a... 4.. is. 33”.) K. 551“ a: .3. .3 .1 .. .5 alu‘. fining,“ . 707 .5 .thzii. g1) . x. {iihnnnuz €‘3!‘o’» A x... : :1!!! ., I... 1...... C 5!“ 5.11.1”... 51. {3‘12}... .rG‘sxzxssv 1v 11.1w ' 2 LIBRARY 5 O 5'; Michigan State ‘- University This is to certify that the dissertation entitled SYNTHESES OF D,L-1,2,4-BUTANETRIOL AND e-CAPROLACTAM FROM D-GLUCOSE-DERIVED STARTING MATERIALS presented by MAPITSO N MOLEFE has been accepted towards fulfillment of the requirements for the DOCTORAL degree in CHEMISTRY QMM; Major Professor’ Signature {/41 as Date MSU is an Affirmative Action/Equal Opportunity Institution u—U- .--.-.-.-.-I-O--‘O-.-I-0-0--"0—C-O—O—O-D-D-n-O-I. PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/CIRC/DateDue.indd—p.1 SYNTHESES OF D,L-1,2,4-BUTANETRIOL AND e-CAPROLACTAM FROM D-GLUCOSE-DERIVED STARTING MATERIALS By Mapitso N. Molefe A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 2005 ABSTRACT SYNTHESES OF D,L-1,2,4-BUTANETRIOL AND e—CAPROLACTAM FROM D—GLUCOSE—DERIVED STARTING MATERIALS By Mapitso N. Molefe Syntheses of D,L-1,2,4-butanetriol and e-caprolactam, from renewable and abundant starting materials using chemical catalysis and biocatalysis are described. The current syntheses of these chemicals rely on the use of stoichiometric reagents or use of non-renewable starting materials. For the synthesis of D,L—1,2,4-butanetriol, two routes involving catalytic hydrogenation as a common step are described. In the first route, D, L-malic acid as well as L-malic acid were hydrogenated over Ru on carbon to afford D,L- l,2,4-butanetriol in 74% yield as part of product mixture. Variation of hydrogen pressure, reaction temperature, time, concentration, catalysts and catalyst loading were evaluated to find optimum reaction conditions. The major contaminants produced were 1,2-propanediol (12%) and 1,4-butanediol (8%). 1,2-Propanediol was shown to result from C—C bond cleavage of D,L-l,2,4-butanetriol whereas 1,4—butanediol resulted from dehydration of 3,4-dihydroxybutyric acid or the corresponding 3-hydroxy-y- butyrolactone followed by hydrogenation. Difficulties associated with purifying D,L- l,2,4-butanetriol from contaminating diols led to the development of an alternative route. In the second route, 2-hydroxy-2—buten-4-olide was hydrogenated to D,L-l,2,4-butanetriol in 96% yield, without accumulation of byproducts. This substrate was obtained from oxidative cleavage of L—ascorbic acid to L-threonate. Dehydration of L-threonate to 4- hydroxy-Z-ketobutyrate was catalyzed by dihydroxy-acid dehydratase. Finally, 4- hydroxy-2~ketobutyrate was cyclized to 2—hydroxy-2—buten-4-olide, which was hydrogenated to afford D,L-l,2,4—butanetriol in 53% overall yield. s-Caprolactam, a monomer for nylon 6 was synthesized from L-lysine. Several routes for deamination of the a-amino group of L-lysine were explored. The strategy involved cyclization of L-lysine followed by deamination of a-aminocaprolactam. Alternatively, e-caprolactam was obtained by deamination of L-lysine followed by cyclization of 6-aminocaproic acid. Catalytic deamination over Pt on Si02 and Mo-Ni-S on A1203 catalysts were attempted to deaminate a-aminocaprolactam. Partial deamination was observed when a-aminocaprolactam was subjected to reduction with sodium metal in 2-propanol (20%). Alternatively, deamination of an isomer of L-lysine, L-B-lysine, was attempted. L-B—Lysine is the first intermediate in the biodegradation of L- lysine by some Clostridium species under anaerobic conditions. L-Lysine-2,3- aminomutase (LAM) catalyzes the conversion. Intact cells and crude extracts of LAM were explored for the isomerization of L-lysine. The resulting product was cyclized to [3- aminocaprolactam and deaminated to s-caprolactam. Copyright by Mapitso N. Molefe 2005 To my family For their constant love and support. ACKNOWLEDGMENTS First and foremost, I would like to thank my supervisor John W. Frost for allowing me to be part of his research group. His passion about scientific research and his vision was certainly inspiring. I admire his dedication and am very grateful for his patience in guiding me throughout my graduate studies. I would also like to thank the members of my graduate committee, Prof. James Jackson, Prof. James Geiger, and Prof. Kris Bergland for their input during the preparation of this thesis. Special thanks to Prof. Babak Borhan and Prof Robert Maleczka, Jr for their help. I am very grateful to Dr Karen Frost for her very invaluable suggestions and advise. I will miss Carolyn Wemple for her kindness and her warmth. My deepest gratitude to the Frost group members, past and present, for their much needed support throughout my time in the group including Dr Sunil Chandran, Dr Jessica Barker, Dr Chad Hansen, Dr Padmesh Venkitasubramanian, Dr Jian Yi, Dr Ningqing Ran, Dr J iantao Gao, Dr Wei Niu, Dr Dongming Xi, Wensheng Li, Xiaofei Jia, Stephen Sing Lee, and Partha Nandi. I am especially grateful to Dr Jihane Achkar, Justas Jancauskas, Man Kit Lau, Heather Steuben, J insong Yang, and Brad Cox whose friendship I will treasure. Finally, I would like to thank my whole clan for their support throughout my graduate studies. I am especially indebted to my parents, brothers and little sister whose love and their constant encouragement throughout my studies kept me going. I owe my sanity, if any, to my friends, Pumza, Kaizer, Phumi, Sindi, Montse, Glenn, Edith, Chrysoula, and Tamiika, who made me laugh whenever I needed to. I dedicate this thesis to the memory of my cousins, Nomakha and Vulindlela, and to a friend Nolwazi. vi TABLE OF CONTENTS LIST OF TABLES ................................................................................................ viii LIST OF FIGURES ................................................................................................ ix LIST OF ABBREVIATIONS ............................................................................... xiii CHAPTER ONE ............................................................................................................. 1 INTRODUCTION ........................................................................................................... l Biomass-derived building blocks ............................................................................. 2 (a) 3-Hydroxypropanoic acid ............................................................................. 5 (b) 2,5—Furandicarboxylic acid ........................................................................... 6 (c) Levulinic acid ............................................................................................... 8 (d) D-Sorbitol ................................................................................................... 10 (e) L-Malic acid ............................................................................................... 11 (f) L-Aspartic acid ............................................................................................ l3 Aromatic Compounds ............................................................................................ 14 The Shikimate Pathway ......................................................................................... 15 (a) Phenol and p-hydroxybenzoic acid from shikimic acid ............................... 17 (b) Vanillin from 3-dehydroshikimic acid ........................................................ 18 (c) Gallic acid and pyrogallol ........................................................................... 20 (d) Catechol and adi pic acid ............................................................................. 22 (e) Hydroquinone from quinic acid .................................................................. 23 Aromatics from non-Shikimate Pathway ................................................................ 25 References ............................................................................................................. 25 CHAPTER TWO .......................................................................................................... 35 SYNTHESES OF D,L-1,2,4—BUTANETRIOL ............................................................... 35 Introduction ............................................................................................................... 35 Catalytic Hydrogenation of Malic Acid. .................................................................... 41 Alternative Substrate for Catalytic Hydrogenation ..................................................... 46 2-Hydroxy-y-butyrolactone 10 ................................................................................... 47 4-Hydroxy-2-ketobutyrate 11 .................................................................................... 49 D-Erythronolactone 14a and L-threonolactone 14b. ................................................... 51 2-Hydroxy-2buten-4—olide 112 from D-eythronolactone 14a ........................................ 52 Chemoenzymatic Synthesis from L—Ascorbic acid. .................................................... 53 Discussion ................................................................................................................. 60 References ................................................................................................................. 69 CHAPTER THREE ....................................................................................................... 73 CONVERSION OF L-LYSINE TO e-CAPROLACT AM .............................................. 73 Introduction ............................................................................................................... 73 Background. .............................................................................................................. 76 vii Catalytic Hydrodenitrogenation ................................................................................. 77 Catalytic Hydrogen Transfer ...................................................................................... 81 Reductive Deamination ............................................................................................. 82 Isomerization of L-Lysine to B-L-Lysine. ................................................................... 85 Fermentation of L-lysine ............................................................................................ 86 Bioconversion with crude extracts of L-lysine-2,3-aminomutase ................................ 91 Bioconversion with intact cells of C lostridium subterminale ...................................... 93 Discussion ................................................................................................................. 97 References ............................................................................................................... 107 CHAPTER FOUR ....................................................................................................... 111 EXPERIMENTAL ...................................................................................................... l l 1 General chemistry .................................................................................................... 1 1 1 Reagents and solvents .............................................................................................. 111 Chromatography ...................................................................................................... 1 12 Spectroscopic and analytical measurements ............................................................. 113 Microbial strains and plasmids ................................................................................. 115 Storage of microbial strains and plasmids ................................................................ 116 Culture medium ....................................................................................................... 116 Fed-batch fermentation ............................................................................................ 119 Genetic manipulations........................... .................................................................. 119 Chapter two ............................................................................................................. 130 Chapter three ........................................................................................................... 141 Reference ................................................................................................................ 150 viii LIST OF TABLES Table 1. Hydrogenation of D, L—Malic Acid as a Function of H2 Pressure, Temperature, and Reaction Time ................................................................................................. 42 Table 2. Hydrogenation of D, L-Malic Acid as a Function of Malate Concentration, Mole Ratio of RuzMalate and the Mole Ratio of Ru:Re .......................................... 44 Table 3. Purification of Dihydroxy-acid Dehydratase from Spinach Leaves ................... 54 Table 4. Plasmid Restriction Maps ................................................................................ 56 Table 5. Substrate Specificity Comparison .................................................................... 58 Table 6. Hydrodenitrogenation of Cyclohexylamine to Cyclohexane. ............................ 80 Table 7. Reductive Deamination of L-Lysine and its Derivatives. .................................. 84 Table 8. Bioconversion of L-Lysine to B-L-Lysine Using Crude Lysate ......................... 92 Table 9. Bioconversion of L-Lysine to B-L-Lysine Using Intact C. subterminale. .......... 93 ix LIST OF FIGURES Figure 1. D—Glucose-derived D,L-l,2,4—butanetriol and e-caprolactam. ............................. 3 Figure 2. Chemical building blocks derived from biomass ............................................... 4 Figure 3. Synthesis of 3-hydroxypropanoic acid and potential derivatives. ...................... 5 Figure 4. Dehydration of D—fructose to versatile derivatives of 2,5-disubstituted furan ..... 7 Figure 5. Levulunic acid derivatives with industrial applications. .................................... 8 Figure 6. Acid—catalyzed dehydration of hexoses to levulinic acid. ................................ 10 Figure 7. Conversion of glucose to sorbitol and its derivatives ....................................... l 1 Figure 8. Malic acid and derivatives with industrial applications. .................................. 12 Figure 9. Synthesis of L-aspartic acid and the ensuing derivatives .................................. 13 Figure 10. The Shikimate pathway for aromatic amino acid biosynthesis. ...................... 16 Figure 11. Synthesis of phenol and p-hydroxybenzoic acid from shikimic acid .............. 18 Figure 12. Synthesis of vanillin from benzene and D-glucose ......................................... 19 Figure 13. Synthesis of gallic acid and pyrogallol from D-glucose. ................................ 21 Figure 14. Chemical and biosynthetic route to catechol and adipic acid. ........................ 22 Figure 15. Synthesis of hydroquinone ............................................................................ 24 Figure 16. Synthesis of polyhydroxy benzenes from myo-inositol. ................................. 25 Figure 17. Syntheses of phloroglucinol and resorcinol from D-glucose. ......................... 26 Figure 18. 1,2,4—Butanetriol trinitrate and trinitroglycerine. ........................................... 35 Figure 19. Biologically active compounds derived from S—1,2,4—butanetriol and its derivatives. ............................................................................................................ 36 Figure 20. Syntheses of 1,2,4—butanetriol ....................................................................... 37 Figure 21. Chemoenzymatic synthesis of D-l,2,4—butanetriol ......................................... 39 Figure 22. Biosynthetic pathway of D—xylose and L-arabinose to D- and L-1,2,4- butanetriol. ............................................................................................................ 40 Figure 23. Catalytic hydrogenation of malic acid. .......................................................... 42 Figure 24. Alternative substrates for catalytic hydrogenation ......................................... 47 Figure 25. Syntheses of 2-hydroxy-y-butyrolactone. ...................................................... 48 Figure 26. Degradation of L-homoserine in mammals .............. _ ...................................... 49 Figure 27. Synthesis of 2-keto-4—hydroxybutyrate from L-aspartic acid. ........................ 50 Figure 28. Syntheses of D-erythronolactone and L-threonolactone. ................................. 51 Figure 29. Attempted synthesis of 2-hydroxy-2-buten-4-olide. ...................................... 52 Figure 30. Chemoenzymatic synthesis of D, L—I,2,4—butanetriol from L-ascorbic acid ........................................................................................................................ 53 Figure 31. Conversion of L-threonate to 4—hydroxy-2-ketobutyrate catalyzed by dihydroxy-acid dehydratase partially purified from spinach leaves ......................... 55 Figure 32. Conversion of L-threonate to 4—hydroxy-2-ketobutyrate and associated specific activity for dihydroxy-acid dehydratase from E. coli JWFl/pWN3. 196A. .............. 57 Figure 33. Conversion of L-threonate to 4-hydroxy-2-ketobutyrate and associated specific activity for dihydroxy-acid dehydratase from E. coli JWFl/pON1.I 18B. ............... 58 Figure 34. Catalytic hydrogenation of malic acid ........................................................... 62 Figure 35. Formation of 1,2—propanediol and 1,2-ethanediol .......................................... 63 Figure 36. Manufacture of s-caprolactam from benzene. .............................................. 73 Figure 37. Syntheses of e—caprolactam from 1,3-butadiene ............................................ 74 Figure 38. Synthetic route to e-caprolactam from D-glucose. ......................................... 76 Figure 39. Mechanistic hydrodenitrogenation catalyzed by Mo-based catalyst ............... 79 Figure 40. Deamination of cyclohexylamine .................................................................. 80 Figure 41. Hydrodenitrogenation of a-amino-e-caprolactam. ........................................ 81 Figure 42. Catalytic hydrogen transfer. .......................................................................... 81 xi Figure 43. Reductive cleavage of acylaziridine with SmI2 .............................................. 82 Figure 44. Reductive deamination of L-lysine derivatives with SmI2 .............................. 83 Figure 45. Reductive deamination with alkali metals. .................................................... 84 Figure 46. Synthesis of caprolactam from L—lysine via L—fi-lysine. ................................. 86 Figure 47. Strickland reaction of amino acids ................................................................ 87 Figure 48. Degradation of L-lysine in Clostridium subterminale .................................... 87 Figure 49. Isomerization of L-Iysine to L-B-lysine catalyzed by L—lysine-2,3- aminomutase .......................................................................................................... 89 Figure 50. Bioconversion of L-Iysine to B-L-lysine catalyzed by E. coli BL21(DE3)/pAF—80/kamA ............................................................................................ 90 Figure 51. Bioconversion of L-lysine to L—B—lysine with crude extracts of L—lysine-2,3- aminomutase. ........................................................................................................ 92 Figure 52. Bioconversion of L-lysine to L-B-lysine using intact C. subterminale. ........... 93 Figure 53. Reductive deamination of L—ornithine to 5-aminovalerate in C lostridium sticklandii .............................................................................................................. 95 Figure 54. Intermediates formed during C-N hydrogenolysis ......................................... 98 Figure 55. Reductive deamination of L-lysine methyl ester with SmI2 ............................ 99 Figure 56. Hypothesized deamination of L-Iysine via a ketene ..................................... 100 Figure 57. Deamination of B-amino group from B-L-lysine. ......................................... 100 Figure 58. L-B-Amino-butyryl CoA deaminase catalyzes B-elimination ....................... 104 Figure 59. Reductive deamination of D-proline to 5-aminovalerate catalyzed by D-proline reductase ............................................................................................... 