40 (m,

2H), 3.20 (t, 2H), 3.74 (d, 2H) ppm. 13C NMR (CDC13): 11.6, 14.8, 19.8, 21.4, 29.5, 36.0, 74.0,

105.9, 168.5, 188.9 ppm.

Synthesis of 2-Alkylcyclohex-2-enone

General procedure using NBS: To a solution of 2-alkylidenecyclohexan-1-one (10

mmol) dissolved in CC14 (50 mL) was added N-bromosuccinimide (10 mmol) and the resulting

suspension was heated in a water bath at 900C for 3 h. The suspension was then cooled to room









temperature, the precipitates filtered off and aniline (10 mmol), was added to the filtrate with

cooling (ice-water). The solution was stirred at room temperature for 15 h, washed with 5% HC1

(3 x 30 mL) and then with 5% NaHCO3 (2 x 30 mL). After drying with MgSO4, the solvent was

evaporated to give brownish oil, which was purified by distillation under reduced pressure. NMR

analysis did not confirm that desired product was obtained.

2-Methylcyclohex-2-enone (5a). A solution of 3-isobutoxy-2-methylcyclohex-2-enone 4a

(1.48 g, 8.14 mmol) and dry ether (6 mL) was added dropwise to LAH (115 mg) in dry ether (6

mL) at such a rate that steady reflux was maintained. The mixture was stirred for a further 1

hour, cooled and 10% H2S04 (6 mL) added. The ether layer was removed and the aqueous layer

extracted with ether (5x20 mL). The combined ether solutions were dried over MgSO4 and

distilled to obtain 2-methylcyclohex-2-enone (0.80 g, 90%) as yellow oil. 1H NMR (CDC13): 6

1.86 (s, 3H,), 1.97 (m, 2H), 2.43 (q, 2H), 2.52 (t, 1H), 5.93 (t, 1H) ppm. 13C NMR (CDC13): 23,

24.5, 39, 45, 116.5, 151.5, 194.4 ppm.

2-Ethylcyclohex-2-enone (5b). A solution of 3-isobutoxy-2-ethylcyclohex-2-enone 4a

(1.0 g, 5.16 mmol) and dry ether (4 mL) was added dropwise to LAH (73 mg) in dry ether (4

mL) at such a rate that steady refluxing was maintained. The mixture was stirred for a further 1

hour, cooled and a 10% H2S04 (4 mL) added. The ether layer was removed and the aqueous

layer extracted with ether (5x10 mL). The combined ether solutions were dried over MgSO4 and

distilled to get 2-ethylcyclohex-2-enone (0.53 g, 84%) as yellow oil. 1H NMR (CDC13): 6 1.1 (t,

3H), 1.96 (m, 2H), 2.0 (m, 2H), 2.44 (q, 2H), 3.1 (t, 2H), 6.2 (t, 1H) ppm. 13C NMR (CDC13):

15.7, 23.2, 24.5, 33.0, 36.0, 134.9, 144.0, 198.0 ppm.

Synthesis of 1,3-Cyclohexanedione

3-Isobutoxy-2-cyclohexenone (7). To a stirred solution of 1,3-cyclohexanedione 6 (10 g,

89 mmol) and p-toluenosulfonic acid (1.44 g) in 314 ml of benzene was added i-BuOH (25 mL).









The mixture was heated at reflux under a Dean-Stark trap for 3 hours. The reaction mixture was

cooled to room temperature and poured into 56 mL of saturated aqueous NaHCO3 solution and

extracted with ether (3x50 mL). The combined extracts were washed with 45 mL of brine and

dried over MgSO4. The solvent was removed under reduced pressure, and the residue was

purified by column chromatography (silica gel, hexane/ethyl acetate = 5/1) to give

3-isobutoxy-2-cyclohexenone as yellow oil (12.71 g, 85%). 1H NMR (CDC13): 6 0.83 (d, 6H,),

1.85 (m, 3H), 2.21 (t, 2H), 2.27 (t, 2H), 3.45 (d, 2H), 5.18 (s, 1H) ppm. 13C NMR (CDC13): 18.7,

20.9, 27.3, 28.6, 36.4, 74.2, 102.3, 177.7, 199.2 ppm.

3-Alkyl-2-Cyclohexen- 1-ones

General procedure: To the solution of the appropriate Grignard reagent (26.7 mmol) and

20 mL of dry ether, a solution of 3-isobutoxy-2-cyclohexenone 7 (2 g, 17.8 mmol) in 10 mL of

dry ether was added and the mixture was stirred for 2 hours. After that time, the Grignard

complex was decomposed with diluted sulfuric acid and the solution was extracted with ether.

The organic phase was washed with diluted NaHCO3, water and dried over MgSO4. After

evaporation of solvent the crude product was purified by silica gel column chromatography

(hexanes/Et20 = 3/1) to give the desired 3-alkyl-2-cyclohexen-1-one.

3-Methyl-2-cyclohexen-l-one (9a). Yellow oil was obtained (1.62 g, 83%). 1H NMR

(CDC13): 6 1.96 (m, 5H,), 2.27 (m, 4H), 5.88 (s, 1H), ppm. 13C NMR (CDC13): 22.3, 24.1, 30.6,

36.7, 126.3, 162.4, 199.2 ppm.

3-Ethyl-2-cyclohexen-l-one (9b). Yellow oil was obtained (1.72 g, 78%). 1HNMR

(CDCl3): 6 1.06 (t, 3H,), 1.96 (q, 2H), 2.1 (m, 6H), 5.79 (s, 1H) ppm. 13C NMR (CDC13): 11.2,

22.7, 29.7, 30.8, 37.4, 124.5, 168.0, 200.1 ppm.









3-n-Propyl-2-cyclohexen-l-one (9c). Yellow oil was obtained (1.84 g, 75%). 1HNMR

(CDC13): 6 0.95 (t, 3H,), 1.1 (m, 10H), 5.85 (s, 1H) ppm. 13C NMR (CDC13): 13.5, 19.9, 22.5,

29.4, 37.7, 39.8, 125.4, 166.4, 199.8 ppm.

3-i-Propyl-2-cyclohexen-l-one (9d). Yellow oil was obtained (1.72 g, 70%). H NMR

(CDC13): 6 1.05 (d, 6H,), 1.97 (m, 2H), 2.33 (m, 5H), 5.83 (s, 1H) ppm. 13C NMR (CDCl3): 21.1,

23.5, 28.2, 36.2, 38.1, 124.0, 172.4, 200.7 ppm.

3-n-Butyl-2-cyclohexen-l-one (9e). Yellow oil was obtained (2.16 g, 80%). 1H NMR

(CDC13): 6 0.94 (t, 3H,), 1.30 (m, 2H), 1.52 (m, 2H), 1.98 (m, 2H), 2.24 (t, 2H), 2.32 (t, 2H),

2.40 (m, 2H), 5.88 (t, 1H) ppm. 13C NMR (CDC13): 13.9, 22.6, 23.0, 29.3, 29.9, 37.4, 37.9,

125.5, 167.0, 199.8 ppm.

2-(1-Hydroxyalkyl)cyclohexanes

General procedure: A solution of cyclohexanone (4.2 mL, 40 mmol) in THF (67 mL)

was added dropwise to a stirred and cooled (-780C) solution of LDA [generated by dropwise

addition of n-BuLi (2.5 M, in hexane, 44 mmol, 17.6 mL) to i-Pr2NH (42 mmol, 5.88 mL) in

THF (200 mL) at 0C, followed, after 15 minutes, by cooling to -780C]. After 1 hour, the

appropriate aldehyde (40 mmol) in THF (100 mL) was added quickly. Stirring was continued for

50 min at -780C, and the reaction was quenched with saturated aqueous NH4Cl (80 mL). The

cooling bath was removed and stirring was continued until the mixture had reached room

temperature. The solution was extracted with ether, washed with water and brine, dried over

MgSO4 and the solvent was evaporated. Flash chromatography (EtOAc/hexane = 1/6) of the

residue gave final products.

2-(1-Hydroxyethyl)cyclohexane (12a). Pale yellow oil was obtained (4.20 g, 75%). 1H

NMR (CDC13): 6 1.2 (d, 3H,), 1.42 (m, 1H), 1.50 (m, 2H), 1.62 (m, 8H), 3.65 (s, 1H), 3.91 (m,

1H) ppm. 13C NMR (CDC13): 20.43, 25.55, 28.32, 31.22, 43.34, 58.24, 68.38, 216.31 ppm.









2-(1-Hydroxybuthyl)cyclohexane (12b).Pale yellow oil was obtained (40.0 g, 60%). 1H

NMR (CDC13): 6 0.85 (t, 3H,), 1.20 (m, 13H), 3.63 (m, 2H), 3.95 (m, 2H) ppm. 13C NMR

(CDC13): 13.1, 18.0, 24.7, 27.6, 30.8, 38.1, 42.5, 55.3, 69.9, 215.8 ppm.

2-Alkylidenecyclohexanones

2-Ethylidenecyclohexanone (13a). Methanesulfonyl chloride (7.33 mL, 93 mmol) was

added dropwise to a stirred and cooled (0C) solution of alcohol 12a (4.3 g, 30 mmol) and Et3N

(21.2 mL, 151 mmol) in CH2C12 (160 mL). The cooling bath was left in place, but was not

recharged. Stirring was continued for 6 hours, the solution was quenched with saturated aqueous

NaHCO3 (37 mL), diluted with Et20, washed with water and brine, dried over MgSO4.

Evaporation of the solvent gave crude mesylate (5.82 g, in 80% yield), which was used

immediately for next step. To a stirred solution of the above mesylate in THF (195 mL), DBU

(7.5 mL, 50 mmol) was added dropwise. After 1 hour, the mixture was diluted with Et2O,

washed with water, 5% hydrochloric acid, and brine, dried over MgSO4 and solvent evaporated.

The obtained crude product was purified by flash chromatography (EtOAc/hexane = 1/10) to

give 13a (2.64 g, 71%) with E isomer in excess (- 80%). 1H NMR (CDC13): 6 6.57 (t, 1H), 2.3

(m, 11H) ppm. 13C NMR (CDC13): 21.6, 23.3, 25.7, 29.8, 38.5, 135.2, 138.5, 199.4 ppm.

2-Butylidenecyclohexanone (13b). a) Methanesulfonyl chloride (5.9 mL, 75 mmol) was

added dropwise to a stirred and cooled (0C) solution of alcohol 12a (4.0 g, 24 mmol) and Et3N

(16.8 mL, 120 mmol) in CH2C2 (128 mL). The cooling bath was left in place, but was not

recharged. Stirring was continued for 6 hours, the solution was quenched with saturated aqueous

NaHCO3 (34 mL), diluted with Et20, washed with water and brine, dried over MgSO4.

Evaporation of the solvent gave crude mesylate (5.43 g, in 73% yield), which was used

immediately for next step; b) to a stirred solution of the above mesylate in THF (178 mL), DBU

(6.9 mL, 46 mmol) was added dropwise. After 1 hour, the mixture was diluted with Et20,









washed with water, 5% hydrochloric acid, and brine, dried over MgSO4 and solvent evaporated.

The obtained crude product was purified by flash chromatography (EtOAc/hexane = 1/10) to

give 13b (7.30 g, 64%) with E isomer in excess (- 80%). 1H NMR (CDC13): 6 2.3 (m, 15H), 6.64

(t, 1H) ppm. 13C NMR (CDC13): 13.3, 21.6, 23.0, 23.2, 25.9, 29.2, 39.5, 135.7, 138.4, 199.8 ppm.

Biotransformation of a,P-Unsaturated Cyclic Enones Using Isolated Old Yellow Enzyme

General procedure: Reaction mixtures contained final concentrations ofNADP+ (10.7

mmol, 8 mg), glucose-6-phosphate (648 mmol, 219.5 mg), glucose-6-phosphate dehydrogenase

(256 pg), enone (0.18 mmol), P-cyclodextrin (around 40 mg), purified OYE (20-40 pg) in 100

mM KPi buffer, pH 7.0 in total volume of 25 mL. Conversions were carried out at room

temperature. All the reaction components (except the buffer) were added portionwise (10 equal

portions during 12 hours) and the mixtures were sampled for GC analysis periodically. After 24

hours the reaction solutions were extracted with Et2O (3x50 mL). The combined organic extracts

were dried over MgSO4. The final products were purified by filtration through silica gel

(EtOAc/hexane = 2/9).

(R)-2-Methylcyclohexan-l-one (14a). Yellow oil; 97% e.e. (14.0 mg, 69.4%). 1H NMR

(CDCl3): 6 1.00 (d, 3H), 1.34 (m, 1H), 1.64 (m, 3H), 2.10 (m, 5H) ppm. 13C NMR (CDC13): 14.6,

25.3, 28.1, 36.1, 41.7, 45.4, 213.7 ppm.

(R)-2-Ethylcyclohexan-l-one (14b). Yellow oil; 92% e.e. (5.0 mg, 22%). 1H NMR

(CDC13): 6 0.96 (m, 3H), 2.60 (m, 11H) ppm.

2-Ethylcyclohexan-l-one (16a). Yellow oil; racemic mixture (4.0 mg, 17.7%). 1H NMR

(CDC13): 6 0.96 (m, 3H), 2.60 (m, 11H) ppm.

(S)-3-Methylcyclohexan-l-one (15a). Yellow oil; 96% e.e. (16.0 mg, 79.3%). H NMR

(CDC13): 6 0.92 (m, 3H), 1.31 (m, 1H), 1.64 (m, 1H), 2.00 (m, 4H), 2.32 (m, 3H) ppm. 13C NMR

(CDC13): 14.6, 25.3, 28.1, 36.1, 41.7, 45.4, 213.7 ppm.









(S)-3-Ethylcyclohexan-l-one (15b). Yellow oil; 95% e.e. (12.3 mg, 54.2%). 1H NMR

(CDC13): 6 0.91 (t, 3H), 1.22 (m, 11H) ppm.

(S)-3-n-Propylcyclohexan-l-one (15c). Yellow oil; 90% e.e. (3.75 mg, 14.88%). 1H NMR

(CDC13): 6 0.89 (t, 3H), 1.34 (m, 5H), 1.95 (m, 3H), 2.00 (m, 1H), 2.05 (m, 1H), 2.25 (m, 1H),

2.34 (m, 1H), 2.42 (m, 1H) ppm.

(S)-3-i-Propylcyclohexan-l-one (15d). Yellow oil; 92% e.e. (3.75 mg, 14.88%). 1H NMR

(CDC13): 6 0.90 (d, 3H), 0.91 (d, 3H), 1.28 (m, 10H) ppm.

Derivatization of Cyclohexanones with (2R,3R)-(-)-2,3-Butanediol

The stereochemical purities of all reduction products were determined by chiral-phase GC

after ketalization with optically pure 2,3-butanediol. Derivatizations were carried out by heating

a mixture of 1.0 equiv. of crude biotransformation product, 2.0 equiv. of

(2R,3R)-(-)-2,3-butanediol and a catalytic amount ofp-TsOH in 1.5 mL of CH2C12 at reflux for 2

hours. A 1 iL aliquot was directly analysed by GC. Samples of racemic ketones 5a, b, 9a-e and

13a, b were derivatized and analyzed by GC to demonstrate baseline resolution of enantiomers.

Biotransformation of a,P-Unsaturated Cyclic Enones Using Whole Cells of E. coli
BL21(DE3)(pOYE-pET3b)

General procedure: A 500 [tL aliquot from an overnight culture ofBL21(DE3)(pOYE-

pET3b) with an OD600 value between 4 and 5 added to 50 mL of LB medium supplemented with

200 [g/mL ampicillin in a 500 mL Erlenmeyer flask. The culture was shaken at 150-200 rpm at

37C until it reached an OD600 value between 0.4 and 0.5, then isopropylthio-P-D-galactoside

(IPTG) was added to final concentration of 0.10 mM. The culture was shaken at 150 rpm at room

temperature for an additional 30 minutes, then the ketone and stoichiometric quantity of

P-cyclodextrin were added and shaking was continued at room temperature at 150 rpm. Samples

for GC analysis were prepared by vortex mixing 50 [L of the reaction mixture with 50 [L of









EtOAc for ca. 30 s. A 1 [L portion of the organic phase was analyzed by GC. At the conclusions

of the reactions, the mixture was extracted with EtOAc (3x30 mL), and then the combined

organic extracts were dried with MgSO4 and concentrated by rotary evaporator. The final

products were purified by filtration through silica gel (EtOAc/hexane = 2/9).

(R)-2-Methylcyclohexan-l-one (14a). Yellow oil; 96% e.e. (12.0 mg, 59.5%). 1H NMR

(CDC13): 6 1.00 (d, 3H), 1.34 (m, 1H), 1.64 (m, 3H), 2.10 (m, 5H) ppm.

(R)-2-Ethylcyclohexan-l-one (14b). Yellow oil; 90% e.e. (3.17 mg, 14%). 1H NMR

(CDC13): 6 0.96 (m, 3H), 2.60 (m, 11H) ppm.

2-Ethylcyclohexan-l-one (16a). Yellow oil; racemic mixture (2.5 mg, 11%). 1H NMR

(CDC13): 6 0.96 (m, 3H), 2.60 (m, 11H) ppm.

(S)-3-Methylcyclohexan-l-one (15a). Yellow oil; 94% e.e. (13.0 mg, 64.4%). H NMR

(CDC13): 6 0.92 (m, 3H), 1.31 (m, 1H), 1.64 (m, 1H), 2.00 (m, 4H), 2.32 (m, 3H) ppm.

(S)-3-Ethylcyclohexan-l-one (15b). Yellow oil; 95% e.e. (12.3 mg, 54.2%). 1H NMR

(CDC13): 6 0.91 (t, 3H), 1.22 (m, 11H) ppm.

(S)-3-n-Propylcyclohexan-l-one (15c). Yellow oil; 89% e.e. (2.5 mg, 9.9%). 1H NMR

(CDC13): 6 0.89 (t, 3H), 1.34 (m, 5H), 1.95 (m, 3H), 2.00 (m, 1H), 2.05 (m, 1H), 2.25 (m, 1H),

2.34 (m, 1H), 2.42 (m, 1H) ppm.

(S)-3-i-Propylcyclohexan-l-one (15d). Yellow oil; 90% e.e. (1.9 mg, 7.4%). 1H NMR

(CDC13): 6 0.90 (d, 3H), 0.91 (d, 3H), 1.28 (m, 10H) ppm.

Synthesis of 2-Alkyloxobutanoates

General procedure: The appropriate Grignard reagent (88.6 mmol) was added dropwise

to a mixture of diethyl oxalate (10 mL, 73.6 mmol), THF (50 mL) and ether (100 mL) at -780C

and the solution was stirred at this temperature for 4 hours. After quenching with saturated

NH4C1 (100 mL), the mixture was extracted with ethyl acetate (3x100 mL). The organic phases









were combined, dried over MgSO4 and concentrated in vacuo to give crude products, which were

purified by flash chromatography.

2-Ethyloxobutanoate (17d). Pale yellow oil (8.13 g, 85%). H NMR (CDC13): 6 1.28 (t,

J=7.2 Hz, 3H), 1.39 (t, J=7.2, 3H), 2.90 (q, J=7.2, 2H), 4.15 (q, J=7.2, 2H) ppm. 13C NMR

(CDC13): 6.45, 13.90, 32.69, 62.24, 161.09, 195.04 ppm.

2-n-Propyloxobutanoate (17e). Pale yellow oil (7.33 g, 69%). 1H NMR (CDC13): 6 0.7

(m, 8H), 2.81 (t, J=7.1 Hz, 2H), 4.31 (q, J=6.3 Hz, 2H) ppm. 13C NMR (CDC13): 14.14, 14.65,

17.15, 41.75, 62.97, 161.86, 195.24 ppm.

Synthesis of Nitro Alcohols

General procedure using CAN: To a stirred solution of an appropriate acrylic ester (1

mmol) in anhydrous CH3CN (5 mL) was added CAN (3 mmol) and NaNO2 (3 mmol) at 0C

under nitrogen. The reaction mixture was vigorously stirred for 24 h at room temperature, diluted

with water and extracted sequentially with saturated solution of NaHCO3, brine and dried over

MgSO4. The residue obtained after evaporation of the solvent was purified by column

chromatography.

General Procedure Using Amberlyst A-21: A 50 mL two necked flask equipped with a

mechanical stirrer was charged with the appropriate nitroalkane (60 mmol) and cooled with

ice-water bath. Amberlyst A-21 (5-7 g) was added and the mixture was stirred for 5 minutes

before the appropriate 2-oxoacid ethyl ester (60 mmol). After stirring overnight at room

temperature the mixture was filtered. The Amberlyst resin was washed with CH2C12 (4x25 mL),

then solvent was evaporated in vacuo to yield crude 0-nitroalcohols, which were purified by

flash chromatography.









Ethyl 2-hydroxy-3-nitrobutanoate (19a). Pale yellow oil (9.45 g, 89%). 1H NMR

(CDC13): 6 4.34 (m, 4H), 3.40 (brs, 1H), 1.64 (d, J=6.9 Hz, 3H), 1.28 (t, J=7.1 Hz, 3H) ppm. 13C

NMR (CDC13): 6 171.0, 83.3, 71.8, 62.9, 14.9, 13.9 ppm.

Ethyl 2-hydroxy-3-nitropentanoate (19b). Yellow oil (10.2 g, 89%). 1H NMR (CDC13):

6 4.74 (m, 1H), 4.40 (m, 4H), 2.32 (m, 2H), 1.36 (m, 3H), 1.11 (m, 3H) ppm. 13C NMR (CDC13):

6 170.8, 90.6, 70.6, 62.8, 22.6, 13.8, 10.3 ppm.

Ethyl 2-hydroxy-2-methyl-3-nitropropanoate (19c). Pale yellow oil (9.63 g, 91%). 1H

NMR (CDC13): 6 4.87 (dd, Ji=13.78 Hz, J2=13.78 Hz, 2H), 4.4 (m, 2H), 1.46 (s, 3H), 1.35 (t,

J=7.13 Hz, 3H) ppm. 13C NMR (CDC13): 6 173.6, 81.1, 72.6, 63.2, 24.0, 14.1 ppm.

Ethyl 2-hydroxy-2-(nitromethyl)butanoate (19d). Yellow oil (11.2 g, 98%). H NMR

(CDC13): 6 4.54 (dd, Ji=13.59 Hz, J2=13.59 Hz, 2H), 4.33 (m, 2H), 3.79 (s, 1H), 1.69 (m, 2H),

1.31 (t, J=7.08 Hz, 3H), 0.90 (t, J=7.37 Hz, 3H) ppm. 13C NMR (CDC13): 6 173.0, 80.9, 75.7,

63.2, 29.2, 14.2, 7.2 ppm.

Ethyl 2-hydroxy-2-(nitromethyl)pentanoate (19e). Yellow oil (10.80 g, 88%). 1H NMR

(CDC13): 6 4.84 (dd, Ji=13.59 Hz, J2=13.59 Hz, 2H), 4.37 (m, 2H), 1.69 (m, 5H), 1.36 (t, J=7.07

Hz, 2H), 0.95 (t, J=7.36 Hz, 3H) ppm. 13C NMR (CDC13): 6 173.1, 81.1, 75.4, 63.2, 38.8, 16.3,

14.4, 14.1 ppm.

Ethyl 2-hydroxy-3-methyl-2-(nitromethyl)butanoate (19f). Yellow oil (12.70 g, 99%).

1HNMR (CDC13): 6 4.85 (dd, J1=13.54 Hz, J2=13.3 Hz, 2H), 4.39 (m, 2H), 4.39 (q, J=7.13 Hz,

2H), 2.00 (m, 1H), 1.36 (t, J=7.13 Hz, 3H), 1.00 (dd, J1=6.89 Hz, J2=6.89 Hz, 6H) ppm. 3C

NMR (CDC13): 6 173.1, 80.0, 63.6, 34.7, 17.4, 16.8, 14.7 ppm.

Ethyl 2-hydroxy-3-nitro-2-phenylpropanoate (19g). Yellow oil (14.97 g, 98%). 1H

NMR (CDC13): 6 7.44 (m, 5H), 5.29 (dd, J1=16.5 Hz, J2=15.0 Hz, 2H), 4.49 (m, 2H), 1.36 (t,









J=7.01 Hz, 3H)ppm. 13C NMR(CDC13): 6 171.8, 136.6, 129.3, 129.0, 125.4, 80.9, 76.1, 63.8,

14.1 ppm.

Synthesis of 3- and 2-Alkyl Substituted P-Nitroacrylates

General procedure: The appropriate P-nitroalcohol (17 mmol) was dissolved in 17 mL of

CH2C12 at -78C under an argon atmosphere, then 1 equiv. of methanesulfonyl chloride (17

mmol) was added in one portion. After 30 minutes, triethylamine (51 mmol) was added

dropwise, and the reaction mixture was stirred for 4 hours at -780C. The reaction mixture was

then transferred to a separatory funnel with the aid of 17 mL of CH2C12, then it was washed with

water, 5% aqueous HC1, and brine. The final product was purified by flash chromatography.

