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Application of Saccharomyces Carlsbergensis Old Yellow Enzyme in the Enantioselective Henry Reaction

Permanent Link: http://ufdc.ufl.edu/UFE0042626/00001

Material Information

Title: Application of Saccharomyces Carlsbergensis Old Yellow Enzyme in the Enantioselective Henry Reaction
Physical Description: 1 online resource (58 p.)
Language: english
Creator: Zhou, Di
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: betanitroacrylate, henryreaction, oldyellowenzyme
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this thesis, we studied the application of Saccharomyces carlsbergensis Old Yellow Enzyme (a kind of flavin dependent enzyme) in the Henry reaction between beta-nitroacrylate and acetylaldehyde. The reaction was monitored every hour until it went to completion after 12 hours, and four different stereoisomers were observed on GC-MS with different ratio. A control reaction between the beta-nitroalkane and acetylaldehyde was conducted and showed that the phosphate buffer solution (KPi, 100mM, pH~7.0/pH~6.0) catalyzed this Henry reaction and that enzyme catalysis was not involved. Further studies should focus on the rate of carbanion protonation and the reaction between the carbanion and acetylaldehyde, modifying the reaction media to inhibit the carbanion protonation, and choosing better substrates.
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 Di Zhou.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042626:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042626/00001

Material Information

Title: Application of Saccharomyces Carlsbergensis Old Yellow Enzyme in the Enantioselective Henry Reaction
Physical Description: 1 online resource (58 p.)
Language: english
Creator: Zhou, Di
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: betanitroacrylate, henryreaction, oldyellowenzyme
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this thesis, we studied the application of Saccharomyces carlsbergensis Old Yellow Enzyme (a kind of flavin dependent enzyme) in the Henry reaction between beta-nitroacrylate and acetylaldehyde. The reaction was monitored every hour until it went to completion after 12 hours, and four different stereoisomers were observed on GC-MS with different ratio. A control reaction between the beta-nitroalkane and acetylaldehyde was conducted and showed that the phosphate buffer solution (KPi, 100mM, pH~7.0/pH~6.0) catalyzed this Henry reaction and that enzyme catalysis was not involved. Further studies should focus on the rate of carbanion protonation and the reaction between the carbanion and acetylaldehyde, modifying the reaction media to inhibit the carbanion protonation, and choosing better substrates.
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 Di Zhou.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Stewart, Jon D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042626:00001


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1 APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN THE ENANTIOSELECTIVE HENRY REACTION By DI ZHOU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Di Zhou

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3 To my loving parents and family

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4 ACKNOWLEDGMENTS I am heartily grateful to those who helped me during my past years in UF, without their kind help, this thesis could not have been completed. First, I would like to thank my advisor, Dr. Jon D. Stewart, for his support and guidance throughout my research, his knowledge, insights and valuable suggestions always saved me from frustrations, his enthusiasm for science and encouragement to students gave me the remarkable confidence and courage to overcome ever y obstacles in my research, I benefited a lot from working with him. My thanks also go to Dr. Ron K. Castellano and Dr. Sukwon Hong, not only for teaching me organic spectroscopy and synthetic organic chemistry, but also for being my committee members. I would also like to extend my appreciation to my colleagues, Dr. Bradford Sullivan, Dr. Dimitri Dascier, Colin Conerly, Adam Wa lton, Yuri Pompeu and Li Zhao for their invaluable advices on my project, as well as assistance in laboratory experiment s. Finally, I want to thank my parents and family for their years of selfless devotion, love and sacrifice they have given in order to prepare me for life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 11 History of Old Yellow Enzyme ................................................................................. 11 The Old Yellow Enzyme Family .............................................................................. 13 Yeas t Homologues of OYE ............................................................................... 13 Plant Homologues of OYE ................................................................................ 13 Bacterial Homologues of OYE .......................................................................... 14 Old Yellow Enzyme Crystal Structure and Mechanism ........................................... 14 Purification of Old Yellow Enzyme .......................................................................... 17 Henry Reaction ....................................................................................................... 17 2 OLD YELLOW ENZYME IN ASYMMETRIC C=C BOND BIOREDUCTIONS ......... 23 Asymmetric C=C Bond Reductions ......................................................................... 23 As Unsaturated Carbonyl Bioreductions ............................................ 25 Asymmetric Cyclohexenone Bioreductions ............................................................. 26 Asymmetric Nitroalkene Bioreductions ................................................................... 30 3 OYE: A POTENTIAL CATALYST FOR ENANTIOSELECTIVE HENRY REACTION ............................................................................................................. 33 Introduction ............................................................................................................. 33 Results and Discussions ......................................................................................... 34 Experimental ........................................................................................................... 39 Reagents and Instrum entations ........................................................................ 39 Nitroalcohol (3). ........................................................................ 40 Nitroacrylate (4a,b). .................................................................. 40 Nitroalkane (5). ......................................................................... 4 1 Synthesis of Thiourea Catalyst (8). .................................................................. 42 Synthesis of Ethyl 4Hydroxy 2 Methyl 3 Nitropentanoate (10). ....................... 42 Nitroacrylate Using Old Yellow Enzyme ................. 43

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6 4 CONCLUSION AND FUTURE WORK .................................................................... 44 APPENDIX A GC ANALYSIS OF HENRY REACTION PRODUCTS ............................................ 45 B 1H NITROACRYLATE AND NITROALKANE ...................................................................................................... 49 C MASS SPECTROMETRY OF THE HENRY REACTION PRODUCT ..................... 53 LIST OF REFERENCES ............................................................................................... 55 BIOGRAPHICAL SKETCH ............................................................................................ 58

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7 LIST OF TABLES Table page 1 1 Hb HNL catalyzed Henry reactions of aldehydes with nitromethane ................... 22 2 1 C onversion and ee value for reduction of different enone with P90 .................... 27 2 2 C onversion and ee value for reduction of different enone with P44 .................... 27 2 3 Asymmetric bioreduction of substituted cyclopetenone and cyclohexenone using OPR1, OPR3 and YqjM. ........................................................................... 28 2 4 Reductions of alkyl substituted 2cyclohexenones by engineered E. coli cells overexpressing S. carlsbergensis old yellow enzyme. ........................................ 29 2 5 Enzymatic reduction of ( Z ) 3 phenyl 2 nitro 2 butene with YNAR I and YNARII .............................................................................................................. 31 2 6 Reductions of nitroacrylates by S. carlsbergensis old yellow enzyme. ............ 32 3 1 nitroacylate ( 4a,b) .. 35 3 2 Different conditions of Henry reaction between nitroalkane ( 5 ) and acetylaldehyde ( 9 ). ............................................................................................. 37 3 3 Conversion and product percentage of chemical and enzymatic Henry reaction products ................................................................................................ 39