104 Figure 60. Hypothesized microbial synthesis of 6-amininovalerate from of L-lysine 105 xii Ac ADP ATP CIAP COMT DAHP DCU DEAE DERA DHAD DHQ DHS D.O. DTT E4P EPSP FBR LIST OF ABBREVIATIONS acetyl adenosine diphosphate adenosine triphosphate ampicillin ampicillin resistance gene base pair bis(trimethylsisly)trifluoroacetamide I ,2,4—butanetrio| benzene, toluene, xylene chorismic acid calf intestinal alkaline phosphatase catechol~0-methyltransferase 3-deoxy-D-arabin0-heptulosonic acid 7-phosphate digital control unit diethylaminoethyl 2-deoxyribose 5-phosphate aldolase dihydroxy-acid dehydratase 3-dehydroquinic acid 3-dehydroshikimic acid dissolved oxygen dithiothreitol D-erythrose 4-phosphate 5—enolpyruvoylshikimate 3-phosphate feedback resistant xiii GA HPLC IPTG Kan kb LAM LPA M9 min mL mM MM OD PBT PCA PEG PEP PHB PID PCR Phe PLP psi PT S gallic acid hour high pressure liquid chromatography isopropyl fi—D-thiogalactopyranoside kanamycin kilobase kilogram lysine aminomutase lysophosphatidic acid minimal salts minute milliliter microliter millimolar micromolar optical density polybutyleneterephthalate protocatechuic acid polyethylene glycol phosphoenolpyruvic acid p-hydroxybenzoic acid proportional-integral-derivative polymerase chain reaction L-phenylalanine pyrodoxaI-S-phosphate pounds per square inch phosphotransferase system xiv PT T PET PVC Trp QA rpm SA SAM SDS S3P Tc TSP TsOH UV polytrimethylene terephthalate polyethylene terephthalate polyvinyl chloride L-tyrosine L-tryptophan quinic acid rotations per minute shikimic acid S-adenosylmethionine sodium dodecyl sulfate Shikimate 3-phosphate tetracycline sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 p—toluenesulfonic acid ultraviolet XV CHAPTER 1 INTR D CTI N Petroleum oil and natural gas are the raw material from which most organic chemicals are derived.1 The current geopolitical instabilities, high demand and declining availability have resulted in steadily increasing crude oil prices.2 Furthermore, these non- renewable resources are slowly dwindling with year 2040 projected to be the end of cheap crude oil.3 For the chemical industry to continue to thrive, integration of renewable feedstock into their manufacturing process will be critical. Nature provides terrestrial biomass such as sugars, amino acids, lipids, biopolymers such as cellulose, hemicellulose, chitin, starch, lignin and proteins in abundance. Carbohydrates constitute two-thirds of the annually renewable biomass even though this feedstock is under-utilized by the chemical industry.4 Systematic development of appropriate methodologies to convert carbohydrates into industrially useful organic building blocks that compete with petroleum-derived building blocks is important. Particularly, the use of biotechnology in which microbial transformation, fermentation and enzymatic conversion of renewable feedstock into commodity chemicals, fine chemicals, pharmaceuticals agents, or enantiopure organic building blocks is gaining popularity.4 For the completion of this dissertation, two chemicals currently derived from petroleum were synthesized from renewable feedstock by incorporating chemical and Chemoenzymatic syntheses. Chapter 2 describes catalytic hydrogenation of petroleum- derived D,L-malic acid and biomass-derived L-malic acid over Ru on C, which afforded D,L-l,2,4-butanetriol in 74% yields as part of a product mixture. The effects of hydrogenation time, pressure, and temperature along with substrate concentration, catalyst loading and catalyst composition were examined. Complications associated with separating product from the reaction mixture by distillation resulted a 28% yield of D,L- l,2,4-butanetriol with significant contamination from 1,4-butanediol. An alternate Chemoenzymatic route to D,L-l,2,4-butanetriol employed D—glucose-derived L-ascorbic acid as the starting material. Chemical oxidation of L-ascorbic acid gave L-threonate, which was dehydrated using dihydroxy-acid dehydratase. Cyclization of the resulting 4- hytlroxy-2—ketobutyrate to 2-hydroxy-2-buten-4-olide was followed by hydrogenation over Ru on C to afford D,L-l,2,4-butanetriol as the only product in 53% overall yield from L-ascorbic acid. Chapter 3 of the thesis explores the use of L-lysine as the starting material for the synthesis of e-caprolactam. L-Lysine is microbially synthesized from D-glucose. Denitrification using catalytic hydrogenation of L-lysine over Pt/C and Mo-based catalysts was evaluated along with single electron reduction of three substrates: (1— aminocaprolactam, the methyl ester of L-lysine, and methyl pipecolinate. To setup removal of the a-amino group of L-lysine, intact Clostridium subterminale cells were utilized to isomerize L-lysine to L-B-lysine. Elimination of the B-amino group and subsequent hydrogenation to afford the s-caprolactam was evaluated over Ru on C. Biomass-derived building blocks Carbohydrate starting materials are heavily functionalized with hydroxyl groups and they possess a high density of stereochemistry while petroleum-derived hydrocarbons are bare of functional groups and stereocenters. By exploiting the structural backbone of carbohydrate starting materials, building blocks such as D,L-1,2,4-butanetriol and s- caprolactam, which have industrial utility, were accessed (Figure l). VII-l HOQC/YCOQH d OH \ HOWOH L-malic acid a OH OH / Ho D,L-1,2,4-butanetriol .\OH b HO $3324) e/‘ s OH HO OH HO OH 0 L-ascorbic acid D-glucose \ + O NH3 NH + - _ e or f H3N W002 ——> L-Iysine s-caprolactam Figure 1. D-GIucose-derived D,L-l,2,4-butanetriol and e-caprolactam. Key: (a) Aspergillus flavus; (b) Reichstein-Griissner Process; (c) Corynebacterium glutamicum; ((1) Catalytic hydrogenation; (e) Chemoenzymatic synthesis; (f) Reductive deamination. For the biomass-based chemical industry to be competitive, replacement of low molecular weight building blocks from petroleum needs to be addressed. The technology for the conversion of petroleum into primary building blocks for the chemical industry such as ethylene, propylene, benzene, toluene, xylene (BTX) and butadiene is very well developed while production of building blocks from biopolymers is still under development.5 D-Glucose is the most abundant monosaccharide industrially produced in pure form and is obtained from enzymatic hydrolysis of starch.6 Cellulose is likely to serve as a cost-efficient source of D-glucose in the future and would avoid competition between food and chemical use of starch-derived D-glucose.7 The cellulose ethanol technology recently developed by Iogen in which 85 gallons of ethanol was obtained from a ton of wheat straws has opened new avenues for the utilization of cellulosic biomass as a source of carbohydrates.8 Twelve value-added chemicals from biomass have recently been identified as potential basic building blocks for the chemical industry (Figure 2).8‘"9 These building blocks along with L-ascorbic acid, L-malic acid and L—lysine discussed in the next chapters, are synthesized from D-glucose. OH OH OH OH OH OH oH OH oH OH 9“ 0” D- sorbitol D-glucarIc 30d QIYCGIOI D- -xOy|Hitol L-arabinitol co H co H /\,co H NH? O 2 /\/ 2 2 - HOzc/E’H HO2C H020 HOZC\/\CozH HON/Km L-malic acid succinic acid fumaric acid L-aspartic acid 3-hydroxypropanoic acid CH2 NH2 Ho 0 0 co H O O o 2 2 HO2C\)LCOZH 1.10sz00sz HOZCVLCH3 g HO itaconic acid L-glutamic acid 2,5-furandicarboxylic acid levulinic acid 3-hydroxy-y-butyrolactone Figure 2. Chemical building blocks derived from biomass. These molecules include glycerol, 3-hydroxypropanoic acid, 3-hydroxy-y- butyrolactone, aspartic acid, itaconic acid, levulinic acid, glutamic acid, D—xylitol and L- arabitol, glucaric acid, 2,5-furandicarboxylic acid, D-sorbitol and 1,4—dicarboxylic acids such as succinic acid, L-malic acid and fumaric acid. Because these building blocks have multiple functional groups, they can serve as platform chemicals from which a variety of products could be derived. Synthesis of selected building blocks and their derivatization is examined from two perspectives. The first part involves transformation of sugars into the basic building blocks while the second part entails derivatization of the basic building block to various secondary building blocks. Biotransformation is an important feature of routes from plant feedstock into sugars through to basic building blocks. Chemical transformation on the other hand predominates in converting basic building blocks into secondary building blocks. A brief overview of challenges associated with conversion of sugar into the selected basic building blocks and subsequent conversion into secondary building blocks is given. 3-Hydroxypropionic acid 3-Hydroxypropanoic acid or B-lactic acid has a potential application as a monomer for (co)-polymerization similar to lactic acid, which affords a biodegradable thermoplastic polylactide polymer.” Furthermore, it can be hydrogenated to 1,3- propanediol, which along with terephthalic acid, is polymerized to polytrimethylene terephthalate (PTT).” PTT is a polymer with desirable properties such as good resilience, stain resistance and low static generation relative to its competitors PET (polyethylene terephthalate) and nylon in fiber and textile applications. Dehydration of B-lactic acid can also result in a family of acrylates such as acrylic acid, methyl acrylate and acrylamide (Figure 3). O VL OH / NH2 AOH 0 acrylamide OH HOVLOH ’ HOMOH i 3-hydroxypropanoic 1 ,3-propanediol OH ' HO aCId \ O D-glucose \JL 0 OH acrylic acid Figure 3. Synthesis of 3-hydroxypropanoic acid and potential derivatives. Acrylic acid is in high demand (1.2 x 109 kg/year in the US) and is currently ‘2 Acrylates are derived from oxidation of propylene via the intermediacy of acrolein. used primarily to prepare emulsion and solution polymers, which have found application in textile, paint, adhesives and paper industry.12 The high price of 3-hydroxypropanoic acid limits its application. Microbial synthesis of 3-hydroxypropanoic acid has not yet been commercialized while synthesis of L-lactic acid from corn starch has been commercialized in a joint venture between Cargill and Dow Chemical with the annual capacity of 140 x 106 kg.13 In 2001, genetically engineered E. coli expressing glycerol dehydratase from Klebsiella pneumoniae and nonspecific aldehyde dehydrogenase from S. cerevisiae was reported to produce 0.2 g/L of 3-hydroxypropanoic acid from glycerol.l4 Since then, five possible biosynthetic routes from D—glucose have been proposed in the patent literature.‘5'16 The microbial synthesis of 3-hydroxypropanoic acid can only be economically competitive if a minimum concentration of 2.5 g/L is achieved in 1 h.8"‘9 Proposed derivatives from 3-hydroxypropanoic acid such as acrylates, which are currently produced from petroleum, must also be accessible via a low cost and hi gh-yielding route. 2,5—Furandicarboxylic acid Acid-catalyzed dehydration of biomass derived pentoses and hexoses affords two furanic compounds, furfural and 5-hydroxymethylfurfural 1 (Figure 4).17 These two compounds and their derivatives represent a class of compounds that are suitable for use as monomers for the preparation of non-petroleum derived polymeric materials. 5- Hydroxymethylfurfural 1 has reportedly been used in the manufacture of phenolic resin.18 5-Hydroxymethylfurfural 1 is readily accessible from acid—catalyzed elimination of 3 mol of water from fructose or inulin hydrolysates.19 This six-carbon commodity chemical is a key intermediate because of various industrially significant chemicals that can be derived from it (Figure 4). / \ o 2 /H Ho . OH Ho /\ b /\ m 1» VQ‘CHdo —> HOQC’U‘ o COQH—> HO OH D-fructose\;2NKU\\M-124\ Figure 4. Dehydration of D-fructose to versatile derivatives of 2,5-disubstituted furan. Key: a) H3O“; b) Pt/Pd, C/ 02; c) BaMnO4; d) i. NH20H, ii. Ni/ H2. These include oxidation to 2,5-furandicarboxaldehyde 2, reduction of aldehyde to 2,5—bis(hydroxymethyl)-furan 6, oxidation to 2,5-furandicarboxylic acid 4 or reductive amination to 2,5-bis(aminomethyI)—furan 3.20 These monomers have been exploited for the preparation of furanoic polymers. In particular, copolymerization of 3 and 4 to furanoic polyamide has a potential of replacing petroleum-based polyamides.&"9"2' Copolymerization of 2,5-furandicarboxylic acid 4 with aliphatic or aromatic diamines has resulted in an analogue of Kelvar®.22 This polyamide was found to have a promising glass transition temperature and thermal stability compared to all furanic polyamide. Unlike furfural, which is produced industrially at 200 000 tons/yr capacity from dehydration of pentoses, 5-hydroxymethylfurfural 1 has only been produced on a pilot plant scale. Hydroxymethylation of furfural in excess formaldehyde has been evaluated for the synthesis of 5-hydroxymethylfurfural 1.21 Due to the deactivating effect of the aldehyde group, the conversion of furfural was only 50%. Reversal of polarity by protecting the aldehyde as 1,3-dithiolane however, resulted in the formation of 5- hydroxymethylfurfural 1 in 90% yield. 2,5-Furandicarboxylic acid 4 has a potential of replacing terephthalic acid, which is widely used in polymers such as polyethylene terephthate (PET) and polybutyleneterephthalate (PBT).23 There is an annual demand of 7 5 billion lb for these polymers.8""9 The increasing price of benzene, from which the aromatic monomers are derived, fueled by high demand and limited availability would favor furanic compounds as alternative building blocks.2 Levulinic acid Levulinic acid has great potential as an inexpensive feedstock for producing a wide variety of industrially important products (Figure 5).:24 Currently, the worldwide market for levulinic acid is ~ 1 million lb/year at a price of $4—6/lb. Levulinic esters and salts are used in the food industry as preservatives, stabilizers and as flavoring agents.25a These esters increase thermal stability of poly(vinylchloride) when used as additives. (3- Amino levulinic acid is a nontoxic, biodegradable broad-spectrum herbicide, which is triggered by light to kill weeds.25b g... 2-methyl tetrahydrofuran H3O COQH H3C\n/\/ COzH —> H2N /\n/\/COZH O O O O levulinic acid 6-amino levulinic acid X X' X = X' = OH diphenolic acid I X = X' = Br X = OH. X. = Br H3Cm/\/C02Fi O Ievulinate esters Figure 5. Levulunic acid derivatives with industrial applications. Diphenolic acid has a potential of replacing bisphenol-A as a monomer in the production of polycarbonate resins. Brominated diphenolic acid on the other hand could serve as an environmentally acceptable marine coating while dibrominated diphenolic acid may find use a fire retardant. The U. S. Department of Energy has approved the use of methyl tetrahydrofuran as a fuel additive, which increases oxygenate levels in gasoline.‘°'5°'d This molecule is derived from catalytic hydrogenation of levulinic acid. The process developed by Biofine Inc. for the synthesis of methyl tetrahydrofuran, together with the high demand for o-amino levulinic acid, may potentially expand the demand of levulininc acid to between 200 million and 400 million lb/year thus decreasing the price to $0.04- $0.10 per pound. The process for the synthesis of levulinic acid relies on feedstocks such as cellulose-containing waste materials from paper mill sludge, waste wood, paper waste, and agricultural residues. Hydrolysis of cellulosic biomass results in hexoses, which are enolized to enediol 8 under acidic conditions (Figure 6).26 The first dehydration affords an enol form of 3-deoxyhexosulose 9, which undergoes further dehydration to 3,4- dideoxyglycosulosene-3 10. The latter is readily converted into dienediol 11, which after cyclization results in 12. Subsequent dehydration affords 5—hydroxymethyl furfural 1. Addition of a molecule of water across C2-C3 of 5-hydroxymethylfurfural results in the ring opening of the furfural into an unstable tricarbonyl intermediate 13. The latter decomposes into levulinic acid and formic acid. 9H0“ CH0 CH0 9H0 C-OH O-OH (3:0 C-OH D- lucose .. . -- D-gnannose = HO H M). C-H i9, 9H —> 9H —> D-fructose H OH H OH CH 9H H OH H OH H—l—OH Q-OH CHon CHZOH CHZOH CHon 8 9 10 11 HO "' -H20 / \ -H20 fl WOW/COW o CHOH—* 0 CH0 —> H30 CHo_, 12 1 13 Figure 6. Acid-catalyzed dehydration of hexoses to levulinic acid. Alternatively, heating of furfuryl alcohol in the presence of HCl has been reported to yield up to 80% of levulinic acid. Both furfuryl alcohol and 5- hydroxymethyl furfural are derived from wood pulp processing.26 Side products consisting of humic compounds reduce the yield of levulinic acid from cellulosic biomass. Development of a more selective dehydration process is the key to the use of levulinic acid as a building block.&“9 D-Sorbitol Batch-hydrogenation of D-glucose to D-sorbitol over Raney nickel is an established route for the synthesis of this sugar alcohol (Figure 7).27 As an alternative to the widely used batch process, a continuous process based on Ru affords an almost quantitative yield of D-sorbitol with a high hourly space velocity.9 Almost all of the 650,000 tons of sorbitol produced annually is used as a food additive. OH HO\/K propylene glycol l (I OH no 0 ..0H a 9” OH OH b o 5 OH OH OH O . HO OH OH D-sorbitol R = R' = H isosorbide D glucose 0 R = R' = N02 isosorbide dinitrate I R =H, R' = N02 isosorbide mononilrate O ..\\ Hon \OH Ho“ OH 1 ,4-sorbitan Figure 7. Conversion of glucose to sorbitol and its derivatives. Key: a) Raney Ni 122 atm H2, 140 °C; b) H30; 0) H30; (1) Ru/C H2. D-Sorbitol is also an intermediate in Reichstein’s synthesis of L-ascorbic acid from D-glucose.28 This large-volume sugar alcohol can potentially be the source of dehydration sugars such as isosorbide or 1,4-sorbitan (Figure 7). Isosorbide mono- and 10 dinitrates are used therapeutically as vasodilators to treat angina pectoris, congestive heart failure and dysphasia.29 Another commercially viable derivative of sorbitol, sorbitan monoesters (SME) obtained from esterification of 1,4-sorbitan are used commercially as non-ionic surfactants, solubilizers and emulsifiers in cosmetics and various other formulations.30 Recent studies have shown that sorbitol is an effective stabilizer of polyvinyl chloride (PVC). This occurs by dehydration of sorbitol to 1,4-anhydrosorbitol. The water formed during the intramolecular dehydration then binds free hydrogen chloride in PVC via hydrogen bonding.31 Isosorbide has also been reported to improve properties of polyethyleneterephthalate when used as a comonomer.9 Catalytic hydrogenolysis of sorbitol to 1,2-propanediol is not selective. An alternative synthesis of propylene glycol from hydrogenation of D-glucose-derived lactic acid over Ru on C has been reported.32 Microbial fermentation of common sugars such as D-glucose and D-xylose have also been shown to afford 1,2-propanediol under anaerobic conditions.33 L-Malic Acid Over 80% of malic acid produced worldwide is used in food and beverages.34a Both L- and D, L-malic acid are generally recognized as safe (GRAS) substances for use as flavor enhancers, pH control agents, flavoring agents and adjuvants (Figure 8). D, L- Malic acid. is obtained from acid—catalyzed hydration of fumaric acid and hydrolysis of maleic anhydride, which is derived from butane oxidation over (VO)2PZO7.34b 11 HO OH OH 1 ,2,4-butanetriol l a b D—glucose —> Hozc’YcozH <— fumaric acid OH L-malic acid 2-butenyI-v-Iactone 3-hydr0xy- y-butyrolactone Figure 8. Malic acid and derivatives with industrial applications. Key: (a) Aspergillusflavus; (b) Brevibacteriumflavum. L-Malic acid is produced by two biocatalytic methods. Enzymatic hydration of fumaric acid is catalyzed by intact cells of Brevibacterium flavum or isolated fumarase activity (Figure 8).”3 The second route involves direct fermentation of D-glucose under aerobic conditions to afford L-malic acid.35b A pilot plant aimed at hydrogenating malic anhydride to 3-hydroxy-y-butyrolactone at 250 atm H2 was constructed by SK Energy and Chemicals in Daeduk, Korea.36 It is anticipated that this plant would supply the pharmaceutical industry with 3-hydroxy-y-butyrolactone, which is currently obtained from oxidation of pentoses and hexoses.37 Dehydration of 3-hydroxy-y-butyrolactone to 2-butenyl-y—lactone, and esterification can yield acrylate lactone, which can potentially find use in the synthesis of new polymers.9 Complete hydrogenation of malic acid affords 1,2,4-butanetriol, a subject of this thesis. Applications of D,L-1,2,4-butanetriol range from application as a synthetic scaffold in organic chemistry and incorporation into energetic plasticizers to treatment of angina pectoris after nitration to D,L-1,2,4- butanetriol trinitrate.38 l2 L-Aspartic acid L-Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of the low-calorie sweetener aspartame (Figure 9).39 It is obtained by amination of fumaric acid catalyzed by immobilized aspartate ammonia— lyase (aspartase) from Bacillus with an annual capacity of 100 ton/yr.40 The major drawback for the development of aspartic acid as a building block is the lack of a direct fermentation route starting from sugar substrate.9 Reduction in the cost of fumaric acid production from D—glucose fermentation could potentially reduce the cost of L-aspartic acid. 0 o g H2N 3-aminobutyrolactone l 0 ”Hz _02C/YLN OCH3 H o D-glucose—> HO2C¢COQH _, HOZCVKCOzH _, EH3 fumaric acid L-aspartic acid aspartame l NHz 2-amino-1,4-butanediol Figure 9. Synthesis of L-aspartic acid and the ensuing derivatives. Hydrogenation of L-aspartic acid to either 3-aminobutyrolactone or 2-amino-1,4- butanediol would provide amino analogs of 3-hydroxy-y—butyrolactone and 1,2,4- butanetriol. These derivatives can potentially find use as intermediates for the synthesis of high value pharmaceutical compounds if their proposed derivatization can be accomplished selectively under mild conditions. L-Aspartic acid is an important 13 monomer for the biodegradable nylon—3 derivative poly(a-isobutyl—L—aspartic acid)“ This polyamide along with other nylon 3 (poly-B-alanine) and nylon 3-derivatives could serve as substitutes for polyacrylic acid and polycarboxylates.9 Use of L-aspartic acid along with derivatives is contingent on the availability of fumaric acid. This unsaturated 1,4-diacid is obtained using byproducts from phthalic anhydride manufacture because the biological production of fumaric acid is too expensive to compete with the petroleum-based manufacture.42 Although many species produce small amount of fumaric acid as a byproduct metabolite during oxidative metabolism, mycelial fungi are capable of producing significant quantities of fumaric acid from D-glucose and C02.43 Fungal fermentation of Rhizopus oryzae has been extensively studied for the production of fumaric acid.44 When the nitrogen source is limited, the growth of Rhizopus is stunted and during this no growth period, fumaric acid is synthesized in a maximum yield of 2 mol/mol of D-glucose.45 Typically, CaCO3 is added during Rhizopus oryzae fermentation for the production of fumaric acid as a neutralizing agent and to aide in the removal of fumarate from the fermentation broth. The process of regenerating free acid from fumarate is complex, tedious and expensive. Several techniques employing adsorbent to bind fumaric acid while it is being produced or use of alternative neutralizing agents are being explored for production of fumaric acid S from D-glucose.44’4' Aromatic Compounds Aromatic compounds such as benzene, toluene and xylenes constitute a major part of the primary building blocks from petroleum and they are the basis for industrial 14 polymers such as nylon, polyester, polystyrene, polyurethane.