(E)-Ethyl 3-nitrobut-2-enoate (E-20a). Yellow oil (1.08 g, 40%). 1H NMR (CDC13): 6

7.00 (s, 1H), 4.20 (q, J=7.2 Hz, 2H), 2.50 (s, 3H), 1.20 (t, J=7.2 Hz, 3H) ppm. 13C NMR

(CDC13): 6 164.2, 160.0, 121.5, 61.8, 14.1, 14.0 ppm. IR Vmax 17351533, 1355,1227 cm1.

(E)-Ethyl 3-nitropent-2-enoate (E-20b). Yellow oil (1.35 g, 46%). H NMR (CDC13): 6

6.97 (s, 1H), 4.32 (q, J=7.08 Hz, 2H), 3.11 (m, 2H), 1.37 (t, J=9.91 Hz, 3H), 1.23 (t, J=8.47 Hz,

3H) ppm. 13C NMR (CDC13): 6 187.0, 185.5, 120.8, 61.9, 21.2, 14.4, 12.5 ppm. IR Vmax 1735,

1533, 1352, 1227 cm1.

(Z)-Ethyl 2-methyl-3-nitroacrylate (Z-20c). Yellow oil (1.46 g, 54%). 1H NMR (CDC13):

6 6.89 (s, 1H), 4.39 (q, J=7.1 Hz, 2H), 2.11 (s, 3H), 1.37 (t, J=7.4 Hz, 3H), ppm. 13C NMR

(CDC13): 6 166.7, 141.5, 136.3, 63.0, 18.2, 14.4 ppm. IR Vmax 1737, 1533, 1355, 1227 cm1.

(Z)-Ethyl 2-(nitromethylene)butanoate (Z-20d). Yellow oil (0.109 g, 37.2%). 1H NMR

(CDC13): 6 6.84 (s, 1H), 4.39 (q, J=7.4 Hz, 2H), 2.48 (m, 2H), 1.37 (t, J=7.4 Hz, 3H), 1.2 (t,

J=7.4 Hz, 3H) ppm. 13C NMR (CDC13): 6 166.1, 146.7, 135.1, 62.5, 25.5, 14.0, 11.2 ppm. IR

Vmax 1735, 1533, 1353, 1222 cm1.









(Z)-Ethyl 2-(nitromethylene)pentanoate (Z-20e). Yellow oil (1.11 g, 35%). 1H NMR

(CDC13): 6 6.85 (s, 1H), 4.39 (m, 2H), 2.41 (m, 2H), 1.66 (m, 2H), 1.37 (t, J=7.4 Hz, 3H), 1.28

(t, J=7.4 Hz, 3H), ppm. 13C NMR (CDC13): 6 166.1, 145.3, 135.6, 62.5, 33.9, 20.2, 14.0, 13.5

ppm. IR vmax 1735, 1532, 1353, 1221 cm1.

(Z)-Ethyl 3-methyl-2-(nitromethylene)butanoate (Z-20f). Yellow oil (0.388 g, 12.2%).

1HNMR (CDC13): 6 6.83 (s, 1H), 4.40 (q, J=7.1 Hz, 2H), 2.77 (m, 1H), 1.39 (t, J=7.4 Hz, 3H),

1.23 (d, J=7.1 Hz, 6H) ppm. 13C NMR (CDC13): 6 165.8, 150.9, 134.9, 62.4, 31.6, 20.5, 14.0

ppm. IR vmax 1736, 1533, 1352, 1221 cm1.

(Z)-Ethyl 3-nitro-2-phenylacrylate (Z-20g). Yellow oil (2.25 g, 60%). 1H NMR (CDC13):

6 7.53 (m, 5H), 7.35 (s, 1H), 4.51 (q, J=7.1 Hz, 2H), 1.42 (t, J=7.1 Hz, 3H) ppm. 13C NMR

(CDC13): 6 164.5, 143.1, 134.2, 129.3, 127.2, 62.6, 13.6 ppm. IR Vmax 1737, 1533, 1355, 1227
-1
cm

Biotransformation of P-Nitro Acrylates Using Isolated Old Yellow Enzyme

General procedure: Reaction mixtures contained final concentrations ofNADP+ (20

tmol, 15 mg), glucose-6-phosphate (1.27 mmol, 429 mg), glucose-6-phosphate dehydrogenase

(500 pg), nitroacrylate (25 mM), and purified OYE (20-40 pg) in 100 mM KPi, pH 6.93 in total

volumes of 50 mL. Conversions were carried out at room temperature. Reaction components

(except for KPi buffer) were added in 10 equal portions every 45 minutes and the mixtures were

sampled for GC analysis periodically. After nearly all of the substrates had been consumed, the

reaction mixture was extracted with Et20 (3 x (5 x reaction volume)). The combined organic

extracts were washed with brine (1 volume) and water (1 volume), dried with MgSO4, and

concentrated in vacuo.

Ethyl 3-nitrobutanoate (21a). Yellow oil (0.201 g, 98%). 1H NMR (CDC13): 6 4.98 (m,

1H), 4.21 (q, J=7.1 Hz, 2H), 3.18 (dd, J1=8.8 Hz, J2=8.8 Hz, 1H), 2.73 (dd, J1=5.0 Hz, J2=5.0









Hz, 1H), 1.63 (d, J=6.9 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H) ppm. 13C NMR (CDC13): 6 169.3, 78.6,

61.4, 38.6, 19.5, 14.1 ppm.

Ethyl 3-nitropentanoate (21b). Yellow oil (0.096 g, 95%). 1H NMR (CDC13): 6 4.89 (m,

1H), 4.20 (q, J=6.5 Hz, 2H), 3.18 (dd, Ji=9.4 Hz, J2=9.4 Hz, 1H), 2.71 (dd, J1=4.2 Hz, J2=4.5

Hz, 1H), 2.02 (m, 2H), 1.28 (t, J=7.1 Hz, 3H), 1.02 (t, J=7.4 Hz, 3H) ppm. 13C NMR (CDC13): 6

169.4, 84.6, 61.5, 36.9, 27.2, 14.2, 10.0 ppm.

(R)-Ethyl 2-methyl-3-nitropropanoate (21c). Yellow oil (0.191 g, 93%). 1H NMR

(CDC13): 6 4.76 (dd, J1=8.1 Hz, J2=8.1 Hz, 1H), 4.44 (dd, J1=5.7 Hz, J2=5.7 Hz, 1H), 4.24 (q,

J=7.0 Hz, 2H), 3.29 (m, 1H), 1.30 (m, 6H) ppm. 13C NMR (CDC13): 6 172.6, 76.6, 61.7, 37.8,

14.5, 14.2 ppm.

(R)-Ethyl 2-(nitromethyl)butanoate (21d). Yellow oil (0.151 g, 75%). 1H NMR (CDC13):

6 4.78 (dd, J1=9.1 Hz, J2=9.4 Hz, 1H), 4.46 (dd, J1=5.1 Hz, J2=4.8 Hz, 1H), 4.22 (q, J=7.1 Hz,

2H), 3.18 (m, 1H), 1.76 (m, 2H), 1.30 (t, J=7.1 Hz, 3H), 1.01 (t, J=7.7 Hz, 3H) ppm. 13C NMR

(CDCl3): 6 172.2, 75.1, 61.5, 44.4, 22.7, 14.3, 11.2 ppm.

(R)-Ethyl 2-(nitromethyl)pentanoate (21e). Yellow oil (0.187 g, 93%). 1H NMR

(CDC13): 6 4.77 (dd, J1=9.3 Hz, J2=9.3 Hz, Hz, 1H), 4.44 (dd, J1=4.8 Hz, J2=4.8 Hz, 2H), 4.20

(q, J=7.4 Hz, 2H), 3.24 (m, 1H), 1.72 (m, 2H), 1.44 (m, 2H), 1.30 (t, J=7.1 Hz, 3H), 0.97 (t,

J=7.4 Hz, 3H) ppm. 13C NMR (CDC13): 6 172.5, 75.4, 61.5, 42.9, 31.5, 20.1, 13.9, 14.3 ppm.

(R)-Ethyl 3-methyl-2-(nitromethyl)butanoate (21f). Yellow oil (0.182 g, 90.5%). 1H

NMR (CDC13): 6 4.83 (dd, J1=10.5 Hz, J2=10.5 Hz, 1H), 4.44 (dd, J1=4.0 Hz, J2=4.0 Hz, 1H),

4.25 (q, J=7.1 Hz, 2H), 3.12 (m, 1H), 2.11 (m, 1H), 1.30 (t, J=7.1 Hz, 3H), 1.01 (m, 6H) ppm.

13C NMR (CDC13): 6 172.4, 74.3, 61.8, 49.4, 29.4, 20.4, 20.3, 14.7 ppm.









Synthesis of p-Amino Acids

General procedure: Crude biotransformation products (ca. 1.5 mmol) were hydrogenated

at 500 psi in the presence of Raney nickel (200 mg) in EtOH (50 mL) at room temperature. After

16 hours, the resulting solution was filtered through Celite and the solvent was evaporated. A

portion of the residue (50 mg) was dissolved in 6 M HC1 and the solution was held on reflux

overnight. The solution was concentrated under reduced pressure to afford yellow oil, which was

washed with EtOAc to remove any non-polar impurities. Water was removed by rotary

evaporator to yield the P-amino acids as hydrochloride salts.

3-Aminobutanoic acid (23a). Ethyl 3-aminobutanoate (22a) was obtained as pale yellow

oil (0.150 g, 74.5%), which was further hydrolyzed to give 23a as a white precipitate (0.105 g,

89%). H NMR (D20): 6 3.88 (m, 1H), 2.8 (dd, J1=17.5 Hz, J2=5.8 Hz, 1H), 2.72 (dd, Ji=17.5

Hz, J2=7.2 Hz, 1H), 1.36 (d, J=6.7 Hz, 3H) ppm. 13C NMR (D20): 6 174.1, 44.4, 37.7, 17.8 ppm.

3-Aminopentanoic acid (23b). Ethyl 3-aminopentanoate (22b) was obtained as pale

yellow oil (0.064 g, 81%), which was further hydrolyzed to give 23b as a white precipitate

(0.047 g, 91%). H NMR (D20): 6 3.63 (m, 1H), 2.88 (dd, Ji=4.53 Hz, J2=4.81 Hz, 1H), 2.74

(dd, J1=8.21 Hz, J2=8.21 Hz, 1H), 1.79 (m, 2H), 1.03 (t, J=7.64 Hz, 3H) ppm. 1C NMR (D20: 6

174.7, 49.7, 35.8, 25.3, 8.9 ppm.

3-(R)-Aminomethylpropanoic acid (23c). Ethyl 3-amino-2-methylpropanoate (22c) was

obtained as pale yellow oil (162.40 g, 85%), which was further hydrolyzed to give 23c as a white

precipitate (0.143 g, 88%). [a]25D=13.0 (c=0.94; 1M HC1). H NMR (D20): 6 3.30 (dd, Ji=8.77

Hz, J2=8.5 Hz, 1H), 3.16 (dd, J1=4.81 Hz, J2=4.81 Hz, 1H), 2.97 (m, 1H), 1.30 (d, J=7.36 Hz,

3H) ppm. 13C NMR (D20): 6 177.7, 41.3, 37.1, 14.3 ppm.

2-(R)-(Aminomethyl)butanoic acid (23d). Ethyl 2-(aminomethyl)butanoate (22d) was

obtained as pale yellow oil (0.106 g, 85%), which was further hydrolyzed to give 23d as a white









precipitate (0.077 g, 90%). [a]25D=-2.7 (c=l, 1M HC1). H NMR (D20): 8 3.33 (dd, J1=9.06 Hz,

J2=9.06 Hz, 1H), 3.19 (dd, J1=4.53 Hz, J2=4.81 Hz, 1H), 2.83 (m, 1H), 1.78 (m, 2H), 0.99 (t,

J=7.36 Hz, 3H) ppm. 13C NMR (D20): 8 177.1, 43.9, 39.6, 22.7, 10.2 ppm.

2-(R)-(Aminomethyl)pentanoic acid (23e). Ethyl 2-(aminomethyl)pentanoate (22e) was

obtained as pale yellow oil (0.131 g, 85%), which was further hydrolyzed to give 23e as a white

precipitate (0.102 g, 95%). [a]25D=1.16 (c=l, 1M HC1). H NMR (D20): 8 3.33 (dd, Ji=9.34 Hz,

J2=9.06 Hz, 1H), 3.21 (dd, J1=4.25 Hz, J2=4.24 Hz, 1H), 2.93 (m, 1H), 1.75 (m, 2H), 1.41 (m,

2H), 0.93 (t, J=7.36 Hz, 3H) ppm. 13C NMR (D20): 6 177.3, 42.4, 39.9, 31.4, 19.3, 13.1 ppm.

2-(R)-(Aminomethyl)-3-methylbutanoic acid (23f). Ethyl 2-(aminomethyl)-3-

methylbutanoate (22f) was obtained as pale yellow oil (0.124 g, 81%), which was further

hydrolyzed to give 23f as a white precipitate (0.095 g, 93%). [a]25D=1.6 (c=1.03, H20). 1H NMR

(CD30D): 6 3.16 (dd, Ji=10.47 Hz, J2=10.15 Hz, 1H), 2.96 (dd, Ji=3.4 Hz, J2=3.68 Hz, 1H),

2.52 (m, 1H), 2.09 (m, 1H), 0.95 (t, J=6.61 Hz, 6H) ppm. 13C NMR (CD30D): 6 175.6, 50.2,

39.3, 30.2, 20.2, 19.7 ppm.

Derivatization of p-Amino Acid Esters with TFAA.

P-Amino acid ester (0.03 mmol) was stirred with TFAA (0.9 mmol) in 60C for 30

minutes. After this time trifluoroacetic acid was evaporated in the stream of argon and the

residue was dissolved in EtOAc (1 mL).

Biotransformation of P-Nitroacrylates Using Cell Extract

General procedure: An overnight cultures of E. coli (BL21(DE3)(pOYE-pET3b)) and E.

coli overexpressing glucose dehydrogenase (GDH) from B. subtilis, grown separately in LB

medium containing 100 tg/mL ampicillin were diluted 1:100 into 1 L of the same medium. The

cultures were shaken in 37C until the optical densities at 600 nm reached 0.8, then the enzymes

overproduction was induced with isopropylthio-P-D-galactoside (IPTG) at a final concentration









of 400 iM and the cultures were stirred for an additional 2.5 hours at room temperature. The

cells were harvested by centrifugation (5,000 rpm for 15 min at 40C), washed twice with cold

sterile water and then resuspended in 25 mL of 100 mM KPi buffer pH 6.93 (with addition of

PMSF to final concentration of 10 iM). The cells were lysed using a French Press and debris

was removed by centrifugation at 20,000 g for 60 min at 40C. The pH of supernatant was

adjusted to 6.93 by adding diluted HC1. The extracts were mixed together and diluted with the

same buffer to the final volume of 100 mL and NADP+ was added (to the final concentration of

0.05 mM). To this mixture were added every 45 minutes portions of: glucose (to the final

concentration of 10 mM), 0-cyclodextrin 40 mg and 20 mg of the substrate (20a, 20c, 20e) (to

the final concentration of 17 mM). The pH of the solution was controlled and kept at 6.93 by

addition of 3M NaOH. The concentration of glucose was monitored by using glucose assay.

When the starting material was completely consumed (after 11 hours) the protein was

precipitated with sodium chloride and the mixture was centrifuged at 15,000 rpm for 15 minutes

at 40C. The supernatant was extracted with Et20, dried over MgSO4 and solvent was evaporated.

Ethyl 3-aminobutanoate (22a). Obtained in 72% yield as a racemic mixture.

Ethyl 3-amino-2-methylpropanoate (22c). Obtained in 75% yield with 73% e.e.

Ethyl 2-(aminomethyl)pentanoate (22e). Obtained in 71% yield with 89% e.e.

Glucose Assay

The glucose concentration in the reaction was determined using a Trinder assay kit

commercially available from Diagnostic Chemicals Limited, Canada. The absorbance of the

reaction, which consists of 5 [iL of the reaction media and 1 mL of the Trinder reagent mixed by

inversion and incubated at 370C for 15 minutes, was measured at 505 nm. The concentration of

the glucose in the reaction was measured by comparing it to the standard reference containing

0.4 g/L glucose.









Incubation of 21a in D20.

Compound 21a (20 mg, 0.012 mmol) was stirred in KPi buffer (prepared in D20) (5 mL)

overnight. Samples for GC/MS analysis were taken every hour. The product of the incubation

was analyzed by 1H NMR spectroscopy: 1H NMR (CDC13): 6 4.21 (q, J=7.1 Hz, 2H), 3.18 (dd,

J1=8.8 Hz, J2=8.8 Hz, 1H), 2.73 (dd, J1=5.0 Hz, J2=5.0 Hz, 1H), 1.63 (s, 3H), 1.29 (t, J=7.1 Hz,

3H) ppm.

Biotransformation of (Z)-20c Using NADPD.

Tris-HCl buffer (250 [L of 1M, pH 8.0) was added to 4.595 mL of KPi buffer, 5 [L of(Z)-

20c in 50 [L of isopropanol-d8 was added to that solution added to that solution and stirred for

few minutes. After that time 9 mg of NADP 3 mg of TBADH and 100 [L of OYE1 were added

to the mixture and stirred. After 6 hours the reaction was completed and the reaction mixture was

extracted with ether (3x8 mL), the solvent was dried over MgSO4 and evaporated. The product

was analyzed by GC-MS and 1H NMR spectroscopy: H NMR (CDC13): 6 4.76 (dd, J1=8.1 Hz,

J2=8.1 Hz, 1H), 4.44 (dd, J1=5.7 Hz, J2=5.7 Hz, 1H), 4.24 (q, J=7.0 Hz, 2H), 1.30 (m, 6H) ppm.







APPENDIX A
GC ANALYSIS OF SUBSTITUTED CYCLOHEXANONES


0


0
cii


0
bj..


Figure A-1. GC chromatogram of 14a, 15b and 15c.


0,


0


-.. ~ -LLII~IIII*IIILIII~


Q.











0


0
^7-

^ ________ ^ _


Figure A-2. GC chromatogram of 14b, 15a and 15d.


0


0
b.I


C,


6---IA"


I











O

F3C N Y
S CH3



4.5 C"rmn 1-
10 mn
402 C
2 min


O
SF3C C02Et
F3C NH
H
CH3


10 Can ." 1CC
1 mmin
80 C
2 rm


(R)


100Clmftn
l ^ n 5 min


(R)


(R)


I II II I Z1FII II III I


Racemic Enzymatic
standard reduction


Racemic
standard


Enzymatic
reduction


Figure A-3. GC chromatogram of TFA derivatives of 22c and 22d.


1l j











0
FC N CO2Et
H
ICH
CHz


1C/rrin

80 C
2 mnw




(R) \ (S)


Ii 1I


Racemic
standard


10 Cw .. 150C


mrn
V'
wiv





1

Ii
(R) ';











-1i bJ III Itan -I=
Enzymatic
reduction


0
O,,UN coEt
F3C HN CNOEt
H
H:C CH3


r


80
21


10 C/Mln


rtn

rim

(S)


standard


1U0Cr"nn

fnnn
T(n


Enzymatic
reduction


Figure A-4. GC chromatogram of TFA derivatives of 22e and 22f.










APPENDIX B
NMR SPECTRA OF B-NITROACRYLATES AND B-AMINO ACIDS


COEt
02Nx CH3


200 180 160


140 120 100 80 60 40 20 0 ppm


Figure B-. 13C NMR of spectrum of 20c.


220


1


PW
















C02Et
02N- CH,


7 5


4 3


1


J 1 .'II

I RPp


I r


Figure B-2. H NMR spectrum of 20c.


a













02N (C CEt


M~ ~ ______ONNO'_OJAI1____ IRM


220 201 180 160 140

Figure B-3. 13C NMR spectrum of 20d.


120 100 80


60 40 20


B pf










02N C02Et


a'. a .


Figure B-4. H NMR spectrum of 20d.


PP.


I


s s
1 1



















Q2N~ l-CO2Et


_-1


r'- w~-wjr f-rr .v.rv r -- w-y ]r --,' F-
220 200 180 160 140 120 100


Figure B-5. 13C NMR spectrum of 20e.


so 60 40 70 0 ppe


i ~-~, ~-- I-,-, r-L.ILI' -, r 1 -1 Wr I'l~ld~ -~ Y-~IYCY~~IY~LY



















C2N J C02Et


8 7 S 5






Figure B-6. H NMR spectrum of 20e.


- U

* U


4 3 2


* *..JJ .J J



III


a
Iii *


U p
1 p


2.130
a r2 21


I,2


t31=
*rrr
nrr*l*r

L


;?~ L
"i
c~i'~













C02Et
02 N;N


220 200 180 160 140


Figure B-7. 13C NMR spectrum of 20f


120 100 80


s0 40 20


LLYrr~k~m(lll~L~


















C02Et
02N -;- I


I I S,


3I+3


4 3


I .II
II,


Figure B-8. H NMR spectrum of 20f.














CO2H
H3C

P NH3
Cl


2?0 ?00 180 160 140 120 1rO 80 60 40 28 0 ipm


CH3 CO2H


cPH3
CP(


220 00O 180 160 140 170 160 80


Figure B-9. 13C NMR spectra of 23a and b.


60 40 70


__~ ~~l ur ~WY) -"W rrLrww""I~LIUL ~w- I~v _LV I I I I IUI L- ~ -Y ~


t--
r r, "


















CH3


I L. A ...,j. i


I770 200 l'A 16r 0 140 1l -- -0 i -
?0o ?00 1 a | 0 140 120 100 80


60 40 21 0 ppm


cP


CH,


r-.-- rw- _a-L-n ~ rrttf~L~) C-UIZ--rt 2>71r-l l K~--.y -i-


220 200 180 160 140 170



Figure B-10. 13C NMR spectra of 23c and d.


100 80


60 40 20 0 ppm


J~aYn -1


A--- -


----LII---l~-----LIL-LII----LI~-- -II~~---LLIIILIIIII













cD



CH3


- -


220 200 180 160 140 120 100 80


60 40 20 0 ppe


cP
H CO2H

H:C CH3


220 200 10 160 140 120



Figure B-11. 13C NMR spectra of 23e and f.


100 80


60 40 20


i W-


0 ppe


1____11_ ____1____ _~________ ____~_______ ~__ ____~__ _


_C~11~1___~111__*l~II-*l-*)*)UUn~UI


- C I __ -I --














El standard naoantersr
Pals, SequgnC.l ipLY


D
J COzEt
OzN


7 6 5 4 3

C'Sl 0.96
2,01 0.95


Figure B-15. H NMR spectrum of 21a incubated in D20.













































118


3.11 3.28











119300 Mar10,. 2100
SOS PIN 0101 Mr lO.na npl9P p
11520*C S~u*..c 52112!h


H

O2N COzEt


-J ;- j


S 362 t1S
t "jz.oz


Figure B-16. H NMR spectrum of 21a.


l.Bo 1.12


IOU


;ii :ii
~I-J
--I--
















D

O2N CO2Et


7.0 E 5 i0 5 0 5


.0 3.
0 J -1


3 D ? 5 : 0 I. pFr.


7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm


Figure B-17. H NMR spectrum of deuterated 21c.









































120









LIST OF REFERENCES

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BIOGRAPHICAL SKETCH

Magdalena Alicja Swiderska was born in Mragowo, Poland, on February 1st, 1978. In 2002

Magdalena graduated from the University of Warsaw, Poland with a master's degree in chemistry

under the supervision of Prof Zbigniew Czarnocki. In 2003 Magdalena moved her scientific

career to University of Florida, joined the Stewart's group and began her Ph.D. studies in the

field of biocatalysis and bioorganic chemistry. After 5 years of research, she graduated from

University of Florida with a Doctor of Philosophy degree in biochemistry.





PAGE 1

1 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL KETONES AND BUILDING BLOCKS FOR -AMINO ACIDS By MAGDALENA ALICJA SWIDERSKA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Magdalena A. Swiderska

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3 To my loving parents, Helenie and Janowi Swiderskim.