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8 LIST OF FIGURES Figure page 1 1 Reaction system of Warburg and Christian. ....................................................... 11 1 2 A ribbon diagram illustrating the structure of a dimer of OYE. ............................ 12 1 3 Active site of OYE in complex with p hydroxybenzaldehyde. ............................. 16 1 4 Kinetic mechanism of OYE. ................................................................................ 16 1 5 Catalytic cycle for old yellow enzyme in the reduction of activated alkene. ........ 18 1 6 Transformation of nitroalcohol to different building blocks. ................................. 18 1 7 Reaction mechanism of the first catalytic asymmetric nitroaldol reaction with the binaphthol/rare earth protocol. ...................................................................... 19 1 8 Zn catalyst developed by Trost and enantioselective nitroaldol reaction with CH3NO2. ............................................................................................................. 20 1 9 Bifunctional Cu(OAc)2 BOX catalyst for broadscope enantioselective Henry reactions developed by Evans, together with the proposed TS model. .............. 21 1 10 A quininederived bifunctional aminethiourea organocatalyst and its performance in Henry reactions. ......................................................................... 22 1 11 Hb HNL catalyzed Henry reactions of aldehydes with nitromethane ................... 22 2 1 First use of a chiral ligand in the hydrogenation of an olefin. .............................. 23 2 2 BINAPRu catalysts that are developed by Noyori. ............................................ 24 2 3 unsaturated aldehydes and ketones using whole microbial cells often shows undesired carbonyl reduction. ....................... 26 2 4 The asymmetric enzymatic reduction of the isomers neral and geranial (citral) to ( S ) citronellal by different OYEs. .................................................................... 26 2 5 Reduction of enone with P90 from Nicotiana tabacum. ...................................... 27 2 6 Reduction of enone with P44 from Nicotiana tabacum. ...................................... 27 2 7 Asymmetric bioreductions of cyclic alkenes by OPR1, OPR3 and YqjM. ........... 28 2 8 A series of 2and 3alkyl substituted cyclohexenones in the asymmetric bioreductions by OYE from S. carlsbergensis. ................................................... 29

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9 2 9 Schematic diagram of cyclohexenone OYE bioreduction. .................................. 29 2 10 Nitroalkene as a precursor to a variety of intermediates. .................................... 31 2 11 Enzymatic reduction of ( Z ) 3 phenyl 2 nitro 2 butene with YNAR I and YNARII .............................................................................................................. 31 2 12 2amino acid. ................. 32 2 13 nitroacrylates OYE bioreduction s. ................................ 32 3 1 Catalytic cycle for biocatalytic Henry reaction using old yellow enzyme. ............ 34 3 2 Synthetic route of mononitroacrylate ( 4a,b). ................................ 35 3 3 nitroacylates ( 4a,b) reduction using NaBH4 and NaBH(OAc)3 ........................ 36 3 4 nitroacylate ( 4 a, b ). ........................ 36 3 5 nitroacrylate ( 4a) and acetylaldehyde ( 9 ). .......................................................... 38 1 A GC chromatogram of 10. .................................................................................... 45 2 A GC chromatogram of 10a. .................................................................................. 46 3 A GC chromatogram of 10b. .................................................................................. 47 4 A GC chromatogram of 10c. .................................................................................. 48 1 B 1H NMR spectra of nitroalcohol ( 3 ). .................................................................... 49 2 B 1H nitroacrylate ( 4a, Z isomer). .............................................. 50 3 B 1H nitroacrylate ( 4b, E isomer). .............................................. 51 4 B 1H nitroalkane ( 5 ). ................................................................. 52 1 C Mass spectrometry of Henry product through chemical synthesis ( 10). ............. 53 2 C Another mass spectrom etry of Henry product through chemical synthesis ( 10). .................................................................................................................... 54

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science APPLICATION OF Saccharomyces carlsbergensis OLD YELLOW ENZYME IN THE ENANTIOSELECTIVE HENRY REACTION Di Zhou December 2010 Chair: Jon D. Stewart Major: Chemistry In this thesis, we studied the application of S accharomyces carlsbergensis Old Yellow Enzyme ( a kind of flavin dependent enzyme) in the nitroacrylate and acetylaldehyde. The reaction was monitored every hour until it went to completion after 12 hours, and four different stereoisomers were observed on GC MS with different ratio A control nitroalkane and acetylaldehyde was conducted and showed that the phosphate buffer solution ( KPi, 100mM, pH ~7.0/ pH~6.0) catalyzed this Henry reaction and that enzyme catalysis was not involved. Further studies should focus on the rate of carbanion protonation and the reaction between t he carbanion and acetylaldehyde, modifying the reaction media to inhibit the carbanion p rotonation, and choosing better substrates.

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11 CHAPTER 1 INTRODUCTION History of Old Yellow Enzyme The flavin dependent old yellow enzyme (OYE) of yeast was the first disco vered flavoprotein. It was is o lated f rom Brewers bottom yeast by War bu rg and Christian in 1932 during their study of biological oxidation.1 In their study, t hey tried to use methylene blue to oxidize glucose 6phosphate in the presence of Zwischenferment which is g lucose6 phosphate dehydrogenase, and a small heat stable Coferment which is NADP+. Wa r b u rg found that the addition of a yellow enzyme could allow the reaction system to form a respiratory chain when m olecular oxygen was introduced (Fig.11) .2 Figure 1 1 Reaction system of Warburg and Christian. T his yellow enzyme was later named old yellow enzyme. The reason for this peculiar name comes from an interesting history: when Wa r b u rg first discovered it, he gave it the name das gelbe Ferment After two years, another yellow enzyme was isolated from yeast by Theorell and named das neue gelbe Ferment Therefore, War burg and Christian creatively dubbed the first one old yellow enzyme, a name that has persisted to today.3

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12 Although War burg and Christian successfully isolated the old yellow enzyme, it was very impure with respect to the highmolecular constituent parts which consisted main ly of polysaccharides Theorell studied t he yellow pigment in OYE and surprisingly found that this color faded away on reduction and returned on oxidation. Therefore, he came to the conclusion that the enzymatic reaction has something to do with the yellow pigment. A fter his purification in 1955, Theorell proved that the old yellow enzyme includes two parts: the yellow part is the flavin mononucleotide (FMN), and the other part is the colorless enzyme protein (Fig 1 2).4 Figure 1 2 A ribbon diagram illustrating the structure of a dimer of O YE.