‘ The recent surge in the price of benzene from $1.40/gal in November 2003 to ~$4.00/gal in November 2004 has resulted in an increase in the price of polymers manufacture from monomer derived from benzene.46 The high demand for benzene and continued constraints on its availability are predicted to continue to drive the price for benzene upward. This may force the chemical industry to explore other sources for benzene-derived starting materials. Replacing benzene with D-glucose thus becomes an appealing Option. The Shikimate Pathway Plants, bacteria and fungi utilize the Shikimate pathway for the biosynthesis of aromatic amino acids and aromatic vitamins.47 There are seven enzymatic reactions that convert phosphoenolpyruvate (PEP) and D—erythrose-4—phosphate (E4P) into chorismic acid (Figure 10). The first committed step in the synthesis of aromatic amino acids is the condensation of (PEP) and (E4P) to afford 3-deoxy-D-arabino-heptusonic acid 7- phosphate (DAHP) catalyzed by DAHP synthase. Three isozymes of DAHP synthase exist in E. coli, and they are sensitive to feedback inhibition by one of the three aromatic amino acids. The genes aroF, aroG and aroH encode for L-tyrosine-sensitive, L- phenylalanine—sensitive, and L-tryptophan-sensitive isozymes of DAHP synthase, respectively. Conversion of DAHP to 3-dehydroquinic acid (DHQ) is catalyzed by aroB- encoded DHQ synthase. The reaction involves an intramolecular oxidation-reduction at C-5 of DAHP with a very tightly bound NAD+ cofactor, a syn elimination of phosphate, and an intramolecular cyclization to afford DHQ.48 syn-Elimination of a molecule of 15 water from 3-dehydroquinate (DHQ) to 3-dehydroshikimate (DHS) is catalyzed by aroD- encoded 3-dehydroquinate dehydratase.49 NADP-dependent Shikimate dehydrogenase, encoded by the aroE gene catalyzes the reduction of DHS to shikimic acid (SA).50 The 3- hydroxyl group of shikimic acid is phosphorylated by aroL- and aroK-encoded Shikimate kinase isozymes to afford shikimate-3-phosphate (S3P), which condenses with the second equivalent of PEP to afford 5-enolpyruvylshikimate-3-phosphate.5x OPO3H2 ” £19” + H PO H PO OH 0 3 4 3 OH 3 4 O , OH H203PO OH 0H ; DHQ DHS H203PO OH DAHP E4P 002H COQH COzH COQH O. . O.IL 9O.IL —> —> HO‘ OH H203 PO‘ OH H2 03 PO‘ OJL COQH OJL 002 H OH OH SA S-3-P EPSP chorismic acid OH HOZC" 2 COZHNH 0 Q 2 OH . ' x - OH PBA 6H 2bgndz'2ic (12“;in X—= NH2 P AB A prephenic acid anthranillic acid enterochelin ubiquinones L-phenylalanine L-tryptophan folic acid L-tyrosine Figure 10. The Shikimate pathway for aromatic amino acid biosynthesis. Key: Abbreviations: PEP, phosphoenolpyruvate; E4P, D-erythrose 4-phosphate; DAHP, 3- deoxy—D-arabino-heptulosonate 7-phosphate, DHQ, 3-dehydroquinate; DHS, 3- dehydroshikimic acid; SA, shikimic acid; S3P, Shikimate 3-phosphate; EPSP, 5- enolpyruvylshikimate 3-phosphate; PBA, p—hydroxybenzoic acid; PABA, p-aminobenzoic acid. Enzymes: a) 3-deoxy-D-arabin0-heptulosonate 7-phosphate synthase (AroF, AroG, AroH); b) 3-dehydroquinate synthase (AroB); c) 3-dehydroquinate dehydratase (AroD); d) Shikimate dehydrogenase (AroE); e) Shikimate kinase (AroK, AroL); f) 5- enolpyruvylshikimate 3-phosphate (AroA); g) chorismate synthase (AroC). l6 This condensation is catalyzed by aroA-encoded 5-enolpyruvylshikimate—3-phosphate synthase? As the final step, chorismate synthase catalyzes the concerted 1,4-trans elimination of phosphate from 5-enolpyruvylshikimate-3-phosphate to afford chorismic acid, which is the common intermediate from which L-phenylalanine, L-tyrosine and L- tryptophan are derived.53 Furthermore, biosynthetic pathways leading to ubiquinone, folic acid and enterochelin branch off at chorismic acid (Figure 10). Folic acid-derived coenzymes are frequently involved in the biosynthetic transfer of one-carbon fragments, ubiquinones are involved in electron transport, and enterochelin is an iron chelator responsible for iron uptake in numerous microorganisms.54 Phenol and p—hydroxybenzoic acid from shikimic acid Shikimic acid is currently used as the starting material for the synthesis of the neuraminidase inhibitor GS-41014, which is used to treat influenza infection and is marketed as TamifluTM by Roche.55 Before shikimic acid was produced from fermentation, it was obtained from extraction of the fruit of Illicium plants, which is commonly known as the anise tree.56 Production of shikimic acid from D-glucose under fed-batch fermentation conditions have been improved from 52 g/L with E. coli SP1.1/pKD12.138 to 87 g/L with SP1.1pts/pSC6.090.57 Shikimic acid and the intermediates in the shikimic acid pathway can be exploited for the synthesis of aromatic compounds. By interfacing biocatalysis with chemical catalysis, value-added aromatic compounds were derived from D-glucose. The next section of this thesis presents a brief overview of benzene-free routes to access some aromatic compounds from D-glucose by exploiting shikimic acid and intermediates in the shikimate pathway. When an aqueous 17 solution of shikimic acid was heated to 350 °C, phenol was isolated in 45% yield (Figure 11).58 O‘“ y phenol OH C02H 0 A0“ a a 0 002H ——-> v ——> ; OH HO‘ i OH H00 HO OH OH . p-hydroxybenzonc D-glucose shikimic acid \ ac'd ' \ COQEt :>-O\v : VNH2’H3PO4 NHAC 68-41014 Figure 11. Synthesis of phenol and p-hydroxybenzoic acid from shikimic acid. Key: a) E. coli SP1.1/pKD12.138; b) H20, 350 °C; c) 1 M H2804 in AcOH. Phenol is used to make synthetic resins, dyes, pharmaceuticals, pesticides, perfumes, lubricating oils and solvents.59 The Hock oxidation of benzene—derived cumene is currently the predominant method used in the production of phenol with an annual production of 5 x 109 kg. It is estimated that 20% of the global benzene production is directed to the manufacture of phenol.59 Benzene, however, is a hazardous organic pollutant whose emission must be reduced as mandated by the Chemical and Manufacturing Rule issued by the U. S. Environmental Protection Agency. Furthermore, exposure to benzene has been linked to acute leukemia and non—Hodgkin’s lymphoma."0 When shikimic acid was heated in the presence of 1 M sulfuric acid and acetic acid at 120 °C under atmospheric pressure, p—hydroxybenzoic acid was obtained in 57% yield.58 p-Hydroxybenzoic acid is a component of liquid crystal polymers such as Xydar, which 18 have attracted considerable attention because of their use in high—performance applicationsf’l Esters of p—hydroxybenzoic acid are also widely used as food preservatives. p-Hydroxybenzoic acid is currently manufactured by Kolbe—Schmitt reaction of dried potassium phenoxide with 20 atm dry CO2 at 180-250 °C. Product potassium p-hydroxybenzoate is converted to its free acid upon addition of mineral acid. Chemicals derived from 3-Dehydroshikimic acid Vanillin is a natural flavoring agent, which is isolated from the dried pods of the orchid Vanillin plam‘folia.62a It accounts for 20 tons of the 1.2 x 105 tons/year world flavor market and is used in food, beverages and perfumes.62b Only 0.2% of vanillin is isolated from the natural sources,62C the rest is obtained from benzene-derived guaiacol by condensation with glyoxylic acid to afford mandelic acid. Oxidation of mandelic acid followed by decarboxylation affords vanillin (Figure 12).623 HO COzH COCOZH ——> OCH3 C d \ CHO Q G MGO -—-> MOO OH OH benzene guaiacol mandelic acid phenylglyoxalic MeO acid OH OH COZH COZH COZH b vanIIIIn OH O " . O _>. O _,. i OH O i OH HO H3CO D-glucose S-hydroxyshikimic protocatechuic vanillic acid acid acid Figure 12. Synthesis of vanillin from benzene and D-glucose. Key: a) KL7/pKL5.26A; b) N. crassa aryl aldehyde dehydrogenase; c) HCOCOzH; d) 02; e) H*. 19 Synthetic vanillin sells for $I2/kg while natural vanilla flavoring extracted from vanilla bean containing 2% vanillin sells for $30—120/kg.62C The high price for natural vanilla flavoring reflects the labor-intensive cultivation, pollination, harvesting and curing of vanilla beans. The demand for natural flavorings has, in turn, prompted the deveIOpment of biocatalytic routes to vanillin. Biocatalytic conversion of D—glucose to vanillin proceeds via the intermediacy of 3-dehydroshikimic acid. Heterologous expression of the aroZ locus in E. coli aroE auxotroph KL7 leads to protocatechuic acidffi Expression of rat-liver COMT-encoded catechol-0-methyltransferase in KL7 resulted in 4.9 g/L of vanillic acid by fed-batch fermentation from D—glucose when the construct was supplemented with L-methionine (Figure 12). COMT catalyzes the methylation of protocatechuic acid (PCA) to a mixture of vanillic acid and isovanillic acid. The in vitro reduction of vanillic acid to vanillin was carried out by aryl aldehyde dehydrogenase purified from the fungus Neurospora crassa in 66% yield.64 This two- step biocatalytic synthesis of vanillin is the one of two biocatalytic synthesis of vanillin using a carbohydrate as a starting material. Recently, Paolis patented a technology (Gly Link) for the synthesis of vanilla by harnessing yeast fermentation of glucose.“35 Gallic Acid and pyrogallol 3-Dehydroshikimic acid can also serve as the starting material for 3,4,5- trihydroxybenzoic acid, which is commonly known as gallic acid. This polyhydroxylated aromatic is currently isolated from gall nuts or from seed pods of Coulteria tinctoria trees found in Peru.66 Thermal decarboxylation of gallic acid in c0pper autoclaves affords pyrogallol.66 Two biocatalytic routes were developed for the synthesis of DHS to 20 supplant isolation of gallic acid and pyrogallol from scarce natural resources. In one route, 3—dehydroshikimic acid in acetic acid solution was oxidized by 02 in the presence of catalytic amounts of Cu2+ and an“ to afford gallic acid in 67% yield.67 Alternatively, gallic acid was obtained directly from D-glucose via the intermediacy of protocatechuic acid (Figure 13).‘53 OH COzH COQH 23L“ 8 £1 a s OH O s OH HO HO OH OH OH D-glucose DHS PCA b\ / a COZH .i. HO OH HO OH OH OH pyrogallol gallic acid Figure 13. Synthesis of gallic acid and pyrogallol from D-glucose. Keys: 3) E. coli KL7/pSK6.16l; b) Oz, Cup, Zn2+, AcOH; (c) E. coli RB79lserA::aroB/pSK6.234. Abbreviations: DHS, 3-dehydroshikimic acid; PCA, protocatechuic acid. E. coli KL7/pSK6.16l expresses a plasmids-localized mutant isozyme of p- hydroxybenzoate hydroxylase encoded by plasmid-localized p0bA* and DHS dehydratase encoded by a genomic copy of mol and feedback-insensitive DAHP synthase encoded by plasmid-localized aroFTBR.69 E. coli KL7/pSK6.16I afforded 20 g/L of gallic acid in 12% (mol/mol) yield from D—glucose under fermentor-controlled conditionsf’8 Decarboxylation of gallic acid to pyrogallol was effected by E. coli RB79lserAzzaroB/pSK6234 expressing aro Yeencoded PCA decarboxylase. Addition of gallic acid to a batch culture of E. coli RB79lserAzzaroB/pSK6.234 during its stationary phase of growth afforded pyrogallol in a concentration of 14 g/L in 97% (mol/mol) yield. 21 The high-yielding biocatalytic decarboxylation of gallic acid to pyrogallol provides an attractive alternative to currently employed chemical decarboxylation process. The toxicity of pyrogallol towards growing E. coli cells precluded the direct synthesis of pyrogallol from D-glucose using a single microbial construct. Catechol and adipic acid Inclusion of a cat/l encoding catechol 1 ,—2 —dioxyg enase in a catechol- -producing E coli strain resulted in the synthesis cis,cis-muconic acid from D-glucose.7O The catA- encoded catechol 1,2-dioxygenase was isolated from Acinetobacter calcoaceticus.71 Catalytic hydrogenation of ci.s,cis-muconic acid under mild conditions afforded adipic acid (Figure 14), a monomer for synthesis of nylon-6,6.72 acetone E) a hydroquinone benzene cumene phenol \th‘“ OH cogH COZH OH O ,.OH d I OH/ catechol —-> , OH 0 , oH HO OH OH d D-glucose 3-dehydro protocatechuic shikimic acid acid : OH cyclohexanol COzH / COZH i2. .1. H020 .1 H020 / benzene (l adipic acid cis, cis-rnuconic O aCId cyclohexanone Figure 14. Chemical and biosynthetic route to catechol and adipic acid. (a) propylene, 400-600 psi., solid H3PO4 catalyst, 200-260°C; (b) 02, 80-130 °C, S02, 60- 100 °C; (0) 70% H202, EDTA, Fe2+ or C0”, 70-80 °C; (d) E. coli WNl/pWN2.248; (e) H2, 50 psi., 10% PVC; (0 Ni-AI2O3, H2, 370-800 psi., 150-250 °C; (g) Co, 02, 120-140 psi., 150-160 °C; (h) Cu, NH4VO3, 60% HNO3, 60-80 °C. 22 An improved route to adipic acid via catechol utilized an aroE auxotroph E. coli WNl expressing aroZ-encoded DHS dehydratase for the conversion of DHS to protocatechuic acid, aroY—encoded PCA decarboxylase for the conversion of PCA to catechol, and cam-encoded catechol 1,2-dioxygenase for the conversion of catechol to ci.s',ci.s'—muconic acid.73 The construct E. coli WNl/pWN2.248 synthesized 37 g/L of cis,cis-muconic acid from D-glucose in 23% yield (mol/mol). Catalytic hydrogenation over Pt/C at 50 psi H2 afforded adipic acid in 97% yield from cis,cis-muconic acid. Adipic acid is one of the top 50 large-volume chemicals produced in the U. S. at 918,000 tons/year. In 1999, ~84% of the adipic acid produced was used in the synthesis of nylon 6,6, with the rest going to the production of polyurethane and plasticizers.74 Most of the syntheses rely on benzene-derived cyclohexane as the chemical feedstock. Air oxidation of cyclohexane over cobalt naphthalene or cobalt octanoate affords a mixture of cyclohexanol and cyclohexanone. Cyclohexanol has also been produced from oxidation of cyclohexene. Conversion of cyclohexanol or cyclohexanone to adipic acid is effected by nitric acid oxidation over copper and vanadium catalysts at 92-96% yield. During this process, NO, NO2 and N20 are produced as byproduct. Nitrous oxide contributes to the depletion of the ozone layer and global warming.75 Other routes for adipic acid involved reaction of butadiene and carbon monoxide or carbonylation of 1,4- butanediol. The biocatalytic route starting from D-glucose to cis, cis-muconic acid offers a viable alternative to the synthesis of adipic acid monomer for nylon 6,6. Hydroquinone from Ouinic Acid Microbial synthesis of quinic acid from D-glucose and the subsequent conversion of quinic acid to hydroquinone provide an example of how renewable feedstocks can be 23 substituted for fossil fuel feedstock in chemical manufacture. Quinic acid is an important chiral synthon isolated from Chicoma bark.76 Hydroquinone is a pseudocommodity chemical used mainly for photographic development with an annual production of approximate 4.5 x 107 kg from benzene-derived aniline, phenol and p— diiSOpropylbenzene.77 Shikimate dehydrogenase, which catalyzes the reduction of 3- dehydroshikimic acid to shikimic acid, also catalyzes in the reduction of 3-dehydroquinic acid to quinic acid. Overexpression of shikimate dehydrogenase in 3-dehydroquinate- synthesizing E. coli strains has resulted in a high-yielding microbial synthesis of quinic acid, which after oxidation with household bleach followed by heating at reflux affords hydroquinone in 87% isolated yield.78 An alternative chlorine-free route was also explored in which oxidation of quinic acid was catalyzed Ag3PO4 (10 mol%) in the presence of K25208 as a co-oxidant at 50 °C to afford hydroquinone in 85% yield after reflux (Figure 15).78 0 Ho“' j]: ‘OH OH 3 OH O 0 HO COQH OH 9 Ho,,_ 0 “ ._____ go“ OH OH HO 5 OH OH OH \ OH f OH OH ’/hydroxyhydro D-glucose quinic acid 2-deoxy-scyllo - quinone inosose OH hydroquinone Figure 15. Synthesis of hydroquinone. Key: (a) E. coli QPl.1/pKD12.l38; (b) i). NaOCl, ii). isopropanol, H“; (c) reflux; (d) i). Ag3PO4 (10 mol%), K25208, 50 °C, ii). reflux; (e) 0.5 M H3PO4, reflux, 39%; (f) 50 psi H2, Rh on A1203, 12 h, 82% 24 Aromatic Compounds from N on-Shikimate Pathway Instead of the shikimate pathway, the myo-inositol biosynthesis has also been exploited for the synthesis of 1,2,3,4-tetrahydroxybenzene (Figure 16).79 Polyhydroxy benzenes along with quinones are known to possess biological activity. Specifically, aurantiogliocladin and fumigatin are antibiotics while coenzyme Q":lo is an essential antioxidant in humans.80 OH OH O. x: s OH \ OH HO O HO OH D-glucose ,. $ 3 HO\ OH 9 OH 9H / myo-inositol HO ’ 0P03H2 1b " Q: OH HO‘ OH ;- HO OH HO OH H O myo-Inosnol 6-phosphate H 0,. O OH (I C OH OH Pyrogallol \ R10 R3 / myoinosose OH Fi1 = R2 = R3 = R4 = H; 1,2,3,4-tetrahydroxybenzene R1 = R2 = R3 = Fl4 = CH3; aurantioagliocladin R1 = R4 = H; R2 = R3 = CH3; fumigatin R1=R2=R3=CH3;R4= \ H CH3” Figure 16. Synthesis of polyhydroxybenzenes from myo-inositol. Key: (a) E. coli JWFl/pAD1.88A, 11%; (b) Gluconobacter oxydans, 95%; (c) H2SO4, HZO, reflux, 66%; (d) 50 psi H2, Rh on A1203, 12 h, 44%; (e) phytase. coenzyme Q n =10 E. coli JWFl/pAD1.88A synthesized 21 g/L of myo-inositol and 4 g/L of myo- inositol-l-phosphate in 11% combined yield from D-glucose under fed-batch fermentor conditions.79 The biocatalyst E coli JWFl/pAD1.88A expressed lNOI-encoded myo- inositol-l-phosphate synthase from Saccharomyces cerevisiae, which catalyzed 25 cyclization of D-glucose 6-phosphate into myo-inositol-l-phosphate. Hydrolysis of the phosphoester into myo—inositol was catalyzed by an unidentified phosphatase within E. croli JWFl/pAD1.88A. Oxidation of myo-inositol into myo-inosose was catalyzed by Gluconobacter oxydans (ATCC 621) in 95% isolated yield. Acid-catalyzed dehydration of myo-inosose in refluxing in 0.5 M H2504 for 9 h under Argon afforded 1,2,3,4— tetrahydroxybenzene in 66% isolated yield. Catalytic hydrogenation of 1,2,3,4- tetrahydroxybenzene over Rh on A1203 afforded pyrogallol in 44% yield.79 Phloroglucinol, which is a substituent for a variety of natural products, was synthesized by two routes and subsequently deoxygenated to resorcinol (Figure 17).8| HO:OH resorcinol d OH OH J30” __,. {:1 HO 5 OH HO OH OH D-glucose phloroglucinol OH R l\ / O O triacetic acid lactone Figure 17. Syntheses of phloroglucinol and resorcinol from D-glucose. Key: (a) E. coli JWFl (DE3)/pJA3.l31A; (b) S. cereviciae; (0) Na, MeOH, 185 °C; ((1) 50 psi H2, Rh on A1203, 12 h, 82%. In the first route, phloroglucinol was synthesized via the intermediacy of triacetic acid lactone by Saccharomyces cereviciae harboring fatty acid synthase mutant (Y2226F).8"‘ In a different route, heterologous expression of pth from Pseudomanas 26 flzmrescens in E. 6011' led to the production of phloroglucinol.81b Catalytic hydrogenation of phloroglucinol over Rh on A1203 afforded resorcinol in 82% yield (Figure 17).80 Hydroquinone was also synthesized from a non-shikimate pathway (Figure 15).8‘)'82 D- Glucose-derived 2-deoxy—scyllo-inosose was dehydrated under acidic conditions to afford hydroxyhydroquinone. Subsequent hydrogenation of hydroxyhydroquinone over Rh on A1203 resulted in hydroquinone in 53% yield. 27 9 Ix) 10 REFERENCES (a) Weissermel, K.; Arpe, H.—J. In Industrial Organic Chemistry, 3rd ed.; VCH: New York, 1997: p. 310. (a) Mullin. R. Chem. Eng. News. 2004,82(42),29. (b) Tullo, A. H. Chem. Eng. News. 2004, 82(11), 20. (a) Campbell, C. J.; Laherrére, J. H. Sci. 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Sci. ‘The Stabilizing Mechanism of Polyol in Heavy Metal-Free PVC Compounds’ manuscript in preparation. (b) Steenwijk, J.; van Es, D. S.; van Haveren, J.; Geus, J. W.; Jenneskens, L. W. J. Appl. Polym. Sci. ‘Compatibility of Polyol with PVC’ manuscript in preparation. Zhang, 2.; Jackson, J. E.; Miller, D. J. Appl. Catal. 2001, 219, 89. Altaras, N. E.; Etzel, M. R.; Cameron, D. C. Biotechnol. Prog. 2001, 17, 52. (a) Kirk-Othmer Encyclopedia of Chemical Technology; Wiley: New York, 2003,; (b) Battat, E.; Peleg, Y.; Bercovitz, A.; Rokem, J. S.; Goldberg Biotechnol. Bioeng. 1991, 37, 1109. (c) Peleg, Y.; Steiglitz, B; Goldberg, 1. Appl. Microbiol. Biotechnol. 1988, 28, 69. (d) Abe, S.; Furuya, A.; Saito,T.; Takayama, K. US Patent 3,063,910, 1962. Wang, X.; Gong, C. S.; Tsao, G. T.; George, T. Biotech. Lett. 1996, 18, 1441. 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Shikimic Acid: Metabolism and Metabolites; Wiley & Sons: New York, 1993; pp 56. Hydroquinone-66: Mervis, S. H.; Walwoth, V. K. Kirk-Othmer Encyclopedia of Chemical Technoloy; Wiley; New York. 2001, http://www.mrw.interscience.wiley.com/kirk/articles/colowalwall1/sect5-fs.html Ran, N.; Knop, D. R.;Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 2001, 123, 10927. Hansen C. A.; Frost, J. W. J. Am. Chem. Soc. 2002, 124, 5926. Hansen C. A.; Dean, A. B.; Frost, J. W. J. Am. Chem. Soc. 1999, 121, 3799. (a) Zha, W.; Shao, 2.; Frost, J. W. J. Am. Chem. Soc. 2004, 126, 4534; (b) Achkar, J.; Xian, M.; Frost, J. W. J. Am. Chem. Soc. 2005 asap. (a) Kakinuma, K.; Nango, E.; Kudo, F.; Matsushima, Y.; Eguchi, T. Tetrahedron Lett. 2000, 41, 1935; (b) Yamauchi, N.; Kakinuma, K. J. Antibiot. 1992, 45, 756. 34 CHAPTER 2 SYNTHESIS OF D,L-1,2,4-BUTANETRIOL Introduction 1,2,4—Butanetriol is an important industrial precursor and a useful synthetic scaffold in organic syntheses.‘ Nitration of racemic D,L-l,2,4—butanetriol leads in 1,2,4- butanetriol trinitrate, which is the energetic plasticizer used in single stage rockets used in the US. military.2 Compared to trinitroglycerine, 1,2,4-butanetriol trinitrate has reduced sensitivity to impact, enhanced stability, and lower volatility (Figure 18).3 The heat of explosion is not compromised in substituting trinitroglycerine (1455 kcal/kg) with 1,2,4— butanetriol trinitrate (1440 kcal/kg).4 HONOH —> OZNO\/\K\QN02 02NO/Y\ONO?_ <— Ho’\(\0H OH OH 0N02 0N02 1 ,2,4-butanetrio| 1,2,4-trinitrobutanetriol trinitroglycerine glycerol Figure 18. 1,2,4-Butanetriol trinitrate and trinitroglycerine. l,2,4-Butanetrioltrinitrate also has potential application as a vasodilator for treatment angina pectoris.5 Coincidentally, trinitroglycerine is the most prescribed nitrate ester for alleviation of angina.6 It is assimilated within a minute and offers relief almost instantaneously. However, the relief is short-lived and longterm use requires increasing doses due to nitrate tolerance.” Furthermore, trinitroglycerine can lead to dilation of other vessels including arterioles and veins, which can lead to side effects such as violent headache that can last for several hour.7b Studies have shown that the rate of hydrolysis of 1,2,4-butanetriol trinitrate is markedly reduced compared to trinitroglycerine and erythrityl trinitrate.7c This slow hydrolysis corresponds to a prolonged effect of the 35 nitrate ester in assuaging angina.7C Replacement of trinitroglycerine, both as an energetic plasticizer and a vasodilator, hinges on the availability of racemic D,L-l,2,4—butanetriol. Enantiomerically pure 1,2,4-butanetriol and its derivatives have been employed in the syntheses of important pharmaceutical compounds (Figure 19).8 Notably, S—1,2,4- butanetriol has been used in the synthesis of lysophosphatidic acid (LPA) analoguesg“ These compounds play a critical role as general growth, survival and pro-angiostenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro.8b Abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Therefore, LPA receptors constitute a potential target for cancer therapy.Ba o O 0 90H 0 . F MeN lysophosphatidic acid analogue (+)-carpaine: n = 3 decarestrictine L (+)-azamine: n = 1 Figure 19. Biologically active compounds derived from S-1,2,4-butanetriol and its derivatives. (+)-Carpaine, which belongs to a novel class of macrocyclic dilactones containing a 2,3,6-trisubstituted piperidine skeleton, exhibits a range of biological activities including antitumor activities at low concentrations.8C These compounds along with (+)- azamine were synthesized from S-1,2,4-butanetriol as a single source of chirality.8d S-3- Hydroxy-y-butyrolactone, a derivative of S-1,2,4-butanetriol, constitutes the core structure of (+)-(2R, 3S, 6R)-decarestrictine L.8° The decarestrictine family is a growing 36 class of natural products with remarkable potential in inhibiting HMG-CoA reductase, the first enzyme involved in the cholesterol biosynthetic pathway.8f Several processes have been developed for the large-scale synthesis of 1,2,4- butanetriol as evident by the patent literature (Figure 20). ' O HO 9 HON OH ' A ’ W OH \/\<(‘) 3-butene-1-ol allyl alcohol I OH O OH a 1 HO OH HOW OH A 1130020\/3\CO2CH3 _. D,L-1,2,4-butanetriol c H b HO OH d HO \ 2 )20 + H : H A, F:_—/ NOH H 2-butyne-1,4-diol 2-butene-1,4-diol dimethyl malate Figure 20. Syntheses of 1,2,4-butanetriol. Key: (a) NaBH4, THF, MeOH; (b) CuCz, 50 atm; (c) i) HgSO4, H2504, ii) 2CuO-CrzO7, H2; ((1) Lindlar’s catalyst, H2; (e) H202, H2WIO4; (f) Pd/C, H2; (g) H202, H2WO4; 0‘) H2804; (i) 1) H202; (I) C02(CO)89 (CO, H2), ii) LiAlH4. The current commercial synthesis of D,L-l,2,4—butanetriol relies on stoichiometric reduction of dimethyl D,L-malate.9 Racemization does not occur during reduction. When dimethyl D— and L-malate is reduced with NaBH,, D- and L-l,2,4—butanetriol is obtained, respectively. 