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4 ACKNOWLEDGMENTS First of all, I would like to acknowledge Prof Jon D. Stewart for giving me an opportunity to carry out my study in his group and for introducing me to the fiel d of biocatalysis. I am deeply thankful for his help, advice and encouragemen t. I thank my committee members: Dr. Nicole Horenstein, Dr. William Dolbier, Dr. Tom Lyons and Dr. Keelnatham Shanmugam for their time and contributions. I also thank the Stewarts group members: Dr. Santosh Kumar Padhi, Neil Stowe, James Melotek. Special acknowledgment goes to Dr. Despina Bougioukou who introduced me to the techniques of molecular biology and always served with great advice. I cannot forget about Dimitri Dascier who once was a good friend and help ed me in bad times, I really appreciate it. I would like to thank my loving m czyzna Dr. Daniel Serra for his love and encouragement. He was there for me both in difficult and happy moments. Lastly and most importantly, I would like to th ank my parents for their belief in me and for their constant support. I thank my mom for unders tanding me and listening to my complaints, and my dad for being proud of me. I cannot forget about my little brother Maciej who I would like to thank for making me laugh when I felt sad. My family is always the most important for me and without you I would not be the person I am today.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............14 CHAPTER 1 HISTORICAL BACKGROUND OF OLD YELLOW ENZYME........................................16 Discovery and Structure of an Old Flavoprotein....................................................................16 Occurrence and Physiological Import ance of OYE Family Members...................................18 Compounds Bound by OYE...................................................................................................20 Catalytic Properties of an Old Flavoprotein...........................................................................20 Mechanism of Old Yellow Enzyme.......................................................................................24 Purification of Old Yellow Enzyme.......................................................................................28 2 CHIRAL CYCLOHEXANONES..........................................................................................32 Introduction................................................................................................................... ..........32 Chemical and Enzymatic Methods toward Chiral Cyclohexanones.......................................33 Old Yellow Enzyme Family Approach to Formation of Chiral Cyclohexanones..................38 3 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL CYCLOHEXANONES..............................................................43 Synthesis of -Unsaturated Cyclic Enones..........................................................................43 General Procedure for Preparation of 2-Alkyl-2-Cyclohexen-1-ones.............................43 General Procedure for Preparation of 3-Alkyl-2-Cyclohexen-1-ones.............................44 General Procedure for Preparation of 2-Alkylidenecyclohexan-1-ones.........................45 Biotransformation of -Unsaturated Cyclic Enones Using Old Yellow Enzyme...............46 Biotransformation Using Isolated OYE..........................................................................46 Biotransformation Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b)...............49 Biotransformation Using Sodium Dithi onite as Reducing Agent for Old Yellow Enzyme........................................................................................................................51 Conclusions.................................................................................................................... .........53 4 CHIRAL -AMINO ACIDS..................................................................................................55 Introduction................................................................................................................... ..........55 Chemical and Enzymatic Routes to -Amino Acids..............................................................57 Old Yellow Enzyme Approach to Chiral -Amino Acids......................................................67

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6 5 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL -AMINO ACIDS......................................................................70 Synthesis of Mono-Substituted -Nitroacrylates....................................................................70 Biotransformation of -Nitroacrylates and Synthesis of -Amino Acids..............................73 Biotransformation Using Isolated Enzyme.....................................................................73 Biotransformation Usi ng Cell-Free Extract.....................................................................79 Conclusions.................................................................................................................... .........80 6 EXPERIMENTAL SECTION................................................................................................82 General Methods and Instrumentation....................................................................................82 Purification of S. carlsbergensis Old Yellow Enzyme...........................................................83 Cell Growth and Extract Prepar ation for Protein Isolation.............................................83 Isolation of Old Yellow Enzyme.....................................................................................84 Regeneration of Affinity Matrix......................................................................................84 Enzyme Activity Assay...................................................................................................84 Synthesis of 2-Alkyl-cyclohexane-1,3-diones........................................................................85 Synthesis of 3-Isobutoxy-2-alkylcyclohex-2-enone...............................................................85 Synthesis of 2-Alkylcyclohex-2-enone...................................................................................86 Synthesis of 1,3-Cyclohexanedione........................................................................................87 3-Alkyl-2-Cyclohexen-1-ones................................................................................................88 2-(1-Hydroxyalkyl)cyclohexanes...........................................................................................89 2-Alkylidenecyclohexanones..................................................................................................90 Biotransformation of -Unsaturated Cyclic Enones Using Isolated Old Yellow Enzyme......................................................................................................................... .......91 Derivatization of Cyclohexanones with (2 R ,3 R )-(-)-2,3-Butanediol.....................................92 Biotransformation of -Unsaturated Cyclic Enones Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b)................................................................................................92 Synthesis of 2-Alkyloxobutanoates........................................................................................93 Synthesis of Nitro Alcohols....................................................................................................94 Synthesis of 3and 2-Alkyl Substituted -Nitroacrylates......................................................96 Biotransformation of -Nitro Acrylates Using Isol ated Old Yellow Enzyme.......................97 Synthesis of -Amino Acids...................................................................................................99 Derivatization of -Amino Acid Esters with TFAA.............................................................100 Biotransformation of -Nitroacrylates Using Cell Extract...................................................100 Glucose Assay...............................................................................................................101 Incubation of 21a in D2O...............................................................................................102 Biotransformation of ( Z )-20c Using NADPD...............................................................102 APPENDIX A GC ANALYSIS OF SUBSTITUTED CYCLOHEXANONES...........................................103 B NMR SPECTRA OF -NITROACRYLATES AND -AMINO ACIDS............................107

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7 LIST OF REFERENCES.............................................................................................................121 BIOGRAPHICAL SKETCH.......................................................................................................129

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8 LIST OF TABLES Table page 2-1 List of OYEs from yeasts a nd corresponding substrate specificity...................................41 2-2 List of OYEs from bacterias a nd corresponding substrate specificity...............................41 2-3 List of OYEs from plants a nd corresponding substrate specificity...................................42 3-1 Substituted cyclohexenones...............................................................................................43 3-2 Reduction of substituted cyclic enon es by isolated old yellow enzyme............................50 3-3 Reduction of substituted cy clic enones by whole cells......................................................51 3-4 Reduction of substituted cyclic enones using sodium dithionite as reducing agent for Old Yellow Enzyme...........................................................................................................53 5-1 Reduction of substituted nitroacrylates by isolated old yellow enzyme and production of -amino acids..............................................................................................75 5-2 Reduction of substituted nitroacrylates using E. coli cell-free extract with over expressed OYE and GDH..................................................................................................81

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9 LIST OF FIGURES Figure page 1-1 Reaction system of Warburg and Christian.......................................................................16 1-2 Ribbon diagram of the oxidized Oy e1 monomer (PDB# Q02899-1OYB)........................17 1-3 Examples of typical ligands of OYE.................................................................................21 1-4 Examples of -unsaturated aldehydes, ketones a nd nitro compounds as substrates for OYE........................................................................................................................ ......22 1-6 Dismutation reaction catalyzed by OYE............................................................................23 1-7 Nitrate reduction by OYE1: Pathway a.............................................................................24 1-8 Nitrate reduction by OYE1: Pathway b.............................................................................25 1-9 Denitration reaction....................................................................................................... ....25 1-10 Interaction between Thr-37 and FMN...............................................................................26 1-11 Interaction between the substrate and Asn 194 and His 191 from OYE1 active site........27 1-12 Reduction mechanism for ketone s and aldehydes catalyzed by OYE...............................27 1-13 Reduction mechanism for nitrocyclohexene catalyzed by OYE.......................................28 1-14 Catalytic cycle for old yellow enzyme...............................................................................29 1-15 Active site of OYE in complex with p -hydroxybenzaldehyde..........................................30 1-16 Kinetic mechanism of OYE...............................................................................................30 1-17 Affinity matrix for OYE purification.................................................................................30 2-1 Application of 3-substitu ted chiral cyclohexanones..........................................................32 2-2 Application of 3and 2-s ubstituted chiral cyclohexanones...............................................33 2-3 Enantioselective conjugate addition of R2Zn compounds to cyclic enones......................34 2-4 Enantioselective co njugate reduction of -substituted cyclic enones................................34 2-5 Chemical reduction of double bond of -substituted cyclohexenones..............................35 2-6 Enzymatic hydrogenation of C-C double bond of enones.................................................35

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10 2-7 Enzymatic hydrogenation of -unsaturated ketones.......................................................36 2-8 Catalytic enantioselective decarboxylative protonation....................................................36 2-9 Enantioselective alkylation of ketones via chiral enamines..............................................37 2-10 Enantioselective hydro lysis of enol esters.........................................................................37 2-11 Enantioselective protonation of proc hiral enolates using chiral imides............................38 2-12 Substituted cyclohexenones as substrates for OYE1.........................................................39 2-13 Two-step conversion of ketoisophorone to (4 R ,6 R )-actinol using old yellow enzyme homologs and LVR............................................................................................................40 3-1 Synthesis of 2-alky l-2-cyclohexen-1-ones.........................................................................44 3-2 General synthesis of th e 2-alkyl-2-cyclohexen-1-ones 5 ...................................................45 3-3 General synthesis of 3alkyl-2-cyclohexen-1-ones 9a-e ....................................................45 3-4 General synthesis of 2alkylidenecyclohexane-1-ones 13a and 13b .................................46 3-5 Biotransformation of cyc lic enones using isolated OYE...................................................47 3-6 Derivatization of cyclic enones..........................................................................................48 3-7 Schematic diagram of the S. carlsbergensis old yellow enzyme active site......................49 3-8 Biotransformation of substituted cyclic enones using isolated old yellow enzyme..........50 3-9 Biotransformation of substituted cyclic enones using whole cells....................................51 3-10 Biotransformation of substituted cyclic enones using sodium dithionite as reducing agent for old yellow enzyme..............................................................................................52 3-11 Reduction of -unsaturated ketones by Na2S2O4...........................................................53 4-1 Linear -amino acids.........................................................................................................55 4-2 Nomenclature proposed by Seebach and co-workers........................................................55 4-3 Enantiopure 2-phenylalanine and 2-homovaline............................................................56 4-4 Cryptophycin and its precursor..........................................................................................56 4-5 Carbocyclic and heterocyclic -amino acids.....................................................................57 4-6 ( R )-(+)-3-Amino-3-phenyl-2,2-dimethylpropa noic acid and its derivative.......................57

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11 4-7 Arndt-Eistert homologation...............................................................................................58 4-8 Arndt-Eistert homologation in 2-amino acids synthesis..................................................58 4-9 Stereoselective acylation of one enantiomer of the racemic -amino esters.....................59 4-10 Hydrolysis of N -phenylacetyl derivatives of -amino esters.............................................59 4-11 Chemical resolution of -amino acids...............................................................................60 4-12 Synthesis of -hydroxy -amino acids...............................................................................60 4-13 Asymmetric synthesis of -amino acids derivatives..........................................................61 4-14 Stereoselective preaparation of ituri nic acid and 2-methyl-3-aminopropanoic acid.........61 4-15 Addition of a chiral ammoni a equivalent to an acceptor................................................62 4-16 Addition of a nitrogen nucleophile to a chiral acceptor.....................................................62 4-17 Conjugate addition of amine nucleophile by asymmetric catalysis...................................62 4-18 Asymmetric synthesis of -amino acids via conjugate addition of chiral metallated amines......................................................................................................................... .......63 4-19 Addition of a nitrogen nucleophile to a chiral acceptor.....................................................63 4-20 Asymmetric catalysis in conjugate addition......................................................................63 4-21 Rhodium catalyzed hydrogena tion of 3-aminoacrylates....................................................65 4-22 Rhodium-catalyzed hydrogenation....................................................................................65 4-23 Hydrogenation of -enamino esters catalyzed by Pearlmans catalyst..............................66 4-24 Direct reductive amination of -keto esters with ( R )-L-Ru catalyst..................................66 4-25 Synthesis of -lactams via modified Staudinger reaction..................................................68 4-26 Reduction of nitro-olefins by S. carlsbergensis old yellow enzyme.................................69 5-1 Synthesis of methyl 3-nitroacrylate using N2O4................................................................70 5-2 Synthesis of -nitroacrylates using NO2Cl or NOCl.........................................................71 5-3 Synthesis of -nitroacrylates using nitrous acid................................................................71 5-4 NaNO2-Ceric ammonium nitrate mediated conversion of acry lic esters into -nitroacrylates................................................................................................................ ..72

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12 5-5 Proposed radical mechanism of 2hydroxy-3-nitroacrylates formation............................72 5-6 Amberlyst A-21 mediated conve rsion of acrylic esters into -nitroacrylates....................73 5-7 Biotransformation of ni troacrylates by OYE towards -amino acids................................75 5-8 Proposed mechanism of reduction of substituted-2-cyclohexenones and nitroacrylates by old yellow enzyme.................................................................................76 5-9 Biotransformation of nitroacrylates in D2O.......................................................................77 5-10 Incorporation of deuterium into 21a ..................................................................................77 5-11 Fragment of the 1H NMR analysis of the product of incubation.......................................78 5-12 Incubation of compounds 21b in D2O...............................................................................78 5-13 Reaction of ( E )20c with NADPD.....................................................................................79 5-14 Biotransformation of ( E )20d by isolated OYE1...............................................................79 5-15 Incubation of 20d in KPi buffer.........................................................................................79 5-16 Biotransformation of nitroacrylates using E. coli cell-free extract with overexpressed OYE and GDH...................................................................................................................81 A-1 GC chromatogram of 14a 15b and 15c ...........................................................................103 A-2 GC chromatogram of 14b 15a and 15d ..........................................................................104 A-3 GC chromatogram of TFA derivatives of 22c and 22d ...................................................105 A-4 GC chromatogram of TFA derivatives of 22e and 22f ....................................................106 B-1 13C NMR of spectrum of 20c ...........................................................................................107 B-3 13C NMR spectrum of 20d ...............................................................................................109 B-4 1H NMR spectrum of 20d ................................................................................................110 B-5 13C NMR spectrum of 20e ...............................................................................................111 B-6 1H NMR spectrum of 20e ................................................................................................112 B-7 13C NMR spectrum of 20f ................................................................................................113 B-8 1H NMR spectrum of 20f .................................................................................................114 B-9 13C NMR spectra of 23a and b ........................................................................................115

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13 B-10 13C NMR spectra of 23c and d .........................................................................................116 B-11 13C NMR spectra of 23e and f ..........................................................................................117 B-16 1H NMR spectrum of 21a ................................................................................................119 B-17 1H NMR spectrum of deuterated 21c ...............................................................................120

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL KETONES AND BUILDING BLOCKS FOR -AMINO ACIDS By Magdalena Alicja Swiderska August 2007 Chair: Jon D. Stewart Major: Chemistry This dissertation descri bes the application of S. carlsbergensis Old Yellow Enzyme (an NADPH dependent yeast oxidoreducta se) in the biotransformation of different types of activated alkenes. Two classes of compounds substituted cyclohexenones and -nitroacrylates, were synthesized and tested as pot ential substrates for OYE. Both 2and 3-substitu ted 2-cyclohexenones ( 5a-b and 9a-e ) were shown to be reactive with the mentioned protein. Chemoand stereose lective alkene reductions were observed and no alcohol products were detectable. In most of the cases, biotransformations proceeded with high optical purities, with the exception 2exo -methylene cyclohexanones ( 13a-b ), which were obtained as racemic mixtures. The enantioselectiviti es of the reactions were determined based on the chiral GC separation of the de rivatized biotransformation products. Enzymatic reductions of 2-substituted-nitroacrylates ( 20c-f ) occurred with 87-96% of enantiomeric excess (e.e.), with larger substrat es providing great er stereoselectivities. The products of the biotransformations were furthe r chemically reduced to amino acid esters ( 22c-f ) and derivatized with TFAA (trifluoroacetic anhydrid e) in order to assess the enantiomeric excess values. The acid hydrolysis of esters gave optically active 2-amino acids ( 23c-f ), important drug

PAGE 15

15 intermediates and subjects of biological studies. In the cas e of 3-substituted-nitroacrylates ( 20a-b ), only racemic products were observed.

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16 CHAPTER 1 HISTORICAL BACKGROUND OF OLD YELLOW ENZYME Discovery and Structure of an Old Flavoprotein Saccharomyces carlsbergensis old yellow enzyme (OYE) is known as the first discovered and characterized flavoprotein.1 It was isolated from brewers bottom yeast by Warburg and Christian2 in 1932 during their studies on the oxidati on of glucose-6-phosphate by methylene blue. They discovered that the reaction takes place only when oxygen is reduced to hydrogen peroxide in the presence of add itional elements called at that time Zwischenferment-identified later as glucose-6-phosphate dehydrogenase, Gel be Ferment-yellow protein, and a heat-stable Coferment-NADP+ ( 1-1). glucose-6phosphate 6-phospho gluconolactoneZwischenfermentglucose-6-phosphatedehydrogenaseCofermentNADP+NADPH H2O2O2 GelbeFermentOldYellowEnzyme Figure 1-1. Reaction system of Warburg and Christian. A few years after this discovery, another yeast flavoenzyme (D-amino acid oxidase) was isolated and named das neue gelbe Ferment.3 This led to the final na me given to the Warburgs protein: old yellow enzyme. In 1935, Hugo Theorell pu rified OYE and showed that it consists of two components: a colorless apop rotein and a yellow dye, both e ssential for the enzyme activity. In 1955, he identified the yellow element as fl avin mononucleotide (FMN), which was later found to bind non-covalently to the enzyme active site (Figure 1-2).4

PAGE 17

17 Figure 1-2. Ribbon diagram of the oxidized Oye1 monomer (PDB# Q02899-1OYB). Despite the fact that OYE had been known since 1932, its first crystallization was not achieved for nearly another 20 years by Hugo Theo rell in 1955. Unfortunately, the quality of the crystals was not sufficient for X -ray studies, which were later e xplained as a probable result of the heterogeneity of natural OYE aris ing from the two genes present in S. carlsbergensis .1 Since then, several X-ray data were published mainly by Fox and Karplus, who solved the crystal structure at a resolution of 2.0 for the oxidized and reduced forms of recombinant intact Oye1 and for its complex with phydroxybenzaldehyde.4 Based on Foxs results, the enzy me was found to exist as an / barrel in the form of a dimer, with a monomer of ~ 45 kDa. Each singl e domain binds non-covalently the molecule of flavin mononucleotide (FMN) that interacts by hydrogen bonds with surrounding it amino acids.5 Fox and Karplus used also spectroscopic tec hniques to obtain some additional information on solvent accessibility to th e active site of the OYE. 13C and 15N NMR together with the X-ray

PAGE 18

18 experiments revealed that it is the si face of the FMN that is exposed to solvent, when the re face is buried by interactions with the protein. Since that time, OYE has been characterized in great detail, especially by Vincent Masseys laboratory, and it has served as a model for studying other flavoproteins.1,6 Unfortunately, despite the exte nsive knowledge on the structur e and reactivity of the old flavoprotein, the physiological ro le of OYE has remained unknown. Occurrence and Physiological Importance of OYE Family Members Several proteins with amino acid sequence hom ologous to OYE have been found in yeasts, plants and bacteria and their postulated physiological function gives some idea on the importance of old yellow enzyme in Nature.7 Based on those reports a number of possible functions for Saccharomyces carlsbergensis OYE were suggested, from asse mbly of the yeast cytoskeleton6 to oxidative stress response in yeast.8 In Saccharomyces cerevisiae two enzymes have been di scovered, OYE2 and OYE3, with sequence closely related to OYE1. The most recent st udies on acrolein toxicity in this strain of yeast ( Saccharomyces cerevisiae ) point to the role of OYE2 as a main agent mediating resistance to small -unsaturated carbonyl com pounds, such as acrolein.9 However, based on the previous reports on the role of OYE in the control of th e redox state of the actin cytoskeleton, the authors suggested that this group of enzymes is likely to have phys iological functions beyond the simple detoxification of ha rmful metabolites. There have been found some other strains of y easts containing enzymes with genes related to OYE, such as Candida albicans : contains estrogen binding protein ( EBP1 ), which possesses desaturase activity and reduces 19-nor-testost erone (OYE1 can only aromatize it).

PAGE 19

19 Hansenula polymorpha : contains hansenula yellow enzymes ( HYE1 HYE2 and HYE3 ), which have been found to increase the resist ance towards high con centrations of allyl alcohol in the presence of alcohol oxidase (AO). Kluyveromyces lactis : contains kluveromyces yellow enzyme ( KYE1 ), whose physiological role is unknown. Yarrowia lipolytica : contains N -ethylmaleimide reductase. Candida macedoniensis : contains old yellow enzyme ( oye ). A number of enzymes related to OYE have been identified in bacteria in the following strains Gluconobacter suboxydans : contains old yellow enzy me, physiological role unknown. Pseudomonas putida M10: contains morphinone reductase ( morB ), which reduces double bonds of both morphinone and codei none, producing important pharmaceutical drugs like hydromorphone and hydrocodone, respectively. Pseudomonas putida II-B and Pseudomonas fluorescenc I-C: contain nitroester reductases ( XenA and XenB respectively), responsible for nitrate ester degradation. Agrobacterium radiobacter : contains glycerol trinitrate reductase ( Ner ), responsible for nitrate ester degradation. Enterobacter cloacae Pb2: contains pentaerythritol tetranitrate reductase ( Orn ), which is known for degradation of explosives, such as nitroglycerine (GTN) and 2,4,6trinitrotoluene (TNT). Escherichia coli JM109: contains N-ethylmaleimide reductase ( NemA ) Bacillus subtilis: contains YqjM ( B14911_08447 ), involved in detoxification. Shewanella oneidensis : contains SYE1, SYE3 and SYE3 ( AAN55488.1 AAN57126.1 AAN56390.1 respectively), physio logical role unknown. Plant homologues of OYE were first identi fied during studies on elucidation of octadecanoid biosynthesis by Vick and Zimmerm an, who isolated 12 -oxophytodienoic acid reductase (OPR) from Zea mays strain found in corn.10 The enzyme was found to catalyze one of the steps in jasmonic acid biosynthes is, the reduction of the double bond of 12-oxo-10,15( Z )-hytodienoic acid (OPDA).11 After this discovery seve ral other plant strains, which contain proteins related to OYE, were identified, such as

PAGE 20

20 Corydalis sempervirens : contains 12-oxophytodienoic acid reductase (OPRI), which reduces (9 R ,13 R )cis and (9 S ,13 R )trans diastereoisomers of OPDA. Arabidopsis thaliana : contains 12-oxophytodien oate reductase 1 ( OPR1 ), which reduces (9 R ,13 R )cis and (9 S ,13 S )cis diastereoisomers of OPDA. Arabidopsis thaliana : contains 12-oxophytodien oate reductase 2 ( OPR2 ), which reduces (9 R ,13 R )cis diastereoisomer of OPDA. Arabidopsis thaliana: contains 12-oxophytodien oate reductase 3 ( OPR3 ), which reduces (9 R ,13 R )cis (9 S ,13 S )cis (9 R ,13 S )trans and (9 S ,13 R )trans diastereoisomer of OPDA. Oryza sativa L. (rice): contains 12-oxophytodeca noic acid reductase ( opda ). Compounds Bound by OYE In the process of searching for the physiologi cal role of old yello w enzyme, researchers identified a number of compounds cap able of inhibiting this protein.12-14 The OYE ligands bind with milimolar to micromolar affinities to the oxidized form of the enzyme. Aromatic compounds with an io nizable hydroxyl substituent ar e one of the most studied group of inhibitors of OYE (Figure 1-3). U pon binding, the phenolic lig and OYE forms deeply colored long wavelength charge -transfer complexes, which can be detected by their green color.15 Two other classes of binding ligands are simple monovalent anions (e.g., acetate, chloride or azide) and pyridine nucleotide derivatives such as the acid hydrolysis products of reduced form of nicotinamide adenine di nucleotide phosphate ( NADPH). All three types of inhibitors, as well as substrates, share a common active site on the si -face of the flavin. Catalytic Properties of an Old Flavoprotein The first reports on the reactivity of old yell ow enzyme can be found in the same studies that helped Warburg discover this protein.1 During the attempts to elucidate the nature of glucose6-phosphate oxidation process, he id entified OYE as the component oxidizing NADPH and reducing oxygen to hydrogen pe roxide. Since then, several experiments proved that NADPH

PAGE 21

21 HO H O p-hydroxybenzaldehyde HO Cl p-chlorophenol O OH HO OH H H H testosterone -estradiol Figure 1-3. Examples of typical ligands of OYE could be the physiological reductant for the ol d flavoprotein. There are some other compounds capable of reducing OYE, like reduced form of nicotinamide adenine dinucleotide (NADH) or sodium dithionite, but the efficiencies of the r eactions are significantly lowered compared to NADPH.16,17 Substrates capable of oxidizi ng OYE include methylene blue, Fe3+, quinones, cytochrome c and ferricyanide.18 The reoxidation of the enzyme can also be effected by molecular oxygen, producing H2O2.19 Recently, it has been found that a number of -unsaturated aldehydes, ketones,20,21 and nitro compounds22 could serve as more efficient substrates for this enzyme (Figure 1-4). These enzymatic reactions ar e chemoselective with exclus ive reduction of the double bond of the olefin, but not th e carbonyl or nitro groups (F igure 1-5). The presence of a stoichiometric amount of NADPH or another reduc tant for OYE is required dur ing the reaction. It has been found by Massey and co-workers that in the absence of an agen t capable of reducing OYE, the oxidative aromatization of the cyclic enones occurs.21 In this dismutation reaction, the substrate

PAGE 22

22 O S O O O CHO O O O O NO2 NO2 O O O O NO2 2-cyclohexenone 3-oxodecalin4-ene 3-oxodecalin4-ene-10-carboxaldehyde 3-methyl2-cyclohexenone cinnamaldehyde mesityloxide 1,2-cyclohexanedioneduroquinone menadione nitrocyclohexenenitrostyrene nitrovinylthiopheneH Figure 1-4. Examples of -unsaturated aldehydes, ketones a nd nitro compounds that serve as substrates for OYE is first dehydrogenated, and subsequently the ol efinic bond of a second substrate molecule is reduced (Figure 1-6). Another interesting class of reactions catalyzed by old yellow enzyme is reduction of nitrate esters.23. The mechanism of the process is still not completely known but two pathways were proposed Pathway a: the reaction involves a h ydride transfer from the reduced flavin to the nitrogen of the nitrate residue (Figure 1-7). This step coul d be followed by or concerted with electron rearrangement, resulting in liberat ion of nitrite and form ation of the alcohol product. Pathway b: the reduction involves sequential electr on and proton transfers as described in Figure 1-8.