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13 The Old Yellow Enzyme Family After the first discovery of OYE, there was a growing interest among scientists in this flavin dependent enzyme and a number of OYE family member s had been found. These homologous proteins have been found in yeasts, plants and bacteria, but not in animals .5 T hrough genomic sequencing, many more uncharacterized relatives were predicted.2 Yeast Homologues of OYE In 1991, Saito cloned a gene encoding an isoform of OYE from Saccharomyces carlsbergensis, and named it OYE1. After two years another OYE from Saccharomyces cerevisiae was cloned by him and given the name OYE2. Both contain 400 amino acid residues and have very similar molecular weights.6,7 In 1995, Miranda did a sequence analysis study on a genomic DNA fragment from the yeast Kluyveromyces lactis fragment, and found a full length open reading frame (ORF) which encodes for a protein homologous to the OYE T he deduced amino ac id sequence of this ORF predicted a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevasiae.8 In addition, Buckman and Miller characterized the estrogenbinding protein ( EBP1 ) of Candida albicans and showed that it was similar in many way s to OYE in 2000.9 Plant Homologues of OYE Plant homologues of OYE were first identifi ed during studies of octadecanoid biosynthesis by Vick and Zimmerman They characterized the 12o xo phytodienoic acid reductase (OPR), which is a protein related to OYE, from the kernel and seedling of corn in 1985.10 After their discovery, several more plant strains that contain homologues of OYE were identified. F. Shaller and Weiler purified OPR to homogeneity from

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14 Corydalis sempervirens in 1997,11 and subsequently cloned the OPR1 from Arabidopsis thaliana.12 Straner and A. Shaller also isolated and characterized an OYE related enzyme LeOPR from tomato in 1999.13 Bacterial Homologues of OYE There are also some report s on bacterial enzymes with homology to OYE Pentaerythritol tetranitrate (PETN) reductase from Enterobacter cloacae PB21 4 can sequentially remove two of the four nitro groups of PETN. Agrobacterium radiobacter that contains glycerol trinitrate (GTN) reductase is also responsible for the nit rate ester degradation through catalyz ing the reductive scission of GTN to glycerol dinitrates T he difference between the two enzymes is their preference for NADPH and NADH cofactor respectively.15 Morphinone reductase produced by Pseudomonas putida M10 can catalyze the NADHdependent saturation of the carboncarbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine.16 OYE 1, OYE2, OYE3 and NAD(P)Hdependent 2 cyclohexen 1 one reductase from Zymomonas mobilis were expressed in Escherichia coli recently and used to reduce the carbon double bond in unsaturated alkenals and alkenones .17 Old Yellow Enzyme Crystal Structure and Mechanism Theorell first obtained crystal s of OYE in 195 5; however, the quality was too low for X ray studies. This was due to heterogeneity of native OYE arising from the two genes present in S. carlsbergensis .2 Fox and Karplus studied the crystal structures of oxidized and reduced form of OYE at 2 resolution.18 T hey came to the conclusion that OYE belonged to t he family which is closely related with trimethylamine de hydrogenase.18 T he overall structure of OYE is an / barrel with

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15 the flavin binding to the protein at the C ter minal end and the isoalloxazine ring perpendicular to the barrel axis. The crystal structure revealed a long solvent channel through the loops at the C terminal end, and this expos es the si face of the flavin to the solvent. By contrast, the re face is completely buried by interactions with protein main chain and side chain groups. The dimethylbenzyl ring of the flavin is solvent accessible both from the si face and along the edge, whereas the other two rings are accessible only from the si face.19 From representation of the active site of OYE in complex with p hydroxybenzaldehyde (Fig 13) it can be seen that the phenolic ring undergoes a stacking i nteraction with the fl avin (grey bonds). The phenol oxygen forms two hydrogen bonds to His 191 and Asn194, displacing a chloride ion bound in the empty oxidized enzyme structure.2 After the determination of OYE crystal structure, scientists began to investigate the catalytic properties of OYE. Masseys studies show ed that OYE could catalyze the NAD(P)Hdependent reduction of quinines. It was also effective in reducing unsaturated carbonyl compounds such as 2cyclohexenone, in which the olefinic double bond is reduced while the carbonyl functional group remains untouched.3 These reactions proved to be faster than the NAD ( P ) H oxidase reaction and are limited in rate by the reduction of enzyme flavin by NAD ( P ) H .3 The NADPH dependent reduction of OYE was found to proceed through a ping pong mechanism, in which the product and NAD(P)+ leave the enzyme before the reaction with acceptor (Figure 14). In this mechanism, the oxidized flavincontaining OYE first bind s with the cofactor NAD(P)H, then hydride transfer happens between NAD(P)H and the flavin in OYE. This forms the EFlox intermediate and releases

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16 NAD(P)+. A fter protonation from the solvent, the intermediate will convert to the reduced flavin containing OYE, and bind with a new substrate. After hydride transfer between the FMNH2 and the substrate, the reduced substrate will be released and the oxi dized flavin containing OYE goes to the next regeneration cycle. In the pingpong mechanism, o nly after the first substrate is released can another substrate bind and react with the modified enzym e, regenerating the unmodified enzyme. Figure 1 3 A cti ve site of OYE in complex with p hydroxybenzaldehyde. Figure 14 Kinetic mechanism of OYE. Since the nicotinamide cofactor NAD(P)H is expensive, an efficient coenzyme recycling system is desired in order to use the cofactor in catalytic amount. In this case, a number of dehydrogenase and reductase are used to regenerate NAD(P)H cofactors

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17 and the overall mechanism could be summarized as Fig 15 An alkene is first activated by an electron withdrawing group attached on it, then the flavin that is contained in OYE deliver s d in the presence of NAD(P)H cofactor while the cofactor itself is regenerated by adopting a hydride from the dehydrogenase. Purification of Old Yellow Enzyme The old yellow enzyme was first is olated as a homogeneous, crystalline protein by Theorell and ke son in 1956, howe ver, this method was very time consuming and the efficiency was low.20 It was later found that the enzyme activity was associated with a distinctly green fraction and that this fraction was a chargetransfe r complex formed by oxidized enzyme and a low molecular weight small molecule T he classical old yellow enzyme could be regenerated from the distinctive green complexes by dialysis of the reduced enzyme and subsequent re oxidation, the old yellow enzyme was therefore isolated as a complex.21 Later, Massey developed a simple method of old yellow enzyme purification by binding enzyme to an affinity matrix containing a phenolic substituent followed by release from the matrix by flavin reduction using sodium dithionite. This strategy was based on the finding that many phenolic compounds bound strongly to old yellow enzy me when the flavin was in the oxidized state, but not in the reduced state.22 Henry Reaction The Henry r eaction, which is also known as the nitroaldol reaction, is a basecatalyzed C C bondforming reaction between nitroalkanes and aldehydes or ketones. The significance of this reaction is that the nitroalcohol formed from this carbonyl

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18 Figure 15 Catalytic cycle for old yellow enzyme in the reduction of activated alkene. addition may be transformed into valuable building blocks23, which can be very useful in pharmaceutical and natural product synthesis24 (Fig. 16) R ecent efforts have therefore focused on the development of catalytic enantioselective reaction variants Figure 16 Transformation of nitroalcohol to different building blocks.