2-Butyn-1,4-diol produced by the reaction of acetylene and formaldehyde (the Reppe process), provides two avenues for D,L—1,2,4-butanetriol production.10 Oxymercuration of 2-butyn-l,4-diol gives 1,4—dihydroxy-2-butanone, which is subsequently hydrogenated to 1,2,4—butanetriol over 2CuO-Cr207 catalyst.10a The catalyst contained 20% copper and 0.5% chromium on silica.lOb Alternatively, 2-butyn-l,4—diol can be hydrogenated to 2-buten-l,4-diol, followed by oxidation to the oxirane-(bis)- methanol.‘1 Hydrogenation of the epoxide over Pd catalyst yields D,L-l,2,4-butanetriol.ll 37 In another route, low yielding dehydration of acetaldehyde-derived 1,3—butanediol leads 3-buten-l-ol.”“ Oxidation to the corresponding epoxide followed by acid-catalyzed hydrolysis results in D,L-1,2,4-butanetriol.12b Hydroformylation of 2,3-epoxy-1-propanol (glycidol) into 3-hydroxy-y-butyrolactone followed by reduction with LiAlH4 results in D,L—l,2,4-butanetriol in low yield.13 Glycidol is obtained from epoxidation of allyl alcohol en route to synthetic glycerol.l4 This reaction is reminiscent of the Prins reaction in which the condensation of allyl alcohol and formaldehyde in the presence of acid affords 1,2,4-butanetriol.15 All the substrates employed thus far for the syntheses of D,L-1,2,4-butanetriol are derived from petroleum. Use of NaBH4 or LiAlH4 in stoichiometric quantities as a reducing agent results in generation of a large amount of salts. Reaction conditions needed to transform 2-butyn-1,4-diol into 1,2,4-butanetriol call for use of mercury and Cr(VI). In addition to being detrimental to the environment, Cr(VI) along with formaldehyde and acetaldehyde are carcinogenic.l6 In light of stricter environmental policies and a long term shift from petroleum-based towards a biomass—derived chemical industry, Chemoenzymatic and biocatalytic routes have been explored for the synthesis of enantiomerically pure 1,2,4—butanetriol. Man Kit Lau in the Frost group has examined a synthesis of D-1,2,4-butanetriol by employing a Chemoenzymatic route starting from glycaldehyde and acetaldehyde (Figure 21).17 2-Deoxyribose 5-phosphate aldolase catalyzed the condensation of glycaldehyde and acetaldehyde to D-3,4-dihydroxybutanal,'8a which was hydrogenated over Ru on C to D-l,2,4-butanetriol in 17% overall yield after vacuum distillation.” 38 b O O a OH O OH )bOH + )LH —» HONH —> HO\/'\/\ OH glycaldehyde acetaldehyde 3,4-dihydroxybutanal D-1 ,2,4-butanetriol 11 a, c HO/IOJrOH OH 2,4-dideoxy- D-hexapyranose Figure 21. Chemoenzymatic synthesis of D-l,2,4-butanetriol. Key: (a) 2- Deoxyribose- 5—phosphate aldolase (20,000 U), pH 7.6, rt, 35%; (b) 1.0 mol% Ru on C, 14 atm H2, 30 °C, 5 h, 99%; (c) acetaldehyde. The double aldol condensation product of one glycaldehyde and two acetaldehyde molecules, 2,4-dideoxy-D-hexapyroanoside, accounted for the majority of the product formed.18b Although this route is very concise, it is low yielding and affords only the D- enantiomer of 1,2,4-butanetriol. Moreover, the starting materials are derived from nonrenewable sources. Glycaldehyde is derived from reaction of formaldehyde with syn gas (CO, H2) over Rh catalyst at 300 atm and 150 °C.19 Acetaldehyde on the other hand is derived from Wacker oxidation of ethylene over PdCl2 and CuCl2 catalysts at 125-130 °C and 11.3 atm.20 Recently, microbes have been constructed that catalyze the synthesis of D-1,2,4- butanetriol and L-l,2,4—butanetriol from D-arabinose and L-xylose, respectively (Figure 22).21 The first step involves oxidation of L-arabinose and D-xylose to L-arabinoic acid and D-xylonic acid catalyzed by L-arabinose dehydrogenase and D-xylose dehydrogenase, respectively. Dehydration of the L-arabinoic acid and D-xylonic acid to the corresponding pentulosonic acid is catalyzed by L—arabinoate dehydratase and D-xylonate dehydratase, respectively. 39 OH OH OH OH OH HO\/'\‘/H'\rrH a HOMOH b HO\/'\H/u\g,0H c Ho\/kH/IOLH dHO\}\/\OH OH O D- xyloseO D- x—ylonic acid 3- --deoxy-D glycero- 3,4- ~dihydroxy- D-1,2,4-butanetriol pentulosonic acid D-butanal OH OH OH OH OH HOMH aa HOMOH bb HoNkan c Ho\/\)LH d HONOH OH O L-arabinose L arabinonicO acid 3- --deoxy -L- g-lycero- 3.4- -dihydroxy- L-1,2,4-butanetriol pentulosonic acid L-butanal Figure 22. Biosynthetic pathway of D-xylose and L-arabinose to D- and L-1,2,4- butanetriol. Enzymes: (a) D—xylose dehydrogenase; (aa) L-arabinose dehydrogenase; (b) D-xylonate dehydratase; (bb) L-arabinonate dehydratase; (c) 2-keto acid decarboxylase; (d) alcohol dehydrogenase. Decarboxylation followed by reduction of D- and L-3-deoxy-glycero-pentulosonic acid catalyzed by benzylformate decarboxylate and alcohol dehydrogenase afforded L- and D—1,2,4-butanetriol in 19% and 18% overall yield. The starting L-arabinose and D— xylose are derived from abundantly available plant hemicellulose. However, inexpensive, pure streams of these pentoses are not commercially available yet.22 Catalytic hydrogenation of D, L-malic acid and L-malic acid were explored as alternatives to stoichiometric reduction of methyl D,L-malate. Racemic D,L-1,2,4-butanetriol was obtained as a product from hydrogenation of both substrates. As an alternative route, a Chemoenzymatic synthesis of D,L-l,2,4-butanetriol starting from L—ascorbic acid is described. The centerpiece in this route is the formation and catalytic hydrogenation of 2-hydroxy-2-buten-4-olide. Syntheses were examined from the perspective of starting material, the number of steps, reaction conditions, byproducts and product stereochemistry. Catalytic Hydrogenation of Malic Acid Based on the successful use of Ru on C in the hydrogenation of lactic acid to 1,2- propanediol in aqueous medium,23 this catalyst was employed in the hydrogenation of D,L-malic acid 2a,b. Variation of pressure, temperature, reaction time, concentration of D,L-malic acid, the D,L-malate/Ru mol ratio and catalyst composition were explored (Table 1-2). A typical hydrogenation was conducted by dissolving D, L-malic acid in distilled, deionized water (100 mL) in a glass liner. The catalyst was suspended in malic acid solution. The liner was inserted into a 500 mL Parr 4575 stainless steel high temperature-high pressure reactor, and the vessel was sealed. The temperature and stirring rate were controlled by a Parr 4842 temperature controller. Hydrogen was bubbled through the reaction mixture for 10-15 min to remove air while stirring at 100 rpm. The vessel was then charged with H2 to a pressure below the desired value. After heating the reaction to desired temperature, the H2 pressure was adjusted to the desired pressure. The reaction was stirred at 200 rpm for 1-10 h at a constant temperature. When the reaction was complete, the reaction vessel was cooled to rt and the pressure was released. After removal of the catalyst by filtration, the reaction mixture was concentrated to dryness under vacuum to afford a colorless oil, which was derivatized with (N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and analyzed by gas chromatography. 4] 0 o H02C/\rC02H RU on C HO/YVOH 5 7? 1:013 HO/\C';\ OH _’ OH 2 b H2 1 b DL HO 3 5 7 a, D,L' - a, a - 2a 0- ”“3150 1a 0- butanetriol HOQC’VOH HO/VVOH HONOH 2b L- 30' 1b L- OH 4 6 8 Figure 23. Catalytic hydrogenation of malic acid. Table 1 a-c. Hydrogenation of D, L-Malic Acid as a Function of H2 pressure, Temperature, and Reaction Time. reaction product, intermediate, byproduct yields (mol %)1 conditions 1 2 3 4 5 6 7 8 a. atm variable: H2 pressure2 68 24 28 22 17 3 4 O 1 136 67 9 6 7 4 3 0 1 204 70 4 3 2 6 6 0 l 272 71 2 0 1 9 8 0 2 340 73 0 0 0 13 9 0 2 b. °C variable: temperature3 125 61 14 1 1 l 4 3 2 O 130 67 1. 10 0 10 7 3 2 135 74 0 0 0 12 8 3 3 140 70 0 O 0 15 9 2 2 c. h variable: time4 1 21 20 46 9 2 5 0 O 5 69 8 5 trace 6 7 2 2 10 72 l l 1 1 1 9 2 2 15 71 1 1 trace 13 10 3 2 20 66 0 0 0 14 10 3 2 ' See Figure 23 for structures. 3 1 M malate, 135 °C, 5 wt % Ru on C, 1.3 mol % Ru/malate, 10 h. 3 l M malate, 340 atm H2, 5 wt % Ru on C, 1.3 mol % Ru/malate, 10 h. 4 1 M malate, 135 °C, 340 atm H2, 5 wt % Ru on C, 1.3 mol % Ru/malate. At 68 atm H2 pressure, 3-hydroxy-y—butyrolactone 3 and 3,4-dihydroxybutyric acid 4 were the dominant products formed along with D,L-1,2,4-butanetriol la,b (Figure 23, Table 1a). Formation of lactone 3 and carboxylate 4 was consistent with a more rapid rate of reduction of the malate C-l carboxylate with its adjacent a—hydroxyl group relative to the malate C-4 carboxylate with its adjacent methylene group. Formation of 3- 42 hydroxy—y-butyrolactone 3 and 3,4-dihydroxybutyric acid 4 declined with increased formation of 1,2,4—butanetriol la,b when the H2 pressure was increased to 136 atm (Table la). Above .136 atm, increasing H2 pressures led to smaller incremental increases in product D,L-1,2,4-butaentriol la,b and increased formation of byproducts 1,2—propanediol 5, 1,4-butanediol 6 (Table la). At all pressures, temperatures, reaction times, malate concentrations, and Ru/malate ratios examined, at least 90% of the molar decline in D,L- malate Za,b starting material could be accounted for by the formation of product D,L- 1,2,4—butanetriol la,b, reaction intermediates 3 and 4, or byproducts 5, 6, 7, and 8 (Figure 23, Table 1a). Increased temperature had a pronounced impact on product and byproduct formation while the reaction pressure was maintained at 340 atm (Table 1 b). The yield of D,L-l,2,4-butanetriol 1 a,b increased as the temperature was increased from 125 °C to 135 °C. Formation of byproducts 1,2-propanediol S and 1,4-‘butanediol 6 increased across the entire range of examined temperatures. Formation of 1,2-butanediol 7 in addition to ethylene glycol 8 was also observed. The maximum yield of 1,2,4—butanetriol 1 a,b was obtained at 135 °C and 340 atm H2 pressure at a reaction time of 10 h (Table 1b). Longer reaction times resulted in reduced yields of D,L-l,2,4—butanetriol la,b and increased yields of byproducts 5 and 6 (Table 1c). A 1 M concentration of D,L-malate 2a,b led to the highest yield of D,L-1,2,4- butanetriol 1 after reaction for 10 h at 340 atm H2 and 135 °C (Table 23). Increasing D,L-malate Za,b concentrations beyond 2 M led to a precipitous decline in the yields of product 1 and byproducts 5-8 (Table 2a). The highest yield of 1,2,4-butanetriol la,b was achieved with a Ru/malate mol ratio of 1.3 mol % when 1 M D,L-malate 2a,b was reacted at 340 atm H2 and 135 °C. 43 Table 2 a-c. Hydrogenation of D, L-Malic Acid as a Function of Malate Concentration, Mole Ratio of RuzMalate and the Mole Ratio of Ru:Re. reaction product, intermediate, byproduct yields (mol %)1 conditions 1 2 3 4 5 6 7 8 a. M variable: malate concentration2 1 72 2 1 1 12 9 2 3 2 63 11 1 4 12 6 2 2 5 3 94 0 0 0 0 0 2 10 0 99 trace trace 0 0 0 2 b. mol% variable: Ru/malate3 0.66 63 l 6 2 3 12 3 0 1.3 73 0 2 0 10 9 2 2 2.0 69 0 1 0 12 6 2 3 3.3 55 O 1 0 15 3 2 2 c. mol% variable: mol Ru/mol Re4 10 55 6 0 0 7 5 5 0 25 51 7 2 1 4 5 3 0 45 58 4 2 1 3 6 6 0 65 70 10 trace 1 2 8 3 0 85 34 13 21 6 1 9 1 0 ‘ See Figure 23 for structures. 2 340 atm H2, 135 °C, 5 wt % Ru on C, 1.3 mol % Ru/malate, 10 h. 3 l M malate, 340 atm H2, 135 °C, 10 h. 4l M malate, 135 °C, 340 atm [-12, 1.3 mol% Ru/Re on C, 10 h. Increasing the Ru/malate mol ratio beyond 1.3 mol % led to a steady decline in the yield of D,L-1,2,4-butanetriol la,b and had a disproportionate impact on increased formation of 1,2-propanediol 5 relative to 1,4—butanediol 6 formation (Table 2b). Full conversion of D,L-malic acid was observed with intermediates 3 and 4 essentially absent in the solution. This however, did not translate to improved conversion to D,L-1,2,4- butanetriol la,b. Instead, the amount of byproducts increased drastically while the yield of D,L-1,2,4-butanetriol la,b declined. As a final parameter, a mixture of Re and Ru ranging from 10-85 wt % was evaluated for hydrogenation of D, L-malic acid (Table 2c). The overall catalyst loading of Re-Ru on C was maintained at 1.3 mol%. When Ru catalyst was spiked with 10 wt % of Re catalyst, there was over a 90% conversion of D, L-malic acid 2 but only 55% of 1,2,4—butanetriol was observed (Table 2c). The catalyst 44 composition of 25 wt % Re relative to Ru resulted in a slight decrease both in .D.L—1,2,4- butanetriol la,b and 1,2-propanediol 5. At the optimum catalyst composition of 65 wt % Re/Ru, the amount of 1,2-propanediol 5 diminished to only 2% while 1,4—butanedi01 6 increased to 8%. At the highest composition of Ru relative to Re, 1,2-propanediol 5 vanished almost completely while 1,4-butanediol 6 formation was at its highest levels (9%). The amount of D,L-1,2,4-butanetriol la,b also declined significantly from 70% when the catalyst composition of 65 wt % Re/Ru was used down to 34% when the catalyst composition of 85 wt % Re/Ru was used (Table 20). Hydrogenation over 1.3 mol% of Re on carbon relative to D, L—malic acid 2 resulted in the recovery of unreacted starting material (results not shown). Other catalysts such as Pt, Pd and Rh were examined for the hydrogenation of D, L-malic acid 2. Unreacted starting material was recovered with 1.3 mol% Pt on C and 1.3 mol% Pd on C while only 12% of D,L—1,2,4- butanetriol la,b and 3-hydroxy-y-butyrolactone 3 (12%) was obtained with the 1.3 mol% Rh on C. Catalytic hydrogenation at the optimized conditions of 340 atm H2 and 135 °C of a 1 M aqueous solution of L-malic acid 2b over 5% R on C using a 1.3% Ru/malate ratio and a reaction time of 10 h was also examined. Formation of Mosher esters24 of the product and analysis by HPLC indicated a 74% yield of racemic D,L-l,2,4—butanetriol la,b. Apparently, Ru-catalyzed racemization of the stereogenic center occurred during the course of the catalytic hydrogenation.25 Hydrogenation of a 1 M aqueous solution of D,L-1,2,4-butanetriol la,b at 340 atm H2 and 135 °C for 10 h over 5% Ru on C using a 1.3 mol % Ru/ D,L-1,2,4-butanetriol la,b mol ratio was also examined to determine if byproducts were formed as a consequence of product reactivity. In addition to a 70% 45 recovery of unreacted D,L-1,2,4—butanetriol la,b, formation of 1,2-propanediol 5 (18%), 1,4-butanediol 6 (3%), 1,3-propanediol (2%), 1,2-butanediol 7 (2%), 1,3—butanediol (2%) and ethylene glycol 8 (3%) was observed. Purification of product D,L-l,2,4-butanetriol la,b was not straightforward. Short path distillation in vacuo of crude product afforded a 28% overall yield from starting D,L- malic acid Za,b of a 95:5 mol/mol mixture of D,L-1,2,4-butanetriol la,b and 1,4- butanediol 6. Trace amounts of other diols could also be detected in the distilled product. The loss of more than half of the product during distillation resulted from decomposition of product D,L-1,2,4-butanetriol la,b. Contamination may reflect azeotropic distillation of the diols with the desired D,L-l,2,4-butanetriol la,b during purification. Alternative Substrates for Catalytic Hydrogenation Byproduct diol formation during catalytic hydrogenation of D, L-malic acid 2 is not readily removed by simple distillation. Intermediates 3 and 4 are poised to undergo dehydration via [ES-elimination to afford 1,4-butanediol 6 after reduction. The remaining diols are presumably derived from CC and C-0 cleavage of product la,b as a result of forcing reaction conditions required for hydrogenation of the unactivated carboxylate of malate. Ideally, substrates that could be hydrogenated to D,L-l,2,4—butanetriol la,b under conditions that do not promote C-C and C-0 cleavage are desired (Figure 24). The presence of an a-hydroxy moiety adjacent to the carboxylic acid reduces the energy barrier required to hydrogenate the carboxylic acid. If intermediates are formed during hydrogenation, they should not undergo secondary reactions such as elimination reactions to afford byproducts. 2,4-Dihydroxybutyric acid 9 or the corresponding 2-hydroxy-y- butyrolactone 10 are would be ideal substrates for catalytic hydrogeantion to afford D,L- 1,2,4—butanetriol. OH O OH Ho’\/S‘cozH HOVLCOZH HO/\{'\COZH 9 11 0” 2,4-dihydroxybutyric acid 4-hydroxy-2-ketobutyric acid D-erythronic acid 133 or L-threonic acid 13b 0 O LOIO fro 10 OH 12 OH HO "OH 2-hydroxy-y-butyrolactone 2-hydroxy-2-buten-4-olide D-erythronolactone 14a or L-threonolactone 14b Figure 24. Alternative substrates for catalytic hydrogenation. They could both be hydrogenated directly to D,L-l,2,4—butanetriol la,b. Under milder reaction conditions, hydrogenolysis of D,L-1,2,4—butanetriol la,b to 1,2-propanediol 5 is not expected to lower the yield of product. Dehydration of the (It-hydroxyl group is not expected to occur because of its proximity to the carboxylate. Another substrate that could be hydrogenated to D,L-1,2,4-butanetriol la,b via the intermediacy of 2,4- dihydroxybutyric acid 9 or 2—hydroxy-y-butyrolactone10 is 4—hydroxy-2-ketobutyric acid 11 or-the corresponding lactone, 2-hydroxy-2-buten-4-olide 12. D-Erythronic acid 13a or L-threonic acid 13b and the corresponding D-erythronolactone or L-threonolactone l4a,b were also evaluated as possible substrate for catalytic hydrogenation to D,L-1,2,4- butanetriol la,b (Figure 24). 2-Hydroxy-y-butyrolactone 10 Two procedures, which involve reduction of the unactivated carboxylic acid group of malic acid 2a,b have been described (Figure 25).26 In the first route, the 01- 47 hydroxyl group along with the carboxylate are simultaneously protected as a 2-t-butyl-5- 25a substituted 1,3-dioxolanone 15 using pivaldehyde in the presence of acid catalyst. of 1 OH /1( O a O b O HO2C\/S\COZH + CHO —> H020 \HJW ' HO/\I"J\\( 2a,b O O 15 16 ld,e / OH OH OH OH HO2C\/S\C02Me —* Ho’\/S‘COZMe —> o O —’ H0 17 18 1o ‘3’” Figure 25. Syntheses of 2-hydroxy-y-butyrolactone. Key: a) pTSA, pentane, Dean- Stark, 79%; b) BH3-SMe3, B(OCH3)3, THF, 97%; c) HCl; (1) Trifluoroacetic anhydride; e) Methanol; f) BH3—SMe3, B(OCH3)3, THF, 81% g) NazCO3, Dowex-SO (H"); (h) 170 atm H2, 125 °C, 1.0 mol % Ru/C, 10 h, 100%. The resulting unprotected carboxylic acid was reduced to the corresponding alcohol 16 using 1.3 equivalents of borane-dimethylsulfide.25b Alcohol 16 residue was redissolved in MeOH and concentrated several times to remove contaminating methyl borate byproduct. Acid deprotection of alcohol 16 afforded 2-hydroxy-y-butyrolactone 10 after removal of solvent. In the second procedure, trifluoroacetic anhydride was suspended in D,L—malic acid 2a,b to afford the anhydride.25° Methanolysis of the anhydride resulted in methyl malate 17 as a sole product. Reduction of the C-4 carboxylic acid required 2 equivalents of borane-dimethyl sulfide.25b The resulting product was a mixture of 2,4-dihydroxy-methy1-butyrate 18 and 2-hydroxy-y- butyrolactone 10. Base hydrolysis of the mixture followed by a cation exchange column resulted in 2-hydroxy-y-butyrolactone 10 after removal of water. Hydrogenation of the 0.5 M aqueous solution of 2-hydroxy-y-butyrolactone 10 at 340 atm H2 for 10 h over 1.3 48 mol % Ru/C at 135 °C was conducted. D,L-1,2,4-Butanetriol la,b was obtained in 82% yield in addition to formation of 1,2-propanediol 5 in 13% yield. D, L-Malic acid 2 hydrogenation studies (Table 1 and 2) that were conducted established that H2 pressures between 136 and 204 atm H2 were sufficient to hydrogenate the carboxylic acid adjacent to the hydroxyl group. To avoid hydrogenolysis of 1,2,4-butanetriol la,b to 1,2- propanediol 5, it was necessary to reduce temperature and catalyst loading. Dehydration of 3,4-dihydroxybutyric acid 4 and subsequent hydrogenation to 1,4-butanediol 6 was shown to occur during catalytic hydrogenation. With the current substrate, 2-hydroxy-y- butyrolactone 10, this type of dehydration is not possible. As predicted, quantitative conversion of 2-hydroxy-y-butyrolactone 10 to D,L—l,2,4-butanetriol la,b was observed under milder reaction conditions (170 atm H2, 125 C, and 1.0 mol% Ru on °C) relative to those employed (340 at Hz, 135 °C, and 1.3 mol% Ru on C) for D,L- and L-malic acid (Figure 25). 4-Hydroxy-2-ketobutyric acid 11 In mammals, 2-keto-4-hydroxybutyrate 11 is an intermediate in L-homoserine degradation (Figure 26).273 This degradation is catalyzed by glutamate-aspartate transaminase, where oxaloacetate or 2-ketoglutarate serve as amino group acceptor. + UHs a O b O HoMco; ——> HO’VLCOQ’ —> HCHO + Ace; Figure 26. Degradation of L-homoserine in mammals. Key: a) glutamate-aspartate or glutamine-alanine transaminase; b) 2-keto-4-hydroxyglutarate aldolase. 49 Glutamine-alanine transaminase also catalyzes transamination of L-homoserine to 2-keto- 4-hydroxybutyrate 11 and either pyruvate or 2-ketoglutarate serve as the amino group acceptor. 26” The a-Keto acid that results is further cleaved to pyruvate and formaldehyde by 2-keto—4-hydroxyglutarate aldolase (Figure 26)?“f In bacteria and fungi, L- homoserine is an intermediate in the biosynthesis of L-threonate 13b from L-aspartate.28 For the synthesis of 2-keto-4—hydroxybutyrate 11, L-aspartic acid was used as the starting material (Figure 27). Methanolysis of the aspartic anhydride afforded a mixture of C-1 and C-4 methyl aspartates.25c These isomers were separated by trituration with diethyl ether and petroleum ether (2:1). The unprotected carboxylic acid of the desired C-l methyl aspartate 19 was reduced with two equivalents of borane to afford a methyl ester of homoserine 20.25b Subsequent base hydrolysis of ester 20 afforded the sodium salt of L-homoserine. o 0 MHz 3 b F3C/ILNH C Fac’lLIyH HO 0 ' ' _ - ,, - 2 $00211 HO2C\/\002<3H3 Ho’\/‘co2CH3 L-asparic acid 19 20 id 0 CH0 NH2 NHL — e'f HO \ OH + 7 H0 002 ‘— | HOMCOQNa 11 ’ N CH3 L-homoserine Figure 27. Synthesis of 2-keto-4-hydroxybutyrate from L-aspartic acid. Key: (a) Trifluoroacetic anhydride; b) Methanol; c) BH3.THF; d) 2 eq. NaOH; e) CuC12.2H20, acetate buffer, pH 5; f) Dowex—SO (Hi), pH 7. Transamination of L-homoserine with pyridoxal-HCI was catalyzed by copper chloride to afford 2—keto-4—hydroxybutyrate 11 in disappointing yield 10-15%.29 The product was contaminated with acetate used in the buffer. This synthesis of 2-keto—4— 50 hydroxybutyrate 11 along with the two syntheses of 2,4—dihydroxybutyrate 9 are not amenable to scale up due to the stoichiometric use of borane reagent. Furthermore, the methyl borate esters generated as by-products are difficult to separate from product. D-Erythronolactone 14a and L-threonolactone 14b Oxidative cleavage of D-isoascorbic acid and L-ascorbic acid in the presence of basic hydrogen peroxide solution afforded D-erythronate 13a and L-threonate 13b, respectively in addition to sodium oxalate (Figure 28).30 Acidification of the product mixture to pH 2.5 with HCl resulted in formation of D—erythronolactone 143 and L- threonolactone 14b. Following D-erythronolactone 14a and 14b D-erythronolactone 143 and 14b. HO O HO O OH _ OH HO OH HO/E/K’ D-isoascorbic acid 9H b O O D Erythritol . C ' HO —a—> HOYCOZNa —> 'I 2 0 OH HO OH OH How D-erythronate 13a 143:9 HO/YK/OH or L-threonate 13b HO OH 0” L-threitol L-ascorbic acid Figure 28. Syntheses of D-erythronolactone and L-threonolactone . Key: (a) N aCO,, H202; (b) 6 M HCl, pH 2.5, 94%; (c) 340 atm H2, 135 C, 1.3 mol% Ru on C, 10 h, 100%. The propensity for elimination of water to occur in D-erythronolactone 14a under hydrogenation conditions was evaluated. This avenue was pursued because of the previous observation that intermediates 3-hydroxy—y-butyrolactone 3 and 3,4- dihydroxybutyrate 4 presumably underwent dehydration-hydrogenation sequence to afford 1,4—butanediol 6 during malate hydrogenation (Figure 23). An aqueous solution of 51 D-erythronolactone 143 was subjected to hydrogenation conditions at 340 atm H2 and 135 °C over 1.3 mol% Ru on C, yielding a quantitative amount of D-erythritol (Figure 28). No 1,2,4—butanetriol la,b, the product of dehydration-hydrogenation, was observed. The hydrogenolysis of L-threonolactone also led to of L-threitol as the sole product. 