PAGE 23

23 O NO2 2-cyclohexenone nitrocyclohexene NO2 nitrocyclohexane O cyclohexanone OYE,NADPH OYE,NADPH Figure 1-5. Example of reduc tion reaction catalyzed by OYE O HO O H H OH O O 3-oxodecalin4-ene 2-cyclohexenone 3-oxodecalin phenolcyclohexanone OYE OYE 3-hydroxy6,7,8,9-tetrahydronaphthalene Figure 1-6. Dismutation reaction catalyzed by OYE

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24 O O N O2N O O O O N O2N O O N N NH N H O O O O N O2N HO O H OH O O2N N N NH N O O NO2 Figure 1-7. Nitrate redu ction by OYE1: Pathway a Two of the compounds were particularly studie d: glycerin trinitrat e (GTN) and propylene dinitrate. The reactions resulted in product mixt ures as the rate of reduction for primary and secondary nitrate was different (Figure 1-9). Mechanism of Old Yellow Enzyme There are several residues that are assumed to have a major role in the catalytic function of OYE. At least one of them, Thr-37, was found to st rongly affect the reactivity of the enzyme by direct interaction with the flavin molecule.19 It was suggested that this residue plays an important

PAGE 25

25 role in controlling the redox potential of the en zyme by stabilizing the negative charge of the reduced flavin by hydrogen bonding with th e C-4 oxygen of the FMN (Figure 1-10). O O N O2N O O O O N O2N O OH O O N O2N HO OH OH O O2N NO2 N N NH N H O O N N NH N O O N N NH N O O H Figure 1-8. Nitrate redu ction by OYE1: Pathway b O2NO ONO2 ONO2 HO ONO2 ONO2 O2NO OH ONO2 O2NO ONO2 HO ONO2 O2NO OH OYE,NADPH OYE,NADPH glycerinetrinitrate GTN propylenedinitrate Figure 1-9. Denitration reaction

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26 The determination of the crystal structure4 of the complex between Oye1 and phydroxybenzaldehyde was critical in identifyi ng two important residu es of the enzyme: histidine 191 and asparagine 194, which direct the positioning of the reactants (substrate or inhibitor) in the active site of OYE by formation of hydrogen bonds (Figure 1-11). These interactions are particularly important in the cat alytic process, since they stabilize the anionic form of the reactant, which acts as an electr on acceptor and is invol ved in charge-transfer interaction with the FMN group. N N NH N O O FMN N O T37 T37 H H 2.91 2.77 Figure 1-10. Interaction between Thr-37 and FM N. In this projection the view is on the si -face of the flavin. Further studies on the mechanistic role of the amino acid residues from the active site of OYE provided some important information a bout the catalytic cycle of this enzyme.24 The investigations were especially focused on tyrosine 196, which was suggested to be a proton donor in the substrate double bond reduction. The e xperiments were performed with different enones, including 2-cyclohexenone and 1-nitr ocyclohexene. The results confirmed the importance of Tyr-196 only in the case of the ke tones, where single mutation of the tyrosine residue to phenylalanine (Y196F) inhibited the process of reduction. Completely different

PAGE 27

27 situation was observed for the nitro compound, wh ere the same mutation (Y196F) almost did not affect the reaction and it was possible to obtain a saturated product withou t a visible inhibition. O Asn194 His191 2.79 2.74 Figure 1-11. Interaction betw een the substrate and Asn 194 a nd His 191 from OYE1 active site. These results suggest different mechanis ms for those two types of compounds Mechanism a: utilized by -unsaturated ketones and alde hydes. This reaction may be best described as a concerted mechanism in which the transfer of a hydride is possible by the presence of Tyr-196 primed for protonation (Figure 1-12). Mechanism b: utilized by 1-nitrocyclohexenones, may involve the formation of an aci nitro intermediate by transfer of hydride to the substrat e followed by its protonation either by Tyr-196 or water. In this case, the process does not depend on the presence of the tyrosine residue (Figure 1-13). O O O H H Asn194 His191 FMNN5H H O-Tyr196 OYE Figure 1-12. Reduction mechanism for ke tones and aldehydes catalyzed by OYE In order to complete the model of the cat alytic cycle for old yellow enzyme, some additional studies were performed to establish the mechanism of the hydride transfer to the flavin from NADPH, which is assumed to be the physiological reductant for OYE.21 According to the

PAGE 28

28 results of this investigation, the proR -hydrogen of NADPH is transf erred as a hydride to the flavin N5, then this is followed by the tr ansfer of the same hydride to the -carbon of the substrate (Figure 1-14). Base on the studies on the interaction between p -hydroxybenzaldehyde ligand and the active site of OYE, the flav in should be always positioned in the re face of the bound substrate that means that the hydride uptake is possible only from this direction, which would make the reduction highly stereospecific (Figure 1-15). N O O N O O His191 Asn194 FMNN5H N O O His191 Asn194 Tyr196 O H N O O N O O OYE nitronate Figure 1-13. Reduction mechanism for nitrocyclohexene catalyzed by OYE It was suggested that NADPH and the substrat e bind to the same site of the enzyme, requiring the protein to act by a ping-pong mechanism, which is c onsistent with the steady state kinetics of all forms of the enzyme studied (Figure 1-16).15,19,24,25 Purification of Old Yellow Enzyme Old yellow enzyme was first isolated in the form of a homogeneous, crystalline protein by Theorell and kerson in 1956 as a result of long studies started by Warburg. However, the original purification method was time-consuming w ith a very low efficiency. Since then, several attempts were made in order to improve this pr ocess. During the develo pment of the procedure, it was discovered that the oxidized enzyme forms green colored charge-tra nsfer complex with a

PAGE 29

29 N N NH N N N O O NH2 O HR HS O O H H HS NH2 O N N NH N O O 5 5HR OYEOxOYERedOxidative half-reaction Reductive half-reaction NADPH NADP+ Figure 1-14. Catalytic cycle for old yellow enzyme low molecular weight compound.12,13 Later, the ligand was identified as any aromatic molecule with an ionizable hydroxyl subst ituent, e.g. naturally occurring p -hydroxybenzaldehyde.14 Additionally, it was found that the green complex dissociates upon reduction of the enzyme. This characteristic interaction between pheno lic compounds and OYE helped Abramovitz and Massey to develop a simple purification procedure based on affinity chromatography.17 They used 4-hydroxyN n -butylbenzamide as an affinity matrix (Figure 1-17). In this method, protein isol ation process consists of 3 step s. First, the solution of crude extract of brewers bottom yeas t is loaded on the column and only OYE binds to the agarose containing phenolic ligand. The formation of th e complex can be easily noticed as the gel changes color from white to green. In order to remove all the unbound proteins the column is washed several times with a Tris-HCl buffer (pH 8.0) that contains (NH4)2SO4 and PMSF. The

PAGE 30

30 presence of the last reagent methylsulfonyl fluor ide proved to increase the efficiency of the process by inhibiting proteolysis. N N N N O O O N O H H N N H H O OH O Asn194 His191 Tyr196 Tyr375 H H Figure 1-15. Active site of OYE in complex with p -hydroxybenzaldehyde. OYEFMNNADPH OYEFMN NADPHOYE NADP+OYE Substrate OYESubstrate FMNH2FMNH2 OYEFMN Substratere d SubstrateredOYEFMN FMN NADP+ Figure 1-16. Kinetic mechanism of OYE OH HN O a g a r ose N-(4-hydroxybenzoyl)aminohexylagarose Figure 1-17. Affinity matrix for OYE purification

PAGE 31

31 The desired protein is eluted from the column by using the same washing buffer but with addition of 3 mM sodium dithion ite, which acts as a reducing agen t for the enzyme and releases it from the complex with the ligand from the ma trix. Upon elution from the column, the protein becomes oxidized and returns to its yellow color. This procedure delivers the enzyme in nearly homogeneous form with high efficiency. Additiona lly, this way of purification is simple and does not require too many steps in comparison to the previous methods.

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32 CHAPTER 2 CHIRAL CYCLOHEXANONES Introduction Optically active cyclic ketones, especi ally those bearing a stereogenic center or to the carbonyl group, are important reaction intermedia tes (synthons) for asymmetric synthesis. Among this group, chiral cyclohexanones are one of the most interesting synthons due to their broad applications in the pr oduction of biologically active su bstances. The synthesis of (-)-agarospirol, (-)-acorenol or (+)-acorenol, a family of sesquiterp enes used as ethereal oils in perfumery, can serve as an in teresting example (Figure 2-1).25,26 H OH (-)-agarospirol OH (-)-acorenol OH (+)-acorenol O R-(+)-3-methylcyclohexanone Figure 2-1. Application of 3-s ubstituted chiral cyclohexanones. Another class of compounds that find their origin in chiral or -substituted cyclohexanones are lactones (Figure 2-2), whic h are present in various forms in numerous naturally occurring substances like antibio tics and essential oils (Figure 2-2).28,29

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33 O R O R O O R O O R O R O O R S S R R S S CHMO m -CPBA/CH2Cl290.5%9.5% 94%(94%ee) 6%(>99%ee) lactones 3-substitutedcyclohexanones 2-substitutedcyclohexanone lactone R=Me,Et, n -Pr Figure 2-2. Application of 3and 2-substituted chiral cyclohexanones. Chemical and Enzymatic Methods toward Chiral Cyclohexanones Considering the variety of applications fo r optically active substituted cyclohexanones, many research groups concentr ate their work on developing methodologies to obtain these important chiral intermediates. To date, severa l procedures, both chemical and biochemical, have been developed. The two most widely used st rategies are: i) the conjugate addition of organometallic reagents to -unsaturated compo unds (Figure 2-3),30-32 and ii) the asymmetric conjugate reduction of cyc lic enones (Figure 2-4).33,34 Unfortunately, these methods are limited only to formation of -alkylated ketones. Another procedure that is commonly applied in the synthesis of optically enriched or -substituted ketones is enantio selective reduction of the CC double bond of the corresponding enone, which can be carried out using both chemical (Figure 2-5) and biochemical (Figure 2-6) catalysts.33-37

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34 O O Et2Zn, toluene,-300C Cu(OTf)2(0.5mol%) catalyst(1.0mol%) 95%yield e.e.>98% N Ph Ph O O P phosphoramiditecatalyst Figure 2-3. Enantioselectiv e conjugate addition of R2Zn compounds to cyclic enones catalyzed by copper phosphoramidite.28-30 O O 1mol%(Ph3P)CuH 0.1-0.5%catalyst 2eqPMHS,PhMe 98%yield e.e.90% ( R )-DTBM-SEGPHOScatalyst PMHS-polymethylhydrosiloxane O O O O P P OMe OMe 2 Figure 2-4. Enantioselectiv e conjugate reduction of -substituted cyclic enones.31,32

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35 O R O R N H EtO2C CO2Et + 5mol%catalystsalt Bu2O, 600C,48h 94-99%yield 94-98%e.e. R=Me,Et, i -Pr 1.2eq O O P O O i -Pr i -Pr i -Pr i -Pr i -Pr i -Pr H3N CO2t -Bu i -Pr valineesterphosphatesaltcatalyst Figure 2-5. Chemical re duction of double bond of -substituted cyclohexenones. O R R=Me,Et, n -Pr O R R O p90,NADPH p44,NADPH 35-95%yield 95-99%e.e. 37-80%yield >99%e.e. p90,p44-reductasesfrom Nicotianatabacum Figure 2-6. Enzymatic hydrogenation of C-C double bond of enones. Most of the enzymatic reductions of -unsaturated cyclic ketone s yield not only saturated cyclohexanones but also the corresponding alcohols37-39 (Figure 2-7). One of the newest methods

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36 leading to chiral -substituted cyclohexanones is enanti oselective decarboxyl ative protonation recently reported by Stoltz and co-workers40 (Figure 2-8). Enantioselective alkylation of ketones via ch iral enamines, first reported by Horeau,41 subsequently modified by othe rs, such as Meyers et al.,42 delivers a highly enantioselective and efficient way of obtaining chiral -substituted cyclohexanones (Figure 2-9). O OH OH O Reductase + + Reductase= Synechococcus sp.PCC7942 Figure 2-7. Enzymatic hydrogenation of -unsaturated ketones. O O O Pd N P O O Ph2P N O t-Bu Pd2(dba)3+L Et2O,rt,10h HCO2H L= Figure 2-8. Catalytic enantioselective decarboxylative protonation. Among other interesting approaches to -alkylated cyclohexanones is enzymatic hydrolysis of prochiral -substituted enol esters catalyzed by a number of esterases. The intermediates of the reaction, -substituted enols, undergo enantio selective rearrangement in the active site of the enzyme to yield optically active ketones.28,43

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37 Ph H2N H OMe N Ph H OMe O N Ph OMe Li N Ph OMe R H O R H LDA/THF -200C RX -780C H3O+RX=MeI,EtI, n -PrI,BzBr 87-99%e.e. Figure 2-9. Enantioselective alkylati on of ketones via chiral enamines. One of the most recent works characterizing this type of reaction describes esterases I and II isolated from cultured plant cells of Marchantia polymorpha (Figure 2-10).44 Another route to optically active substituted cyclohexanones is en antioselective protonation of prochiral enolates using chiral imides. An interesting example of this type of reaction was reported by Yamamoto et al. (Figure 2-11).45 OAc esteraseI KPibuffer(pH7.0) 350C,0.5h O R O R R=Me,Et >99%yield 14-99%e.e. R R=Me,Et, i -Pr,n-Pr R= i -Pr, n -Pr 10-99%yield 17-99%e.e. Figure 2-10. Enantioselective hydrolysis of enol esters. Despite the variety of existing methods that l ead to optically active cyclohexanones, there is constant demand for easier, environmentally friendly and universal for both alkyl substitutions

PAGE 38

38 ( and ) synthetic routes. This fact forces chemists to explore new chemical and biochemical procedures. H N O O O N Ph Ph ( S S )-imide OLi O R R ( S S )-imide R=Me, n -Bu,Ph,Bz 4-67%e.e. Figure 2-11. Enantioselective protonation of prochiral enolates using chiral imides. Old Yellow Enzyme Family Approach to Formation of Chiral Cyclohexanones Asymmetric hydrogenation by chiral rhodium or ruthenium phosphines has resulted in an impressive number of enantioselective alke ne reductions during the last 20 years.46,47 Despite the tremendous progress in this area, high stereo selectivities nearly always depend on olefin proximity to highly polar groups su ch as amides, acids and alcohols.48 Attempts to generalize these procedures to aprotic oxyge n functionalities such as aldehyde s, ketones, esters or nitro groups have been much less su ccessful; although some exceptions were reported (those examples were mentioned in the previous paragraph of this chapter). More over, the fact that organometallic approach requires preparation of complex chiral reducing agents and extreme reaction conditions makes this methodology unattr active, especially w ith respect to its environmental issues. Enzymatic alkene reductions might be one useful solution to problem.

PAGE 39

39 While several isolated enzymes have been reported to reduce -unsaturated cyclic ketones producing optically active saturated ketones,49,50 those of the old yellow enzyme (OYE) family have been characterized most thoroughly. Th e Stott et al. study repo rted several enones as good substrates for NADPH-mediate d reduction by OYE1, among those many -unsaturated cyclic ketones were found as quite reactive specie s (Figure 2-12).21 Based on preliminary results on the reactivity of OYE1, many research groups extended their studies to the search for the other OYEs from different organism s. OYE2 and OYE3 were isolated from S. cerevisiae and both of them showed activity towards -unsaturated cyclic ketones.51,52 Similar results were obtained with old yellow enzyme from Candida macedoniensis The protein was discovered during the screening of different funga l species for the ability to reduce stereoselectively C-C double bond of ketoisophorone (KIP) to produce ( 6R )-levodione, a biologically important chir al synthon (Table 2-1).53 Reduction of unsaturated cyclic ketones was also reported among some of the bacter ial relatives of old yellow enzyme. O O O O OH O O O O O H O 2-cyclohexenone O duroquinone cyclohexane-1,2-dione4-oxo-isophoronemenadione 3-oxo-decalin-4-ene-10carboxaldehyde 3-methyl-2-cyclohexenone> >>> > Figure 2-12. Substituted cyclohexenones as substrates for OYE1 ordered from the least to the most reactive enone.

PAGE 40

40 In 1994, Bruces laboratory isolat ed morphinone reductase from Pseudomonas putida M10, enzyme responsible for reduction of ol efin double bonds of morphinone and codeinone.54,55 The same group demonstrated the ability of th is enzyme to reduce 2-cyclohexenone in an NADH-dependent manner. OYEs have been also detected in other Pseudomonas species54 and other bacteria, like: Enterobacter cloacae ,56 Escherichia coli ,57 Bacillus subtilis,58 or Shewanella oneidensis .59 All of them showed some reactivity towards 2-cycloh exenone (Table 2-2). Several OYEs were also isolated from plants. Among those, some de monstrated ability to reduce double bond of 2-cyclohexenone (Table 2-3).60-63 Despite the extensive studies on the crystal stru cture and substrate specificity of proteins from the old yellow enzyme family, the stereo selectivities of reducti ons catalyzed by those enzymes were never determined. There are only two reported examples that employed homologs of the S. carlsbergensis old yellow enzyme, both of which described production of (6 R )-levodione used in the synthesis of (4 R ,6 R )-actinol (Figure 2-13).52,64 The above results suggested a new view on old yellow enzyme as a stereoselective catalyst that may be used in multi step reaction. O O O O ketoisophorone (6 R )-levodione S.cerevisiae, OYE2 or C.macedoniensis, OYE O OH (4 R ,6 R )-actinol Levodionereductase Figure 2-13. Two-step convers ion of ketoisophorone to (4 R ,6 R )-actinol using old yellow enzyme homologs and LVR. Combining all these results and properties, th e protein appears to be an effective and inexpensive catalyst. In this work, we examined the substrate specificity and stereoselectivity of

PAGE 41

41 the S. carlsbergensis old yellow enzyme and point to ways in which it can be employed in chiral building block production. Our approach was base d on the chemical synthesis of the starting materials for the OYE1, in this case we concentrated on -unsaturated cyclohexenones, followed by their biohydrogenation using isolated enzyme or whole cells overexpressing OYE1. Table 2-1. List of OYEs from yeasts and corresponding substrate specificity. Organism and Protein Substrate Saccharomyces cerevisiae Old Yellow Enzyme 2 Old Yellow Enzyme 3 Candida macedoniensis Old Yellow Enzyme O O O O O O O 2-cyclohexenone menadione duroquinone4-oxo-isophorone Table 2-2. List of OYEs from bacteria s and corresponding substrate specificity. Organism and Protein Substrate Pseudomonas putida II-B Nitroester reductase Pseudomonas fluorescens I-C Nitroester reductase Enterobacter cloacae Pentaerythritol reductase Escherichia coli N-ethylmaleimide reductase Bacillus subtilis YqjM Shewanella oneidensis SYE1, SYE3, SYE4 O O NCH3 RO O R=H,morphinone R=Me,codeinone 2-cyclohexenone

PAGE 42

42 Table 2-3. List of OYEs from plants and corresponding substrate specificity. Organism and Protein Substrate Corydalis sempervirens OPRI Arabidopsis thaliana 12-oxophytodienoate reductase 2 and 3 Lecopersicon esculentum 12-oxophytodienoate reductase 1, LeOPR1 Pisum sativum PsOPR1-6 O 2-cyclohexenone

PAGE 43

43 CHAPTER 3 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL CYCLOHEXANONES Synthesis of -Unsaturated Cyclic Enones A number of compounds were synthesized in or der to serve as starting materials for the enzymatic reductions with S. carlsbergensis old yellow enzyme. Early experiments on ligand binding to OYE suggested that the en zyme should be reactive with some -unsaturated cyclohexenones.21,25 Moreover, the studies on the mechanism of ligand binding and interaction with the active site of OYE1 suggested that th e process may be highly stereoselective. Based on already published data, we proposed three gr oups of substituted cyclohexenones that were examined as substrates for OYE, as described in Table 3-1. Table 3-1. Substituted cyclohexenones. Cyclohexenones Substituents 2-alkyl-2-cyclohexen-1-ones O R R = Me, Et 3-alkyl-2-cyclohexen-1-ones O R R = Me, Et, n -Pr, i -Pr, n -Bu 2-alkylidenecyclohexan-1-ones O R R = Me, Et General Procedure for Preparation of 2-Alkyl-2-Cyclohexen-1-ones The first effort to obtain this class of compounds was concentrated on the procedure reported by Ohta and coworkers,65 which consisted of two steps: a) bromination of 2-alkyl-cyclohexan-1-one with N -bromosuccinimide and b) subs equent dehydrobromination with aniline to give the corresponding 2-alkyl-2-cyclo hexen-1-one (Figure 3-1) Unfortunately, after several attempts no positiv e result was obtained.

PAGE 44

44 O R O R a) N -bromosuccinimide,CCl4b)PhNH2R=Me,Et Figure 3-1. Synthesis of 2alkyl-2-cyclohexen-1-ones base d on method by Ohta et al. We turned our attention to another procedure, which consisted of three-steps resulting in the desired product. Although the first step of this method proceeded with very low yield, all the reagents were readily available and inexpensiv e. The synthesis of 2alkyl-2-cyclohexen-1-ones began with a Friedel-Crafts acylation of the appropriate carboxylic acid with glutaryl chloride 1 in the presence of aluminum chloride to yield the 2-alkylcyc lohexane-1,3-diones 2a and 2b ,66,67 which were converted to the corresp onding 2-alkyl-3-isobutoxy-2-cyclohexenones 4a and 4b by reaction with i -BuOH and p -TsOH.68,69 The resulting vinylogous esters were reduced by lithium aluminum hydride (LAH) to afford th e desired 2-alkyl-2cyclohexen-1-ones 5 (Figure 3-2).70,71,72 General Procedure for Preparation of 3-Alkyl-2-Cyclohexen-1-ones Our approach to 3-alkyl-2-cyclohexen-1-one s was based on a widely applied Grignard reaction.73 The reason why we chose this route was practic al. First of all, the reagents used were readily available; second, the intermediates fo r all the 3-alkyl-2-cyclohexen-1-ones were the same (cyclohexane-1,2-dione 6 and 3-isobutoxy-2-cyclohexenone 7 ).74,75 The synthesis began by reacting 6 with 1-isobutanol to yield 7 This reaction was followed by addition of the appropriate Grignard reagent 8a-e to 7 and then the crude product was hydrolyzed in aqueous acid. The final enones 9a-e were purified by silica gel column chromatography (Figure 3-3).76-79

PAGE 45

45 HO O R O O R O R O O R i -BuOH/ p -TsOH 12a R=Me b R=Et 3a,b 4a,b LAH Et2O 5a,b AlCl3MeNO215-20% 80-95% 84-90% Cl Cl O O Figure 3-2. General synthesis of the 2-alkyl-2-cyclohexen-1-ones 5 O O O 67 9a-e O O i -BuOH/ p -TsOH 85% RMgBr(8a-e) Et2O 70-83% RMgBr 8a R=Me b R=Et c R= n -Pr d R= i -Pr e R= n -Bu R Figure 3-3. General synthesis of 3-alkyl-2-cyclohexen-1-ones 9a-e General Procedure for Preparation of 2-Alkylidenecyclohexan-1-ones The synthesis was based on proce dure developed by Huang et al.80 which gave acceptable chemical yields. Although the auth ors reported that the method resu lts in a mixture of cisand trans-isomers, always one of them is obtained in high excess. The procedure began with aldol condensation of cyclohexanone with readily available aldehydes a nd gave expected aldols which were dehydrated by mesylation, followed by treatment with DBU.81,82 Enones were obtained as a mixture of isomers (80% of E ; 20% of Z ) (Figure 3-4).83,84 Unfortunately, attempts to separate the isomers did not give positive results.