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19 The first example of a catalytic asymmetric Henry reaction was reported by Shibasaki In t his reaction, ( S ) ( ) binaphthol in conjunction with a lanthanum alkoxide were us ed as the reagent system, and an enantiomeric excess of 73% 90% was obtained (Fig. 1 7) .25 After the first successful trial using the chiral binaphthol/rare earth reagent system, a series of enantioselective Henry reactions were developed by Shibasaki using catalysts derived from parent binaphthol/lanthanum alkoxide.2629 In 2002, Trost reported a novel type of asymmetric zinc catalyst which involves a dinuclear zinc complex center with a chiral semi azacrown ligand (Fi g. 18). T his was the first time zinc catalyst had been applied in enantioselective Henry reaction, and this catalyst proved to be successful when applied to enantioselective, direct nitro aldol reactions involving various ketone nucleophiles and aldehyde electrophiles even when aldehyde substitutes are bulky .30 (Fig. 18). Figure 17 Reaction mechanism of the first ca talytic asymmetric nitroald ol reaction with the binaphthol/rare earth protocol

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20 Figure 18 Zn catalyst developed by Trost and enantioselective nitroaldol reaction with CH3NO2. Chiral copper complexes have also found wide applications in the catalytic Henry reactions. A notable example was developed by Evans, which involved the copper acetatebis(oxazoline) catalyzed enantioselective Henry reaction between nitromethane and aldehydes .23 This method is very general for a range of both aliphatic and aromatic aldehydes and works well under mild reaction conditions (room temperature, ethanol as solvent) A transition state model involving a Jahn Teller effect on CuII coordination and positioning of reactants in the most favorable orientations according to steric and electronic considerations has been proposed .23 Besides inorganic catalysts, research on small organic molecules in the organocatalytic Henry reactions has also a ttracted c onsiderable attention. There have been reports on using chiral guanidine species, but the enantiomeric excess obtained

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21 Figure 1 9 Bifunctional Cu(OAc)2 BOX catalyst for broadscope enantioselective Henry reactions developed by Evans, together with the proposed TS model was relatively low (20% 56%).3133 The minimum requirement for effective catalytic activity in a nitroaldol reaction was found t o involve organic molecules bearing both a thiourea and an amine residue. Hiemstra s catalyst se rve s as a good example. It catal yzed the nitroaldol reactions between nitromethane and aromatic aldehydes with 5099% yields and 8592% ee values (Fig. 110) .34 Although the stereoselective chemical catalyzed Henry reaction is relatively mature, the enzymatic catalyzed Henry reaction is still in the stage of development. The first biocatalytic asymmetric Henry reaction was not reported until 2007, when Griengl used hydroxynitrile lyase from Hevea brasiliensis to conduct the Henry reaction between aldehyde and nitromethane (Fig. 111, Table 1 1) .35 His idea was based on the fact that the hydroxynitrile lyase can not only catalyze the formation and cleavage of cyanohydrins but al so the reaction of nitroalkanes with aldehydes (Henry reaction)

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22 Figure 11 0 A quinine derived bifunctional aminethiourea organocatalyst and its performance in Henry reactions Figure 11 1 Hb HNL catalyzed Henry reactions of aldehydes with nitromethane Table 11. Hb HNL catalyzed Henry reactions of aldehydes with nitromethane R pH 7.0 Yield (%) pH 7.0 ee (%) pH 5.5 Yield (%) pH 5.5 ee (%) a. Ph 63 92 32 97 b. 3 HOC 6 H 4 46 18 c. 4 NO 2 C 6 H 4 77 28 57 64 d. n hexyl 25 89 34 96 e. Ph(CH 2 ) 2 9 66 13 66 f. 2 furyl 57 72 43 88 g. CH 3 (CH 2 ) 4 CHCH 3 2 88 h. thienyl 29 98 i. 3 furyl 16 89 j. 2 ClC 6 H 4 23 95 k. 3 ClC 6 H 4 36 98 l. 4 ClC 6 H 4 25 97 m. 4 MeOC 6 H 4 20 99 n. cyclohexyl 18 99

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23 CHAPTER 2 OLD YELLOW ENZYME IN ASYMMETRIC C=C BOND BIORED U CTIONS Asymmetric C=C Bond Reductions A symmetric C=C bond reductions offer especially attractive routes to chiral building blocks since up to two adjacent stereocenters could be established by a single reaction. This technique is of great importance due to the increasing demand to produce enantiomerically pure p harmaceuticals agrochemicals, flavors, and other fine chemicals .3 6 The start of the development of catalysts for asymmetric hydrogenation was the concept of replacing the triphenylphosphi ne ligand of the Wilkinson catalyst with a chiral ligand.37 Knowles and Horner reported the earliest examples of enantioselective hydrogenation, however the optical purity was very low (Fig 21).38,39 Figure 21. First use of a chiral ligand in the hydrogenation of an olefin. After the initiation of asymmetric hydrogenation, studies on making chiral ligands to control the enantioselectivity began to attract scientists interest. A notable work was Noyoris research on BINAP Ru catalysts for asymmetric hydrogenation, which opened up opportunities for efficient hy drogenations of a variety of substrates The enantiomeric

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24 excess could reach up to 100% in the presence of the BINAP Ru catalysts (Fig 22).40,41 His distinguished work on chirally catalyzed hydrogenation reactions won him the Nobel Prize in chemistry in 2001. Figure 22 BINAPRu catalysts that are developed by Noyori. Despite of the tremendous development in this area, high stereoselectivity nearly always depends on the polarity of the functional group that is attached to the alkene. H igh ly polar groups such as amides, acid s and alcohol s can lead to higher stereoselectivity while l ess polar groups such as ketones, esters or nitro groups usually result in lower enantiomeri c excess values. In addition, because complex chiral ligands for chemical reduction are usual ly required, they are expensive, toxic and difficult to