2—Hydroxy—2-buten-4-olide 12 from D-erythronolactone 14a With D-erythronolactone 14a in hand, synthesis of 2-hydroxy-2-buten-4-olide 12 was attempted with hopes to ultimately reduce the butenolide 12 to D,L-l,2,4—butanetriol la,b (Figure 29). The D-erythronolactone 14a was benzoylated or acetylated and subsequent B-elimination with a base afforded 2-O-acetyl~D-erythronolactone or 2-0- 3‘32 Deprotection of the acetyl or benzoyl benzoly—D-erythronolactone, respectively. group using in situ generated lithium hydroxide resulted in the degradation of the product and 2-hydroxy—2-buten-4-olide was not isolated (Figure 29).30 o o C o o a g ——-> (J / AcO¢ bAc 21a OAc \ HO“. "OH b 12 OH 14a \ O o d O 0 9" LT —+ LT 1320* 6132 21b 082 Figure 29. Attempted synthesis of 2-hydroxy-2-buten-4-olide. Key: a) Acetic anhydride; b) pyridine, benzoyl chloride; c) pyridine; d) triethylamine; e) LiOH. Successful elimination was achieved with trifluoroacetic anhydride in the presence of pyridine as both a solvent and base. 2-Hydroxy-2-buten-4—olide was obtained in quantitative yields after hydrolysis. However, large-scale purification of the 52 butenolide 12 from pyridine and pyridinium trifluoroacetate using multiple cation exchange columns was tedious. This route was ultimately abandoned for the synthesis of butenolide 12. An alternative synthesis of this substrate starting from L—ascorbic acid via the intermediacy of L-threonate 13b and 4-hydroxy-2-ketobutyrate 11 was pursued (Figure 30). HQ 0 HO ’ 0 a 9“ b _ —> HOWCOZNa —> HO\/\n,C02Na HO OH OH O L-ascorbic acid 13" 11 c O 0 d HO ’ 1:25 —" NOH OH OH 12 1a,b Figure 30. Chemoenzymatic synthesis of D, L-l,2,4-butanetriol from L-ascorbic acid. Key: (a) NaZCO3, 30% H202, 74%; (b) JWFl/pON 1.1 18B, pH 8.2, 37 °C, N2, 80%; (c) HCI, pH 1.5, 93%; (d) 1.0 mol% Ru on C, 170 atm H2, 125 °C, 96%. Chemoenzymatic Synthesis of D,L-l,2,4-Butanetriol To obtain large quantities of L-threonate 13a, a literature procedure was modified.29a Instead of a 0.2 M solution of L-ascorbic acid, a 1 M solution was used. Because of its elevated concentration, byproduct sodium oxalate precipitated out of solution when the reaction mixture was allowed to stand at 4 °C. This eliminated the need to concentrate large volumes of water along with the multi step procedure (acidification, EtOAc extraction, concentration and base hydrolysis) previously required to obtain L—threonate 13b. Reaction of L-ascorbate at 1 M concentration with H202 afforded L-threonate 13b in 74% yield (Figure 30). In a Chemoenzymatic synthetic study of 2-keto-3—deoxy-aldonic acid starting from aldonic acids, dihydroxy-acid dehydratase was shown to accept different substrates.30 53 This enzyme catalyzes the dehydration of 2,3-dihydroxyisovalerate and 2,3-dihydroxy-3- methylvalerate during the biosyntheses of L-leucine and L-isoleucine the dehydration, respectively.33 Spinach and Escherichia coli were explored as sources for this enzyme. The spinach dihydroxy-acid dehydratase with its active site [2Fe-2S] cluster was stable to air but overexpression was not an option since the encoding gene had not yet been identified.34 Dihydroxy-acid dehydratase from E. coli with its active site [4Fe-4S] cluster was unstable when exposed to air.35 However, the encoding ilvD gene was known and thus was amenable to overexpression. Dihydroxy-acid dehydratase was partially purified by a modified literature procedure from a homogenate prepared from spinach leaves using protamine sulfate precipitation followed by DEAE-cellulose anion exchange chromatography (Table 3).30 Table 3. Purification of Dihydroxy-acid Dehydratase from Spinach Leaves. Specific Total Step Protein Activity Activity Yield Purification (mg) (U/mg) (U) (%) (fold) Crude lysate 6044 0.89 538 100 1 Protamine sulfate 3198 0.13 494 92 1.4 DEAE cellulose 98 3.4 332 60 38 U/mg = 1 umol/min/mg. This short purification scheme afforded an enzyme preparation with a specific activity of 3.4 U/mg, which constituted a 38-fold increase in specific activity and a 60% yield in total units relative to the dihydroxy-acid dehydratase activity assayed in crude spinach homogenate. Conversion of L-threonate 13b (152 mM) to 4—hydroxy-2— ketobutyrate 11 using the spinach dihydroxy—acid dehydratase (630 units) was carried out at 37 °C and pH 8.0 under N2 (Figure 31). The spinach dihydroxy—acid dehydratase activity was relatively stable (~3.3 U/mg) during the 48 h reaction time but only a 22% yield of 4—hydroxy-2-ketobutyrate 11 was obtained. 54 160 4 120 + A 0 concentration (mM) 00 o 1 1 16 specific activity (U/mg) o . 11].]. 0 612182430364248 time(h) Figure 31. Conversion of L-threonate to 4-hydroxy-2-ketobutyrate catalyzed by dihydroxy-acid dehydratase partially purified from spinach leaves. Legends: L-threonate (white bar), 2-keto-4—hydroxybutyrate (black bar), specific activity (open circle). Due to the lack of chromosomal DNA sequence for spinach dihydroxy-acid dehydratase gene, attention was focused on overexpressing the ilvD gene locus in Escherichia coli. Construct Design and Culturing Conditions E. coli JWFl, which was used as the host strain, was derived from E. coli RB791 (W31 10 lale). Homologous recombination was used to replace the serA gene from the RB791 genome with the non-functional serA gene.36a Consequently, E. coli JWFl encoded catalytically inactive D-3-phosphoglycerate dehydrogenase, which is an enzyme required for L-serine biosynthesis. Dihydroxy-acid dehydratase was encoded by ilvD in plasmid pWN3. 196A and transcribed from a PM. promoter while in plasmid pON1.118B ilvD was transcribed from a PM. promoter (Table 4). Both plasmids used in this study contained the serA gene, which encodes D-3-phosphoglycerate dehydrogenase (Table 4). This provided the basis for plasmid maintenance when E. coli JWFI was grown in minimum salts medium L-serine supplementation. 36*" The additional plasmid-localized 55 lac]Q gene in pON1.118B allowed ilvD transcription to be controlled by the concentration of isopropyl B-D-thioglactopyranoside (IPTG) in the growth medium. Table 4. Plasmid Restriction Maps. Plasmid (size) Plasmid Mapa pWN3 196A H E E H (50.143) 1 >H>J CmR P,alc ilvD H E E B B H pON1.11BB 1 ) ( ) (9.0-kb) >h-I1-H < Ap lale Ptalc ilvD serA “ Restriction enzyme sites are abbreviated as follows: H = HindIII, E = EcoRI, B = BamHI. Parentheses indicate that the designated enzyme site has been eliminated. Lightface line indicates vector DNA; boldface line indicates insert DNA. Both E. coli JWFl/pWN.196A and JWFl/pON1.118B were cultured under fed- batch fermentor conditions at 33 °C and pH 7.0. Dissolved 02 levels were maintained at a set point of 20% air saturation. D-Glucose addition was controlled by dissolved 02 concentration with the rate of D-glucose addition dictated by a proportional—integral- derivative (PID) control loop. When dissolved 02 level exceeded the set point value indicating decreased microbial metabolism, the rate of D—glucose addition was increased. Conversely, the rate of D-glucose addition was decreased when the dissolved 02 level declined below the set point value indicating increased microbial metabolism. Transcription of dihydroxy—acid dehydratase in E. coli JWFl/pON1.118B was induced by addition of IPTG to a final concentration of 218 mg/L when both the impeller speed and airflow had reached the maximum settings of 1100 rpm and 1.0 L/min, respectively. The respective E. coli were cultured for 36 h and the cells were harvested by centrifugation of the fermentation broth. 56 After E. coli JWFl/pWN3.l96A cells were harvested, they were resuspended phosphate buffer previously purged with N2 and lysed by passage through French press. After centrifugation, the resulting crude lysate containing 65,000 U of dihydroxy-acid dehydratase was combined with L-threonate (155 mM) inside a fermentor. A fermentor was used to maintain a reaction temperature of 37 °C, the solution at pH 8.2, and a constant N2 atmosphere during the enzyme-catalyzed dehydration. 180 10 150-c- _. _. 0“" -—8 120 I 90~ 60- I concentration (mM) specific activity (U/mg) 30- I. 0 1 2 3 time (h) Figure 32. Conversion of L-threonate to 4-hydroxy-2-ketobutyrate and associated specific activity for dihydroxy-acid dehydratase from E. coli JWFl/pWN3.l96A. Legends: L-threonate (white bar), 2-keto-4-hydroxybutyrate (black bar), specific activity (open circle). Incubation with crude lysate of E. coli JWFl/pWN3.196A resulted in the conversion of 60% of L-threonate to 2-keto-4-hydroxybutyrate in 5 h (Figure 32). About 70% of the initial activity of E. coli dihydroxy-acid dehydratase activity was lost after 5 h of bioconversion. Comparison of dihydroxy-acid dehydratase specific activity from spinach leaves and E. coli JWFl/pWN196A for the dehydration of native substrate and L- threonate is shown in Table 5. 57 Table 5. Substrate Specificity Comparison. Specific activity‘ Substrate Dihydroxy acid dehydratase Spinach dihydroxy acid of J WFl/pWN3. 196A2 dehydratase’ 2,3-dihydroxy isovalerate 9.04 3.39 L-threonate 0. 17 0.49 specific activity ratio 53:1 7:1 ‘ pmol/min/mg. 2crude cell lysate. 3 partially purified dihydroxy-acid dehydratase. The spinach dihydroxy-acid dehydratase had a 1/7 ratio of specific activities for dehydration of L-threonate 13b/dehydration 2,3-dihydroxy isovalerate (native substrate). E. coli dihydroxy-acid dehydratase had a substantially lower 1/53 ratio of specific activities for dehydration of L-threonate 13b/dehydration of 2,3-dihydroxy isovalerate (native substrate). 1000 250 O A g 800 -~ ' ~- 200 g s o a z > 9 e g 4001— , - 100 g s '5 0 200 ~ - 50 E21 0 4. I . , ' , : ~ 0 0 2 3 4 5 6 time (h) Figure 33. Conversion of L-threonate to 4-hydroxy-2-ketobutyrate and associated specific activity for dihydroxy-acid dehydratase from E. coli JWFl/pONl.ll8B. Legends: L-threonate (white bar), 2-keto-4- hydroxybutyrate (black bar), specific activity (open circle). The ability to overexpress plasmid-localized ilvD from a Pm promoter in E. coli JWF 1/pON1.1 188 more than compensated for the instability of E. coli dihydroxy-acid dehydratase in air and its lower activity towards L-threonate 13b relative to spinach dihydroxy-acid dehydratase and expressed by E. coli JWFl/pWN3.196A, which 58 transcribed ilvD from a PM. promoter. Harvested E. coli JWFl/pON1.1 18B cells were resuspended in the concentrated solution of L-threonate 13b after precipitation/removal of sodium oxalate, addition of phosphate buffer, and purging of the reaction solution with N2. After lysis of the cells and centrifugation, the reaction solution contained 2.4 x 106 U of dihydroxy-acid dehydratase. A fermentor was used to maintain a reaction temperature of 37 °C, the solution at pH 8.2, and a constant N2 atmosphere during the enzyme- catalyzed dehydration. This led to the conversion of L-threonate 13 into 4-hydroxy-2- ketobutyrate 11 in 80% yield after 5 h (Figure 33). Approximately half of the DVD specific activity was lost during the enzyme-catalyzed dehydration. Allowing the enzyme-catalyzed dehydration to continue beyond 5 h did not lead to conversion of the residual amount of L-threonate 13b into additional concentrations of 4-hydroxy-2- ketobutyrate 11. The crude reaction mixture was acidified to pH 1.5 and the precipitated protein removed by centrifugation. Lactonization of 2-keto—4hydroxybutyric acid and selective extraction away from L-threonolactone 14b using liquid-liquid extraction37 with EtOAc afforded a 93% yield of 2-hydroxy-2-buten-4—olide 12 that was free of contamination by L-threonolactone. Subsequent hydrogenation at 170 atm H2 and 125 °C of a 0.5 M aqueous solution of 2-hydroxy-2-buten-4-olide 12 over 5 wt % Ru on C at a 1.0 mol% Ru/2-hydroxy-2-buten-4-olide mol ratio for 10 h afforded D,L-l,2,4—butanetriol la,b in 96% yield. The overall yield of D,L-1,2,4-butanetriol from L-ascorbic acid was 53%. No byproducts such as those resulting from C-C and C-0 bond cleavage during hydrogenation of D,L-malic acid Za,b could be detected. As a consequence, purification of product D,L-1,2,4-butanetriol la,b by distillation was not required.17 59 DISCUSSION The current commercial manufacture of D,L-1,2,4-butanetriol la,b relies on a stoichiometric NaBH4 reduction of dimethyl D,L-malate (Figure 20).9 Liquefiable petroleum gas, which is produced from petroleum and natural gas, is the source of the carbon atoms in D,L-malic acid (2a,b) and the derived dimethyl D,L-malate (Figure 20).38 Petroleum-derived acetylene is the starting material for 2-butyne-l,4—diol and 2-buten- 1,4-diol used for the synthesis of D,L-1,2,4-butanetriol la,b. Allyl alcohol and acetaldehyde were required for the syntheses of glycidol and 3-buten-1—ol. By contrast, the syntheses described in this chapter employed starting materials derived from abundant and inexpensive D-glucose derived from renewable feedstocks. The source of carbon atoms is an important consideration given the increasing prices of petroleum and natural gas and the projected decline of domestic reserves of fossil fuels.39 L—Malic acid (2b, Figure 23) can be microbially synthesized from D-glucose using Aspergillusflavus.40 L-Ascorbic acid (Figure 30) is manufactured from D-glucose-derived D-sorbitol via the Reichstein-Grussner process.41 Stereochemical considerations are also important. 1,2,4—Butanetriol trinitrate that has been used as an energetic material has been a racemic D,L-mixture, which reflects the use of methyl D,L-malate (Figure 20) as the starting material for commercial synthesis of precursor D,L—1,2,4-butantriol la,b. The difference in the melting point of the pure enantiomers relative to the different melting point for the racemic mixture. of enantiomers is an important consideration in the utilization of energetic materials. Biocatalytic routes typically lead to either the D- or L-enantiomer of 1,2,4-butanetriol. For example, D-1,2,4- butanetriol la has been microbially synthesized from D-xylose while a different microbe 60 ' Condensation of was used to synthesize L-l,2,4-butanetriol lb from L-arabinose.2 glycolaldehyde and acetaldehyde catalyzed by 2-deoxyribose—S-phosphate aldolase gave D-l,2,4-butanetriol 1a (Figure 21).‘7 No enzyme is currently known that is capable of catalyzing the condensation of glycolaldehyde and acetaldehyde to afford L-l,2,4— butanetriol 1b. By contrast, L-malic acid 23 (Figure 23) underwent racemization during its catalytic hydrogenation to afford D,L-1,2,4-butanetriol la,b. Racemic D,L-l,2,4- butanetriol la,b was the product of the Chemoenzymatic synthesis using L-ascorbic acid as the starting material (Figure 30).” Catalytic hydrogenation of D,L-malic acid 2a,b and L-malic acid 2b was the shortest route for synthesis of D,L-l,2,4-butanetriol la,b. Optimized hydrogenation of a 1 M aqueous solution of D,L—malic acid 23,b for 10 h at 340 atm H2 and 135 °C over 5% Ru on C using a 1.3% Ru/malate ratio led to a 74% yield of D,L-1,2,4-butanetriol la,b along with a complex mixture of byproducts. Distillation of this crude product mixture afforded only a 28% yield of impure D,L-l,2,4-butanetriol la,b. Notably, the 340 atm H2 used in these hydrogenations was at the maximum pressure rating for the reactor used for these experiments. Such elevated H2 pressures and temperatures are typical for catalytic hydrogenation of unactivated carboxylates.42 Adkins first reported the synthesis of 1,2,4- butanetriol from hydrogenation of neat dimethyl malate over 2CuO-CrzO3.43 High catalyst loading relative to substrate, 150 °C reaction temperatures, and H2 pressures of 340 atm were required to obtain a 67% yield of 1,2,4-butanet’riol and a 20% yield of 1,4- butanediol. Anton reported hydrogenation of aqueous malic acid at 200 atm H2 and 60 °C over Ru-Re to afford D,L-1,2,4-butanetriol la,b in 80% yield."4 61 Hydrogenation of malic acid (Figure 23) was the obvious catalytic alternative to stoichiometric reduction of D,L-malic acid 23,b using NaBH4. However, the elevated H2 pressures and temperatures required for catalytic hydrogenation of D,L-malic acid 2a,b and the separation of D,L-1,2,4-butanetriol la,b from byproducts compromise the synthetic directness of this route. Malic acid possesses two chemically different carboxylates. One carboxylate is inductively activated by an (Jr-hydroxyl group while the second carboxylate remains unactivated. The first step in malic acid 2a,b hydrogenation proceeds under relatively mild conditions to afford 3,4-dihydroxybutyric acid 4 and the corresponding 3-hydroxy-y-butyrolactone 3 (Figure 34). This is presumably due to the presence of an electron-withdrawing hydroxyl group at the alpha position, which eases the hydrogenation barrier.45 Hydrogenation of the remaining unactivated carboxylate results in D,L-1,2,4-butanetriol la,b. CO H a O HO 0 2 , HO C/Y\OH __ O a HO 2 /\O/H 2 0H ‘_ 71—2 ' \/\O(H\OH OH 1 1 / \ 071:7 Ho/\/OH + Y9“ OH 11 HO/\/\/OH 4— HOZC/VOH Figure 34. Catalytic hydrogenation of malic acid. Key: 340 atm Hz, 1.3 mol% Ru/malate, 135 °C, 10 h. Byproduct formation was shown to result mainly from C-C and C-0 bond cleavage of D,L-1,2,4-butanetriol la,b. The major byproduct, 1,2-propanediol is derived from C-C bond cleavage of D,L-l,2,4—butanetriol la,b. Ru-catalyzed dehydrogenation of 1,2,4-butanetriol leads to 1,4-dihydroxy-2-butanone or 3,4-dihydroxybutanal, which ultimately undergo retro-aldol followed by hydrogenation to afford 1,2-propanediol and ethylene glycol, respectively (Figure 35).46 62 OH Ho’\{”\/ HO/\{/\yo Ho’fi] Ho’\] OH a OH retro- 0 0 OH ‘-;- ama OH . HO/\{/\/ Ho’\m”\/OH Ho/\f’ HO/\T/ OH 0 OH OH Figure 35. Formation of 1,2-propanediol and 1,2-ethanediol. 1,4-Butanediol, which accounted for 32% of the byproduct formed, did not originate from 1,2,4-butanetriol. Presumably, it was formed from dehydration of the partially reduced intermediates 3,4-dihydroxybutyric acid 4 and 3-hydroxy-y- butyrolactone 3 followed by hydrogenation (Figure 34).47 Both 3,4-dihydroxybutyric acid 4 and 3-hydroxy-y-butyrolactone 3 have a high propensity to undergo B-elimination. An interesting observation was the complete racemization of the L-malic acid 2b to D,L- 1,2,4-butanetriol la,b during the course of hydrogenation. This can be attributed to the high temperatures and length of reaction time employed in this study. Racemization was an advantage because it allowed D,L-l,2,4-butanetriol la,b to be synthesized from D- glucose-derived L-malic acid instead of petroleum-derived sources. However, difficulties associated with purifying product from contaminating byproducts significantly reduced the yield of D,L-1,2,4-butanetriol la,b. In order to make this route viable as a source of D,L-l,2,4-butanetriol la,b, a practical method for the removal of contaminants is needed. Acetalization of the mixture of polyols in the presence of acetaldehyde has been used to separate ethylene glycol from propylene glycol.48"‘d The resulting 1,3-dioxolanes of ethylene glycol and propylene glycol are more volatile than the corresponding polyols and can be easily separated from each other by fractional distillation. This methodology might be employable for the purification of D,L-l,2,4-butanetriol la,b from contaminating 1,2-glycols such as 1,2-propanediol 5, 1,2-butanediol 7 and ethylene 63 glycol 8. The 1,2-O-ethylidenebutane-1,2,4-triol could then be hydrolyzed to afford pure racemic 1,2,4-butanetriol and acetaldehyde, which would need to be recycled.37 Catalytic hydrogenation of L-malic acid to D,L-l,2,4-butanetriol la,b route is concise, employs a starting material derived from renewable feedstock and affords the desired racemic mixture. The forcing reaction conditions result in the formation of undesired byproducts, which reduce the yield from 74% to 28% after distillation. Furthermore, the intermediates formed during the hydrogenation process are poised to undergo 1.3-elimination, giving rise to undesired 1,4-butanediol. To circumvent these forcing reaction conditions and reactive intermediates, new substrates for catalytic hydrogenation were explored (Figure 24). The key consideration in choosing the starting material was the carbon source. Carbohydrate-derived starting materials, which afford the desired stereochemistry of product were ideal. Syntheses of three substrates, 2- hydroxy-y-butyrolactone 10, 2-hydroxy-2-buten-4-olide 12 and D-erythronolactone 14a, were undertaken. Because catalytic hydrogenation was to be used, the presence of an activating hydroxyl group at the (at-position was imperative. The absence of B-hydroxyl group eliminated the possibility of forming 1,4-butanediol. Although the synthesis of 2-hydroxy-y-butyrolactone 10 starts from D,L-malic acid, it was necessary to protect the unactivated carboxylic acid as an ester 17 or 1,3- dioxolanone 15 (Figure 25). Stoichiometric reduction of the unactivated carboxylic acid with borane results in the alcohol (16 or 18), which was subsequently deprotected to afford the desired 2—hydroxy-y-butyrolactone 10. The protection/deprotection sequence increases the length of the synthesis and requires the use of volatile and toxic reagents. Borane reduction was almost quantitative, but 1 to 1.3 equivalents of borane were required to transform the acid to the alcohol. Subsequently, methyl borate by-product is generated as with the current commercial synthesis of 1,2,4-butanetriol (Figure 20). Purification of products from methyl borate is tedious. This synthesis cannot be scaled- up but it was useful to generate enough substrate to evaluate hydrogenation to 1,2,4— butanetriol la,b. As predicted, hydrogenation of 2-hydroxy-y-butyrolactone 10 afforded 1,2,4-butanetriol la,b in quantitative yields (Figure 25). This was because an a-hydroxy carboxylic acid group requires milder hydrogenation conditions as was established during hydrogenation studies of malic acid (Figure 23). No intermediates or diol by—products were observed during the hydrogenation under mild conditions. Having established that hydrogenation of 2-hydroxy-y-butyrolactone 10 is more efficient than malic acid in affording 1,2,4-butanetriol, a more practical synthetic route was investigated. Several procedures for making 4-hydroxy-2-ketobutyrate 11 have been reported starting from L-aspartic acid, L-ascorbic acid or D-isoascorbic acid. In the L- aspartic acid route, protection and borane reduction sequence similar to 2-hydroxy-y- butyrolactone 10 synthetic route was carried out to obtain L-homoserine (Figure 27). The transamination of homoserine to 4—hydroxy—2-ketobutyric acid proceeded in an acetate buffer in the presence of pyridoxal and cuprate. However, low yields, use of stoichiometric borane and tedious purification processes limited application of this process. In an alternative route, intermediates to 4-hydroxy-2-ketobutyrate 11 or the corresponding lactone 12 were obtained from L-ascorbic acid. A synthetic route for D- erythronate 13a and L-threonate 13b was reported by Isbel and Frush,30 which took advantage of the known product of vitamin C oxidation. Hydrogen peroxide was consumed by L-ascorbic acid or D-isoascorbic acid to afford an inert 01,13-diketone adduct, 65 which under basic conditions was cleaved to L-threonate 13b or D-erythronate 13a and oxalic acid (Figure 28).30 Acid-catalyzed lactonization of D-erythronate 13a afforded the corresponding D-erythronolactone 14a. Having obtained both L-threonolactone 14a and D-erythronolactone 14a, attempts were made to dehydrate and hydrogenate these intermediates to obtain 1,2,4-butanetriol (Figure 28). Although the presence of the B- hydroxyl makes these substrates potentially susceptible to B—elimination, quantitative yields of L-threitol or D-erythritol were obtained even under reaction conditions employing high pressures and temperatures. This suggested that hydrogenation of the carboxylate is faster than the dehydration process. L-Erythritol or D -threitol is not poised to dehydrate under hydrogenation conditions used. By taking advantage of the trans-relationship of the a-hydrogen and the [3- hydroxyl group in D—erythronolactone, B-elimination of the benzoyloxy-, acetyloxy- or trifloroacetyloxy- was effected under in the presence of triethylamine or pyridine (Figure 29). Subsequent deprotection of the benzoyloxy and acetyloxy groups was problematic under basic conditions. This was likely due to the degradation of product 2-hydroxy-2- buten—4-olide under basic conditions.31 When trifluoroacetic anhydride was used to activate the hydroxyl group, excess pyridine was used. Quantitative conversion of D- erythronolactone 14a to 2-hydroxy—2-buten-4-olide 12 was observed by 1H NMR. However, removal of pyridine was unsuccessful, which compromised this route for the synthesis of 2-hydroxy—2-buten-4-olide 12. In the last and final route, L-threonate 13b was dehydrated to 4-hydroxy-2-keto- butyrate 11 in 80% yield at ambient conditions (Figure 30). Dihydroxy-acid dehydratase obtained from overexpression of the E. coli ilvD locus catalyzed the dehydration. 