PAGE 46

46 Biotransformation of -Unsaturated Cyclic Enones Using Old Yellow Enzyme There are two major approaches in biotechnol ogy when it comes to the physical state of biocatalyst, which can be applied either as isolated enzyme or in the form of whole microorganism. The final decision as to which of them should be used depends on many factors, such as: (i) the type of reaction, (ii) if there are cofactors to be recycled and (iii) the scale in which the biotransformation has to be performed.85 H R O 1.LDA2.RCHO(11a,b) 60-75% 1.MeSO2Cl/Et3N O R OH R O O 2.DBU/THF 64-71% 11a R=Me b R= n -Pr 10 12a,b 13a,b Figure 3-4. General synthesis of 2-alkylidenecyclohexane-1-ones 13a and 13b In our studies, we first needed to examine the reactivity and stereoselectivity of the enzyme. The most secure way to accomplish this task was by using purified protein. In this way, we ensured that OYE is the source of chirality in the bioreduction products. The next step was to scale up (up to 1 gram) the characterized react ions by using whole cells and determine their efficiency. Biotransformation Using Isolated OYE The old yellow enzyme plasmid (pOYE-pET3b) (the plasmid was a gift from Professor Masseys Laboratory) was transformed into BL 21 (DE3) cells. The overexpressed protein was purified based on the procedure developed by Massey and coworkers.17 Each of the ketones was tested as a s ubstrate for the OYE using NADPH, which was supplied by a cofactor regeneration system. This was based on the conversion of NADP+ and

PAGE 47

47 glucose-6-phosphate into NADPH and 6-phosphoglucono-lactone, respectively, catalyzed by glucose-6-phosphate dehydrogenase (Figure 3-5). The presence of NADPH was required in order to avoid the dismutation reaction, catalyzed by OYE in the absence of this cofactor, which results in formation of phenol compounds.21 O R R' R O NADPH 6-phosphogluconolactone NADP+glucose-6-phosphate OldYellowEnzyme Glucose-6-phosphateDehydrogenase O O R R R' a R=alkyl,R'=H b R=H,R'=alkyl c Figure 3-5. Biotransformation of cyclic enones using isolat ed OYE and NADPH regeneration system. The first step in the biotransformation of enone s was to test them as potential substrates for the enzyme. This procedure was performed on sm all scales, with substr ate concentration of 3 mM, and the ketones were introduced to the re action in the form of stock solutions with ethanol, which increased the solubility of orga nic compounds in aqueous buffer. The reductions were carried out at room temperature. The progr ess of the biotransformations was monitored by taking 30 L samples and extracting them w ith 30 L of EtOAc. The organic phase was analyzed by non-chiral-phase gas chromatography and mass spectrometry. Each of the reactions went to completion, except 9e and 13b which were not reduced. The next step was to define the stereosele ctivity of the enzyme. This was achieved by scaling up the reactions to 20 mg of substrate, an amount suff icient for further analysis. A stoichiometric quantity of -cyclodextrin was added to promote the substrate solubility in the

PAGE 48

48 reaction solution. The enantiomeric excess of the products obtained was assessed by chiral-phase gas chromatography. The complete separation of th e chiral ketones was possible only after their derivatization with (2 R ,3 R )(-)-2,3-butanediol.86 The same procedure was applied to the corresponding racemic cyclohexanones to demonstrate baseline resolution of enantiomers (Figure 3-6). Product absolute configurations were revealed by comparison with authentic standards available from earlier studies.87 HO OH (2 R ,3 R )-butane-2,3-diol O R R substituted cyclohexanone p -TsOH CH2Cl2 R R O O cyclohexanone derivative Figure 3-6. Derivatizati on of cyclic enones. According to the results of th e scaled up biotransformation w ith isolated enzyme, after 24 hours, only the least substituted substrates ( 5a and 9a ) were completely reduced. Larger ketones were not fully reduced, even after 24 h and the co nversion did not proceed fu rther after this time (Table 3-2). Based on GC analysis, the conversion decreased with the leng th of the substituent, with two examples of no conversion ( 9e and 13b ). The enzyme displayed high enantioselectivity towards 2and 3-subs tituted cyclohexenones (series of 5 and 9 ) with enantiomeric excess values raging from 90 to 97% (Table 3-2). Moreover, the results of the absolute conf iguration assessment support the OYE reduction mechanism proposed by Massey and coworkers. According to this model, reduction of 3-substituted cyclohexenones should deliver the S enantiomers, if we assume that the double bond is at the right hand side as illustrated in Figure 3-7. This is a result of hydride donation by reduced FMN from the re -face. With simultaneous -face protonation by the phenol of Tyr 196,

PAGE 49

49 which can be seen as formation of 2-subs tituted cyclohexanone w ith excess of its R enantiomer (Figure 3-7). Attempted reductions of 2-alkylid enecyclohexanones resulted in very low extents of conversions and almost racemic mixtures of products (Figure 3-8). Biotransformation Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b) The application of isolated enzymes in the biotransformation of or ganic compounds has its pros and cons. One of the major disadvantages of this approach is the re quirement for cofactor recycling. In the case of old ye llow enzyme, the regeneration of NADPH becomes very costly, especially when applied to bigge r scale reactions. One useful so lution to this problem may be biocatalysis mediated by whole cells. Our strategy was to increase the scale of the enone reductions using whole cells of E. coli BL21(DE3)(pOYE-pET3b) that overprodu ced the old yellow enzyme from S. carlsbergensis Flavoprotein expression was i nduced by adding isopropylthio-D-galactoside (IPTG) when the cultures reached the early logar ithmic phase of growth. After 30 min, the appropriate ketone and stoichiometric amount of -cyclodextrin (to increase substr ate solubility) were added. The reactions were allowed to proceed at room temperature until the bioconversions ceased. O R R' N N H O H N O H H H O H R R' H Tyr196 His191 Asn194 Figure 3-7. Schematic diagram of the S. carlsbergensis old yellow enzyme active site.

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50 O R O R O R O R 5a,b 9a-e ( R )-14a,b ( S )-15a-e NADPHNADP+OYE NADPHNADP+OYE O O 13a,b ( R )-16a,b NADPHNADP+OYE O ( S )-16a,b O R ( S )-14a,b O R ( R )-15a-e R R R Figure 3-8. Biotransformation of substituted cyc lic enones using isolated old yellow enzyme. Table 3-2. Reduction of substituted cyclic enones by isolated old yellow enzyme. Ketone R Conversion (%) ee (%) Configuration 5a Me 100 97 R 5b Et 40 92 R 9a Me 100 96 S 9b Et 81 95 S 9c n -Pr 30 90 S 9d i -Pr 23 92 S 9e n -Bu NRa 13a Me 40 13b n -Pr NRa a No reaction Experiments suggested that the enones were ulti mately toxic to one or more of the reaction components after extended periods. The biotrans formation with whole cells delivered similar results to those obtained with isolated enzyme (T able 3-3). The decrease in substrate conversion was observed as the size of the substituents increased. 2-Exo-methylene cyclohexanones gave either very low or no conversion. This was also the case for pure enzyme reactions. Additionally,

PAGE 51

51 the enantioselectivity of the biotransformations mediated by whole cells wa s just slightly lower than with isolated protein. One of the reasons for this may be the presence of other reductases in the cells E. coli that could to small extent affect the reduction by OYE (Figure 3-9), (Table 3-3). O R O R O R O R 5a,b 9a-e ( R )-14a,b ( S )-15a-e O O 13a,b ( R )-16a,b O ( S )-16a,b O R ( S )-14a,b O R ( R )-15a-e R R R Engineered E.coli cells Engineered E.coli cells Engineered E.coli cells Figure 3-9. Biotransformation of substitu ted cyclic enones using whole cells. Table 3-3. Reduction of substitute d cyclic enones by whole cells Ketone R Conversion (%) ee (%) Configuration 5a Me 100 96 R 5b Et 16 90 R 9a Me 100 94 S 9b Et 76 95 S 9c n -Pr 25 89 S 9d i -Pr 18 90 S 9e n -Bu NRa 13a Me 40 13b n -Pr NRa a No reaction Biotransformation Using Sodium Dithionite as Reducing Agent for Old Yellow Enzyme The concept of using sodium dithionite as a reductant for the enzyme, instead of NADPH, was based on the fact that the hydride that re duces the substrate does not come directly from

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52 nicotinamide cofactor but from the flavin a nd so any chemical reagent (in this case Na2S2O4) that can reduce the FMN can substitute for NADPH (Figure 3-10). O R O R O R O R 5a 9a-d ( R )-14a ( S )-15a-d O R ( S )-14a O R ( R )-15a-d OYE/Na2S2O4OYE/Na2S2O4 Figure 3-10. Biotransformation of substituted cyc lic enones using sodium dithionite as reducing agent for old yellow enzyme. Since reactions with sodium dithionite re quire basic conditions and exclusion of oxygen, which causes autooxidation of Na2S2O4, the buffer used for the biotransformations was degassed and its pH increased from 7.0 to 8.0. Substrates ( 5a 5b and 9a-e ) and -cyclodextrin were added in a 1:1 ratio in the presence of the excess Na2S2O4. After 24 hours, although product formation was detected, none of the reactions proceeded to completion (Table 3-4). Two major issues that might have contributed to th e decreased conversion are: Oxidation of sodium dithionite by oxygen from the air that was not completely eliminated. Reaction between the substrat es and sodium dithionite. It has been reported in the literature that Na2S2O4 can serve as a str ong reducing agent for cyclohexenones (Figure 3-11).88 In this case, dithionite may reduce both enzyme and starting material. The GC/MS spectra suggested that this may be the reason for the loss of substrate without product conversion. On th e other hand in the re action analysis data there was no sign of

PAGE 53

53 phenol compounds formation which could be the re sult of the dismutation reaction between OYE and the cyclohexenones. S2O4 2-+H2O HSO2 -+ HSO3 HSO2 -+S2O4 2-HSO3 -S2O3 2-+ R4O R1 R2 R3 R4O R1 R2 R3 R4O R1 R2 R3 SO2H R4SO2H R1 R2 R3 O H or HSO2 S H O O OH Figure 3-11. Reduction of -unsaturated ketones by Na2S2O4. Table 3-4. Reduction of substituted cyclic enones using sodium dithionite as reducing agent for Old Yellow Enzyme. Ketone R Conversion (%) ee (%) Configuration 5a Me 60 97 R 9a Me 60 96 S 9b Et 20 95 S 9c n -Pr 5 90 S 9d i -Pr NRa 92 S a No reaction Conclusions Sketching the rough outlines of old yellow enzy mes substrateand stereoselecivities was the goal of this study, which employed a homologous series of simple alkyl-substituted enones.48 The enzyme displayed gratifying enantioselectivity. Moreover, the absolute configurations of the products could be predicted reliably from a si mple model derived from X-ray crystallography

PAGE 54

54 data (Figure 3-7). Because of hydrogen bonding with the carbonyl oxygen and the requirement that the -carbon lie above N5 of the flavin, reduced FMN must deliver hydride to the re face of the bound cyclohexenones while the pr otonation must occur from the si face. Increasing the extents of convers ion, particularly for larger substrates, is a key challenge the must be overcome before the enzyme can be considered synthetically useful. Nonetheless, the present results underscore the high potential of S. carlsbergensis old yellow enzyme and probably related proteins in ster eoselective organic synthesis.

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55 CHAPTER 4 CHIRAL -AMINO ACIDS Introduction -Amino acids occur in nature both in free and bound form, and even though they are less abundant than their -analogues, they have become one of the most investigated subjects in chemistry and biology. Especially interesting fo r scientists are the properties of oligomers composed exclusively of -amino acids (so called -peptides), which are stable to metabolism, exhibit slow microbial degradation and ar e stable to proteases and peptidases.89 There are three general types of open-chain chiral -amino acids, depending on whether the substitution takes pl ace at the carbon bear ing the carboxyl group ( -position), the carbon bearing the amino group ( -position), or at both positions ( -disubstitution) (Figure 4-1).90 Recently, Seebach and co-workers91,92 proposed the terms 2and 3-amino acid, where the numbers indicate the position of the side chains in order to distinguish positional isomers (Figure 4-2). H2N OH O R H2N OH O R H2N OH R R' O -substituted -substituted -substituted Figure 4-1. Linear -amino acids H2N OH R2 R 3 O -aminoacid,R3=H 3-aminoacid,R 2 =H1 2 3 Figure 4-2. Nomenclature proposed by Seebach and co-workers.

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56 Enantiopure 2-phenylalanine and 2-homovaline were recently synthesized by Gellman and co-workers93 to provide access to new -peptides with specific conformations and particular functions (Figure 4-3). Besides their importance for peptidomimetic studies, -amino acids are also gaining attention as potential precursors for natural products and pharmaceuticals. Several examples of this kind of amino acid structur e can be found as an e ssential component of biologically active compounds.94 One of the simplest -amino acids, ( R )-2-methyl-3-aminopropionic acid, is a resi due in cryptophyci n, a potent antitumor depsipeptide (Figure 4-4).95 OH H2N O OH H2N O ( S )2-phenylalanine( R )2-homovaline Figure 4-3. Enantiopure 2-phenylalanine and 2-homovaline. N H O O O HN O O O Cl OMe H2N OH O ( R )-2-methyl-3-aminopropionicacid cryptophycin Figure 4-4. Cryptophycin and its precursor. Carbocyclic -amino acids, like cispentacin, and their heterocyclic analogues, such as methylphenidate, are useful intermediates fo r the enantioselective synthesis of antifungal antibiotics96 and mental disorder medicati ons, respectively (Figure 4-5).97

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57 ( R )-(+)-3-Amino-3-phenyl-2,2-dimet hylpropionyl derivative NSL95301 is a novel trisubstituted -amino acid exhibiting potent inhi bition of platelet aggregation, which makes it promising antithrombotic agent (Figure 4-6).98 NH2 CO2H cispentacin NH CO2CH3 methylphenidate Figure 4-5. Carbocycli c and heterocyclic -amino acids. H2N NH N H N O O CO2H H2N OH O ( R )-(+)-3-amino-3-phenyl2,2-dimethylpropanoicacid ( R )-(+)-NSL-95301 Figure 4-6. ( R )-(+)-3-Amino-3-phenyl-2,2-dimethylpropa noic acid and its derivative. Chemical and Enzymatic Routes to -Amino Acids The importance of -amino acids and their derivatives in the field of pharmacology and in peptide chemistry is well represented by the multitude of reports that have been published in the past decades. Additionally, sin ce the far-reaching discovery that -peptides form much more stable structures than their -peptidic natural counterparts, there has been an ever-growing interest in synthesizing -amino acids with various substitu tion patterns. In particular, the preparation of enatiomerically pure -amino acids has become an important and challenging endeavor for organic chemists.89 There are eight main approaches available till date for

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58 stereoselective synthesis of -amino acids: homologation of -amino acids, enzymatic resolution, addition of enolates to imines, Curtius rearrangement, conjugate addition of a nitrogen nucleophile to -unsaturated esters or imides, hydrogenation, amino hydroxylation and -lactam synthesis.99 A number of homologation reaction examples in the synthesis of -amino acids have been reported,100-102 since this method is consid ered to be the best for one carbon chain elongation of carboxylic acid. Seebach and co-workers have utilized the Arndt-Eistert procedure in -peptide synthesis to produce -amino acid derivatives in enantiome rically pure form (Figure 4-7).103 Unfortunately, this method is limited to the synthesis of 3-amino acids, with only few exceptions to their -substituted equivalents. One of the examples of the application of the Arndt-Eistert homologation in the synthesis of 2-amino acids is proposed by Yang and co-workers104 synthesis of -substituted-amino esters (Figure 8). N H OH P R O N H P R O H N2 N H P R O OMe P=BocorCbz 88-98%yield 1.Et3N/ClCO2Et 2.CH2N2 UVlight orcat.PhCO2Ag inEt3N/MeOH R=Me, i -Pr, i -PrCH2 Figure 4-7. Arndt-Eistert homologation. OH NHBoc R1 O N2 R2 BocHN R1 OMe R1 R2 BocHN O O 1. i -BuOCOCl/Et3N 2.CH2N23.KHMDS/HMPA,R2X h,MeOH/CH2Cl2R1=alkyl R2=Horalkyl Figure 4-8. Arndt-Eistert homologation in 2-amino acids synthesis. Enzymatic resolution of -amino acids is a cheap and environmentally friendly approach to their optically active derivatives This method can be applied in several ways, but two commonly

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59 used strategies are stereoselective acyl ation of one enantiomer of the racemic -amino esters105 and hydrolysis of N -phenylacetyl derivatives, both of which are catalyzed by lipases106 (Figure 4-9 and 4-10). R OEt NH2 O R OEt NH2 O R OEt NH O Pr O lipase PrCOOR1R=Me,Et, n -Pr, i -Pr lipase-lipaseAfrom Candidaantarctica R1=CH2CF3orBu Figure 4-9. Stereoselective acylation of one enantiomer of the racemic -amino esters. R OH NH2 O R OH NH2 O R OH NH O O R=C6H5,4-F-C6H4, 2-F-C6H4,4-MeO-C6H4acylase-penicillinacylasefrom E.coli ATCC9637 Et3N Ph R OH NH O O Ph Cl Ph O acylase H2O 6NHCl 500C R O H NH2 O Figure 4-10. Hydrolysis of N -phenylacetyl derivatives of -amino esters. Similar chemical procedures may be achieved by formation of diastereomeric salts via complexation with a chiral base, for example (-)-ephedrine (Figure 4-11).107 The diastereomeric salts a and b can be separated by fractiona l crystallization due to their difference in solubility in

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60 a suitable solvent (Figure 4-11). However, the pr ocess of multistep recrysta llizations is long and tedious. Enzymatic resolution was also applied as one of the steps in the synthesis of -hydroxy -amino acids, which are important class of co mpounds that possess interesting bioactivities.94 Cardillo and Gentilucci reported a two-st ep approach to the production of syn-hydroxy -amino acids (Figure 4-12).108 The two key steps in this pro cess were PGA catalyzed kinetic resolution of a racemic amino acid ester, followed by the highly diastereoselective formation of trans -oxazoline. Escalante and Juaristi have demons trated the utilization of pyrimidinones in the synthesis of -hydroxy -amino acids (Figure 4-13).109 Another interesti ng way of obtaining R OH NH2 O Ph OH NHMe R O O Ph OH H2N R O O NH2 Ph OH H2N NH2 + + a b (-)-ephedrine Figure 4-11. Chemical resolution of -amino acids. Ph OH NH O Ph OH NH2 O Ph OMe NH O Ph O PGA PGA-penicillinGacylase Bn O 1.Et3N,PhCOCl 2.SOCl2/MeOH LiHMDS/I2Ph OMe NH O Ph O I NO Ph Ph CO2Me 1MHCl Ph OMe NH O Ph O OH Figure 4-12. Synthesis of -hydroxy -amino acids.

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61 chiral -amino acids is transformation of the carboxy into an amino group by means of a Curtius rearrangement (Figure 4-14). Sibi and Deshpa nde used this methodology in stereoselective preparation of iturinic acid and 2-methyl-3-a minopropanoic acid, componen ts of biologically important peptides itu rin and cryptophycin.110 Among various strategies available to date, co njugate addition of an amine nucleophile to -unsaturated carboxylic acid deri vatives represents one of the most attractive methods for the stereoselective synthesis of -amino acids.94,111 There are basically three ways to achieve asymmetric induction using this methodology NN NN Bz Bz Ph O Ph O OH Ph OMe NH OH O B z S N O O O LDA 1.6NHCl 2.TMSCl/MeOH 3.BzCl/Et3N perhydropyrimidinone (2 R ,3 R )-methyl3-benzamido2-hydroxy-3-phenylpropanoate Figure 4-13. Asymmetric synthesis of -amino acids derivatives. N O O OtBu O O Ph Ph Xc OtBu O O R NaHMDS RBr Xc81-97%d.e. HO OtBu O O R BocHN OtBu O R BocHN OH O 10Xc OH O O R Xc NHBoc O R HO NHBoc O R R=Me componentof cryptophycins LiOH/H2O2TFA Curtius one-pot* Curtius one-pot* 1.Pd/C/H22.TFA 3.Boc2O/Et3N R=undec-3-ene LiOOH Figure 4-14. Stereoselective preaparation of iturinic acid and 2-met hyl-3-aminopropanoic acid using Curtius rearrangement. 1) Et3N, ClCO2Et, acetone, 00C, 1h; 2) NaN3, H2O, acetone, 00C, 1h; 3) toluene, heat, 1h; 4) t -BuOH, heat, 12-24h

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62 Method 1: addition of a chiral ammonia equiva lent to an acceptor (Figure 4-15). Method 2: addition of a nitrogen nucleophile to a chiral acceptor (Figure 4-16). Method 3: asymmetric catalysis (Figure 4-17).99 The first case is well represented by the pr otocol developed by Da vies and co-workers,112 who used lithium amides as synthetic equiva lents of ammonia (Figure 4-18). The second case can be described by the addition of diphenylmeth anamine to chiral crotonates, reported by Chiaroni and co-workers (Figure 4-19).113 The synthesis of -aryl-amino acid derivatives using catalytic amounts of a chiral Lewi s acid can serve as an example fo r the third case of conjugate addition (Figure 4-20).114 Z R O Z R O Chiral"NH3" Z=achiraltemplate NH2 Figure 4-15. Addition of a chiral ammonia equivalent to an acceptor. Xc R O Xc R O Chiral"NH3" NH2 Xc=chiralauxiliary Figure 4-16. Addition of a nitrogen nucleophile to a chiral acceptor. Z R O Z R O NH2 Z=achiraltemplate NitrogenNu: chiralLewisacid Figure 4-17. Conjugate addition of amin e nucleophile by asymmetric catalysis.

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63 R1 OR R2 O R1 OR R2 O R1 OH R2 O N NH2 Ph Ph Ph N Li Ph R1=PhorMe R2=HorMe 1.Pd(OH)2/C/H22.TFA 3.Dowex >95%d.e. N H H H Li O R O t -Bu Proposedmodel Figure 4-18. Asymmetric synthesis of -amino acids via conjugate addition of chiral metallated amines. O O Ar OR* HN O Ph Ph Ph2CHNH2 14-15bar R*-chiralauxiliary O O H2NCHPh2 Proposedmodel Figure 4-19. Addition of a nitrogen nu cleophile to a chiral acceptor. N Ar O O N O Ph O Ar HO Ar NH2 O 2.2eqBnNHOH,-600C 0.3eqMg(ClO4)2orMgI2 H2/Pd O N N O Figure 4-20. Asymmetric catal ysis in conjugate addition. Reductions of -unsaturated esters or ni triles can serve as anothe r interesting approach to enantiopure -amino acids, which can be achieved in tw o ways: 1) catalytic reduction and 2) reductive amination. These two strategies are exemplified by

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64 Synthesis of -amino esters and synthesis of 2-amino acids: proceed through rhodium catalyzed hydrogenation of 3aminoacrylates (Figure 4-21)115and rhodium-catalyzed hydrogenation of -phthalimide acrylates (Figure 4-22).116 Synthesis of -aryl-amino esters and synthesis of -amino esters: proceed through hydrogenation of -enamino esters catalyzed by P earlmans catalyst (Figure 4-23)117 and direct reductive amination of -keto esters with NH4OAc and H2 in the presence of ( R )-L-Ru catalyst (Figure 4-24).118 One of the most important derivatives of -amino acids are -lactams with a broad application in the production of bi ologically active substances. De spite their significance, there are only few protocols for their construction. The development of the efficient methods for their asymmetric synthesis is still a very active area of research.119 The Staudinger reaction, which involves [2+2] ketene-imine cycloaddition, is one of the most reliable methods available for the construction of -lactam rings.120 Lecka et al. developed a modi fied Staudinger reaction, where nucleophilic ketene was generated in the presence of 10 mol% of benzoylquinine (BQ) (Figure 4-25).121 Each of mentioned above approaches has advantages along with limitations. The most important advantages of available approaches are: Arndt-Eistert homologation uses read y available, inexpensive and high enantiomerically pure -amino acids as starting materials. Asymmetric addition of enolates or sily l enolates to imines gives very high e.e., around 98%. Catalytic hydrogenation process mostly uses the systems that tolerate an E / Z mixture of the substrates (derivatives of acrylic acid or nitrile), which simplifies the starting material preparation process. The major disadvantages of mentioned methods are: Arndt-Eistert homologation is not suitable for large scale synthesis due to the high cost of the silver catalyst and danger of working with the hazardous reagent CH2N2.

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65 R AcHN CO2Me R AcHN CO2Me R AcHN CO2Me 1mol%Rh(I)+L toluene,rt H2pressure: 40psi, E 294psi, Z E Z or R=alkyl,90-99%e.e. R=Ph,65-66%e.e. PPh2 Ph2P H H P P ( R R )-BICP ( R R )-Me-DuPhos L Figure 4-21. Rhodium catalyzed hyd rogenation of 3-aminoacrylates. H CO2Et HCHO CO2Et HO O CO2Et O OEt O N O O OEt O N O O NK O O HO NH2 O DABCO THF/H2O CH3COCl + [Rh(COD)2]BF4+L H2(10atm),CH2Cl2rt HCl resin ethylacrylate hydoxyacrylate acetoxyacrylate -phthalimideacrylate phthalimido-protected -aminoacid (R)-(-)-methyl-alanine O O P O O O O O Ph Ph OR L=ManniPhos Figure 4-22. Rhodium-catalyzed hydrogenation.