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25 make. Also, h igh hydrogen pressures are often required for the optimal results These drawbacks have prompted chemists to explore biocatalytic alternatives and enzymatic reduction proved to be a useful solution to this problem especially the whole cell mediated reductions.42 45 Asymmetric Unsaturated Carbonyl Bio r eductions The use of whole cell s for alkene reductions avoids the need for external cofactor recycling. A lthough the stereoselectivities achieved were often excellent, chemoselectivities of whole cell bioreductions with respect to C=C versus C=O bond reduction are often poor, which is due to the presence of competing alcohol d ehydrogenase. As a result the selective asymmetric bioreduction of conjugated enals or enones to furnish the corresponding saturated aldehydes or ketones is severely impeded by side reactions (Fig 23).46 Finally the overall reduction yield is low because native cofactor regeneration pathways in whole cells are often inefficient All of these drawbacks could be avoided by using flavincontaining old yellow enzyme with suitable dehydrogenase in the pres ence of nicot inamide cofactors.47 T he asymmetric synthesis of citral is a challenging task in terms of chemo, regio and stereoselectivity. Therefore, this reaction was chosen to investigate the potential of the flavin dependent OYE in chemical synthesis Citral, a mi xture of the geometric isomers geranial (trans isomer ) and neral (cis isomer ), is an important chiral building block Citral is reduced by the OYE from Gluconobacter oxy dans ,48 OPR1 and OPR3 from Lycopersicon esculentum (tomato), and YqjM from Bacillus subtilis into citronellal (Fig 24).47 T he reduction was performed with high regioand chemoselectivity, only the activated C=C bond was reduced while the other isolated C=C bound and the carbonyl moiety stayed untouched. Also, the reaction conversion reached up to 100%, and for

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26 the stereoselectivity of the product the enzymes produced the ( S ) citronella l with an excellent ee of >99%. Figure 23. Asymmetric bioreduction of unsaturated aldehydes and ketones using whole microbial cells often shows undesired carbonyl reduction. Figure 24. The asymmetric enzymatic reduction of the isomers neral and geranial (citral) to ( S ) citronellal by different OYEs. Asymmetric Cyclohexenone Bio r eductions Cyclohexenone is an organic compound which is a versatile intermediate used in the synthesis of a variety of chemical products such as phar maceuticals and fragrances

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27 When or position of cyclohexenone is substituted, C=C bond reduction lead s to chiral cyclohexanones, which are one of the most interesting synthons due to their applications in the production of biologically active substances. Up to now, both chemical and biochemical reductions for the cyclohexenone have been developed. One is the c onjugate a dditions of o rganometallic reagent to cycloalkenone, the other is the asymmetric conjugate reduction of conjugate enones. Also e nzymatic reduction using P90 and P44 reductases from Nicotiana tabacum demonstrates that this enzyme can enantiotropically reduce the C= C double bond of enones to afford optically active 2 alkylated cycl ic ketones with high enantiomeric excess ( Fig 25, Table 21, Table 22 ) .49 Figure 2 5 Reduction of enone with P90 from Nicotiana tabacum Figure 2 6 Reduction of enone with P44 from Nicotiana tabacum Table 21. Conversion and ee value for reduction of different enone with P90 R conv.% ee% Me 95 99 Et 57 98 n Pr 35 95 Table 22 Conversion and ee value for reduction of different enone with P44 R conv.% ee% Me 80 > 99 Et 45 > 9 9 n Pr 3 7 > 9 9

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28 A variety of flavin containing old yellow enzyme catalyzed bioreduction for cycl ic ketones have also been studied, and the catalytic mechanism has been investigated in great detail Faber used OPR1, OPR3, YqjM to reduce substituted cyclopentenone and cyclohexenone, and found that the ring size of the substrate had a tremendous effect on the stereochemical outcome of the bioreduction (Fig 27, Table 23) .47 However, this transformation only applied to the substituted compounds. F or the corresponding substituted cycloalkenones conversions were not observed for both substrates using OPR1 and YqjM. OPR3 showed low activities, but with excellent stereoselectivity Stewart used OYE of Saccharomyces carlsbergensis expressed in Escherichia coli cells and conducted the chemoand stereoselective alkene reductions using a series of both and substituted 2cyclohexenones ( Fig 28) T he proposed mechanism includes an anti addition of H2, h ydrogen bonds contributed by H is 191 and Asn 194 position activate the carbonyl oxygen, then hydride transfer from the flavin with concomitant protonation by the sidechain of Tyr 196 (Fig 29) .50 C hemoand stereoselectivity are generally excellent and the product configuration depends on substituted pattern of the 2cyclohexenone ( Table 24) Figure 27 Asymmetric bioreductions of cyclic alkenes by OPR1, OPR3 and YqjM. Table 2 3 Asymmetric bioreduction of substituted cyclopetenone and cyclohexenone using OPR1, OPR3 and YqjM. Entry Cofactor OPR1 OPR3 YqjM Conv.% ee% Conv.% ee% Conv.% ee% 1 NADP + /G6PDH 14 (S)61 10 (S)58 72 (S)94 2 NADP + /G6DPH 82 (R)66 92 (R)71 95 (R)93

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29 Figure 28 A series of 2and 3alkyl substituted cyclohexenones in the asymmetric bioreductions by OYE from S. carlsbergensis Table 2 4 Reductions of alkyl substituted 2cyclohexenones by engineered E. coli cells overexpressing S. carlsbergensis old yellow enzym e. ketone R Conversion(%) ee(%) Configuration a Me 100 94 S b Et 76 94 S c n Pr 25 89 S d i Pr 18 90 S e n Bu f Me 100 96 R g Et 16 90 R Figure 2 9 Schematic diagram of cyclohexenon e OYE bioreduction.

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30 Asymmetric Nitroa lkene Bio r eductions Optically active nitroalkanes are valuable building blocks in synthetic chemistry; they could be easily converted into chiral amines, which are very useful compound in organic synthesis,51 they are valuable precursors to a variety of intermediates, some of which are summarized in Fig. 210. Commonly used reduction methods for nitro alkene include borohydride derivative based reduction,52 catalytic transfer hydrogenation53 and metal halides based reduction.54 However, the selective reduction of the conjugated double bond is rather difficult because nitrogroup reduction often takes place simultaneously S odium borohydride is an effective reductant but the reaction is often accompanied by the formation of polymeric side products through Michael addition of the nitronate intermediate to the starting nitroalkene. Metal catalyzed hydrogenations and transfer hydrogenation are also very effective, but disposal of the toxic metal ions aroused another difficulty.55 Whole cell bioreduction using bakers yeast can avoid the drawbacks. Kawai used two different nitroalkene reductases YNARI and YNARII that were isolated from bakers yeast to reduce a trisubstituted nitroalkene, and the products showed excelle nt enantioselectivity, moderate diastereoselectivity, and good yield (Fig. 2 11) .56 Nitroacrylates are a class of electronpoor nitroalkenes having two electronand positions. This peculiarity makes their chemistry behavior more interesting with respect to the classical conjugated nitroalkenes The flavin containing old yellow enzyme with the NADPH cofactor recycling system was 2amino acids. These enzymatic reductions occur with 8796% ee, with larger substrate providing greater

