SYNTHESIS AND AROMATIZATION OF BIOBASED CYCLOHEXENES By Yukari Nishizawa-Brennen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry-Doctor of Philosophy 2017 ABSTRACT SYNTHESIS AND AROMATIZATION OF BIOBASED CYCLOHEXENES By Yukari Nishizawa-Brennen In recent years, the cost of petrochemicals has declined by increasing production of unconventional gases such as shale gas, tight gas and coalbed methane. The main component of the unconventional gases is methane. Aromatic compounds are ubiquitous in commodity, specialty and pharmaceutical chemicals. Many aromatic chemicals are derived from benzene, toluene and xylenes (BTX), which could be derived from methane. Biobased aromatic chemicals production may appear unnecessary since we can produce BTX from methane. However, other factors need to be considered such as climate change, avoidance of toxic starting materials and/or byproducts, and significant consumer preference for biobased products. Because aromatics are generally toxic to microbes, biobased aromatic compounds are often synthesized from nontoxic biobased intermediates. An ideal strategy is a synthesis of biobased hydroaromatics that can be either dehydrated or dehydrogenated to aromatics in a single step. We can perform elimination reactions on biobased cyclohexenes possessing appropriately placed leaving groups. In the absence of leaving groups, dehydrogenation reactions need to be employed. This thesis focuses on biobased cyclohexenes that are aromatized either by dehydration reactions or by dehydrogenation reactions. Copyright by YUKARI NISHIZAWA-BRENNEN 2017 This dissertation is dedicated to Mike and Ike Thank you for coming to Michigan with me. iv ACKNOWLEDGMENTS First, I would like to thank my research adviser, Professor John W. Frost, for his patience and guidance. Even though, I was clueless in chemistry laboratory, he never gave up on me. I would like to thank the members of my guidance committee, Professor Babak Borhan, Professor Xuefei Huang and Professor Gary J. Blanchard for their intellectual input throughout the graduate school. I am grateful for Dr. Karen Draths for detail guidance in experimental techniques and intelligent input. I extremely appreciate all of former group members for their research and strain collections. Lastly, I am deeply indebted to my lab mate, Kelly K. Miller, for help during the writing of this thesis. v TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ..................................................................................................................... xiii LIST OF SCHEMES ................................................................................................................... xiv KEY TO ABBREVIATIONS ..................................................................................................... xvii CHAPTER ONE ..............................................................................................................................1 1. Introduction ..................................................................................................................................1 REFERENCES ..............................................................................................................................14 CHAPTER TWO ...........................................................................................................................20 Synthesis of Biobased p-Hydroxybenzoic Acid ............................................................................20 1. Introduction ................................................................................................................................20 2. p-Hydroxybenzoic Acid Production ..........................................................................................21 3. Toxicity of Benzene and Phenol ................................................................................................24 4. Biocatalytic Routes to p-Hydroxybenzoic Acid ........................................................................26 5. Results ........................................................................................................................................33 5.1. Conversion of Shikimic Acid to p-Hydroxybenzoic Acid in Ionic Liquid Bromides .........33 5.2. Dehydration of Shikimic Acid in non-Bromide Ionic Liquids ............................................38 5.3. A Possible Mechanisum for Bromide-Catalyzed Dehydration of Shikimic Acid ...............39 5.4. Lewis Acid Catalyzed Conversion of Shikimic Acid to p-Hydroxybenzoic Acid in 1Butyl-3-methylimidazolium Salts .........................................................................................41 5.5. Conversion of (3R,4S,5R)-Triacetoxy-1-cyclohexene-1-carboxylic Acid to m-Hydroxybenzoic Acid in 1-Butyl-3-methylimidazolium Salts .........................................44 6. Discussion ..................................................................................................................................47 7. Experimental ..............................................................................................................................49 7.1. General .................................................................................................................................49 7.2. Product Analysis ..................................................................................................................50 7.3. Screening of Ionic Liquid for Dehydration of Shikimic Acid .............................................50 7.4. Comparison of Shikimic Acid Concentration in Dehydration of Shikimic Acid ................50 7.5. Synthesis of 1-Butyl-3-methylimidazolium Bromide ..........................................................51 7.6. Recycling 1-Butyl-3-methylimidazolium Bromide .............................................................51 7.7. Lewis Acid Catalysts Screening for Dehydration of Shikimic Acid ...................................53 7.8. Synthesis of Tetra-(n)-butylphosphonium Iodide ................................................................54 7.9. (3R,4S,5R)-Triacetoxy-1-cyclohexenecarboxylic Acid .......................................................55 7.10. Screening of Ionic Liquid for m-Hydroxybenzoic Acid Synthesis from (3R,4S,5R)Triacetoxy-1-cyclohexenecarboxylic Acid ........................................................................55 7.11. 