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66 Ar CO2Me O H2N OMe AcOH/toluene 650C,reduced pressure HN OMe Ar MeO2C HN OMe Ar CO2Me ZE 2eq.BF3 .Et2O Ar MeO2C NH2 Pd(OH)2/C/H2 Figure 4-23. Hydrogenation of -enamino esters catalyzed by Pearlmans catalyst. R OEt O O R R OEt NH2 O OH O ( R )-L-Ru(1mol%) NH4OAc,TFE H2R=alkyl,aryl -aminoester -hydroxyester + PPh2 PPh2 Cl MeO MeO Cl L=( R )-ClMeOBIPHEP Figure 4-24. Direct re ductive amination of -keto esters with ( R )-L-Ru catalyst

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67 Classical resolution of -amino acids requires multistep fractional recrystalization, and therefore the sequence is long and tedious. The enzymatic resolution reactions n eed to be stopped at 50% of the conversion and so yield is usually low; another problem is a narrow tolerance of the enzymes toward racemic -amino acids. Oppolzers sultam-chiral auxiliary is uns table at temperatur es greater than 45C. Evans chiral auxiliary is too expe nsive for the large-scale synthesis. Catalytic hydrogenation requires the us e of expensive rhodium catalysts. All the mentioned methods (besides enzymatic resolution) are environmentally unfriendly chemical processes. Based on above information on the synthesis of chiral -amino acids, the development of an efficient, easy to operate, inexpensive and su itable for large-scale synthesis process, still remains a significant issue. Old Yellow Enzyme Approach to Chiral -Amino Acids Old yellow enzyme is well known for its reduction of the olefinic bond of -unsaturated carbonyl compounds by using NADPH as a cofactor. Moreover, we proved that these reactions are highly stereospecific.122 Another class of similar compounds is that of unsaturated nitro compounds. Like a carbonyl group, a nitro group exerts a strong electron attracting in fluence within the molecule, enhancing the acidity of the hydroge n atoms attached to the carbon to the substituent group. Nitro compounds also exhibit a tautomerism an alogous to keto-enol tautomerism. Massey and co-workers found that old yellow enzyme is cap able of reducing double bond conjugated with a nitro group (Figure 4-26).18

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68 Similar reactions can be catalyzed by other me mbers of old yellow enzyme family, such as old yellow enzyme from Candida macedoniensis N -ethylmaleimide reductase from Escherichia coli or YqjM from Bacillus subtilis .49,53,54 It was discovered that the reduction cataly zed by OYE1 proceeds in a stepwise manner, with formation of a nitronate intermediate wh ich is the result of hydr ide transfer to the -carbon of the olefin. This process is follo wed by protonation of the nitronate at -carbon to form the saturated nitroalkane. Both steps are catalyzed by the enzyme. Meah and Massey suggested, based on result s from theoretical model of interaction between active site of the protein and nitrocyc lohexenone, that reduction of nitro-olefins by old yellow enzyme may proceed via a trans -addition across the double bond.18 If that is the case, it seemed reasonable to conclude that the reaction may be stereoselective, like it was showed for unsaturated cyclohexenones. Based on this information, we extended our st udies on the stereoselectivity and substrate specificity of old yellow enzyme to the substitu ted nitro acrylates. The re duction products served as intermediates for -amino acids synthesis. R Cl O N EtO2C H Ts C O R H O BQ R H N O EtO2C R Ts N N OCOPh OMe BQ protonsponge 10mol%BQ 10mol%In(OTf)3 BQ 96-98%e.e. Figure 4-25. Synthesis of -lactams via modified Staudinger reaction.

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69 S N N N O O O O O O N O O N O O S N O O S.carlsbergensis ,OYE1 NADPH S.carlsbergensis ,OYE1 NADPH S.carlsbergensis ,OYE1 NADPH nitrocyclohexenenitrocyclohexane nitrostyrene nitrovinylthiophene nitroethylbenzene nitroethylthiophene Figure 4-26. Reduction of nitro-olefins by S. carlsbergensis old yellow enzyme.

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70 CHAPTER 5 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN SYNTHESIS OF CHIRAL -AMINO ACIDS Synthesis of Mono-Substituted -Nitroacrylates The preparation of -nitro acrylic esters has been re ported by several groups. Shechter et al. proposed their method based on nitration of corresponding acrylic es ters with dinitrogen tetroxide (Figure 5-1).123 The reaction usually yielded a mixt ure of products in the form of methyl 3-nitrocrylate, methyl 2-hydroxy-3nitropropionate, oxa lic acid dihydrate and nitrogen-containing polymer s of methyl acrylate. O O N2O4 O2 O NO2 ONO O O NO2 ONO2 O 1.H2O 2.CO(NH2)23.Dist. O O NO2 O NO2 OH HO2CCO2H.H2O O + 13% 27%80% ++ methyl 3-nitroacrylate 2-hydroxy3-nitropropionate oxalicacid dihydrate methylacrylate Figure 5-1. Synthesis of met hyl 3-nitroacrylate using N2O4 Besides this, nitryl chloride (Figure 5-2),123 nitrosyl chloride (Figure 5-2)124 and NaNO2 in aqueous solution of CH3CO2H (Figure 5-3) have also been employed to synthesize

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71 -nitroacrylates from the corresponding acrylic esters.125 Unfortunately, the low boiling point of some of these reagents makes them inconvenient, especially if the react ions are carried out on small scales. R O O R O ONO O NO2 R O O NO2 Cl R O Cl Cl O R O NO2 O R O OH O NO2 R O OCOCH3 O NO2 NO2Cl or NOCl Et3N H2OAc2O + + R=CH3,C2H5, n -C3H7,i-C3H7,C6H5ethyl -unsaturated carboxylate -nitritonitrocarboxylate ethyl -hydroxy-nitrocarboxylate ethyl -acetoxynitrocarboxylate -chloronitrocarboxylate ethyl -unsaturated -nitroacrylate -dichlorocarboxylate base Figure 5-2. Synthesis of -nitroacrylates using NO2Cl or NOCl. O O O O NO2 NaNO2-aqueousAcOH orN2O3methylmethacrylate methyl2-methyl-3nitroacrylate Figure 5-3. Synthesis of -nitroacrylates using nitrous acid. Recently, Vankar et al. reporte d their approach using NaNO2-ceric ammonium nitrate (CAN) in CH3CN in order to convert acrylic esters into -nitro alcohols that were dehydrated via their mesylates by following modified McMurrys method (Figure 5-4).126 The reaction is believed to proceed via radical intermediates, which may be the reason for formation of side

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72 products and low yield of reacti on (13-25%) (Figure 5-5). Despite our attempts to improve the efficiency (10-13% average yield) of the Vanka rs procedure, we were not able to obtain sufficient amount of products for further application. R H R1 CO2R2 O2N R R1 CO2R2 R1 CO2R2 R H OH NO2 CAN-NaNO2CH3CN 00C-r.t. CH3SO2Cl,Et3N -200C R=H,Me,Ph R1=H,Me R2=Me,Et Figure 5-4. NaNO2-Ceric ammonium nitrate mediated conversion of acry lic esters into nitroacrylates. MeO2C MeO2C MeO2C NO2 MeO2C ONO2 NO2 MeO2C NO2 MeO2C OH NO2 NO2 NaNO2CAN CAN ONO2 Hydrolysis or Figure 5-5. Proposed radical mechanism of 2-hydroxy-3-nitroacrylates formation. After considering severa l synthetic methods to -nitro acrylic esters, we decided to follow the protocol developed by Palmieri.127 The first step of this appr oach was a nitroaldol (Henry) reaction carried out under heteroge neous catalysis using a solidphase base (Amberlyst A-21) along with the appropriate -keto-ester (either comme rcially available or prepared by the method of Macritchie et al.128) and nitroalkane. The NMR spectral data for nitroaldol adducts 19a-e g matched those reported previously.127,129-131 These were converted to the corresponding

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73 nitroacrylates via mesylate derivatives (Figur e 5-6). Both 3-alkyl-substituted nitroacrylates 19a and b were obtained predominantly in the ( E )-form (~90%)127 whereas the ( Z )-isomers predominated for 2-substituted alkenes (~80%) 19c-g possibly as a result of E1cB reaction on the corresponding mesylates. Because olefin geomet ry may directly impact the stereoselectivity of enzymatic reactions, the major alkene isom ers were chromatographically enriched (>95% geometric purity). CO2Et R2 O O2N R2 CO2Et HO R1 O2N CO2Et R2 R1 O2N R2 CO2Et R1 R1CH2NO2(18) AmberlystA-21 MsCl,Et3N 17 19a R1=Me,R2=H b R1=Et,R2=H c R1=H,R2=Me d R1=H,R2=Et e R1=H,R2= n -Pr f R1=H,R2= i -Pr g R1=H,R2=Ph E -20a,b Z -20c-g Figure 5-6. Amberlyst A-21 mediated conversion of acry lic esters into -nitroacrylates. Biotransformation of -Nitroacrylates and Synthesis of -Amino Acids We studied two approaches towards the bioreduction of -nitroacrylic esters: i) biotransformation using isolated enzyme and ii) in the form of enzyme extract. The first method served for determination of the stereoa nd enantioselectivity of the protein towards nitroacrylates. The reactions with extract were used to scale up the process. Biotransformation Using Isolated Enzyme Biocatalytic reductions utili zed old yellow enzyme that had been purified by affinity chromatography. NADPH was supplied by a cofactor regeneration system (glucose-6-phosphate / bakers yeast glucose-6-phosphate dehydrogenase). Preliminary stud ies had revealed that olefin isomerization was more rapid under alkaline co nditions, and pH 6.93 was selected to minimize this side-reaction while maintaining acceptable en zyme efficiency. A two-fold molar excess of

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74 -cyclodextrin (relative to the nitroacrylate) was also include d to enhance subs trate solubility under aqueous conditions. Unfortuna tely, the solubility of the -cyclodextrin / 20g complex was still too low for efficient reduction, and the obser ved conversion was too low for further analysis of the product. Both the substrates ( 20a-f and glucose-6-phosphate) and the two enzymes were added portionwise to enhance the longevity of the processes, which were monitored by GC/MS and complete substrate cons umption was observed after ca. 8 hr in all ca ses except for ( E )20b The NMR and GC analysis of the crude products verified that only the double bond of the olefins had been reduced and the nitro groups remained in tact. No significant leve ls of side products were observed and yields after purification ra nged from 74-98%. Because it was not possible to determine the optical purities of the obtained products by ch iral-phase chromatography, the crude materials were hydrogenated in the pres ence of Raney-Ni to the corresponding amines ( 23a-g ) (75-85% yield) (Figure 5-7) Enantiomer separations were then possible by chiral-phase GC following derivatization with trifluoroacetic anhydride. The racemic standards, required for enantioselectivity assignment, were obtained by hydrogenation of corresponding -nitroacrylates catalyzed by Raney-Ni. Good optical purities were obtained from 2-alkyl-substituted nitroalkenes 20c-f ; by contrast, 3-alkyl-substituted products were obtained in essentially racemic form.105,132-135 The absolute configurations were assigned by the direction of op tical rotations of the free 2-amino acids as their hydrochloride salts (obtained by acid hydrolysis in 88-95% yields). Overall yields of 2-amino acids from -nitroalkenes ranged from 57-73% (Table 5-1). The results of our previous studies of al kyl-substituted-2-cyclohexenone reductions by old yellow enzyme122 were consistent with the net trans -hydrogenation mechanism elucidated by Massey and Karplus.4,15,24 Hydride -addition (from reduced FMN) occurs from the re -face

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75 while -protonation likely involves th e phenol side-chain of Ty r-196. Carbonyl activation is achieved via hydrogen bonding by the side-chain s of His-191 and Asn-194. For the acyclic -nitroacrylates investigated here, analogous binding could o ccur in which one nitro-oxygen occupies the same location as the carbonyl oxygen and the alkene is positioned similarly (Figure 5-8). O2N CO2Et R2 R1 O2N R2 CO2Et R1 E -20a,b Z -20c-g H2N CO2H R2 R1 1.OYE,NADP+,cofactor regenerationsystem 2.H2,Ra-Ni 3.HCl, 23a-g Figure 5-7. Biotransformation of nitroacrylates by OYE towards -amino acids. Table 5-1. Reduction of substituted nitroacrylates by isolated old yellow enzyme and production of -amino acids. Nitroolefin Conversion (%) ee (%) [ ]D a ( E )20a >98 8( R ) -1.1o c 1.0 (-39.5o c 0.56)136 ( E )20b 50 ----( Z )20c >98 87 ( R ) -13.0o c 0.94 (-12.6o c 1)137 ( Z )20d >98 91 ( R ) -2.7o c 1.0 (-2.9o c 1)137 ( Z )20e >98 94 ( R ) +1.2o c 1.0 (3.5o c 1)137 ( Z )20f >98 96 ( R ) -1.6o c 1.0 (-14.4o c 1)138 a Measured in aqueous solution at the indicated concentrations fr om hydrochloride salts at room temperature. This substrate binding orientation was verified by carrying out the en zymatic reductions of ( E )20b and ( Z )20c in D2O (Figure 4-9) All MS and NMR data were consistent with deuterium incorporation only on nitro-bearing carbon in bo th cases, although they did not eliminate the possibility of the hydrogen exchange with water after the biotransformation, which could explain the racemization at the -carbon.

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76 In order to determine whether the mentioned hydrogen incorporation occurs we examined the deuterium exchange between the enzymatic reduction product 21a and deuterated phosphate buffer. After only five hours the deuterium incorpor ation could be detected in high levels in MS chromatogram by measuring the ra tio of peak 117 to 116 as observed in Figures 5-10 and Figure 5-11. Based on the above results, the forma tion of the racemic center at the -position was attributed to the process of the epimerization related to the presen ce of the acidic hydrogen at the asymmetric -carbon (Figure 5-12). Unfortunately, this data was not sufficient to confirm proposed mechanism of the biotransformation and additional tests us ing deuterated NADPH were required. R R' O O H R R' H EtO2C N H H R2 H EtO2C N H R2 O O O O R2N H CO2Et O O R2N H H CO2Et H O O H H: OYE, NADPH OYE, NADPH OYE, NADPH EtO2C N H R2 O O R2N H CO2Et O O H: H: EtO2C N H R2 O O R2N H CO2Et O O H H H H Figure 5-8. Proposed mechanism of reducti on of substituted-2-cyclohexenones and nitroacrylates by old yellow enzyme.

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77 O2N CO2Et O2N CO2Et H D O2N CO2Et O2N CO2Et H D H H OYE NADPH OYE NADPH D2O D2O 20b21b 20c21c Figure 5-9. Biotransformati on of nitroacrylates in D2O. Figure 5-10. Incorporation of deuterium into 21a

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78 Figure 5-11. Fragment of the 1H NMR analysis of the product of incubation. O2N CO2Et O2N CO2Et H D H D2O 21b21b H H H Figure 5-12. Incuba tion of compounds 21b in D2O. There are two different approaches to the re actions with isotope labeled nicotinamide: either using isolated NADPD or applying NADPD regeneration system directly to the analyzed reaction. The first method seems to be rather unattractive considering the high cost of the reagents and the time consuming purification pro cess. Instead, we turned our attention to the second method by using Thermoanaerobium brockii alcohol dehydrogena se (TBADH) and isopropanol-d8 in OYE reduction of ( Z )20c (Figure 5-13).139 The MS and NMR data supported proposed net trans -addition of hydrogen to 20c-f that leads to the observed ( R )-products. Additionally, in a preliminary experiment, ( E )20d was reduced by the OYE with largely ( S )-stereoselectivity, as would be expected from the model

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79 described in Figure 5-14. With in 7 hours, initially pure ( Z )20d incubated in buffer alone afforded three by-products in a combined conversion of ca 50%. They were identified by GC/MS as ( Z )20d the alternate acrylate alkene regioisomer of 20d and the water addition product from 20d (in racemic form) (Figure 5-15). These observations underscore the need to reduce -nitroacrylates rapidly and minimize their e xposure to the aqueous medium conditions. O2N OC2H5 H O O2N OC2H5 O H H H D NADPD acetone-d6 NADP+isopropanol-d8 OldYellowEnzyme AlcoholDehydrogenase Figure 5-13. Reaction of ( E )20c with NADPD. O2N CO2Et E -20d O2N CO2Et OYE NADPH ( S )-21d Figure 5-14. Biotransformation of ( E )20d by isolated OYE1. O2N CO2Et Z -20d O2N CO2Et KPibuffer pH7.0 ( Z )-20d O2N CO2Et O2N CO2Et H H OH ( E )-20d19d 7h Figure 5-15. Incubation of 20d in KPi buffer. Biotransformation Using Cell-Free Extract Old yellow enzyme requires NADPH as cofactor which in purified form is very expensive in big amounts. The need to scale up the bioreductions forced us to look for

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80 regeneration of NADPH by other means. One solution to this problem is application of the coupled-enzyme regeneration protocol.140 The procedure is based on combining cell extracts of glucose dehydrogenase from B. subtilis responsible for cofactor re generation and old yellow enzyme S. carlsbergensis both over expressed in E. coli Cells over expressing those two proteins were grown separately in 1 liter of LB medium and harv ested just before they reached the stationary phase. Next, they were lysed and centrifuged in order to remove cell debris. The extracts were then mixed together in 100 ml KPi buffer (100 mM) and the biotransformation performed by portionwise addition of the appropriate nitroacrylate ( 20a c e at final concentrations of 13 mM), a two-fold molar excess of -cyclodextrin (relative to the nitroacrylate) and glucose (at a final concentration of 10 mM). The pH of the solution was constantly controlled and kept at 6.93. The reactions were allowed to proceed in room temperature with gentle stirring until th e bioconversions ceased after 11 hours. Although the chemical yields of the biore ductions were satisfactory (72-85%), the enantioselectivity of th e transformations of 20c and 20e was lower than in the case of isolated enzyme reactions (Table 5-2). The reason for th is could be the presen ce of small amounts of other reductases in the extract (e.g. old yellow enzyme from E. coli : NemA) that to some extent affected the final optical purity of the product. Another problem may be insuffici ent amount of in tercellular NADP+ and decreased production of the NADPH required by OYE1 as a cofactor. Additionally, the MS and NMR analysis identified by-products: ( E )20a 20c 20e and the water addition product from 20a 20c and 20e (in racemic form) (Figure 5-16). Conclusions Old yellow enzyme-mediated reduc tions of 3-alkylsubstituted -nitroacrylates 20a and 20b yielded essentially racemic products.141 Given the highly stereoselective -protonation observed

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81 in 2-cyclohexenone reductions, this result was surprising. The isotope wash-in following reduction analysis revealed chiral instability of the -carbon of the biotransformation product, which undergoes spontaneous epimerization in aque ous media. The above explanation is further supported by Ohta and co-workers who reported racemization of similar -substituted nitro compounds occurring both in ba sic and acidic water solutions.142 Further studies with deuterium labeled NADPH confirmed proposed net transaddition mechanism by incorporation of deuterium at the -carbon. O2N CO2Et R2 R1 O2N R2 CO2Et R1 E -20a Z -20c,e H2N CO2H R2 R1 1. E.coli cellextract withoverexpressed OYEandGDH 2.H2,Ra-Ni 3.HCl, 23a,c,e Figure 5-16. Biotransformati on of nitroacrylates using E. coli cell-free extract with overexpressed OYE and GDH. Table 5-2. Reduction of substituted nitroacrylates using E. coli cell-free extract with over expressed OYE and GDH. Nitroolefin Conversion (%) e.e. (%) Configurationa ( E )20a 96 ----( Z )20c 98 73 R ( Z )20e 97 89 R a Determined by chiral-phase GC following n itro group reduction and derivatization with trifluoroacetic anhydride. In conclusion, our results have uncovered a new application for S. carlsbergensis old yellow enzyme in synthe sizing optically active 2-amino acids. The synthetic route is concise and utilizes inexpensive starting materials. Th e major difficulties lie in suppressing alkene isomerization prior to reduction and ensuring active site protonation of the nitronate intermediate.

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82 CHAPTER 6 EXPERIMENTAL SECTION General Methods and Instrumentation Standard media and techniques for growth and maintenance of E. coli were applied. Luria-Bertani (LB) medium used for bacteria l cultivation contained 1% Bacto-Tryptone, 0.5% Bacto-Yeast Extract and 1% NaCl Synthetic reactions were carried out u nder argon atmosphere, with the exception of water containing r eactions. Dichloromethan e, diethyl ether and tetrahydrofuran were dried on an MBRAUN solv ent purification system using a double 4.8 L activated alumina columns type A2. Triethylamine was dried by distillation in the atmosphere of argon and stored over molecular sieves in -20C. An ion-exchange resin Amberlyst A-21 was used in the water-moist free base form in nitroaldol reaction procedures. Reactions were monitored by thin-layer chromatography (TLC), using precoated silica gel plates (EMD Chemicals), or by gas chromatography using DB-17 column (0.25 mm x 25 m x 0.25 m thickness) with a flame ionization detector. Products were purified by flash chroma tography on Purasil silica gel 230400 mesh (Whatman). For chiral separation, gas chromatography was applied which utilized a Chirasil-Dex CB column (0.25 mm x 25 m x 0.25 m thickness) with mass spectrometric detection. NMR spectra were measured in CDCl3, CDOD or D2O solutions and recorded at room temperature on a Varian Mercury 300 spectrometer operating at 300 MHz for 1H and 75 MHz for 13C, respectively, with chemical shifts ( ppm) reported relative to tetramethylsilane (1H NMR) or residual solvent (13C NMR). IR spectra were obtained as neat films on NaCl plates using a Perkin-Elmer Spectrum One FT-IR spectrophotometer. Racemic -amino acid esters were prepared from the corresponding nitroacrylates by hydrogenation at 500 psi in the presence of Raney nickel. A solution of 6M HCl was used in hydrolysis procedures of -amino acid esters.