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31 Figure 2 10 Nitroalkene as a precursor to a variety of intermediates. Figure 2 11 Enzymatic reduction of ( Z ) 3 phenyl 2 nitro 2 butene with YNAR I and YNARIIa Table 2 5 Enzymatic reduction of ( Z ) 3 phenyl 2 nitro 2 butene with YNARI and YNARIIa Enzyme Coenzyme Relative activity d.e. b (%) e.e. c (%) e.e. d (%) YNAR I NAD P H 1.00 31 >98 97 NADH 0.60 29 >98 97 YNAR II NAD P H 1.00 35 >98 97 NADH 0.54 34 >98 97 a Condition: acetate buffer, 100mM, pH =5.0. b Excess of 2 over 3. c e.e. of 2 d e.e. of 3 stereoselectivities (Fig 212, Table 26) .57 A m echanism for the reduction was also propos ed. Compared with 2 cyclohexenone, one nitro oxygen occupies the same

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32 location as the carbonyl oxygen and the alkene is positioned similarly. H ydrogen bonds contributed by His 191 and Asn 194 position activate the nitro oxygen, then hydride transfer happens from the flavin with concomitant protonation by the sidechain of Tyr 196, the net result is that H2 trans addition was achieved (Fig 213) .57 Figure 21 2 2amino acid. Table 26 Reductions of nitroacrylates by S. carlsbergensis old yellow enzym e. R1 R2 Starting Cmpd Conv. after 8h(%) e.e. c (%) Config. a Me H ( E ) a >98 8 R b Et H ( E ) b 50 c H Me ( Z ) c >98 98 R d H Et ( Z ) d >98 91 R e H n Pr ( Z ) e >98 94 R f H i Pr ( Z ) f >98 96 R Figure 21 3 nitroacrylates OYE bioreductions.

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33 CHAPTER 3 OYE: A POTENTIAL CATALYST FOR ENANTIO SELECTIVE HENRY REACTION Introduction The addition reaction between nitroalkanes and carbonyl compounds to yield a nitroalcohol, namely the nitroaldol or Henry reaction, it provides chemists a powerful strategy for C C bond formation. In addition, the nitro alcohol products obtain ed from Henry reaction could be transformed into many important derivatives, such as niroalkene, amino alcohol, amino acids etc. Although significant success has been achieved in asymmetric version s of the Henry reaction, control of the stereoselectivity still remains difficult. The use of chiral metal catalysts has proved to be effective in the stereocontrol, but the long reaction time s, the cost of the complex catalysts, and their disposal has prompt ed chemists to find simple r and environmental friendly w ay s. Based on the mechanis m of the Henry reaction, as well as the asymmetric bioreduction of nitroalkene s, we hypothesized that the same enzymatic catalysis could also be applied to Henry reaction. S ince the nitronate carbanion intermediate formed after the hydride has a long life time, if we add a small carbonyl compound like acetylaldehyde, nucleophilic attack might happen to the acetylaldehyde with in the OYE, with a subsequent protonation by the solvent By contrast, the nitronate carbanion protonation should be inhibited. The Henry reaction product is interesting because it contains three different chiral centers, we can see if the formation of one chiral center influences the chirality of another, also, due to the fact that enzymatic reduction is highly selective, we could see whether OYE catalyzed Henry reaction can improve the stereoselectivity or not. Fig. 31 shows the catalytic cycle for biocatalytic Henry reaction using old yellow enzyme.

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34 Figure 31 Catalytic cycle for biocatalytic Henry reaction using old yellow enzyme. Results and Discussions To test the hypothesis we first needed to prepare a nitro acrylic ester. After comparing several synthetic methods, we decided to follow Ballinis protocol since this method avoids drastic conditions, low yields and low selectivity (Fig 32 ).58 The first step o f the sequence is a nitroaldol Henry reaction between nitromethane and ethyl pyruvate, performed under heterogeneous catalysis using ion exchange resin ( Amberlyst A 21) The reaction proceeds under room temperature without the protection of argon, and the yield after purification can vary from 64% 8 8 %. NMR spectra of the nitroaldol product

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35 matched the previous reports.58 This alcohol wa s further converted to the dehydration produc nitroacrylate through mesylation and elimination.59 Since this step usually nitroacrylate, we explored the reaction conditions, including temperature s of 70oC 20oC, 0oC along wit h a variety of reactant stoichiomet ries. The optimal reaction condition proved to be nitroalcohol, MsCl and Et3N with a 1:3:3 ratio under 0oC for 2 4 hours (Table 31) Figure 32 Synthetic route of mononitroacrylate ( 4a,b) Table 31. nitroacylate ( 4a,b) Starting Material Reagent Ratio (SM:MsCl:Et3N) Temp Rxn Time Total Yield% Yield( E )% Yield( Z )% Alcohol MsCl,Et 3 N 1:3:3 0 o C 2 4h 53.8 4.2 49.6 Alcohol MsCl,Et 3 N 1:3:3 20 o C 2 4h 3 2.1 5.5 26.6 Alcohol MsCl,Et 3 N 1:3:3 70 o C 2 4h 49.2 4.8 44.4 nitroacrylate is a necess ary step in making the standard. W e have used NaBH4 as a reducing agent to make the nitroalkane but the ester group w as overreduced at the same time. A fter purification, the yield of the nitroalkane was only 20% 32%. A milder reducing agent NaBH(OAc)3 was substituted. Unfortunately there was no significant improvement and the yield of the nitroalkane was

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36 23% 30% (Fig 33 ). The organocatal ytic transfer hydrogenation using Hantzsch ester as the hydride source and thiourea catalyst can effectively increase the yield (up to 77% in our case, Fig. 3 4 ),60,61 but purification was difficult since t he pyridine by product and the thiourea catalyst were always detected in the purified product. After weighing the advantages and disadvantages of different reducing agents, NaBH4 was fin ally chosen due to its low cost, product purity and the facile reaction process Figure 33 nitroacylates ( 4a,b) reduction using NaBH4 and NaBH(OAc)3 Figure 34 O rganocatal nitroacylate ( 4a, b ) The last step to make the standard is the Henry reaction between the nitroalkane and aldehyde. A cetyla l d e hyde was chosen because bigger molecule might