66 Dihydroxy-acid dehydratase catalyzes the third step of branched-chain amino acid biosynthesis where it catalyzes the dehydration and tautormerization of two naturally occurring 2R, 3R-dihydroxycarboxylic acids to the corresponding 2-keto acids. When nonnative substrates were used, the threo-dihydroxy acid isomer was dehydrated more rapidly relative to the erythreo-dihydroxy isomer.31 Dihydroxy-acid dehydratase is utilizes the Fe-S cluster. Mechanistically related enzymes include aconitase, which catalyzes the reversible dehydration of citrate to cis-aconitase, and L-serine dehydratase, which catalyzes the irreversible deamination of serine to pyruvate.”50 All of these enzymes along with dihydroxy-acid dehydratase from E. coli possess a [4Fe-4S], which in the presence of oxygen is oxidized and partially or completely degraded. Dihydroxy- acid dehydratase from spinach leaves on the other hand, is stable to O2 and possesses a [2Fe-ZS] cluster at its active site. By expanding its coordination sphere, the iron of the cluster is able to accommodate the 3R-hydroxy of the substrate as a ligandf’0 This allows the Fe-S cluster to function as a Lewis acid and activates the 3—hydroxyl leaving group. Removal of the proton trans to the hydroxyl furnishes the enol intermediate, which tautormerizes to the 2-keto acid.31 The catalytic turnover of the spinach dihydroxy—acid dehydratase for L— threonate is much higher relative to E. coli dihydroxy-acid dehydratase.” Spinach dihydroxy-acid dehydratase stable over 2 days (Figure 31) of reaction while the specific activity of E. coli dihydroxy-acid dehydratase drops by 60-70% in 5 h (Figure 32,33). Utilization of the stronger PM. promoter improved expression of E. coli dihydroxy—acid dehydratase, which facilitated the conversion of L-threonate 11 to 4-hydroxy-2- ketobutyrate 11 (Figure 30). 4-Hydroxy-2-ketobutyrate 11 was not isolated due to 67 previous reports of its instability. Instead, acid-catalyzed lactonization furnished 2- hydroxy-2—buten-4-olide 12, which is stable. Direct hydrogenation via the intermediacy of 2-hydroxy-y-butyrolactone afforded D,L-1,2,4-butanetriol la,b without concomitant formation of 1,2-propanediol or 1,4-butanediol. This last route provides an alternative to the current commercial synthesis of D,L- l,2,4-butanetriol la,b. Glucose-derived ascorbic acid is used as the starting material to afford D,L-l,2,4-butanetriol la,b in 53% overall yield. Byproduct formation is completely avoided by hydrogenating under mild conditions. Unlike the current commercial synthesis of D,L-1,2,4-butanetriol la,b, this proceeds in water while deriving its carbon source from renewable and abundant starting materials. On the other hand, the L-ascorbic acid route requires more synthetic steps relative hydrogenation of malic acid. 68 10 References (21) Hanessian, S.; Ugolini, A.; Dube, D.; Glamyn, A. Can. J. Chem. 1984, 62, 2146; (b) Drager, G.; Garrning, A.; Maul, C.; Noltemeyer, M.; Thiericke, R.; Zerlin, M.; Kirschinig, A. Chem. Eur. 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(b) Micard, V.; Renard, C. M. G. C.; Thibault, J .-F. Enzyme Microb. Technol. 1996, I 9, 162. Zhang, 2.; Jackson, J. E.; Miller, D. Appl. Catal. 2003, 219, 89. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512. Jere, F. T.; Miller, D. J.; Jackson, J. E. Org. Lett. 2003, 5, 527. (a) Lynn, D. G.; Gong, B. J. Org. Chem. 1990, 55, 4763; (b) Baldwin, J. E.; North, M.; Flinn, A. Tetrahedron Lett. 1987, 28, 3167; (c) Lane, R. S.; Shapley, A.; Dekker, E. E. Biochemistry 1971, 10, 1353; (d) Hift, H.; Mahler, H. R. J. Biol. Chem. 1952, I98, 901; N ishihara, H. and Dekker, E. E. J. Biol. Chem. 1972, 247, 5079. 70 28 3O 31 33 35 36 37 38 39 Cooper, A. J. L.; Gonos, J. 2.; Meister, A. Chem. Rev. 1983, 83, 387 Meister, A. J. Biol. Chem. 1954, 206, 577. Lane, R. S.; Dekker, E. E. Biochem. 1969, 8, 2958. (a) Isbel, H. S.;Frush,H. L. Carb. Res. 1979, 72,301. b) Kvernberg, P. O.; Pederson, B. Acta Chem. Scand. 1994, 48, 646. c) Wei,C. C.; De Bernardo, S.; Tenji,J. P.; Borgese, J.; Weigele, M. J. Org. Chem. 1985,50, 3462. d) Jung, M. E.; Shaw,T. J. J. Am. Chem. Soc. 1980, 102, 6304. Limberg, G.; Klafflte, W.; Thiem, J. Bioorg. Med. Chem. 1995, 3, 487. (a) Limberg,G.;Klaffke, W. Carb. Res. 1995,275,107. (b) Cotterhil, I. C.; Henderson, D. P.;Shelton, M. C.;Toone, S. J. J. Mol. Cat. B 19985, 103. (a) Myers, J. W. J. Biol. Chem. 1961, 236, 1414. (b) Wixon, R. L.; Wikman, J. H. Biochim. Biophys. Acta. 1960, 45, 618. (c) Wixom, R. L; Shaton J. B.; Strassmann, M. J. Biol. Chem. 1960, 235, 128. (d) Myers, J. W.; Adelberg, E. A. Proc. Natl. Acad. Sci. U.S.A. 1954, 40, 468. Flint, D. H.;Empatage. M. H. J. Biol. Chem. 1988, 263, 3558. Flint, D. H.; Empatage, M. H.; Finnegan, F. G.; Fu, W.; Johnson, M. K. J. Biol. Chem. 1993, 268, 14732. (a) Hamilton, C. M.; Aldea, M.; Washburn, B. K.; Babitzke, P.; Kushner, S. R. J. Bacteriol. 1989, 171, 4617. Kambourakis, 8.; Frost, J.W. J. Org. Chem. 2000, 65,6904. Szmant, H. H. Organic Building Blocks of the Chemical Industry; Wiley: New York, 1989; p 362. (a) Lovins, A. B.; Datta, E. K.; Bustnes, O. D.; Koomey, J. G.; Glasgow, N. J. Winning the Oil Endgame; Rocky Mountain Institute: Snowmass, Colorado, 2004. (b) Campbell, C. J.; Laherrére, J. H. Sci. Am. 1998, 278(3), 78. (a) Battat, E.; Peleg, Y.; Bercovitz, A.; Rokem, J. S.; Goldberg, I. Biotechnol. Bioeng. 1991, 37, 1109. (b) Peleg, Y.; Steiglitz, B; Goldberg, 1. Appl. Microbial. Biotechnol. 1988, 28, 69. (c) Abe, S.; Furuya, A.; Saito,T.; Takayama, K. US Patent 3,063,910, 1962 71 41 42 43 45 47 49 (a) Reichstein, T.; Griissner, A. Helv. Chim. Acta 1934, I 7, 311; (b) Lazarus, R.; Seymour, J. L.; Stafford, R. K.; Dennis, M. S.; Lazarus, M. G.; Marks, C. B.; Anderson, S. In Biocatalysis; Abramowitcz, D. Ed.; van Nostrand Reinhold; New York, 1990; p. 135-155. Nishimura, S. Handbook of Heterogeneous Catalytic Hydrogenation for Organic Synthesis; Wiley: New York, 2001 , p 170. Adkins, H.; Billica, H. R. J. 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FEBS Lett. 1994, 351, 416. 72 W CONVERSION OF L-LYSINE TO e-CAPROLACTAM Introduction e-Caprolactam is the monomer for the synthesis of Nylon 6.1 Over 90% of the annually produced c-caprolactam (4 billion kg/year) is polymerized to nylon 6, which along with nylon 6,6 account for 98% of nylon fibers produced worldwide.2 Most commercial processes for the manufacture of e-caprolactam start from either toluene or benzene.3 Oxidation of benzene followed by catalytic hydrogenation of the resulting phenol results in cyclohexanone (Figure 36). OH O NOH a,b C c —» r: —- 6 4- CI ~2- Ow NH benzene phenol cyclohexanone cyclohexanone e-caprolactam oxime Figure 36. Manufacture of e-caprolactam from benzene. Key: (a) 2-propene, HZSM-12; (b) 02, HZSO4; (c) Pd, H2; ((1) (NHZOH)2H2SO4, NH3; (e) 112804-803, N H3. Coupling of the cyclohexanone with hydroxylamine sulfate affords cyclohexanone oxime, along with ammonium sulfate byproduct. Several processes have been developed to minimize formation of ammonium sulfate, which is also used in the manufacture of fertilizers. These include NO reduction and use of phosphate oxime processes licensed and operated by DSM.4 Alternatively, cyclohexanone oxime is produced by reaction with NH3 and H202 in the presence of titanosilicate catalysts. This so-called ammoxidation reaction commercialized by Sumimoto completely eliminates production of ammonium sulfate.5 Alternatively, cyclohexane is converted into cyclohexanone oxime in the presence of nitrous oxide and HCl. Both cyclohexanone and 73 hydroxylamine are eliminated in this process.6 Beckman rearrangement of the cyclohexanone oxime in the presence of oleum results in e-caprolactam (Figure 36).7 During this step, formation of ammonium sulfate byproduct results from neutralization of oleum with ammonia. Sumimoto has successfully commercialized an oleum-free Beckman rearrangement process. The process uses a fluid-bed reactor operated at 350 °C in the presence of methanol to effect rearrangement of cyclohexanone oxime to e- caprolactam.5 Other than the Sumimoto process in which no ammonium sulfate is produced, other commercial processes produce 1.4-4.3 tons of ammonium sulfate/ton of e—caprolactam.3 petroleum 1 O methyl-3-pentenoate 1 ,3-butadiene adiponitrile 1” 1‘ , OCH 0 O/W\n/ 3 NCWNH2 O OH 5-formyl valerate 6-aminocapronitrile lo e-caprolactam d HzN/VWOCHS / O 6-aminocaproate Figure 37. Syntheses of e-caprolactam from 1,3-butadiene. Key: (a) CO, MeOH, Ni- zeolites; (b) CO, H2, Co, Rh; (c) NH3/H2, Ru; (d) 250 °C; (e) HCN; (f) Ni/Co, H2; (g) TiOz, HZO. Non-aromatic feedstocks have been used for the syntheses of e-caprolactam (Figure 37). The DSM-DuPont process employed 1,3-butadiene as the starting material.8 Carbonylation over Ni-based zeolites or Pd borosilicates catalyst in the presence of 74 methanol affords pentenoate ester (Figure 37). Hydroformylation followed by reductive amination of the valerate ester results in 6-aminocaproate ester, which is ultimately cyclized to e-caprolactam at 250 °C. BASF on the other hand converted adiponitrile, which could also be derived from 1,3-butadiene, to 6-aminocapronitrile over Ni-Co catalysts (Figure 37). Hydrolysis of 6-aminocapronitrile over TiO2 at 240 °C results in e- caprolactam (Figure 37).9 With the synthesis of adipic acid, one of the monomers required for the synthesis of nylon 6,6 from D—glucose delineated, a complementary synthesis of e-caprolactam starting material derived from renewable feedstocks sought.lo This chapter explores D- glucose-derived L-lysine as the starting material for the synthesis of s-caprolactam. After masking the e-amino group of L-lysine as an amide by cyclization, several strategies were employed to remove the or-amino group. The first approach employed catalytic hydrodenitrogenation employing Mo-based catalysts used in the denitrification of petroleum. A single-electron reduction-deamination sequence was also explored to remove the a-amino group from the methyl ester of L-lysine, methyl pipecolinate and 01- amino e-caprolactam. With limited success in removing the a-amino group, attention was focused on isomerizing L-lysine to B-L-lysine in order to make the amino group susceptible to B-elimination. Although B-L-lysine can be chemically synthesized from L- ornithine by homologation using an Arndt-Eistert reaction, it is not commercially available. Efforts were directed at optimizing the bioconversion of L-lysine to B-L-lysine using C lostridium subterminale. 75 Background L-Lysine is an ideal precursor for the synthesis of s-caprolactam because of its carbon skeleton. It is trails behind L-glutamic acid as the second highest produced amino acid at 7.5 x 108 kg/year.” The increased demand for L-lysine and other amino acids stems from their utilization in food additives, feed supplements, therapeutic agents and precursors for the syntheses of peptides or agrochemicals.‘2 Specifically, L-lysine is required as a feed additive for poultry and swine production.13 The microbial production of L-lysine by Corynebacterium glutamicum was introduced in 1957 and has since been improved to afford titer of 170 g/L from D-glucose (Figure 38).14 + OH H3N O O OH H N NH NH O .\ 2 I. i OH co— HO OH 2 , L-Iysine a-amlno- s-caprolactam D-glucose e-caprolactam Figure 38. Synthetic route of e-caprolactam from D -glucose. Key: (a) Corynebacterium glutamicum; (b) refluxing 1,2-propanediol, 2 h, 96%; (c) NHZOSO3H, KOH, 75%; H“, 250 °C. L-Lysine is an ideal precursor for the synthesis of e-caprolactam because it possesses the C-1 carboxylate and the e-amine necessary for the amide linkage. Amide formation, which consumes these two functionalities, avoids the need for derivatization. Selective removal of the secondary a-amine group is then required to afford the desired product. To mask the e-amino group, L-lysine was cyclized in refluxing 1,2-propanediol to afford a-amino-s-caprolactam (Figure 38). This cyclization was optimized in the Frost group to afford 96% of the desired 7-membered ring lactam.15 1,2-Propanediol is derived from lactic acid hydrogenation, which in turn is derived from microbial fermentation of D-glucose. Reported literature procedures for the cyclization of L-lysine employed 76 petroleum-derived toluene as a solvent for its ability to remove water. Reaction of a- amino-s-caprolactam with hydroxylamine-O-sulfonic acid under basic conditions resulted in the deamination to afford s-caprolactam in 75% yield.” Ironically, traditional synthesis of e-caprolactam from cyclohexanone employs hydroxylamine-O-sulfonic acid (Figure 36). This route constitutes a fundamental departure from reported syntheses of e- caprolactam, which rely on petroleum-derived benzene or 1,3-butadiene as carbon sources. However, similar to the current synthesis, preparation of the inorganic reagent required to effect the deamination results in the generation of a byproduct salt stream. Ideally, a catalytic deamination process would be more desirable to afford e-caprolactam from a-amino-e-caprolactam. To this end, catalytic deamination and catalytic hydrogen transfer were explored along with reductive deamination with single electron transfer reagents. Catalytic Hydrodenitrogenation Removal of sulfur, nitrogen and metals is one of the most important processes during the refining of petroleum.” This process reduces emission of sulfur and nitrogen oxides which are formed when these component of oil are combusted in car and truck engines. This process is referred to as hydrotreating, where crude oil is treated with hydrogen in the presence of a catalyst at high temperature without reducing the boiling point of the oil fractions. Hydrotreating is the largest application in industry. Consequently, hydrotreating catalysts rank third after exhaust gas catalysts and fluid cracking catalysts on the basis of the amount of catalysts sold per year.” 77 The industrial catalysts used to remove sulfur (hydrodesulfurization) from crude oil contain molybdenum and cobalt supported on y-alumina whereas for removal of nitrogen (hydrodenitrification) a combination of molybdenum and nickel is used.18 The formation of H28, which immediately sulfidates the metal or metal oxide catalyst, is inevitable during hydrotreatment. Catalyst suflidation under controlled conditions prior to the hydrotreating process is therefore necessary. Consequently, molybdenum sulfide is considered the actual catalyst. Nickel and cobalt function as promoters, which increase the activity of molybdenum for the removal of nitrogen, sulfur and oxygen. The hydrodenitrogenation catalysts were prepared by a two-step pore volume impregnation procedure.” In the first step, y-alumina was impregnated with an aqueous solution of ammonium molybdenate followed by drying and heating (calcine) at 400 °C. In a second step, the resulting Mo-alumina mixture was impregnated with nickel (II) nitrate, dried and calcined. Finally, the Mo-Ni/AIZO3 catalyst was activated by treatment with a mixture of H25 (10%) and H2 (90%) at 400 °C and 34 atm for 4 h to afford Ni-Mo- S/A1203. Two mechanisms have been proposed for catalytic hydrodenitrogenation of aliphatic amines (Figure 39). In Hoffmann elimination, the acidic group protonates the amine making it a good leaving and the basic sulfur abstracts the B-hydrogen simultaneously resulting in trans-elimination of the amine (Figure 39).'7 78 Hoffmann Elimination I I / H+ I I +/ +8‘ \ H H-C-C-N ——> H-C-C-NH —-—-> CC + N I I \ I I \ -BH I Nucleophilic Substitution I H28 I +/ _ I I —C—N —> —C—NH + SH ——> H—s|—c|;— l | \ | 1 H—S —C — '1' H2 ——> H28 H—C- 1 1 Figure 39. Mechanistic hydrodenitrogenation catalyzed by Mo-based catalyst. Amines, which have no B-hydrogens are denitrogenated by nucleophilic substitution of the amine group by an SH group followed by hydrogenolysis of the weak C—S bond (Figure 39). As an alternative, a Pt/SiO2 catalyst was also been explored for the deamination of or-amino-e-caprolactam.20 The catalyst was prepared by impregnating 3102 with chloroplatinic acid followed by activation at 350 °C and 1 atm H2. The activated catalyst contained 40% Pt and 60% silica.20 Cyclohexylamine was used as a model substrate for catalytic demamination using Mo-Ni and Pt catalysts. Catalytic hydrodenitrogenation was conducted in a flow apparatus consisting of three U-tubes. Cyclohexylamine was places in the first U-tube, which was connected to a second U-tube containing reduced Pt/SiOz. The last U-tube served as a product chamber and was maintained at -78 °C with dry ice-acetone cooling. Substrate and catalyst U-tubes were heated to 200 °C. H2 was allowed to flow through the three U-tubes. The vaporized cyclohexylamine was swept through the catalyst and the resulting cyclohexane was trapped in the third U-tube. After all the cyclohexylamine 79 was evaporated, the sand bath heating was removed. Batch hydrodenitrogenation was also employed with Mo-based catalysts inside a 500 mL 4575 Parr reaction vessel. All catalysts were active in deaminating the cyclohexylamine to cyclohexane. Mo-based catalysts on A1203, SiO2 and C along with 40% Pt on SiO2 were first explored for the deamination of cyclohexylamine (Figure 40, Table 6). NH2 6 catalyst, 6-10 h 0 HzSsz pressure 7 Figure 40. Deamination of cyclohexylamine. Table 6. Hydrodenitrogenation of cyclohexylamine to cyclohexane. Entry Catalyst Temp Pressure (atm) Yield1 (°C) (°/o) 1 Pt/Si02 200 1 90 2 Ni-Mo/Si02 360 g 68 “86 3 7 Ni-Mo/A1203 350 68 93 4 Ni-MO/C 350 68 90 lYield are based on response factor obtained on the GC Higher temperatures and pressures were necessary with Ni-Mo catalysts to deaminate cyclohexylamine compared to Pt/SiOz. These reactions with cyclohexylamine were conducted to establish that the catalysts were active. Attempted catalytic denitrogenation of a-amino-e-caprolactam over Pt/SiO2 in a U-tube resulted in the recovery of unreacted starting material. The low vapor pressure of the lactam prevented the use of the flow apparatus for deamination. Batch hydrodenitrogenation was then evaluated with Mo-based catalysts and Pt/SiOz. a-Amino-e-caprolactam-HCl, a-amino-e-caprolactam, L-lysine and methyl L- 80 lysine in glyme and diglyme were hydrodenitrogenated over Ni-Mo-S (20-40 mol%) under varying conditions of pressures (68-204 atm H2) and temperatures (100-350 °C). H201 O O O H Ni-MO-S / Ni-Mo-S 15 NH --------- -> NH --------- -> NH -NH3 H2 Figure 41. Hydrodenitrogenation of a-amino-e-caprolactam. The desired deamination to form e-caprolactam was not observed. Instead, unreacted starting material was recovered. Oligomerization of a-amino-e-caprolactam was observed when the temperature was increased beyond 160 °C even with thorough drying of a-amino-c-caprolactam prior to deamination. A drying pistol was used to dry or- amino-e-caprolactam with refluxing EtOAc. Catalytic hydrogen transfer Hydrogenolysis of the C-N bond has previously been achieved with catalytic hydrogen transfer. This method offers a viable alternative to selective hydrogenation in the presence of sensitive functionalities without the use of special apparatus. Several compounds such as ammonium formate, formic acid, phosphoric acid and cyclohexadiene may serve as a hydrogen source in the presence of metal catalysts like Pd or Ni.21 Extension of this procedure using ammonium formate as the hydrogen source in methanol over Pd/C was conducted in an attempt to deaminate a-amino-e-caprolactam (Figure 42). 