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83 Purification of S. carlsbergensis Old Yellow Enzyme All enzyme purification steps were carri ed out in 4C. The plasmid encoding S. carlsbergensis old yellow enzyme (pOYE-pET3b) was a gift from Professor V. Masseys laboratory. Routine E. coli strain BL21(DE3) (Novagen) tr ansformations with pOYE-pET3b were performed by electroporation. The affi nity matrix (N-(4-hydroxybenzoyl)aminohexyl agarose) was synthesized based on reported procedure.14 Cell Growth and Extract Preparation for Protein Isolation An overnight culture of E. coli (BL21(DE3)(pOYE-pET3b)) grown in LB medium containing 100 g/ml ampicillin was diluted 1: 100 into 4 L of the same medium in a New Brunswick M19 fermenter. The culture was stirre d at 700 rpm with aeration at 4 L/min in 37C until the optical density at 600 nm reached 0.8, then the enzyme overproduction was induced with isopropylthio-D-galactoside (IPTG) at a final c oncentration of 400 M and the culture was stirred for an additional 2.5 hours at room temperature. The cells were harvested by centrifugation (5,000 rpm for 15 min at 4C), washed twice with cold sterile water and then resuspended in 15 mL of buffer (40 mM Tris-Cl, 10 mM MgCl2, 10 mM DTT, 200 mM KCl, 1 mM PMSF and 10% glycerol, pH 8.0). The cells were lysed using a French Press and debris was removed by centrifugation at 20,000 rpm for 60 min at 4C. The pH of supernatant was adjusted to 8.5 by adding concentrated ammoni um hydroxide and the solution was made to 78% saturation with ammonium sulfat e added portionwise over an hour. Then it was left for another 30 min stirring and the mixture was centrifuged at 20,000 rpm for 60 min at 4C. The pellet was resuspended in 20 mL of buffer (0.1 M Tris-Cl, 0.1 M ammonium sulfate, 10 M PMSF, 10 M sodium dithionite, pH 8.0). The resulting so lution was dialyzed against Tris-buffer (0.1 M Tris-Cl, 0.1 M ammonium sulfate, 10 M PMSF, 10 M sodium dithionite, pH 8.0) which was

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84 changed three times over 20 hours, the second and th ird time without sodium dithionite. After 20 hours of dialysis the crude material was cen trifuged at 20,000 rpm for 10 min at 4C. Isolation of Old Yellow Enzyme A 10 mL affinity column was washed with 600 mL of the starting buffer (0.1 M Tris-Cl, 0.1 M ammonium sulfate, 10 M PMSF at pH 8.0) at a flow rate of 0.5 mL/min. The crude extract was applied and the column was washed w ith 1 L of the same buffer until the absorbance at 280 nm was less than 0.2. The enzyme was elut ed with washing buffer (0.1 M Tris-Cl, 0.1 M ammonium sulfate, 10 M PMSF, pH 8.0) whic h had been degassed and flashed with oxygenfree argon in a 500 ml suction flask and then supp lemented with 3 mM sodium dithionite. The enzyme was collected in three or four 4 mL fractions in test tubes, concentrated by ultrafiltration and stored at -20C. Regeneration of Affinity Matrix The N -(4-hydroxybenzoyl)aminohexyl agarose was regenerated by washing with 0.2 M sodium acetate buffer, pH 5.0, containing 6 M guanid ine HCl. Storage of the gel in Tris buffer, pH 8.0, with 1 mM sodium azide prevented microbial damage. Enzyme Activity Assay Old yellow enzyme activity was assayed by measuring the rate of NADPH oxidation at 340 nm at 25C. The standard assay system c ontained 0.2 mM NADPH (10 L of 20 mM stock solution prepared immediately before use in 0.1 M KPi buffer, pH 7.0), 2.5 mM 2-cyclohexenone (100 L of 25 mM stock solution in EtOH) and 1 L of enzyme in a total volume of 1 mL. Glucose Dehydrogenase activity was a ssayed by measuring the rate of NADP+ reduction at 340 nm at 25C. The standard assay system contai ned 0.5 mM of glucose (0.5 L of 1 M stock solution in water), 0.1 mM NADP+ (5 L of 20 mM stock solution in KPi buffer) and 1 L of GDH. The slope was calculated and used to fi nd the specific activit y. The background NADPH

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85 oxidation was measured using an identical to ab ove mentioned solution in which the amount of substrate was replaced by phosphate buffer. A unit of enzyme activity was defined as the quantity sufficient to oxidize 1 mol of NADPH per minute in th e mixture described above. Unit per ml of enzyme preparation were ca lculated based on the following equation: Units / mL = [(dA / dt x 1000) / ( 340 l)] x (Vassay / Venzyme) x Dilution, where dA / dt is the slope in AU / min, 340 = 6270 L / mol cm, l = 1 cm, Vassay = 1 mL, Venzyme = volume of enzyme added in mL, Dilution = dilution factor. Synthesis of 2-Alkyl-cy clohexane-1,3-diones General procedure: Glutaryl chloride (6.7 g, 40 mmol ) and 80 mmol of the appropriate acid were added to a suspension of 13.58 g (103 mM) of AlCl3 in 13 mL nitromethane with cooling in the atmosphere of argon. The mixture wa s heated for 3 hours at 80C, then cooled to 10C and poured onto 20 g of ice. After cooli ng to about 0C, the crude product that had separated was filtered off, washed with 5 ml of cold water, and recrystalized from water (with charcoal). The aqueous phase of the filtrate was bo iled with charcoal, filtered and extracted with ether. A second fraction crystallized afte r concentration of the ether extract. 2-Methylcyclohexane-1,3-dione (3a). White solid (1.0 g, 20%). 1H NMR (CDCl3): 0.75 (s, 3H,), 1.80 (m, 2H), 2.29 (t, 2H), 3.29 (t, 1H) ppm. 13C NMR (CDCl3): 7.7, 21.0, 33.0, 110.0 ppm (C2 signal not observed). 2-Ethylcyclohexane-1,3-dione (3b). Beige solid (0.84 g, 15%). 1H NMR (CDCl3): 0.75 (s, 3H,), 1.76 (m, 2H), 2.08 (q, 2H ), 2.24 (t, 2H), 3.18 (t, 1H) ppm. 13C NMR (CDCl3): 12, 17.5, 21.5, 32.5, 108.0 ppm (C2 signal not observed). Synthesis of 3-Isobutoxy-2-alkylcyclohex-2-enone 3-Isobutoxy-2-methylcyclohex-2-enone (4a). To a stirred solution of 2-methyl-1,3-cyclohexanedione ( 3a ) (1.08 g, 8.57 mmol) and p-tolue nosulfonic acid (108 mg) in

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86 9 ml of benzene was added i -BuOH (2.6 mL). The mixture was heated at reflux under a DeanStark trap for 3 hours. The reaction mixture wa s cooled down to room temperature and poured into 6.5 mL of saturated aqueous NaHCO3 solution and extracted with ether (3 x 5 mL). The combined extracts were washed with 5 mL of brine and dried over MgSO4. The solvent was removed under reduced pressure, solution purif ied by column chromatography (silica gel, hexane/ethyl acetate = 5/1) to give 3-isobutoxy2-methylcyclohex-2-enone as yellow oil (1.48 g, 95%). 1H NMR (CDCl3): 0.97 (d, 6H,), 1.71 (s, 3H), 1.93 (m, 1H ), 2.31 (t, 2H), 2.51 (t, 2H), 3.39 (t, 2H), 3.73 (d, 2H) ppm. 13C NMR (CDCl3): 7.0, 18.7, 21.3, 27.3, 32.2, 72.3, 105.8, 167.4, 185.9 ppm. 3-Isobutoxy-2-ethylcyclohex-2-enone (4b). To a stirred solution of 2-methyl1,3-cyclohexanedione ( 3a ) (0.90 g, 6.45 mmol) and p -toluenosulfonic acid (80 mg) in 7 mL of benzene was added i -BuOH (1.95 mL). The mixture was he ated at reflux under a Dean-Stark trap for 3 hours. The reaction mixture was cooled down to room temperature and poured into 5 mL of saturated aqueous NaHCO3 solution and extracted with 3 x 5mL of ether. The combined extracts were washed with 4 mL of brine and dried over MgSO4. The solvent was removed under reduced pressure and the residue was purif ied by column chromatography (silica gel, hexane/ethyl acetate = 5/1) to give 3-isobut oxy-2-ethylcyclohex-2-enone as yellow oil (1.01 g, 80% yield. 1H NMR (CDCl3): 0.96 (d, 6H,), 1.1 (t, 3H), 1. 67 (m, 3H), 2.25 (t, 2H), 2.40 (m, 2H), 3.20 (t, 2H), 3.74 (d, 2H) ppm. 13C NMR (CDCl3): 11.6, 14.8, 19.8, 21.4, 29.5, 36.0, 74.0, 105.9, 168.5, 188.9 ppm. Synthesis of 2-Alkylcyclohex-2-enone General procedure using NBS: To a solution of 2-alky lidenecyclohexan-1-one (10 mmol) dissolved in CCl4 (50 mL) was added N -bromosuccinimide (10 mmol) and the resulting suspension was heated in a water bath at 90C for 3 h. The suspension was then cooled to room

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87 temperature, the precipitates filtered off and anili ne (10 mmol), was added to the filtrate with cooling (ice-water). The solution was stirred at room temperature for 15 h, washed with 5% HCl (3 x 30 mL) and then with 5% NaHCO3 (2 x 30 mL). After drying with MgSO4, the solvent was evaporated to give brownish oil, which was purified by distillation under reduced pressure. NMR analysis did not confirm that desired product was obtained. 2-Methylcyclohex-2-enone (5a). A solution of 3-isobutoxy-2 -methylcyclohex-2-enone 4a (1.48 g, 8.14 mmol) and dry ether (6 mL) was adde d dropwise to LAH (115 mg) in dry ether (6 mL) at such a rate that steady reflux was main tained. The mixture was stirred for a further 1 hour, cooled and 10% H2SO4 (6 mL) added. The ether layer wa s removed and the aqueous layer extracted with ether (5x20 mL). The comb ined ether solutions were dried over MgSO4 and distilled to obtain 2-methylcyclohex -2-enone (0.80 g, 90%) as yellow oil. 1H NMR (CDCl3): 1.86 (s, 3H,), 1.97 (m, 2H), 2.43 (q, 2H), 2.52 (t, 1H), 5.93 (t, 1H) ppm. 13C NMR (CDCl3): 23, 24.5, 39, 45, 116.5, 151.5, 194.4 ppm. 2-Ethylcyclohex-2-enone (5b). A solution of 3-isobutoxy-2-ethylcyclohex-2-enone 4a (1.0 g, 5.16 mmol) and dry ether (4 mL) was adde d dropwise to LAH (73 mg) in dry ether (4 mL) at such a rate that steady refluxing was main tained. The mixture was stirred for a further 1 hour, cooled and a 10% H2SO4 (4 mL) added. The ether layer was removed and the aqueous layer extracted with ether (5x10 mL). The co mbined ether solutions were dried over MgSO4 and distilled to get 2-ethyl cyclohex-2-enone (0.53 g, 84%) as yellow oil. 1H NMR (CDCl3): 1.1 (t, 3H), 1.96 (m, 2H), 2.0 (m, 2H), 2.44 (q, 2H), 3.1 (t, 2H), 6.2 (t, 1H) ppm. 13C NMR (CDCl3): 15.7, 23.2, 24.5, 33.0, 36.0, 134.9, 144.0, 198.0 ppm. Synthesis of 1,3-Cyclohexanedione 3-Isobutoxy-2-cyclohexenone (7). To a stirred solution of 1,3-cyclohexanedione 6 (10 g, 89 mmol) and p -toluenosulfonic acid (1.44 g) in 314 ml of benzene was added i -BuOH (25 mL).

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88 The mixture was heated at reflux under a Dean-S tark trap for 3 hours. The reaction mixture was cooled to room temperature and poured into 56 mL of saturated aqueous NaHCO3 solution and extracted with ether (3x50 mL). The combined ex tracts were washed with 45 mL of brine and dried over MgSO4. The solvent was removed under redu ced pressure, and the residue was purified by column chromatography (silica ge l, hexane/ethyl acetate = 5/1) to give 3-isobutoxy-2-cyclohexenone as yellow oil (12.71 g, 85%). 1H NMR (CDCl3): 0.83 (d, 6H,), 1.85 (m, 3H), 2.21 (t, 2H), 2.27 (t, 2H), 3.45 (d, 2H), 5.18 (s, 1H) ppm. 13C NMR (CDCl3): 18.7, 20.9, 27.3, 28.6, 36.4, 74.2, 102.3, 177.7, 199.2 ppm. 3-Alkyl-2-Cyclohexen-1-ones General procedure: To the solution of the appropriat e Grignard reagent (26.7 mmol) and 20 mL of dry ether, a soluti on of 3-isobutoxy-2-cyclohexenone 7 (2 g, 17.8 mmol) in 10 mL of dry ether was added and the mixt ure was stirred for 2 hours. Af ter that time, the Grignard complex was decomposed with diluted sulfuric ac id and the solution was extracted with ether. The organic phase was washed with diluted NaHCO3, water and dried over MgSO4. After evaporation of solvent the cr ude product was purified by silica gel column chromatography (hexanes/Et2O = 3/1) to give the desire d 3-alkyl-2-cyclohexen-1-one. 3-Methyl-2-cyclohexen-1-one (9a). Yellow oil was obtained (1.62 g, 83%). 1H NMR (CDCl3): 1.96 (m, 5H,), 2.27 (m, 4H), 5.88 (s, 1H), ppm. 13C NMR (CDCl3): 22.3, 24.1, 30.6, 36.7, 126.3, 162.4, 199.2 ppm. 3-Ethyl-2-cyclohexen-1-one (9b). Yellow oil was obtained (1.72 g, 78%). 1H NMR (CDCl3): 1.06 (t, 3H,), 1.96 (q, 2H), 2.1 (m, 6H), 5.79 (s, 1H) ppm. 13C NMR (CDCl3): 11.2, 22.7, 29.7, 30.8, 37.4, 124.5, 168.0, 200.1 ppm.

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89 3n -Propyl-2-cyclohexen-1-one (9c). Yellow oil was obtained (1.84 g, 75%). 1H NMR (CDCl3): 0.95 (t, 3H,), 1.1 (m, 10H), 5.85 (s, 1H) ppm. 13C NMR (CDCl3): 13.5, 19.9, 22.5, 29.4, 37.7, 39.8, 125.4, 166.4, 199.8 ppm. 3i -Propyl-2-cyclohexen-1-one (9d). Yellow oil was obtained (1.72 g, 70%). 1H NMR (CDCl3): 1.05 (d, 6H,), 1.97 (m, 2H), 2.33 (m, 5H), 5.83 (s, 1H) ppm. 13C NMR (CDCl3): 21.1, 23.5, 28.2, 36.2, 38.1, 124.0, 172.4, 200.7 ppm. 3n -Butyl-2-cyclohexen-1-one (9e). Yellow oil was obtained (2.16 g, 80%). 1H NMR (CDCl3): 0.94 (t, 3H,), 1.30 (m, 2H), 1.52 (m, 2H), 1.98 (m, 2H), 2.24 (t, 2H), 2.32 (t, 2H), 2.40 (m, 2H), 5.88 (t, 1H) ppm. 13C NMR (CDCl3): 13.9, 22.6, 23.0, 29.3, 29.9, 37.4, 37.9, 125.5, 167.0, 199.8 ppm. 2-(1-Hydroxyalkyl) cyclohexanes General procedure: A solution of cyclohexanone (4.2 mL, 40 mmol) in THF (67 mL) was added dropwise to a stirred and cooled (78C) solution of LDA [generated by dropwise addition of n -BuLi (2.5 M, in hexane, 44 mmol, 17.6 mL) to i -Pr2NH (42 mmol, 5.88 mL) in THF (200 mL) at 0C, followed, after 15 minut es, by cooling to -78 C]. After 1 hour, the appropriate aldehyde (40 mmol) in THF (100 mL) was added quick ly. Stirring was continued for 50 min at -78C, and the reaction wa s quenched with saturated aqueous NH4Cl (80 mL). The cooling bath was removed and stirring was c ontinued until the mixture had reached room temperature. The solution was extracted with et her, washed with water and brine, dried over MgSO4 and the solvent was evaporated. Flash ch romatography (EtOAc/hexane = 1/6) of the residue gave final products. 2-(1-Hydroxyethyl)cyclohexane (12a). Pale yellow oil was obtained (4.20 g, 75%). 1H NMR (CDCl3): 1.2 (d, 3H,), 1.42 (m, 1H), 1.50 (m, 2H ), 1.62 (m, 8H), 3.65 (s, 1H), 3.91 (m, 1H) ppm. 13C NMR (CDCl3): 20.43, 25.55, 28.32, 31.22, 43.34, 58.24, 68.38, 216.31 ppm.

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90 2-(1-Hydroxybuthyl)cyclohexane (12b). Pale yellow oil was obtained (40.0 g, 60%). 1H NMR (CDCl3): 0.85 (t, 3H,), 1.20 (m, 13H), 3.63 (m, 2H), 3.95 (m, 2H) ppm. 13C NMR (CDCl3): 13.1, 18.0, 24.7, 27.6, 30.8, 38.1, 42.5, 55.3, 69.9, 215.8 ppm. 2-Alkylidenecyclohexanones 2-Ethylidenecyclohexanone (13a). Methanesulfonyl chlori de (7.33 mL, 93 mmol) was added dropwise to a stirred and cooled (0C) solution of alcohol 12a (4.3 g, 30 mmol) and Et3N (21.2 mL, 151 mmol) in CH2Cl2 (160 mL). The cooling bath was left in place, but was not recharged. Stirring was continued for 6 hours, th e solution was quenched with saturated aqueous NaHCO3 (37 mL), diluted with Et2O, washed with water and brine, dried over MgSO4. Evaporation of the solvent gave crude mesy late (5.82 g, in 80% yield), which was used immediately for next step. To a stirred solutio n of the above mesylate in THF (195 mL), DBU (7.5 mL, 50 mmol) was added dropwise. Afte r 1 hour, the mixture was diluted with Et2O, washed with water, 5% hydrochlor ic acid, and brine, dried over MgSO4 and solvent evaporated. The obtained crude product was purified by flas h chromatography (EtOAc/hexane = 1/10) to give 13a (2.64 g, 71%) with E isomer in excess (~ 80%). 1H NMR (CDCl3): 6.57 (t, 1H), 2.3 (m, 11H) ppm. 13C NMR (CDCl3): 21.6, 23.3, 25.7, 29.8, 38.5, 135.2, 138.5, 199.4 ppm. 2-Butylidenecyclohexanone (13b). a) Methanesulfonyl chlo ride (5.9 mL, 75 mmol) was added dropwise to a stirred and cooled (0C) solution of alcohol 12a (4.0 g, 24 mmol) and Et3N (16.8 mL, 120 mmol) in CH2Cl2 (128 mL). The cooling bath was left in place, but was not recharged. Stirring was continued for 6 hours, th e solution was quenched with saturated aqueous NaHCO3 (34 mL), diluted with Et2O, washed with water and brine, dried over MgSO4. Evaporation of the solvent gave crude mesy late (5.43 g, in 73% yield), which was used immediately for next step; b) to a stirred solu tion of the above mesylate in THF (178 mL), DBU (6.9 mL, 46 mmol) was added dropwise. Afte r 1 hour, the mixture was diluted with Et2O,

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91 washed with water, 5% hydrochlor ic acid, and brine, dried over MgSO4 and solvent evaporated. The obtained crude product was purified by flas h chromatography (EtOAc/hexane = 1/10) to give 13b (7.30 g, 64%) with E isomer in excess (~ 80%). 1H NMR (CDCl3): 2.3 (m, 15H), 6.64 (t, 1H) ppm. 13C NMR (CDCl3): 13.3, 21.6, 23.0, 23.2, 25.9, 29.2, 39.5, 135.7, 138.4, 199.8 ppm. Biotransformation of -Unsaturated Cyclic Enones Using Isolated Old Yellow Enzyme General procedure: Reaction mixtures contained final concentrations of NADP+ (10.7 mmol, 8 mg), glucose-6-phosphate (648 mmol, 2 19.5 mg), glucose-6-phosphate dehydrogenase (256 g), enone (0.18 mmol), -cyclodextrin (around 40 mg), purified OYE (20-40 g) in 100 mM KPi buffer, pH 7.0 in total volume of 25 mL. Conversions were carried out at room temperature. All the reaction components (except the buffer) we re added portionwise (10 equal portions during 12 hours) and the mi xtures were sampled for GC an alysis periodically. After 24 hours the reaction solutions were extracted with Et2O (3x50 mL). The combined organic extracts were dried over MgSO4. The final products were purifie d by filtration th rough silica gel (EtOAc/hexane = 2/9). ( R )-2-Methylcyclohexan-1-one (14a). Yellow oil; 97% e.e. (14.0 mg, 69.4%). 1H NMR (CDCl3): 1.00 (d, 3H), 1.34 (m, 1H), 1.64 (m, 3H), 2.10 (m, 5H) ppm. 13C NMR (CDCl3): 14.6, 25.3, 28.1, 36.1, 41.7, 45.4, 213.7 ppm. ( R )-2-Ethylcyclohexan-1-one (14b). Yellow oil; 92% e.e. (5.0 mg, 22%). 1H NMR (CDCl3): 0.96 (m, 3H), 2.60 (m, 11H) ppm. 2-Ethylcyclohexan-1-one (16a). Yellow oil; racemic mi xture (4.0 mg, 17.7%). 1H NMR (CDCl3): 0.96 (m, 3H), 2.60 (m, 11H) ppm. ( S )-3-Methylcyclohexan-1-one (15a). Yellow oil; 96% e.e. (16.0 mg, 79.3%). 1H NMR (CDCl3): 0.92 (m, 3H), 1.31 (m, 1H), 1.64 (m, 1H), 2.00 (m, 4H), 2.32 (m, 3H) ppm. 13C NMR (CDCl3): 14.6, 25.3, 28.1, 36.1, 41.7, 45.4, 213.7 ppm.

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92 ( S )-3-Ethylcyclohexan-1-one (15b). Yellow oil; 95% e.e. (12.3 mg, 54.2%). 1H NMR (CDCl3): 0.91 (t, 3H), 1.22 (m, 11H) ppm. ( S )-3n -Propylcyclohexan-1-one (15c). Yellow oil; 90% e.e. (3.75 mg, 14.88%). 1H NMR (CDCl3): 0.89 (t, 3H), 1.34 (m, 5H), 1.95 (m, 3H), 2.00 (m, 1H), 2.05 (m, 1H), 2.25 (m, 1H), 2.34 (m, 1H), 2.42 (m, 1H) ppm. ( S )-3i -Propylcyclohexan-1-one (15d). Yellow oil; 92% e.e. (3.75 mg, 14.88%). 1H NMR (CDCl3): 0.90 (d, 3H), 0.91 (d, 3H), 1.28 (m, 10H) ppm. Derivatization of Cyclohexanones with (2 R ,3 R )-(-)-2,3-Butanediol The stereochemical purities of all reduction pr oducts were determined by chiral-phase GC after ketalization with optically pure 2,3-butanediol. Derivatizatio ns were carried out by heating a mixture of 1.0 equiv. of crude biot ransformation product, 2.0 equiv. of (2 R ,3 R )-(-)-2,3-butanediol and a catalytic amount of p -TsOH in 1.5 mL of CH2Cl2 at reflux for 2 hours. A 1 L aliquot was directly anal ysed by GC. Samples of racemic ketones 5a b 9a e and 13a b were derivatized and analyzed by GC to de monstrate baseline resolution of enantiomers. Biotransformation of -Unsaturated Cyclic Enones Using Whole Cells of E. coli BL21(DE3)(pOYE-pET3b) General procedure: A 500 L aliquot from an overn ight culture of BL21(DE3)(pOYEpET3b) with an OD600 value between 4 and 5 added to 50 mL of LB medium supplemented with 200 g/mL ampicillin in a 500 mL Erlenmeyer flas k. The culture was shaken at 150-200 rpm at 37C until it reached an OD600 value between 0.4 and 0.5, then isopropylthio-D-galactoside (IPTG) was added to final concentration of 0.10 mM The culture was shaken at 150 rpm at room temperature for an additional 30 minutes, then the ketone and stoich iometric quantity of -cyclodextrin were added and shaking was contin ued at room temperature at 150 rpm. Samples for GC analysis were prepared by vortex mixi ng 50 L of the reaction mixture with 50 L of

PAGE 93

93 EtOAc for ca. 30 s. A 1 L portion of the organi c phase was analyzed by GC. At the conclusions of the reactions, the mixture was extracted w ith EtOAc (3x30 mL), and then the combined organic extracts were dried with MgSO4 and concentrated by rota ry evaporator. The final products were purified by filtration th rough silica gel (EtOAc/hexane = 2/9). ( R )-2-Methylcyclohexan-1-one (14a). Yellow oil; 96% e.e. (12.0 mg, 59.5%). 1H NMR (CDCl3): 1.00 (d, 3H), 1.34 (m, 1H), 1.64 (m, 3H), 2.10 (m, 5H) ppm. ( R )-2-Ethylcyclohexan-1-one (14b). Yellow oil; 90% e.e. (3.17 mg, 14%). 1H NMR (CDCl3): 0.96 (m, 3H), 2.60 (m, 11H) ppm. 2-Ethylcyclohexan-1-one (16a). Yellow oil; racemic mixture (2.5 mg, 11%). 1H NMR (CDCl3): 0.96 (m, 3H), 2.60 (m, 11H) ppm. ( S )-3-Methylcyclohexan-1-one (15a). Yellow oil; 94% e.e. (13.0 mg, 64.4%). 1H NMR (CDCl3): 0.92 (m, 3H), 1.31 (m, 1H), 1.64 (m, 1H), 2.00 (m, 4H), 2.32 (m, 3H) ppm. ( S )-3-Ethylcyclohexan-1-one (15b). Yellow oil; 95% e.e. (12.3 mg, 54.2%). 1H NMR (CDCl3): 0.91 (t, 3H), 1.22 (m, 11H) ppm. ( S )-3n -Propylcyclohexan-1-one (15c). Yellow oil; 89% e.e. (2.5 mg, 9.9%). 1H NMR (CDCl3): 0.89 (t, 3H), 1.34 (m, 5H), 1.95 (m, 3H), 2.00 (m, 1H), 2.05 (m, 1H), 2.25 (m, 1H), 2.34 (m, 1H), 2.42 (m, 1H) ppm. ( S )-3i -Propylcyclohexan-1-one (15d). Yellow oil; 90% e.e. (1.9 mg, 7.4%). 1H NMR (CDCl3): 0.90 (d, 3H), 0.91 (d, 3H), 1.28 (m, 10H) ppm. Synthesis of 2-Alkyloxobutanoates General procedure: The appropriate Grignard reagen t (88.6 mmol) was added dropwise to a mixture of diethyl oxalate (10 mL, 73.6 mm ol), THF (50 mL) and ether (100 mL) at -78C and the solution was stirred at this temperat ure for 4 hours. After que nching with saturated NH4Cl (100 mL), the mixture was extracted with ethyl acetate (3x100 mL). The organic phases

PAGE 94

94 were combined, dried over MgSO4 and concentrated in vacuo to give crude products, which were purified by flash chromatography. 2-Ethyloxobutanoate (17d). Pale yellow oil (8.13 g, 85%). 1H NMR (CDCl3): 1.28 (t, J =7.2 Hz, 3H), 1.39 (t, J =7.2, 3H), 2.90 (q, J =7.2, 2H), 4.15 (q, J =7.2, 2H) ppm. 13C NMR (CDCl3): 6.45, 13.90, 32.69, 62.24, 161.09, 195.04 ppm. 2n -Propyloxobutanoate (17e). Pale yellow oil (7.33 g, 69%). 1H NMR (CDCl3): 0.7 (m, 8H), 2.81 (t, J =7.1 Hz, 2H), 4.31 (q, J =6.3 Hz, 2H) ppm. 13C NMR (CDCl3): 14.14, 14.65, 17.15, 41.75, 62.97, 161.86, 195.24 ppm. Synthesis of Nitro Alcohols General procedure using CAN: To a stirred solution of an appropriate acrylic ester (1 mmol) in anhydrous CH3CN (5 mL) was added CAN (3 mmol) and NaNO2 (3 mmol) at 0C under nitrogen. The reaction mixture was vigorously stirred for 24 h at room temperature, diluted with water and extracted sequentiall y with saturated solution of NaHCO3, brine and dried over MgSO4. The residue obtained afte r evaporation of the solv ent was purified by column chromatography. General Procedure Using Amberlyst A-21: A 50 mL two necked flask equipped with a mechanical stirrer was charged with the approp riate nitroalkane (60 mmol) and cooled with ice-water bath. Amberlyst A-21 (5-7 g) was a dded and the mixture was stirred for 5 minutes before the appropriate 2-oxoacid ethyl ester (60 mmol). After stirri ng overnight at room temperature the mixture was filtered. The Amberlyst resin was washed with CH2Cl2 (4x25 mL), then solvent was evaporated in vacuo to yield crude -nitroalcohols, which were purified by flash chromatography.