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37 impede the enzymatic reduction that occurs in the enzyme. Several bases were used to deprotonate the acidic proton, and the addition of 4 molecular sieve was helpful but the ion exchange resin (Amberlyst A 21) proved to be the best choice (Table 32) G as chromatographyMass spectrometry demonstrated the existence of four stereoisomers in the product and they are proved to be the Henry reaction products ( 10) T here is also one impurity among th ese peaks which we were unable to remove. The ratio of eac h isomer is #1: 20.5%, #2: 13.9%, #3: 23.2% and #4: 42.4% individually. The total conversion of this reaction was 48.2%. Table 32 Different conditions of Henry reaction between nitroalkane ( 5 ) and acetylaldehyde ( 9 ) Reagent Solvent Temp. Rxn Time Yield% TBAF THF rt 10h No reaction NaOEt EtOH 0 o C/r t 4h No reaction Et 3 N, MS 4 CH 2 Cl 2 0 o C/rt 6 10h 41.7% Et 3 N, MS 4 Solvent free 0 o C/r t overnight 54.3% Amberlyst A 21 Solvent free 5 o C overnight 81.3% The enzymatic reduction was conducted using flavincontaining w ell t ype OYE1 in the presence of NAD P H cofactor and a glucosebased regeneration system in KPi 100mM, pH ~7 .0 The reaction was monitored by GC MS every hour until it went to completion after 12 hours ( 10a) four stereoisomers were observed on GC with conversion of 48.1% t he ratio of each stereoisomer is: #1: 23.7%, #2: 2.6%, #3: 21.0% and #4: 52.8%. To see if the reaction was also catalyzed by buffer, nitroalkane nitroacrylat e was used in the same reaction. T he same four stereoisomers were still observed on GC with a conversion of 53.3% ( 10b ) and the ratio of each isomer is: #1: 1 8 4 %, #2: 4 7 %, #3: 2 1 2 % and #4: 55. 7 %. Then, a reaction medium without enzyme and regeneration s nitroacrylate and acetylaldehyde was dissolved directly into the buffer and the reaction was monitored,

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38 after 12 hours, four stereoisomers were observed again ( 10c) and the ratio is: #1: 18.1%, #2: 3.3%, #3: 22.5% and #4: 56.1%, with a total conversion of 48.2%. Based on these result s summarized in Table 33 it appears that the Henry reaction w as ac tually catalyzed by the buffer. OYE reduced nitroacrylate and formed a carbanion, which was protonated. After release from the active site, the subsequent Henry reaction occurred in solution. To testify this explanation, we carried out the same reaction, with all conditions the same except that it was conducted at pH 6.0. No Henry product was nitroacrylate and acetyl aldehyde were used. By contrast, nitroalkane and acetylaldehyde, the Henry product was detected, but at much lower conversion (15.7%) Therefore, we came to the conclusion that this Henry reaction was catalyzed by buffer instead of OYE 1 Presumably protonation of the carbanion is faster than the nucleophilic attack Further work might focus on the studies on the rate of carbanion protonation and nucleophilic attack optimization of reaction media and exploration of other substrates. Figure 35 Reaction and m ni troacrylate ( 4a) and acetylaldehyde ( 9 )

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39 Table 3 3 Conversion and product percentage of chemical and enzymatic Henry reaction products Reagent KP i 100mM, PH~7.0 OYE1, GDH, NADP + Conv. % Product Percentage No, chemical synthesis No, Amberlyst A 21 48.2 Yes Yes 48.1 Yes Yes No Yes No 53.3 Yes No 48.2 Experimental Reagents and Instrumentations S olvents were purchased from Fisher Scientific and dichloromethane and tetrahydrofuran were dried on an MBRAUN solvent purification system using a double 4.8 L ac tivated alumina columns type A2. Triethylamine was distilled from CaH2 under atmospheric pressure and methanesulfonyl chloride was distilled from CaH2 under vacuum. Most of the reactions were performed under argon, except for those that are not sensitive to water and air. Reactions were monitored by thin layer chromatography ( Whatman, with fluorescent indicator ) and gas chromatography (DB17 column, 0.25 mm x 25m x 0.25 m thickness) with mass spectrometric detection, products were

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40 purified by column chromatography (Purasil silica gel 230 400 mech, Whatman) NMR spectra were measured in CDCl3 solution and recorded at room temperature on a Varian Gemini 300 spectrometer operating at 300 MHz for 1H ppm) reported relative to tetramethylsilane. Infrared Radiation (IR) spectra were obtained from Perkin Elme r Spectrum One FTIR spectrophotometer. Mass spectrometry were run on ThermoFinnigan (San Jose, CA) LCQ with electrospray ionization (ESI). S ynthesis of Nitro a lcohol (3). In a 50 m L 2 necked flask equipped with a mechanical stirrer, 60mmol (3.66 g) nitromethane was charged and cooled with ice bath. Amberlyst A 21 57 g was added and the mixture was stirred for 10 minutes, t hen 60 mmol ( 6.97 g ) ethyl pyruvate was added. The reaction temperature was raised to room temperature and the mixture was stirre d overnight. The reaction mixture was filtered and the Amberlyst resin was washed with CH2Cl2 (4 x 25 mL ), then the solvent was evaporated in vacuo to yield nitroalcohol. The crude product was purified by flash chromatography. (ethyl acetate: hexane= 1:3, yield : 6488% ) NMR data was consistent with Ballinis report. 58 1H NMR (CDCl3): 4.83 (d, J=6 Hz 1H), 4.54 (d, J=6 Hz 1H), 4.35 (q, J=6 Hz, 2 H), 3.73 (s, 1H), 1.46 (s, 3H), 1.34 ( t J =6 Hz, 3H ) ppm. Nitroacrylate (4a, b). The method of McMurry s was used with som e modifications.59 nitroalcohol ( 3 ) ( 17 mmol, 3.0 g ) was dissolved in 17 mL CH2Cl2 at 0oC under argon atmosphere. Dry methanesulfony l chloride (51 mmol, 3.95 mL ) was added dropwise, and then the mixture was stirred under 0oC for 2 hours. D ry triethylamine (51 mmol, 7.1 mL) wa s added to the mixture dropwise and the reaction mixture was stirred for another 2 hours

PAGE 41

41 at 0oC. The reaction mixture was then transferred to a separ a tory funnel with the aid of 40 mL CH2Cl2, and then washed with water, 5% HCl, and brine. The mixture was dried over anhydrous MgSO4 and gravity filtered. After concentration at the rotary evaporator, the crude product was purified by flash chromatography. (ethyl acetate: hexane=1:10) NMR data was c onsistent with Stewarts report.57 ( Z ) isomer 1H NMR (CDCl3): (q, J=3 Hz, 1H), 4.35 (q, J=6 Hz, 2H), 2.11 (d, J=3 Hz, 3H), 1.35 (t, J=6 Hz, 3H) ppm. ( E ) isomer 1H NMR (CDCl3): J=3 Hz 1H ), 4.31 (q, J=6 Hz, 2H), 2.32 (d, J= 3 Hz, 3H) 1.36 (t, J= 6 Hz, 3H) ppm. Synthesis of Nitroalkane (5). The method of Lists was used with some modifications.61 Reduction with NaBH4: nitroacrylate ( 4a,b) (2.67 mmol, 0.425 g ) in ethanol (0.6 mL) at 0C, NaBH4 ( 4.0 mmol, 0 .151 g, 3.0 equiv) was added in one portion. The reaction mixture was stirred at 0C for 1h and then treated at 0C with a saturated aqueous NH4Cl solution. The two phases were separated and the aqueous phase extracted with Et OAc The combined organic phases were dried over anhydrous MgSO4. The volatile compounds were removed in vacuo and the crude product was purifi ed by flash chromatography (15 % Et2O in hex ane, yield : 2028% ) R eduction with Hantzsch ester nitroacrylate ( 4a,b) ( 1.98 mmol 0.315 g) in toluene ( 1.6 mL), thiourea catalyst (0.396 mmol, 0.186 g, 0.2 equiv) and Hantzsch ester (0.6 g, 2.38 mmol, 1.2 equiv) were added. The reaction mixture was stirred at 40C for 2448 h under argon atmosphere until completion of the reaction. The solvent was removed in vacuo and the resulting mixture purified by flash column chromatography (15 % Et2O in hex ane, yi eld : 6277% ). NMR data was consistent with Lists report.61 1H NMR (CDCl3): J= 15 and 9 Hz 1H ), 4.42 (dd, J=15 and 9