81 H2N ’- 0 HCOzNH4, MeOH 0 ................... § Gm 10 mol % Pd/C Gm rt-reflux Figure 42. Catalytic hydrogen transfer. The temperature was varied from rt to refluxing MeOH with an excess of hydrogen source. In all cases, unreacted starting material was recovered. Methylation of the amine in a—amino-e-caprolactam with formaldehyde and hydrogen and subsequent hydrogenolysis did not yield the desired e-caprolactam.22 Reductive Deamination Samarium (II) iodine is widely used in organic chemistry as a single-electron reductant. This reagent was first introduced by Kagan and has been used extensively in reductive cleavage of a-heterosubtituted carbonyl substrates.23 For instance, an important subgroup of compounds possessing a small ring adjacent to the carbonyl such as epoxy ketones and esters and cyclopropyl ketones have been reductively cleaved to afford ring— opened product.24 Reductive cleavage of 2-acylaziridines has also been observed (Figure 43).25 O O T7)L smlz /\/U\ N R THF, proton source' TSHN R Ts R = Me. OEt Figure 43. Reductive cleavage of acylaziridine with Smlz. 82 This work was extended to the reductive deamination of a variety of amino acids with primary, secondary and tertiary amines.26 Typically, a stoichiometric amount of Sml2 and HMPA is required to effect the deamination. A proton source such as methanol, pivalic acid or N,N-dimethylaminoethanol (DMAE) is also present during the reaction. Since two electrons are required for the deamination, an excess of SmI2 as well as hexamethylphosphamide are typically used. This reagent is generated in situ from the reaction of Sm powder and CHZIZ.27 Following a literature procedure, exploratory reductive deamination of methyl lysine ester with SmI2 resulted in deamination to 6- amino-methyl-caproate in only 20% yields (Figure 44). + + + NHa NH3 + W ' _ a + 7 c H N CO Me H3NWC02 —> H3NWCOQM8 __> 3 2 L—Iysine L-Iysine methyl ester 6-aminocaproate Q H fl» (1 _°_. ACHN Wcone H COZ RIC COZCH3 N-acetyl 6-aminocaproate pipecolinic acid N—acetyl methyl pipecolinate Figure 44. Reductive deamination of L-lysine derivatives with Smlz. Key: (a) 2,2- dimethoxypropane, refluxing MeOH, HCl; (b) pyridine, Ac20; (c) 10 eq SmIz-HMPA, MeOH, THF, rt. Excess SmI2 and a reaction time of 6 h was necessary to effect the deamination. If HMPA was not used, the reaction time was prolonged. This was contrary to the reported procedure in which N-acetyl methyl pipecolinate was reductively deaminated to N-acetyl methyl 6-aminocaproate in moderate yields (63%). Pipecolinic acid (65%) can be derived from L-lysine by oxidative deamination followed by hydrogenation in the presence of 5 mol% Ru on C at 100 psi H2 and 200 °C.15 Methylation of the carboxylate followed by acetylation of the amine resulted in N-acetyl methyl pipecolinate. Reductive 83 deamination of which in THF and MeOH required 10 equivalent of Smlz. The limited solubility of L-lysine ester'HCl in MeOH was the source of limited deamination of this substrate. Obviating protection-deprotection of the L-lysine starting material is ideal if this process is to be used industrially for the synthesis of caprolactam. Since single electron transfer reaction with SmI2 showed promising results, other inexpensive reductants were explored for the deamination of L-lysine, methyl L-lysine ester and aminocaprolactam (Figure 45, Table 7). Single-electron reducing agents, such as Li/NH3, Na, K, Zn powder and activated Mg were investigated for deamination in MeOH, EtOH and i-PrOH (Figure 45).28 ”2'3, 0 o N . NH a, I-PrOH 6H 20% + NHs Hfi Woo; \ H2N W002R R=H R=CH3 Figure 45. Reductive deamination with alkali metals. Table 7. Reductive Deamination of L-Lysine and its Derivatives. Reductant Proton source Temp (°C) Na i-PrOH rt Na i—PrOH, EtOH rt K ’ i-PrOH' ‘ ‘ -10 Zn i-PrOH, NaOH reflux Mg ’ i-PtOH ’ ‘ ’ reflux ' Li NH,” " -78 84 Typically, small pieces of the reductant metal were added to a stirring solution of the substrate in alcoholic solvents. When MeOH and EtOH were used as solvents, respective alkoxides were formed and reductive deamination was not observed. Due to the lack of solubility of L-lysine and L—lysine methyl esters in alcoholic solvents, attention was focused on reductive deamination of a-amino e-caprolactam in i-PrOH. Unreacted starting material was recovered when Li/NH3 or refluxing i-PrOH with Zn or Mg were used. Addition of K metal to a solution of a-amino s—caprolactam in i-PrOH was rather violent and resulted in degradation of a-amino e-caprolactam to unknown products. Reductive deamination was observed with Na metal in i-PrOH to afford the desired a-amino s-caprolactam in 20% as observed by 'H NMR. When the reaction was allowed to stir long after addition of Na, the mixture thickened. The reaction was quenched by addition of MeOH with 2.5% HCl. Half of the unreacted a-amino e- caprolactam was hydrolyzed to L-lysine and L-lysine ester. Isomerization of L—lysine tofi-L-lysine With limited success in deaminating the a-amino group of L-lysine, a new strategy in which the amine group was made more susceptible to elimination by isomerization of L-lysine to B-L-lysine was targeted. In this approach, the synthesis of e- caprolactam from L—lysine exploited an enzymatic reaction exploited by Clostridium species during fermenation of L-lysine.29 In the first step of L-lysine fermentation, the a- amino group is isomerized to the B—position by L-lysine-2,3-aminomutase (Figure 46).30 85 + NH3 L-lysine-2,3— O + 7 _ aminomutase + _ l - HQNI. H3N W C 02 H3N W002 cyc odehydration: CM” L-Iysine EH3 O O B—elimination / hydrogenation ——’ NH > NH Figure 46. Synthesis of caprolactam from L-lysine via L-B-lysine. In order to deaminate the secondary amine, the e—amino group was masked as an amide by cyclization to fi-amino e-caprolactam. It was anticipated that the resulting L-B-lysine would be deaminated to a,B-unsaturated moiety with relative ease compared to L-lysine, which would then afford the desired e-caprolactam after hydrogenation. Fermentation of L-lysine Amino acids can serve as major energy sources for selected species under 1 The fermentation always involves simultaneous oxidation and anaerobic conditions.3 reduction of a single amino acids or different amino acids. Oxidation reactions in anaerobic bacteria (oxidative deamination, transamination and a-keto acid oxidation) are analogous to the reactions that occur in aerobic bacteria, except for the absence of molecular oxygen and oxidants with high potential?”32 The reduction step however, is unique since suitable electron acceptor(s) of appropriate potential such as a- and B-keto acids, a,[3-unsaturated acids or their CoA thioesters and protons must be generated by the anaerobe. Reduction of these substrates afford short fatty acid chains, succinic acid, 6- aminovaleric acid and molecular hydrogen.“ 86 NHs Donor/ Reductant RACO; = “002H + CO2 + NHa + NHs Acceptor/ Oxidant R'ACOQ > R'ACOZH + NH3 Figure 47. Strickland reaction of amino acids. In cases where a single amino acid cannot serve as both the oxidant and reductant, certain pairs of amino acids are coupled in a redox reaction to allow for ATP generation (Strickland reaction) in Clostridium species (Figure 47).29 Not all amino acids can serve as donor or acceptor in a Strickland reaction. Branched amino acids typically serve as donors while aromatic amino acids and glycine can function as both electron acceptor and donor.31 In Clostridium sporogenes, ornithine serves as an electron donor and forms 5-aminovalerate via D-proline. In this organism, proline is preferred as an electron acceptor over glycine whereas glycine inhibits proline reduction in Clostridium sticklandii.33 L-Lysine, its ornithine homologue, is metabolized by completely different pathways. In C. sticklandii and C. subterminale L—lysine is degraded to acetate, butyrate and ammonia via two pathways. + We a + b _ c + - _ —— — —— CO —_ ‘ H3N W002 H3N Wcoz m 2 “(302 L-lysine L-B—lysine EH3 + 3+ 3 3 3,5-diamino- 5-amino-3-keto- hexanoate hexanoate d SCA e / SCAf 9 =___ W 0 = fl 0 = WSCOA= V\C02— O NHO +3 but rate 3-aminobutyryl CoA Crotonyl CoA butyryl COA V Figure 48. Degradation of L-Iysine in Clostridium subterminale. Key: (a) L-lysine- 2,3-aminomutase; (b) B-L-lysine-5,6-aminomutase; (c) oxidative deamination; (d) 3-keto- 5-aminohexanoate cleavage enzyme; (e) 3-aminobutyrl CoA deaminase; (0 reductase; (g) hydrolysis. 87 Degradation of L—lysine is an example of a single amino acid serving as both an oxidant and reductant with concomitant generation of ATP (Figure 48). In C. 3‘ L-lysine is first isomerized to L-fi-lysine by pyridoxal 5-phosphate subterminale, dependent L-lysine-2,3-aminomutase (Figure 48).34 This is followed by migration of the terminal amino group to position 5 catalyzed by cobalamide coenzyme-dependent L-[3- lysine-5,6-aminomutase.35 The resulting intermediate, 3,5-diaminohexanoate, is poised to undergo oxidative deamination at position 3 to afford 5-amino-3-ketohexanoate. This oxidation is catalyzed by L-erythr0-3,5-diaminohexanoate dehydrogenase.36 By forming the 5-amino-3-ketohexanoate intermediate, spontaneous cyclization is avoided. Subsequent cleavage with acetyl CoA catalyzed by 3-keto-5-aminohexanoate cleavage enzyme results in L-3-aminobutyryl-CoA (C3-C6 of lysine) and acetoacetate formation from acetyl CoA. This reaction is said to occur by a concerted mechanism since neither CoA nor intermediates could be detected by several group exchange reactions. L-3-Aminobutyryl-C0A is reversibly deaminated to crotonyl-CoA and ammonia by 3-aminobutyrl CoA deaminase.37 Reduction of crotonyl CoA to butyryl CoA, which then transfer its CoA moiety to acetoacetate to afford butyrate and acetoacetate CoA is catalyzed by CoA transferase. Finally, thiolase-catalyzed cleavage of acetoacetate CoA results in two moles of acetyl CoA. One molecule of acetyl CoA participates in the 5- amino—3-ketohexanoate~cleavage reaction while the second one is used to form ATP and acetate via the phosphate acetyltransferase and acetate kinase reactions. Alternatively, crotonyl CoA is converted to 3-hydroxybutyryl CoA by crotonase, which is oxidized to acetoacetate CoA by action of 3-hydroxybutyryl CoA dehydrogenase.31 This pathway provides additional NADH and ATP that may be needed for biosynthetic reactions. 88 The first step in the degradation of L-lysine in C. sticklandii is initiated by a racemase, which converts L-lysine to D-lysine. Migration of e—amino group to C-5 is catalyzed by a BIZ-dependent D-lysine-S,6-aminomutase. Subsequent degradation of the resulting 2,5-diaminohexanoic acid results in butyrate derived from C1-C4 of D-lysine and acetate.29 H3Q+ Cyss —“+ Cyss‘ —‘|+ S Fe—S. Fer—S. H ;7/4 go: I s—s-fe-S'Cys | S-E-Fe-S'Cys 020 HO Fe—S —— Se—S S—adenosyl-L-methionine (SAM) HaC.S+ + O H3N HO Ad “OZC HO CYSS. 2+ Fe—S, l S—E-Fe-S'CyS O I ' 9S I S::"Fe. ‘Cys .Fe—S + H0 Ad (Ado-CH2) HQC'S HO . + NH3 CO? H I H _ + ' co2 NH3 . R H R + .. + ' - :SN/ H3NWC02 HaN’WCOE H20‘me $602 OH EH3 V \ =03Po ,\ ~ ‘ =o,po |\ O” N CH3 N’ CH3 1‘ H20‘Ado “ H H3C. - Ado . CO H30. R H C, H . R/kr 2 Ado H 3 Ado RY\C02— OH = ' C02 \ : _ OH O3PO I \/ :OSPO I \ OH _03PO I \ N CH3 N’ CH3 N CH3 NH2 + N \ R = CHZCHZCHzNHa Ad: (I l )N CHO IN N’ _ OH — \ OSPO l I =pyrodoxal-5-phopshate(PLP) N CH3 Figure 49. Isomerization of L-Iysine to L-B-lysine catalyzed by L-lysine-2,3- aminomutase. 89 L-Lysine-2,3-aminomutase, which catalyzes the first step in lysine catabolism in C. subterminale is a well-characterized enzyme. It possesses an iron-sulfur cluster, [4Fe- 4S], and is S-adenosylmethionine (SAM) and pyridoxal 5-phosphate (PLP) dependent.38 The isomerization is initiated by the adenosyl radical, which is generated by homolytic cleavage of S—adenosylmethionine by the iron-sulfur cluster (Figure 49).39 The resulting 5’-deoxynosine-5’-yl abstracts a proton from carbon 3 of the imine formed between PLP and L-lysine. The resulting substrate-related radical imine undergoes a 1,2-imino shift via an azacyclopropylcarbinyl radical intermediate to afford a product—related radical imine. Finally, the product-related radical imine abstracts hydrogen from 5’- deoxyadenosine to produce L-B-lysine and regenerate 5’-deoxyadenosyl radical (Figure 49).40 The L-lysine—2,3-aminomutase gene locus (kamA) from C. subterminale SB4 has previously been overexpressed in E. coli.40 The construct E. coli BL21(DE3)/pAF- 80/kamA was cultured under anaerobic conditions following literature procedure.41 Cells were harvested by centrifugation, lysed by passage through a French press (12,000 psi). After removal of cellular debris, the extract was kept under N2 and sodium dithionite was added to scrub Oz. Pyridoxal 5-phosphate and glutathione were added to activate L- lysine-2,3-aminomutase prior to addition of S-adenosylmethionine and dithionite to initiate the isomerization of L-lysine to B-L—lysine (Figure 50).“ §H3 BL21(DE3)/pAF-80/kamA + T __ crude cell lysate H3N WCOZ . . _ ? SAM, PLP, sodium dithionite 37 °C, pH 8.0, N2 20 % Figure 50. Bioconversion of L-lysine to B-L-lysine catalyzed by E. coli BL21(DE3)/pAF-80/kamA. + _ H3N ’VY‘coz 5H3 90 The enzyme was unstable as measured by the specific activity, which dropped from 0.02 U/mg to undetectable levels within 1 h. The isomerization of L-lysine ceased after a 20% conversion of starting material. Crude extracts of L-lysine-2,3—aminomutase from C. subterminale was explored in search of better conversion?“41 C lostrz'dium SB4 was obtained from American Type Culture Collection (ATCC 29748). It was grown in a medium containing L-lysine, yeast extract, sodium dithionite, K2CO3 and phosphate buffer previously purged with N2. The cells were cultured in 5 L Pyrex bottle at 37 °C for 3-5 h (A600 - 0.5) for experiments with cell crude extracts and 12—18 h (A600 ~ 2.0-3.2) for experiments with intact cells. Manipulations were conducted in a Coy chamber to avoid exposure of cells to Oz. Cells were harvested by centrifugation and used without washing. The yield of wet, packed-cells was 04—07 g/L from a 5 h cultures and approximately 2-4 g/L from 18 h cultures. Bioconversion with crude extracts of L-lysine—2.3-aminomutase Cell extracts were prepared by suspending the cell pellet in phosphate buffer (0.1 M, pH 7.5) and treated with a 60-watt ultrasonic homogenizer for 10 minutes or by passage through a French press (12,000 psi). After removal of cell debris, the protein concentration was between 8-15 mg/mL. A 10 mL reaction mixture contained 60 mM L- lysine, 25 mM tris-HCl (le 7.5), 5 mM potassium phosphate (pH 7.5), 10 mM or- ketoglutarate, 10 mg pyridoxal-S-phosphate, 0.2 mM CoA, 0.2 mM S - adenosylmethionine, 5-20 mM glutathione, and 10 mg of protein. 91 + NH3 + 7 _ crude lysate + _ pH 7.5, 37 °C NH3 8 h, 150 rpm + Figure 51. Bioconversion with crude extracts of L-lysine-2,3-aminomutase. Table 8. Bioconversion of L-Iysine to B-L-lysine Using Crude Lysate. Entry Additives L-lysine L-fi-Iysine (°/o) (°/oL 1 none 100 0 2 a-ketoglutarate, CoA, glutathione 11 77 3 PLP, CoA, glutathione 15 75 4 PLP, SAM, glutathione 17 68 5 a-ketoglutarate, SAM, glutathione 24 64 6 PLP, glutathione 67 q 21 7 cat-ketoglutarate, glutathione 61 28 l Yields were determined by 'H NMR. The reaction mixture was incubated at 37 °C and reached equilibrium after 8 h, at which point it was acidified to pH 3 to precipitate the protein. The supernatant was dried and submitted for 1H NMR (Figure 51, Table 8). Contrary to the enzyme heterologously expressed in E. coli, which only gave 20% of the L-B-lysine, crude extracts resulted in moderate isomerization of L-lysine. PLP or ketoglutarate could be interchanged without decreasing the yield (Entry 2-6). However, in the absence of CoA or SAM the yield of L- B-lysine was decreased (1, 6 and 7). Although crude extracts have reportedly been used for the preparative synthesis of L-B-lysine, this route has several disadvantages. When experiments employing crude lysates were conducted with cells harvested after 18 h, isomerization of L-lysine to L-B-lysine was greatly reduced. Because the [4Fe-4S] cluster is deactivated during preparation of crude extracts, activation of the enzyme prior to bioconversion is required. The need to replenish the crude extracts with PLP, SAM and glutathione would require large quantities of these expensive reagents to be added. 92 Furthermore, it was also necessary to freeze the crude extracts for 2-14 days prior to use. Presumably, heat shock also deactivates L-B-lysine-S.6-aminomuatse. These factors forced us to pursue use of intact of Clostridium SB4 cells for the preparative synthesis of L-B-lysine from L-lysine. Bioconversion with intact cells of Clostridium subterminale In order to accumulate L-B-lysine with intact cells in solution, L-B-lysine-5,6- aminomutase had to be deactivated (Figure 48b). Unlike L-lysine-2,3-aminomutase, L-B- lysine-5,6-aminomutase is not a [4Fe—4S] enzyme. It is a cobalamine dependent enzyme.35 Therefore it could be selectively deactivated without affecting L-lysine—2,3- aminomutase. C. subterminale cells were cultured and harvested after 18 h. After cells were harvested by centrifugation, they were resuspended in a reaction mixture containing 50 mM L-lysine, 80 mM Tris-HCl (pH 7.5), 5 mM potassium phosphate (pH 7.5), and FeSO4 (3 mM). The bioconversion mixture contained 1.4-2.1 g of cells in 100 mL. The reactions were incubated at 37 °C in 250 mL Pyrex bottles, which were flushed with N2 and sealed with screw caps. Variation in the amount of FeSO4 was adjusted to optimize the yield of L-B-lysine. The Pyrex bottles were wrapped with aluminum foil to prevent exposing cells to light. Alternatively, cells were exposed to intense light from a tungsten lamp irradiation in a Pyrex reaction bottle for 12 h. + W. + - _ intact cells + — _ — H3NW002 or HaNWCC’z —’—> CH co + “002 pH7.5,37 c NH3 3 2 L-Iysme 250 rpm L-B—Iysine + acetate butyrate Figure 52. Bioconversion of L-lysine to L-B-lysine using intact C. subterminale. 93 Table 9. Bioconversion of L-lysine fi-L-lysine using intact C. subterminale. Entry FeSO4 light L-Iysine L-B-Iysine Acetate & butyrate (m M) We)1 (°/°)1 (°/<>)1 1 0 no 20 O 80 2 3 no trace trace 90 32 3 yes 25 60 O 4 3 yes 50 33 1 5 5 1 0 yes >99 0 O 6 3 yes3 trace >95 0 1 Yields were estimated by 1H NMR integrations. 2 glutathione was used instead of sodium dithionite. 3 Exposure to light for 24 h. In the absence of light and FeSO4, 80% of L-lysine was converted to acetate and butyrate (Entry 1). However, addition of FeSO4 in the absence of light resulted in an approximately 100% conversion of L-lysine to acetate and butyrate without the accumulation of L-B-lysine (Entry 2). When cells were exposed to intense light for 12 h, accumulation of L-B-lysine increased at the optimum FeSO4 concentration (Entry 3). Glutathione was effective in keeping the reduced atmosphere and resulted in the higher yield of L-B-lysine than when dithionite was used (Entry 4). A high concentration of FeSO4 inhibited the bioconversion of L-lysine completely (Entry 5). Allowing the reaction to proceed for 24 h resulted in the complete conversion of L-lysine to L-B-lysine (Entry 6). Acetate and butyrate were not observed but there was a doublet centered at 1.2 ppm, which presumably belongs to 3,5—diaminobutyrate (Figure 48). The bioconversion has successfully been scaled-up such that cells harvested from 10 L cultures can convert L-lysine (200 mM) to L-B-lysine (180 mM) within 24 h in 500 mL. L-B-Lysine has been purified from unreacted L-lysine using Dowex 50 (H) Sodium formate buffer (0.2 M, pH 2.75) containing NaCl (0.35 M) was used to differentially elute L-lysine and L-B- lysine, with the former eluting first followed by L-B-lysine. L-B-Lysine was then loaded on the second Dowex 50 (H+) column, washed with water to remove sodium formate. L- 94 [ES—Lysine was eluted with l N NI-LOH and the eluant was concentrated under vacuum to afford 43% of L-B-lysine based on starting materials. Cyclization of L-B-lysine to [3— aminocaprolactam in EtOH at 200 °C was shown to proceed to completion. Preliminary studies showed 13% of caprolactam could be obtained when L-B-lysine in EtOH was heated to 200 °C in the presence of Ru on AIZO3 at 1,000 psi H2. The rest of the starting material was degraded to unidentified products. Detailed evaluations of catalysts and deamination conditions need to be explored. As a slight departure from Chemoenzymatic synthesis of e—caprolactam from L- lysine, deamination of L-lysine and its derivatives in the presence of mercaptans was also explored. This route was inspired by the mechanism of D-proline reductase, which catalyzes reductive deamination of D-proline to 5-aminovalerate (Figure 53).42 Presumably, the sulfide anion can replace a hydroxyl group adjacent to the ketone.43 + - . _ . HaN/VYCOZ omithlne 4,! S _ L-prollne (loo- D-Qroline HzN/VVCOZH N 2 H . "’CO racemase [EH3 cyclodeaminase N 2 reductase . . _ 5-aminovaleric acid L-ornithine L-proline D-proline Figure 53. Reductive deamination of L-ornithine to 5-aminovalerate in Clostridium sticklandii. A typical reaction mixture contained methyl L-pipecolinate ester or on- aminocaprolactam in either 1,2-ethylene dithiol or 1,3-propylene dithiol as a solvent in the presence of tetrabutylammonium fluoride (TBAF) or tetraammonium chloride (TBACl).44 The reaction temperature was increased from rt to refluxing dithiol and resulted in the recovery of unreacted starting materials. Replacement of TBAF with 95 ZnCl2 or acetylating the amine group of the starting material did not result in any deamination. 96 DISCUSSION The recent surge in benzene prices due to high demand and limited supply of benzene had a direct impact on the price of nylon 6 and other polyamides.7 This development is not surprising because current commercial syntheses of a—caprolactam utilizes benzene as the main starting material. Synthesis of e-caprolactam from 1,3— butadiene has also been described. The ultimate source of carbon for all syntheses of e- caprolactam is petroleum. D—Glucose-derived L-lysine, on the other hand, provides for a renewable carbon source for e-caprolactam synthesis. L-Lysine is inexpensive and abundant Deamination of L-lysine to e-caprolactam with hydroxylamine-O-sulfonic acid under basic conditions is stoichiometric. Catalytic deamination of the a-amino group surveyed with Mo-Ni-S, Pt/SiO2 and catalytic hydrogen transfer were unsuccessful. Successful deamination of cyclohexylamine with both Mo-Ni-S and Pt/SiO2 proved that the catalysts employed during those reactions were active. The activity of Ni-Mo-S catalyst was not responsible for the lack of deaminated product. Probably, Hoffmann elimination of the a-amine is not thermodynamically favorable since it requires abstraction of a B-hydrogen, which is not acidic (Figure 39). Similarly, nucleophilic substitution at the or—carbon of a-aminocaprolactam was not favored. It has been reported that C-N bond cleavage starts by insertion of the catalyst between or-C-H to form the o-complex or the a,B-adsorbed intermediate (Figure 54).20 97 \\ I, \ _ / H C—N H ‘C—N’ | \ / | | Pt Pt Pt Pt Pt Pt o-complex a,B-adsorbed intermediate Figure S4. Intermediates formed during C-N hydrogenolysis. Typically, high temperatures are required to effect C—N hydrogenolysis. Under these forcing conditions on the metal surface, C-C bond cleavage competes with C—N bond cleavage. Presumably, the becomes less basic on the metal surface making chemisorbed N and C to behave similarly, which, in turn, accounts for the lack of selectivity between C-C and C-N hydrogenolysis at high reaction temperatures. The fact that C-C and C-N bond cleavage products were not observed with Mo-Ni and Pt catalysts could mean that a-aminocaprolactam did not form the presumed complexes (Figure 54). High pressures employed during catalytic hydrodenitrogenation may have potentially inhibited deamination of a-aminocaprolactam. Unpublished results in the Frost research groups have shown that catalytic deamination of a-aminocaprolactam over Pt/C at 50 psi H2 resulted in a 23% caprolactam and the yield was reduced to 12% when H2 pressure was increased 100 psi H2. Catalytic transfer hydrogenation did not result in deamination of a—aminocaprolactam. Single electron reduction with SmI2 resulted in the deamination of the a-amino— group of L-lysine methyl ester and N-acetyl methyl pipecolinate. The low yield observed with the L-lysine methyl ester can be attributed to its limited solubility in THF-MeOH solvent. 