PAGE 95

95 Ethyl 2-hydroxy-3-nitrobutanoate (19a). Pale yellow oil (9.45 g, 89%). 1H NMR (CDCl3): 4.34 (m, 4H), 3.40 (brs, 1H), 1.64 (d, J =6.9 Hz, 3H), 1.28 (t, J =7.1 Hz, 3H) ppm. 13C NMR (CDCl3): 171.0, 83.3, 71.8, 62.9, 14.9, 13.9 ppm. Ethyl 2-hydroxy-3-nitropentanoate (19b). Yellow oil (10.2 g, 89%). 1H NMR (CDCl3): 4.74 (m, 1H), 4.40 (m, 4H), 2.32 (m, 2H ), 1.36 (m, 3H), 1.11 (m, 3H) ppm. 13C NMR (CDCl3): 170.8, 90.6, 70.6, 62.8, 22.6, 13.8, 10.3 ppm. Ethyl 2-hydroxy-2-methyl-3-nitropropanoate (19c). Pale yellow oil (9.63 g, 91%). 1H NMR (CDCl3): 4.87 (dd, J1=13.78 Hz, J2=13.78 Hz, 2H), 4.4 (m, 2H), 1.46 (s, 3H), 1.35 (t, J =7.13 Hz, 3H) ppm. 13C NMR (CDCl3): 173.6, 81.1, 72.6, 63.2, 24.0, 14.1 ppm. Ethyl 2-hydroxy-2-(nitromethyl)butanoate (19d). Yellow oil (11.2 g, 98%). 1H NMR (CDCl3): 4.54 (dd, J1=13.59 Hz, J2=13.59 Hz, 2H), 4.33 (m, 2H), 3.79 (s, 1H), 1.69 (m, 2H), 1.31 (t, J =7.08 Hz, 3H), 0.90 (t, J =7.37 Hz, 3H) ppm. 13C NMR (CDCl3): 173.0, 80.9, 75.7, 63.2, 29.2, 14.2, 7.2 ppm. Ethyl 2-hydroxy-2-(nitromethyl)pentanoate (19e). Yellow oil (10.80 g, 88%). 1H NMR (CDCl3): 4.84 (dd, J1=13.59 Hz, J2=13.59 Hz, 2H), 4.37 (m, 2H), 1.69 (m, 5H), 1.36 (t, J =7.07 Hz, 2H), 0.95 (t, J =7.36 Hz, 3H) ppm. 13C NMR (CDCl3): 173.1, 81.1, 75.4, 63.2, 38.8, 16.3, 14.4, 14.1 ppm. Ethyl 2-hydroxy-3-methyl-2-(n itromethyl)butanoate (19f). Yellow oil (12.70 g, 99%). 1H NMR (CDCl3): 4.85 (dd, J1=13.54 Hz, J2=13.3 Hz, 2H), 4.39 (m, 2H), 4.39 (q, J =7.13 Hz, 2H), 2.00 (m, 1H), 1.36 (t, J =7.13 Hz, 3H), 1.00 (dd, J1=6.89 Hz, J2=6.89 Hz, 6H) ppm. 13C NMR (CDCl3): 173.1, 80.0, 63.6, 34.7, 17.4, 16.8, 14.7 ppm. Ethyl 2-hydroxy-3-nitro-2-phenylpropanoate (19g). Yellow oil (14.97 g, 98%). 1H NMR (CDCl3): 7.44 (m, 5H), 5.29 (dd, J1=16.5 Hz, J2=15.0 Hz, 2H), 4.49 (m, 2H), 1.36 (t,

PAGE 96

96 J =7.01 Hz, 3H) ppm. 13C NMR (CDCl3): 171.8, 136.6, 129.3, 129.0, 125.4, 80.9, 76.1, 63.8, 14.1 ppm. Synthesis of 3and 2-Alkyl Substituted -Nitroacrylates General procedure: The appropriate -nitroalcohol (17 mmol) was dissolved in 17 mL of CH2Cl2 at -78C under an argon atmosphere, then 1 equiv. of methanesulfonyl chloride (17 mmol) was added in one portion. After 30 mi nutes, triethylamine (51 mmol) was added dropwise, and the reactio n mixture was stirred for 4 hours at -78C. The reaction mixture was then transferred to a separatory funnel with the ai d of 17 mL of CH2Cl2, then it was washed with water, 5% aqueous HCl, and brine. The fina l product was purified by flash chromatography. ( E )-Ethyl 3-nitrobut-2-enoate ( E -20a). Yellow oil (1.08 g, 40%). 1H NMR (CDCl3): 7.00 (s, 1H), 4.20 (q, J =7.2 Hz, 2H), 2.50 (s, 3H), 1.20 (t, J =7.2 Hz, 3H) ppm. 13C NMR (CDCl3): 164.2, 160.0, 121.5, 61.8, 14.1, 14.0 ppm. IR max 17351533, 1355,1227 cm-1. ( E )-Ethyl 3-nitropent-2-enoate ( E -20b). Yellow oil (1.35 g, 46%). 1H NMR (CDCl3): 6.97 (s, 1H), 4.32 (q, J =7.08 Hz, 2H), 3.11 (m, 2H), 1.37 (t, J =9.91 Hz, 3H), 1.23 (t, J =8.47 Hz, 3H) ppm. 13C NMR (CDCl3): 187.0, 185.5, 120.8, 61.9, 21.2, 14.4, 12.5 ppm. IR max 1735, 1533, 1352, 1227 cm-1. ( Z )-Ethyl 2-methyl-3-nitroacrylate ( Z -20c). Yellow oil (1.46 g, 54%). 1H NMR (CDCl3): 6.89 (s, 1H), 4.39 (q, J =7.1 Hz, 2H), 2.11 (s, 3H), 1.37 (t, J =7.4 Hz, 3H), ppm. 13C NMR (CDCl3): 166.7, 141.5, 136.3, 63.0, 18.2, 14.4 ppm. IR max 1737, 1533, 1355, 1227 cm-1. ( Z )-Ethyl 2-(nitromethylene)butanoate ( Z -20d). Yellow oil (0.109 g, 37.2%). 1H NMR (CDCl3): 6.84 (s, 1H), 4.39 (q, J =7.4 Hz, 2H), 2.48 (m, 2H), 1.37 (t, J =7.4 Hz, 3H), 1.2 (t, J =7.4 Hz, 3H) ppm. 13C NMR (CDCl3): 166.1, 146.7, 135.1, 62.5, 25.5, 14.0, 11.2 ppm. IR max 1735, 1533, 1353, 1222 cm-1.

PAGE 97

97 ( Z )-Ethyl 2-(nitromethylene)pentanoate ( Z -20e). Yellow oil (1.11 g, 35%). 1H NMR (CDCl3): 6.85 (s, 1H), 4.39 (m, 2H), 2.41 (m, 2H), 1.66 (m, 2H), 1.37 (t, J =7.4 Hz, 3H), 1.28 (t, J =7.4 Hz, 3H), ppm. 13C NMR (CDCl3): 166.1, 145.3, 135.6, 62.5, 33.9, 20.2, 14.0, 13.5 ppm. IR max 1735, 1532, 1353, 1221 cm-1. ( Z )-Ethyl 3-methyl-2-(nitromethylene)butanoate ( Z -20f). Yellow oil (0.388 g, 12.2%). 1H NMR (CDCl3): 6.83 (s, 1H), 4.40 (q, J =7.1 Hz, 2H), 2.77 (m, 1H), 1.39 (t, J =7.4 Hz, 3H), 1.23 (d, J =7.1 Hz, 6H) ppm. 13C NMR (CDCl3): 165.8, 150.9, 134.9, 62.4, 31.6, 20.5, 14.0 ppm. IR max 1736, 1533, 1352, 1221 cm-1. ( Z )-Ethyl 3-nitro-2-phenylacrylate ( Z -20g). Yellow oil (2.25 g, 60%). 1H NMR (CDCl3): 7.53 (m, 5H), 7.35 (s, 1H), 4.51 (q, J =7.1 Hz, 2H), 1.42 (t, J =7.1 Hz, 3H) ppm. 13C NMR (CDCl3): 164.5, 143.1, 134.2, 129.3, 127.2, 62.6, 13.6 ppm. IR max 1737, 1533, 1355, 1227 cm-1. Biotransformation of -Nitro Acrylates Using Iso lated Old Yellow Enzyme General procedure: Reaction mixtures contained final concentrations of NADP+ (20 mol, 15 mg), glucose-6-phosphate (1.27 mmol, 429 mg), glucose-6-phosphate dehydrogenase (500 g), nitroacrylate (25 mM), a nd purified OYE (20-40 g) in 100 mM KPi, pH 6.93 in total volumes of 50 mL. Conversions were carried ou t at room temperature. Reaction components (except for KPi buffer) were added in 10 equal portions every 45 minutes and the mixtures were sampled for GC analysis periodically. After nearly all of the substrates had been consumed, the reaction mixture was extracted with Et2O (3 x (5 x reaction volume)). The combined organic extracts were washed with brine (1 volu me) and water (1 volume), dried with MgSO4, and concentrated in vacuo. Ethyl 3-nitrobutanoate (21a). Yellow oil (0.201 g, 98%). 1H NMR (CDCl3): 4.98 (m, 1H), 4.21 (q, J =7.1 Hz, 2H), 3.18 (dd, J1=8.8 Hz, J2=8.8 Hz, 1H), 2.73 (dd, J1=5.0 Hz, J2=5.0

PAGE 98

98 Hz, 1H), 1.63 (d, J =6.9 Hz, 3H), 1.29 (t, J =7.1 Hz, 3H) ppm. 13C NMR (CDCl3): 169.3, 78.6, 61.4, 38.6, 19.5, 14.1 ppm. Ethyl 3-nitropentanoate (21b). Yellow oil (0.096 g, 95%). 1H NMR (CDCl3): 4.89 (m, 1H), 4.20 (q, J =6.5 Hz, 2H), 3.18 (dd, J1=9.4 Hz, J2=9.4 Hz, 1H), 2.71 (dd, J1=4.2 Hz, J2=4.5 Hz, 1H), 2.02 (m, 2H), 1.28 (t, J =7.1 Hz, 3H), 1.02 (t, J =7.4 Hz, 3H) ppm. 13C NMR (CDCl3): 169.4, 84.6, 61.5, 36.9, 27.2, 14.2, 10.0 ppm. ( R )-Ethyl 2-methyl-3-n itropropanoate (21c). Yellow oil (0.191 g, 93%). 1H NMR (CDCl3): 4.76 (dd, J1=8.1 Hz, J2=8.1 Hz, 1H), 4.44 (dd, J1=5.7 Hz, J2=5.7 Hz, 1H), 4.24 (q, J =7.0 Hz, 2H), 3.29 (m, 1H), 1.30 (m, 6H) ppm. 13C NMR (CDCl3): 172.6, 76.6, 61.7, 37.8, 14.5, 14.2 ppm. ( R )-Ethyl 2-(nitromethyl)butanoate (21d). Yellow oil (0.151 g, 75%). 1H NMR (CDCl3): 4.78 (dd, J1=9.1 Hz, J2=9.4 Hz, 1H), 4.46 (dd, J1=5.1 Hz, J2=4.8 Hz, 1H), 4.22 (q, J =7.1 Hz, 2H), 3.18 (m, 1H), 1.76 (m, 2H), 1.30 (t, J =7.1 Hz, 3H), 1.01 (t, J =7.7 Hz, 3H) ppm. 13C NMR (CDCl3): 172.2, 75.1, 61.5, 44.4, 22.7, 14.3, 11.2 ppm. ( R )-Ethyl 2-(nitrometh yl)pentanoate (21e). Yellow oil (0.187 g, 93%). 1H NMR (CDCl3): 4.77 (dd, J1=9.3 Hz, J2=9.3 Hz, Hz, 1H), 4.44 (dd, J1=4.8 Hz, J2=4.8 Hz, 2H), 4.20 (q, J =7.4 Hz, 2H), 3.24 (m, 1H), 1.72 (m, 2H), 1.44 (m, 2H), 1.30 (t, J =7.1 Hz, 3H), 0.97 (t, J =7.4 Hz, 3H) ppm. 13C NMR (CDCl3): 172.5, 75.4, 61.5, 42.9, 31.5, 20.1, 13.9, 14.3 ppm. ( R )-Ethyl 3-methyl-2-(nitro methyl)butanoate (21f). Yellow oil (0.182 g, 90.5%). 1H NMR (CDCl3): 4.83 (dd, J1=10.5 Hz, J2=10.5 Hz, 1H), 4.44 (dd, J1=4.0 Hz, J2=4.0 Hz, 1H), 4.25 (q, J =7.1 Hz, 2H), 3.12 (m, 1H), 2.11 (m, 1H), 1.30 (t, J =7.1 Hz, 3H), 1.01 (m, 6H) ppm. 13C NMR (CDCl3): 172.4, 74.3, 61.8, 49.4, 29.4, 20.4, 20.3, 14.7 ppm.

PAGE 99

99 Synthesis of -Amino Acids General procedure: Crude biotransformation products (ca. 1.5 mmol) were hydrogenated at 500 psi in the presence of Ra ney nickel (200 mg) in EtOH (50 mL) at room temperature. After 16 hours, the resulting solution was filtered th rough Celite and the solvent was evaporated. A portion of the residue (5 0 mg) was dissolved in 6 M HCl a nd the solution was held on reflux overnight. The solution was concentrated under redu ced pressure to afford yellow oil, which was washed with EtOAc to remove any non-polar impurities. Water was removed by rotary evaporator to yield the -amino acids as hydrochloride salts. 3-Aminobutanoic acid (23a). Ethyl 3-aminobutanoate ( 22a ) was obtained as pale yellow oil (0.150 g, 74.5%), which was further hydrolyzed to give 23a as a white precipitate (0.105 g, 89%). 1H NMR (D2O): 3.88 (m, 1H), 2.8 (dd, J1=17.5 Hz, J2=5.8 Hz, 1H), 2.72 (dd, J1=17.5 Hz, J2=7.2 Hz, 1H), 1.36 (d, J =6.7 Hz, 3H) ppm. 13C NMR (D2O): 174.1, 44.4, 37.7, 17.8 ppm. 3-Aminopentanoic acid (23b). Ethyl 3-aminopentanoate ( 22b ) was obtained as pale yellow oil (0.064 g, 81%), which was further hydrolyzed to give 23b as a white precipitate (0.047 g, 91%). 1H NMR (D2O): 3.63 (m, 1H), 2.88 (dd, J1=4.53 Hz, J2=4.81 Hz, 1H), 2.74 (dd, J1=8.21 Hz, J2=8.21 Hz, 1H), 1.79 (m, 2H), 1.03 (t, J =7.64 Hz, 3H) ppm. 13C NMR (D2O: 174.7, 49.7, 35.8, 25.3, 8.9 ppm. 3-( R )-Aminomethylpropanoic acid (23c). Ethyl 3-amino-2-methylpropanoate ( 22c ) was obtained as pale yellow oil (162.40 g, 85%), which was further hydrolyzed to give 23c as a white precipitate (0.143 g, 88%). [ ]25 D=13.0 (c=0.94; 1M HCl). 1H NMR (D2O): 3.30 (dd, J1=8.77 Hz, J2=8.5 Hz, 1H), 3.16 (dd, J1=4.81 Hz, J2=4.81 Hz, 1H), 2.97 (m, 1H), 1.30 (d, J =7.36 Hz, 3H) ppm. 13C NMR (D2O): 177.7, 41.3, 37.1, 14.3 ppm. 2-( R )-(Aminomethyl)butanoic acid (23d). Ethyl 2-(aminomethyl)butanoate ( 22d ) was obtained as pale yellow oil (0.106 g, 85%), which was further hydrolyzed to give 23d as a white

PAGE 100

100 precipitate (0.077 g, 90%). [ ]25 D=-2.7 (c=1, 1M HCl). 1H NMR (D2O): 3.33 (dd, J1=9.06 Hz, J2=9.06 Hz, 1H), 3.19 (dd, J1=4.53 Hz, J2=4.81 Hz, 1H), 2.83 (m, 1H), 1.78 (m, 2H), 0.99 (t, J =7.36 Hz, 3H) ppm. 13C NMR (D2O): 177.1, 43.9, 39.6, 22.7, 10.2 ppm. 2-( R )-(Aminomethyl)pentanoic acid (23e). Ethyl 2-(aminomethyl)pentanoate ( 22e ) was obtained as pale yellow oil (0.131 g, 85%), which was further hydrolyzed to give 23e as a white precipitate (0.102 g, 95%). [ ]25 D=1.16 (c=1, 1M HCl). 1H NMR (D2O): 3.33 (dd, J1=9.34 Hz, J2=9.06 Hz, 1H), 3.21 (dd, J1=4.25 Hz, J2=4.24 Hz, 1H), 2.93 (m, 1H), 1.75 (m, 2H), 1.41 (m, 2H), 0.93 (t, J =7.36 Hz, 3H) ppm. 13C NMR (D2O): 177.3, 42.4, 39.9, 31.4, 19.3, 13.1 ppm. 2-( R )-(Aminomethyl)-3-methylbutanoic acid (23f). Ethyl 2-(aminomethyl)-3methylbutanoate ( 22f ) was obtained as pale yellow o il (0.124 g, 81%), which was further hydrolyzed to give 23f as a white precipitate (0.095 g, 93%). [ ]25 D=1.6 (c=1.03, H2O). 1H NMR (CD3OD): 3.16 (dd, J1=10.47 Hz, J2=10.15 Hz, 1H), 2.96 (dd, J1=3.4 Hz, J2=3.68 Hz, 1H), 2.52 (m, 1H), 2.09 (m, 1H), 0.95 (t, J =6.61 Hz, 6H) ppm. 13C NMR (CD3OD): 175.6, 50.2, 39.3, 30.2, 20.2, 19.7 ppm. Derivatization of -Amino Acid Esters with TFAA. -Amino acid ester (0.03 mmol) was stirre d with TFAA (0.9 mmol) in 60C for 30 minutes. After this time trifluoroacetic acid wa s evaporated in the stream of argon and the residue was dissolved in EtOAc (1 mL). Biotransformation of -Nitroacrylates Using Cell Extract General procedure: An overnight cultures of E. coli (BL21(DE3)(pOYE-pET3b)) and E. coli overexpressing glucose dehydrogenase (GDH) from B. subtilis grown separately in LB medium containing 100 g/mL ampicillin were d iluted 1:100 into 1 L of the same medium. The cultures were shaken in 37C until the optical densities at 600 nm reached 0.8, then the enzymes overproduction was induced with isopropylthio-D-galactoside (IPTG) at a final concentration

PAGE 101

101 of 400 M and the cultures were stirred for an additional 2.5 hours at room temperature. The cells were harvested by centrifugation (5,000 rpm fo r 15 min at 4C), washed twice with cold sterile water and then resusp ended in 25 mL of 100 mM KPi buffer pH 6.93 (with addition of PMSF to final concentration of 10 M). The cell s were lysed using a French Press and debris was removed by centrifugation at 20,000 g for 60 min at 4C. The pH of supernatant was adjusted to 6.93 by adding diluted HCl. The extr acts were mixed together and diluted with the same buffer to the final vo lume of 100 mL and NADP+ was added (to the fi nal concentration of 0.05 mM). To this mixture were added every 45 minutes portions of: glucose (to the final concentration of 10 mM), -cyclodextrin 40 mg and 20 mg of the substrate ( 20a 20c 20e ) (to the final concentration of 17 mM ). The pH of the solution was controlled and kept at 6.93 by addition of 3M NaOH. The concentration of glucose was monitored by using glucose assay. When the starting material was completely consumed (after 11 hours) the protein was precipitated with sodium chlori de and the mixture was centrif uged at 15,000 rpm for 15 minutes at 4C. The supernatant was extracted with Et2O, dried over MgSO4 and solvent was evaporated. Ethyl 3-aminobutanoate (22a). Obtained in 72% yield as a racemic mixture. Ethyl 3-amino-2-methylpropanoate (22c). Obtained in 75% yield with 73% e.e. Ethyl 2-(aminomethyl)pentanoate (22e). Obtained in 71% yield with 89% e.e. Glucose Assay The glucose concentration in the reaction was determined using a Trinder assay kit commercially available from Diagnostic Chemi cals Limited, Canada. The absorbance of the reaction, which consists of 5 L of the reaction media and 1 mL of the Trinder reagent mixed by inversion and incubated at 37C for 15 minutes, was measured at 505 nm. The concentration of the glucose in the reaction was measured by comp aring it to the standard reference containing 0.4 g/L glucose.

PAGE 102

102 Incubation of 21a in D2O. Compound 21a (20 mg, 0.012 mmol) was stirred in KPi buffer (prepared in D2O) (5 mL) overnight. Samples for GC/MS analysis were ta ken every hour. The product of the incubation was analyzed by 1H NMR spectroscopy: 1H NMR (CDCl3): 4.21 (q, J =7.1 Hz, 2H), 3.18 (dd, J1=8.8 Hz, J2=8.8 Hz, 1H), 2.73 (dd, J1=5.0 Hz, J2=5.0 Hz, 1H), 1.63 (s, 3H), 1.29 (t, J =7.1 Hz, 3H) ppm. Biotransformation of ( Z )-20c Using NADPD. Tris-HCl buffer (250 L of 1M, pH 8.0) was added to 4.595 mL of KPi buffer, 5 L of ( Z )20c in 50 L of isopropanol-d8 was added to that solution added to that solution and stirred for few minutes. After that time 9 mg of NADP+, 3 mg of TBADH and 100 L of OYE1 were added to the mixture and stirred. Afte r 6 hours the reaction was complete d and the reaction mixture was extracted with ether (3x8 mL), the solvent was dried over MgSO4 and evaporated. The product was analyzed by GC-MS and 1H NMR spectroscopy: 1H NMR (CDCl3): 4.76 (dd, J1=8.1 Hz, J2=8.1 Hz, 1H), 4.44 (dd, J1=5.7 Hz, J2=5.7 Hz, 1H), 4.24 (q, J =7.0 Hz, 2H), 1.30 (m, 6H) ppm.

PAGE 103

103 APPENDIX A GC ANALYSIS OF SUBSTITUTED CYCLOHEXANONES Figure A-1. GC chromatogram of 14a 15b and 15c.

PAGE 104

104 Figure A-2. GC chromatogram of 14b 15a and 15d.

PAGE 105

105 Figure A-3. GC chromatogram of TFA derivatives of 22c and 22d

PAGE 106

106 Figure A-4. GC chromatogram of TFA derivatives of 22e and 22f

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107 APPENDIX B NMR SPECTRA OF -NITROACRYLATES AND -AMINO ACIDS Figure B-1. 13C NMR of spectrum of 20c

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108 Figure B-2. 1H NMR spectrum of 20c

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109 Figure B-3. 13C NMR spectrum of 20d

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110 Figure B-4. 1H NMR spectrum of 20d

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111 Figure B-5. 13C NMR spectrum of 20e

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112 Figure B-6. 1H NMR spectrum of 20e

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113 Figure B-7. 13C NMR spectrum of 20f

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114 Figure B-8. 1H NMR spectrum of 20f

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115 Figure B-9. 13C NMR spectra of 23a and b

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116 Figure B-10. 13C NMR spectra of 23c and d

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117 Figure B-11. 13C NMR spectra of 23e and f

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118 Figure B-15. 1H NMR spectrum of 21a incubated in D2O.

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119 Figure B-16. 1H NMR spectrum of 21a

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120 Figure B-17. 1H NMR spectrum of deuterated 21c

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129 BIOGRAPHICAL SKETCH Magdalena Alicja Swiderska was born in Mr gowo, Poland, on February 1st, 1978. In 2002 Magdalena graduated from the University of Wars aw, Poland with a master's degree in chemistry under the supervision of Prof. Zbigniew Czarnocki. In 2003 Magda lena moved her scientific career to University of Florida, joined the Stewarts group and began her Ph.D. studies in the field of biocatalysis and bioor ganic chemistry. After 5 years of research, she graduated from University of Florida with a Doct or of Philosophy degree in biochemistry.