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42 Hz, 1H), 4.19 (q, J=6 Hz, 2H), 3.25 (m, 1H), 1.28 (t, J =6 Hz, 3H), 1.30 (d, J= 9 Hz, 3H) ppm. Synthesis of Thiourea Catalyst (8). The method of making Schreiners catalyst was used.62 In a dry 100 ml roundbottom flask, a mixture of 3,5bis(trifluoromethyl)aniline (10 mmol, 2.34 g, 1,59 mL) and triethylamine (11.9 mmol, 1.66 mL ) in THF (72 mL ) was prepared. Under Argon atmosphere, a mixture of thiophosgene ( 4.3 mmol, 0.33 mL ) in THF (7 mL) was added dropwise to the stirred solution at 5oC 0oC. After additio n, the yellow suspension was allowed to stir at room temperature for 24 hours. After the reaction, the bulk of the solvent was removed in a rotary evaporator, the concentrated blown color residue was added to 45 mL water and the aqueous layer was extracted with ether (2 x 15 mL). The combined organic layers were washed with brine (1 x 10 mL ), and dried over anhydrous MgSO4. After filtration and evaporation of the solvent. The red brown solid crude product was purified by recrystallization from chloroform once, and the resulting slightly yellow solid was dissolved in ether to be reprecipitated by addition of hexane as newly white solid that was dried over Sicapent in a dessicator. (yield : 35% ). Melting Point, IR and MS data were consistent with Schreiners r eport.62 MP : 170171oC; IR (KBr) : 3205, 3048, 2985, 1550, 1464, 1371 1326, 1287 1180, 1131, 932, 887, 711, 698.; HRMS calcd for [ C17H8N2SF12] : 500.02; found [ C17H8N2SF12+H+] : 501.0210. Synthesis of Ethyl 4 H ydroxy 2 M ethyl 3 N itropentanoate (10). In a 5 mL tip bottom flask, 0.68 mmol nitro alk ane ( 5 ) was charged and cooled with ice bath. Amberlyst A 21 0.0 5 0.07 g was added and the mixture was stirred for 10 minutes, then 2.0 mmol acetylaldehyde ( 9 ) was added. The reaction temperature was

PAGE 43

43 raised up to 5oC and stirred overnight, the reaction mixture was filtered and the Amberlyst resin was washed with CH2Cl2 (4 x 2 mL ), then the solvent was evaporated in vacuo to yield the desired product ( yield : 8098% ) ( HRMS: calcd for [C8H5NO5]: 205.10 and found [ C8H5NO5+H+] : 206.1013. GC MS showed the existence of four different stereoisomers with reasonable fragmentations.) Enzymatic Reduction N itroacrylate Using Old Yellow Enzyme A reaction mixture contained 25 mM nitroacrylate ( 4a) 25 mM acetyladehyde (9) NADP+ (4 M, 3 mg), glucose (0.254 mmol, 85.8 mg), glucose dehydrogenase (100 g), and purified OYE1(5 mg) in a total volume of 10 mL of 100 mM KPi pH 7.0. The mixture contained 5% isopropanol in order to increase substrates solubility. The reaction mixture were separated in 10 centrifuge tubes (1.5 mL) with 1 mL each, and left on a rotisserie under 30oC. Samples were extracted every hour with EtO Ac (3 x 0.5 mL), and the combined organic layer s were washed with brine, water, dried over MgSO4, and concentrated in vacuo. The sample was diluted with EtO Ac for GC MS analysis.

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44 CHAPTER 4 CONCLUSION AND FUTUR E WORK The flavincontaining old yellow enzyme was used in the Henry reaction between nitroacrylate and acetylaldehyde in the presence of NADP+ cofactor and glucose regeneration system. Because the oxygen on nitro group could form hydrogen bond with His 191 and Asn 194, as well as the requirement that the carbon lies above N5 of the flavin, reduced FMN always deliver s h ydride to the re face of the bound nitroacrylate, and form a reactive nitronate carbanion. We expected this carbanion could proceed through Henry reaction when acetylaldehyde was introduced and achieve a high stereoselectivity however, protonation by the solvent derived hydrogen always seems to be predominant, with a subsequent buffer catalyzed Henry reaction. Future work might be focused on t he kinetic study on the rate of carbanion protonation and reaction between carbanion and acetylaldehyde, modifying the reaction media to inhibit the carbanion protonation, and choosing bet ter substrates.

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45 APPENDIX A GC ANALYSIS OF HENRY REACTION PRODUCTS Figure 1A GC chromatogram of 10.

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46 Figure 2A GC chromatogram of 10a.

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47 Figure 3A GC chromatogram of 10b.

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48 Figure 4A GC chromatogram of 1 0 c

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49 APPENDIX B 1H NMR SPECTRA OF NITRO NITROACRYLATE AND NI TROALKANE Figure 1 B 1H NMR spectra of nitroalcohol ( 3 )

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50 Figure 2B 1H nitroacrylate ( 4a, Z isomer).

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51 Figure 3B 1H nitroacrylate ( 4b, E isomer).

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52 Figure 4B 1H NMR spectra of nitroalkane ( 5 ).

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53 APPENDIX C MASS SPECT R OMETR Y OF THE HENRY REACT ION PRODUCT Figure 1C Mass spectrometry of Henry product through chemical synthesis ( 10)

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54 Figure 2C Another m ass spectrometry of Henry product through chemical synthesis ( 10)

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58 BIOGRAPHICAL SKETCH Di Zhou was born and brought up in Beijing, China. In 2004, he attended Beihang University (formerly Beijing University of Aeron autics and Astronautics) for his undergraduate studies in chemistry. After graduation in 2008, he moved to University of Florida for graduate studies and joined the Stewarts group. His research focused on the organic and biocatalytic chemistry.