98 NW NW IZJHz - Sml - MeOH - , Sml HZNWCOZCH3 ______g> H2NMOCH3 I HQNMOCHS ___2’ O . _ OH L-lysme methyl ester NHz ‘ _ M OH HZNMOCHa —> HZNWOCH3 __e__> HzN/VWOCHS + NH3 OH OH O Figure 55. Reductive deamination of L-lysine methyl ester with Smlz. Acetylation of the pipecolinate ester improved the yield of the deaminated product by 40%. The expected mechanism for deamination of an amine adjacent to a carboxyl involves the reaction of the carbonyl with SmI2 to afford a radical anion, which is rapidly protonated MeOH (Figure 55).45 The second equivalent of SmI2 reduces the protonated radical to afford a carbanion, which is followed by elimination of the amine. Tautomerization of the intermediate enol then leads to the observed product. The same number of equivalents of HMPA as SmI2 are required because HMPA serves as a chelator of Lewis acidic Sm(III) species formed during deamination. Although a catalytic reductive deamination with SmI2 is known in the presence of misch metal, the need to protect the amines is not appealing for large-scale synthesis of e—caprolactam. Misch metal is an alloy of light lanthanides (La 33%, Ce 50%, Nd 12%, Pr 4%, Sm and other lanthanides 1%) and is available at a low cost.46 When this alloy is used, catalytic amount of SmI2 (0.2 eq, 10 mol % ) has been effective as a single electron reductant. Reductive deamination of a 125 mM solution a—aminocaprolactam in i-PrOH with Na afforded e-caprolactam in only 20% yield. Formation of sodium isopropoxide was responsible for consumption of Na. Circumventing protection of the a-amine group 99 makes this route more viable than reductive deamination with SmIz. However, the reaction is stoichiometric with respect to Na. Is the deamination of L-lysine via a ketene a possible route for the synthesis of caprolactam (Figure 56)?47 NH2 NH? ()C02H2 NH .......... ", a--- COzH pyrolysis CZO O Figure 56. Hypothesized deamination of L-lysine to caprolactam via a ketene. Hypothetically, the a-amino acid can be simultaneously protected with oxalic acid, which would form an amide and a mixed anhydride with the amine and carboxylic acid, respectively. Decarboxylation would then result in a 6-aminocaproketene along with formamide, which would cyclize to afford caprolactam. As an alternative strategy to accomplish elimination, L-lysine was isomerized to B-L-lysine. Unlike L-lysine, the a-H in B—L-lysine is acidic and after abstraction would presumably lead to a,[3-unsaturated 6-amine hexanoate via or-carbanionic species (Figure 57). NH NH NH 2 [B] - 2 :0 R = 'CH2CH2CH2NH2 Figure 57. Deamination of B-amino group from fi-L-lysine. The chemical synthesis of B-L- does not utilize L-lysine as the starting material, which is the key goal for the synthesis of caprolactam based on starting materials derived from renewable feedstocks.48 By recruiting L-lysine-2,3-aminomutase from Clostridium 100 subterminale, an unprecedented preparative synthesis of B-L-lysine was accomplished. Reported syntheses of B-L-lysine employed crude extracts. Approximately, 100 mg of protein is required to convert 100 mg L-lysine to 77 mg of L-B-lysine under optimum conditions. This much protein is obtained from 12 L of cultures incubated for 5 h. Use of crude extracts from stationary phase (12-18 r incubation period) results in a 10-15 % yield of desired L-B—lysine. In addition, additives are imperative for the bioconversion in crude cell lysates. a-Ketoglutarate and PLP are required to make the imine with the lysine whereas SAM is required to generate the 5’-deoxyadenosyl radical necessary to initiate the 1,2-migration reaction of the amino group. Glutathione or mercaptoethanol is required to reduce the iron cluster. This implies that scaling up the isomerization in crude cell lysate would also requires scaling-up of these expensive additives. Deactivation of the second enzyme in L-lysine biodegradation, L-B-lysine—5,6- aminomutase, was key in accumulating L—[S-lysine using intact cells. Fortunately, this enzyme could be differentiated from L-lysine-2,3-aminomutase because it is a cobalamine—dependent enzyme. It has been reported that exposure to intense light inactivate cobalamide-dependent enzyme activity and especially L-B-lysine—5,6- aminomutase.49 Nitrous oxide, charcoal as well as ornithine, which is a competitive inhibitor, have also been shown to inhibit L-[3-lysine-5,6-aminomutase.50 Intense light in the presence of FeSO4 was effective in blocking L-B-lysine-S,6-aminomutase and resulted in the accumulation of L-B-lysine. The presence of Fez‘“ is necessary because it is required for the biosynthesis of the [4Fe-4S] cluster of L-lysine-2,3-aminomutase. Irradiation with light (E = 52 kcal/mol for a wavelength of 550 nm) irreversibly 101 deactivated the L-B-lysine-5,6-aminomutase presumably by cleaving the Co-C, which has been reportedly weak (25—40 kcal/mol).5' The advantage of using intact cells instead of crude cell lysate is the elimination of additives such as PLP and SAM. By allowing the bioconversion to proceed for 24 h, a l00% conversion of L-lysine was observed. High concentrations of FeSO4 inhibited the intact cell bioconversion completely. Freezing of cells Clostridium SB4 cell at -20 °C overnight improved their ability to catalyze isomerization of L-lysine to L-B-lysine. This heat-shock presumably inactivated the L-B-lysine-S,6-aminomutase. Previous reports showed that the crude cell lysate used for preparative synthesis of L-B-lysine was heated to 55-70 °C to precipitate unwanted proteins.52 This would suggest that frozen intact cells of C lostridium SB4 should be able to catalyze isomerization without irradiation of L-lysine to L-B-lysine. This avenue remains to be pursued along with systematic evaluation of the freezing-thawing cycles of Clostridium SB4 at -20 °C or -80 °C. Activity of intact cells to catalyze the isomerization of L-lysine to L-B-lysine with and without irradiation can then be evaluated. Irradiation of intact cells at various wavelengths can also be explored to obtain maximum inactivation of the L-B-lysine-5,6— aminomutase. When C lostria’ium SB4 cells were cultured in a medium containing more than 18 g/L of L-lysine, the cell mass declined indicating the toxicity of acetate and butyrate to the cells. To overcome this limitation for large-scale production of L-B-lysine from L- lysine, plasmid-localized kamA gene encoding L-lysine-2,3-aminomutase could be expressed in Clostridium subterminale. The resting cells could then be harvested and used to catalyze the isomerization of L-lysine to L-[3-lysine in large quantities. 102 Six homologous proteins of unknown functions have been revealed to have a sequence identity of 37—72% to L-lysine-2,3-aminomutase from Clostridium subterminale. These prokaryotic organisms include Porphyromanas gingivalus, Bacillus subtilis, Deinococcus radiodurans, Aquzflex aeolicus, Treponema pallidum, Haemophilus influenza, and E. coli. The B. subtilis protein, YodO, is 62% identical to C. subterminale L-lysine-2,3-aminomutase. The yodO gene has been cloned from the B. subtilis and heterologously expressed in E. coli.” The resulting protein was reported to catalyze the isomerization of L-lysine to L-B-lysine under anaerobic conditions as well. Unlike the Clostridium LAM, Bacillus was found to be stable in air and was not irreversibly deactivated. Attempts to heterologoulsy express the yodO gene in E. coli did not result in a viable construct. Intact cells of Bacillus subtilis were never explored for the conversion of L-lysine to L-B-lysine during this study. Given the activity of Clostridium SB4 to catalyze the isomerization reaction, exploring intact cells of Bacillus subtilis would be valuable. Enzyme-catalyzed B-elimination of L-[3-lysine followed by enzymatic reduction is another avenue that can be explored in addition to chemical deamination. Within the degradation pathway of L-lysine in Clostridium (Figure 48), L-B-amino-butyryl CoA deaminase catalyzes B-elimination of L—B-amino—butyryl CoA to an enoate CoA. Substrate analogues of L-B-amino—butyryl CoA have never been explored (Figure 58). L- B-Lysine or its cyclic analogue can be evaluated using this L—B-amino-butyryl CoA. This enzyme has previously been purified from C lostridz'um subterminale. 103 + _ - H3N W002 CoA H3N +’\/Y\n/ SCOA L3aminobuter H NM SCoA EH3 3 o NHao CoA deaminase 5 amino 1 ; acid : 5 oxndase : L-3-aminobutyrl- v i O O : CoA deaminase + _ -H - : H3N W002 __-2__* C02 CoA MSCOA -------------- j 0 [H] N N H H Figure 58. L-B-Amino-butyryl CoA deaminase catalyzes B-elimination. D-Proline reductase, which is found in Clostridium sticklanalii catalyzes C-N bond cleavage of D-proline to 5-aminovalerate (Figure 59). H30 QCOOH [ELCOOH HH 0 H _ 0 Se 0 HS [45kDa l 1 1: Figure 59. Reductive deamination of D-proline to 5-aminovalerate catalyzed by D- proline reductase. This enzyme contains three subunits with molecular masses of 23, 26 and 45 kDa.95 The 23 kDa subunits contains a pyruvoyl moiety which binds the substrate. The 26 kDa subunits houses the selenocysteine moiety (Figure 58).95c An enzyme-bound 104 pyruvoyl group first activates the L-ornithine-derived D-proline substrate. N ucleophilic attack of the a-carbon by the selenol anion of the selenocysteine results in the cleavage C—N bond of proline. Subsequent hydrolysis of the resulting intermediate gives 5- aminovalerate. With the advent of molecular evolution, reductive deamination of L- lysine-derived D-pipecolinate to 6-aminocaproic acid, which can then be cyclized to e- caprolactam is not a far-fetched idea (Figure 60). L-Pipecolinate reductase is not a known enzyme but L-proline reductase has been purified and characterized. C02 002H 0 HANEaior—‘Lai’ffldw HJN fl "’cozH fl COZH H2N _. L-Iysine L-pipecolinic acid D-pipecolinic acid 6-aminocaproic acid s-caprolactam Figure 60. Hypothesized microbial syntheses of 6-aminocaprolactam from L-lysine. Key: (a) L-Lysine cyclase; (b) racemase; (c) L-pipecolinate reductase; (d) EtOH, 200 °C. Production of L-lysine by Corynobacterium glutamicum proceeds under aerobic conditions while its isomerization to L-B-lysine in C lostridium proceeds under anaerobic conditions. Given this challenge, a single microbe capable of converting D-glucose to L- lysine may seem unlikely. However, the Frey group has successfully overexpressed L- lysine-2,3-aminomutase in E. coli. The resulting construct was cultured under aerobic conditions and expression of L-lysine-2,3-aminomutase was induced by degassing the media to remove 02. A similar strategy can be adopted for the production of L-fi-lysine from D-glucose. The use of intact cells for the preparative synthesis of L-B-lysine is unmatched. This route relies solely on D—glucose-derived L-lysine as the starting material, without the 105 need for cofactors. B—Elimination of the B-amino group in the 7-membered ring lactam derived from cyclization of L-B-lysine, followed by hydrogenation hold a promising process for the production of s-caprolactam from renewable starting materials. L-B- Lysine is not commercially available yet, this route can be a potential source of L-B- lysine. 106 h) REFERENCES Anton, A. Kirk-Othmer Encyclopedia of Chemical Technology," Wiley: New Y o r k , 2 O O 4 , http://www.mrw.intcrscience.wiley.com/kirk/articles/fibeanto.aOl/sect2—fs.htm|. Chemical Week 2003, 165 (29), 26. Fisher, W. B.; Crescentini, L. Kirk-OIhmer Encyclopedia of Chemical Technology; Wiley: New York, 2004, http://www.mrw.interscience.wiIeycom/ki rklarticles/caprfi sh.a0 1/sect4-fs.html. (a) Dahlhoff, G.; Niederer, J. P. M.; Hoelderich, W. F. Catal. Rev. 2001, 43, 381; (b)Morgan, M. European Chem. News 2005, 81, 2128. 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M.; Edmunds, H. N.; Baltimore, B. G.; Castilow, R.N.; Barker, H. A. Prep. Biochem. 1973, 3, 4752. Chen, D,; Ruzicka, F. J .; Frey, P. A. Biochem. J. 2000, 248, 539. 110 CHAPTER 4 EXPERIMENTAL GENERAL METHODS General chemistry All air and moisture sensitive reactions were carried out in oven and/or flame- dried glassware under positive argon pressure. Air or moisture sensitive reagents and solvents were transferred to reaction flasks fitted with rubber septa via syringes or cannula. Solvents were removed using either a Biichi rotary evaporator at water aspirator pressure or under high vacuum. Hydrogenations were performed in the Parr 4575 stainless steel high temperature-high pressure reactor equipped with the Parr 4842 temperature controller, which controlled the temperature and stirring rate. Reagents and solvents Tetrahydrofuran and diethyl ether were distilled under nitrogen from sodium benzophenone ketyl. Dichloromethane, pyridine, triethylamine and benzene were distilled from calcium hydride under nitrogen. Methanol and hexanol were distilled from sodium metal under argon and stored over Linde 4 A molecular sieves under argon. DMF, DMSO, hexanes and acetone were dried over activated Linde 4 A molecular sieves under nitrogen. Water was glass distilled and deionized. Charcoal (Darco® G-60 ~ 100 mesh) was used for discoloration of solutions. All reagents and solvents were used as available from commercial sources or purified according to published procedures. Organic solutions of products were dried over anhydrous MgSO4. All chemicals were 111 purchased from Aldrich. Ammonium molybdate and nickel nitrate were purchased from Strem. Sodium salt of 3-(trimethylsilyl)-propionic2,2,3,3-d4 acid (TSP) was purchased from Lancaster Synthesis Inc. Misch metal was purchased from Alfa Aesar. 3-Hydroxy— y-butyrolactone, 2-hydroxy-y-butyrolactone, methyl malate were synthesized from malic acid following a literature procedure.X D-Erythronolactone and L-threonolactone were synthesized from D-isoascorbic acid and L-ascorbic acid using a modified procedure. Diazomethane was generated from Diazald® following a literature procedure. S- Adenosylmethionine was purified on a (1.2 cm x 6 mL) carboxymethylcellulose (CM) column. The CM column was equilibrated with acetate buffer before 10 mL of 10 mg/mL of S-adenosylmethionine iodide was loaded to the column. The column was washed with 6 mL of acetate buffer before eluting with 40 mM HCl. 0.6 mL fractions were collected and those with an absorbance maxima at A260 were pooled and the concentration was determined. SAM was frozen at —20 °C. Chromatography Radial chromatography was carried out on a Harrison Associates Chromatotron using 1, 2 or 4 mm layers of silica gel 60 PF254 containing gypsum (E. Merck). Silica gel 60 (40-63 mm, B. Merck or Spectrum Chemicals) was used for flash chromatography. Analytical thin—layer chromatography (TLC) utilized pre-coated plates of silica gel K6F 60 A (0.25 mm, Whatman). TLC plates were visualized by immersion in anisaldehyde stain (by volume: 93% ethanol, 3.5% sulfuric acid, 1% acetic acid and 2.5% anisaldehyde) or phosphomolybdic acid stain (7% 12-molybdophosphoric acid in ethanol, w/v) followed by heating. Amino acids were visualized by staining with ninhydrin stain 112 prepared by dissolving 15 g of NaOAc in water (40 mL) and adjusting to pH 5-6. Tetramethylene sulfone (40 mL) and ninhydrin (2.0 g) were added followed by hydrindantin (36 mg) after 15 min. The total volume was made up to 100 mL with water. Diethylaminoethyl cellulose (DEAE) was purchased from Whatman, Dowex 50 (W) from Sigma and AG-1X8 from Bio-Rad. Dowex 50 was recycled by first rinsing with 10 column volume of water followed by suspending the resin in 10 N KOH to final pH of 14. The base was decanted and the resin was rinsed with three column volumes of water. Bromine was added to the resin in small portions until the color change stopped. The suspension was allowed to stand for 3 h before bromine before the resin was filtered and washed with 6 N HCl. Finally, the clean Dowex 50 was washed with water to remove all excess acid and was stored 4 °C. AG-1X8 Cl' was converted to the hydroxide form by washing with twenty column volumes of 1 N NaOH. The column was then washed with distilled deionized water until all the chloride was displaced as determined by silver nitrate test. Carboxymethylcellulose (CM) column was equilibrated with sodium acetate buffer before use. Spectroscopic and analytical measurements 1H NMR and 13C NMR spectra were recorded on a Varian VX-300 FT-NMR spectrometer or a Varian VXR-500 FT -N MR spectrometer. Chemical shifts for 1H N MR spectra are reported in parts per million (ppm) relative to internal tetramethylsilane (Me4Si, 6 = 0.0 ppm) with CDCl3 as the solvent and to internal sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP, 6 = 0.0 ppm) when D20 was the solvent. ”C NMR spectra 113 were recorded at 75 MHz on a Varian VX-300 FT -N MR spectrometer or at 125 MHz on a Varian VXR-500 FT-NMR spectrometer. Chemical shifts for 13C NMR spectra were reported in parts per million (ppm) relative to CDCI3 (6 = 77.0 ppm) or CD3OD (6 = 49.0 ppm) in D20. To determine molar concentrations during L—threonate 10 conversion to 4- hydroxy-2-ketobutyrate 11, a portion (1.0-3.0 mL) of the bioconversion mixture was taken every h and the protein was precipitated with 10% HCl and spun down in a microfuge. The supernatant was concentrated to dryness, the residue redissolved in 1 mL D20, concentrated again to dryness and the residue redissolved in 1 mL D20 containing 10 mM TSP. The concentrations of L-threonate 10 and 4-hydroxy-2-ketobutyrate 11 were determined by the ratios of the integrated 1H NMR resonances at 6 3.66 and 6 3.00, respectively, with the integrated resonance corresponding to TSP at 6 0.00. Similarly, the molar concentration during the bioconversion of L-lysine to fi—L-lysine was determined as mentioned above. The concentration of B-L-lysine and L-lysine were determined by the ratios of the integrated 1H NMR resonances at 6 2.8 and 6 2.0, respectively, with the integrated resonance corresponding to TSP at 6 0.00. Gas chromatography was used to determine the product yields obtained during the hydrogenations of D,L-malic acid 2a,b, L-malic acid 2b and 2—hydroxy-2-buten-4-olide 12. Chromatograms were obtained using an Agilent 6890N equipped with an HP—5 column (30 m x 0.25 mm x 0.25 am) after samples were derivatized with bis(trimethylsilyl)trifluoroacetamide. Analysis was optimized by using a temperature program in the range from 120 °C to 210 °C at 15 °C/min. GC derivatization was initiated by dissolving the dry sample (~50 mg) in pyridine (1 mL, 12.4 mmol) followed by the addition of dodecane (0.10 mL, 0.44 mmol) and bis(trimethylsilyl)- 114 trifluoroacetamide (2.0 mL, 7.5 mmol). The reaction was stirred at room temperature for 3 h. Samples were injected (2-5 uL) using an Agilent 7683 series injector. Yields were determined with response factors obtained from authentic samples that were quantified relative to dodecane internal standard. 1,2,4-Butanetriol enantiomers obtained from hydrogenation of L-malic acid were derivatized and the resulting Mosher esters analyzed using Agilent 1100 HPLC interfaced with ChemStation acquisition software (Rev. A.08.03).'5 To 1,2,4—butanetriol (0.003 g, 2.8 x 10'5 mol) in pyridine (0.20 mL) was added CH2C12 (0.30 mL), p- dimethylaminopyridine (0.005 g, 4 x 10'5 mol) and (S)-(+)-a-methoxy-a- (trifluoromethyl)phenylacetyl chloride (0.0040 g, 17 x 10‘5 mol). The reaction mixture was allowed to stir at rt for 8 h. The crude product was passed through a silica gel column and eluted with CHZCI2 (3 mL). After removal of CHzCl2 in vacuo, the resulting residue was redissolved in CH2C12 and washed with 1% NaHCO3 (5 mL) followed by H20 (2 x 5 mL). The organic layer containing the Mosher esters1 of 1,2,4—butanetriol enantiomers was concentrated and loaded on the Chiralpak AD column (Daicel Chemical, 4.6 mm x 250 mm) previously equilibrated with 2-propanolzhexanes (2:98, v/v). The column was eluted with a linear gradient of hexanes with 2-15% 2-propanol (v/v) for 28 min. The solvent was eluted at 1.25 mL/min while the eluant was monitored at 260 nm. Bacterial Strains and plasmids E. coli JWFl was prepared by homologous recombination of a non-functional serA gene into E. coli RB791 (W3110 lacl").2 Linearization of the 1.9 kb serA fragment 115 obtained from pD2625 into pMAK705 provided pLZl.68A. Linearization of pLZl.68A at the unique BamHI site internal to serA followed by treatment with Klenow fragment and dNTP’s and religation afforded pLZl.71A.3 Homologous recombination of the resulting non-functional serA locus of the pLZl.7lA into RB791 afforded JWFl. E. coli BL21(DE3)/pAF-80-kamA + pAlterEX2-argU was obtained from Professor P. Frey at the University of Wisconsin, Madison.4 Plasmid pET-23(a) was purchased from Novagen. Plasmids pMM4.166B, pMM4.202 and pMM4.257 were constructed following a literature procedure.5 Bacillus subtilis, Clostridium subterminale and C lastridiun sticklandii were obtained from ATCC. Storage of microbial strains and plasmids All bacterial strains were stored at -78 °C in glycerol. Plasmids were transformed into DHSa for long-term storage. Glycerol samples were prepared by adding 0.75 mL of an overnight culture to a sterile vial containing 0.25 mL of 80% (v/v) glycerol. The solution was mixed, left at room temperature for 2 h, and then stored at -78 °C. For Bacillus subtilis, Clostridium subterminale and Clostridium sticklandii, glycerol was purged with N2 before the overnight culture was introduced. Culture medium All solutions were prepared in distilled, deionized water. LB medium (1 L) contained Bacto tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g). Bacillus medium (1 L) was obtained by soaking 50 g of soybean in 1 L of distilled, deionized water overnight. NaOH (2.0 g) was dissolved and neutralized with HCl. The mixture 116 ‘1. was steamed for 1 h and filtered through a cheesecloth. The volumes was made up to l L and soluble starch (15 g), (NH4)2HPO3 (10.0 g) and KCl (0.2 g) were added and the solution adjusted to pH 7 prior to autoclaving. After the solution cooled to rt, a previously autoclaved solution of MgSO4 was added to final concentration of 0.2 g/mL. YT Medium (1 L) contained Bacto-tryptone (16 g), Bacto-yeast extracts (10 g) and NaCl (5 g) in distilled deionized water. TB medium (1 L) contained tryptone (10 g) and NaCl (5 g). After autoclaving and directly before use, MgSO4 (10 mL of l M stock) was added to the TB medium. M9 salts (1 L) contained NazHPO4 (6 g), KH2P04 (3 g), NH4C1 (1 g), and NaCl (0.5 g). M9 medium contained D-glucose (10 g), MgSO4 (0.12 g), and thiamine (0.001 g) in l L of M9 salts. CAYE medium (100 mL) contained casamino acids (2 g) and yeast extract (10 g). Minimum salts (1L) contained (NH4)ZSO4 (0,2 g), KH2P04 (0.6 g), KZHPO4 (1.4 g), sodium citrate (0.1 g) and magnesium sulphate (0,02 g). Terrific Broth (1 L) contained Bacto tryptone (12 g), Bacto yeast extract (24 g), glycerol (4 mL) in 900 mL. In a separate flask KH2P04 (2.31 g) and KzHPO4 (12.54 g) were dissolved in 90 mL of distilled deionized water. Each was autoclaved separately and the phophates were added to the YTG base after it cooled to 60 °C. SOC medium (1 L) contained Bacto tryptone (20 g), Bacto yeast extract (5 g), NaCl (10 mL, 1 M), KCl (2.5 mL, 1 M), MgCl2 (10 mL, 1 M), MgSO4 (10 mL, 1 M) and glucose (10 mL, 2 M). 2>