1-Butyl-3-methylimidazolium Chloride ............................................................................56 7.12. m-Hydroxybenzoic Acid Synthesis from (3R,4S,5R)-Triacetoxy-1-cyclohexenecarboxylic Acid .....................................................56 vi REFERENCES ..............................................................................................................................58 CHAPTER THREE .......................................................................................................................66 Scale-Up of the Synthesis of Biobased p-Hydroxybenzoic Acid ..................................................66 1. Introduction ................................................................................................................................66 2. E. coli SP1.1/pKD15.071B ........................................................................................................67 3. Countercurrent Flow Extraction ................................................................................................69 4. Tangential (Cross) Flow Filtration.............................................................................................73 5. Results ........................................................................................................................................75 5.1. Shikimic Acid Titers and Yield ...........................................................................................75 5.2. Shikimic Acid Extraction Solvent Screening ......................................................................75 5.3. Countercurent Extraction of Shikimic Acid ........................................................................76 5.4. Comparison of Shikimic Acid Crystallization Solvents ......................................................79 5.5. Purification of Shikimic Acid ..............................................................................................80 5.6. Scale Up Synthesis of p-Hydroxybenzoic Acid...................................................................81 6. Discussion ..................................................................................................................................82 7. Experimental ..............................................................................................................................84 7.1. General .................................................................................................................................84 7.2. Product Analysis ..................................................................................................................84 7.3. Culture Medium ...................................................................................................................85 7.4. Bacterial Strain and Plasmid ................................................................................................86 7.5. Fed-Batch Fermentation (General) ......................................................................................86 7.6. Fed-Batch Fermentation of Shikimic Acid ..........................................................................87 7.7. Cell Removal .......................................................................................................................89 7.8. Protein Removal...................................................................................................................90 7.9. Shikimic Acid Extraction Solvent Screening in Vials .........................................................90 7.10. KARR® Column General ..................................................................................................90 7.11. Comparison of Shikimic Acid Extraction Solvents in KARR® Column ..........................91 7.12. Comparison of Shikimic Acid Crystallization Solvents ....................................................91 7.13. Decolorizing Isopropanol/Water (9:1 v/v) Extract and Evaporative Crystallization from n-Butanol of Shikimic Acid .........................................92 7.14. Scale-up Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide ........93 REFERENCES ..............................................................................................................................95 CHAPTER FOUR..........................................................................................................................99 Synthesis of Biobased Terephthalic Acid ......................................................................................99 1. Introduction ................................................................................................................................99 2. Terephthalic Acid and Isophthalic Acid Production ................................................................100 3. Biobased Terephthalic Acid .....................................................................................................102 4. Results ......................................................................................................................................107 4.1. Cycloaddition of Acrylic Acid and Isoprene .....................................................................107 4.2. Aromatization of 4-Methyl-3-cyclohexenecarboxylic Acid and 3-Methyl-3-cyclohexenecarboxylic Acid........................................................................110 4.2.1. H2SO4 Oxidation .......................................................................................................110 4.2.2. Dehydrogenation Catalyzed by Pd/C in Solvents .....................................................112 4.2.3. Catalytic Oxidative Dehydrogenation.......................................................................113 vii 4.2.4. Aerobic Dehydrogenation .........................................................................................114 4.2.5. Aerobic Oxidative Dehydrogenation ........................................................................114 4.2.6. Vapor Phase Dehydrogenation .................................................................................116 5. Discussion ................................................................................................................................119 6. Experimental ............................................................................................................................120 6.1. General ...............................................................................................................................120 6.2. Cycloaddition Products 39 and 40 Analysis ......................................................................120 6.3. Analysis of Aromatization Products: p-Toluic Acid 41 and 4-Methyl-3-cyclohexenecarboxylic Acid 44 .....................................................................121 6.4. Analysis of Aromatization Products: p-Toluic Acid 41, m-Toluic Acid 42, 4-Methylcyclohexanecaroboxylic Acid 44, 3-Methylcyclohexanecarboxylic Acid 47, Unreacted 4-Methyl-3-cyclohexenecarboxylic Acid 39, and Unreacted 3-Methyl-3-Cyclohexenecarboxylic Acid 40 ...................................................122 6.5. Uncatalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at rt ...............................123 6.6. Uncatalyzed Cycloaddition of Acrylic Acid and Isoprene in a High Pressure Reactor ....124 6.7. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 25 °C ...........124 6.8. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 50 °C ...........125 6.9. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 95 °C ...........125 6.10. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 6) .......................................................................................126 6.11. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 7) .......................................................................................126 6.12. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 8) .......................................................................................127 6.13. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 9) .......................................................................................127 6.14. H2SO4 Oxidation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 to p-Toluic Acid 41 ..128 6.15. H2SO4 Oxidation of a Mixture of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3Methyl-3-cyclohexenecarboxylic Acid 40 to p-Toluic Acid 41 and m-Toluic Acid 42..128 6.16. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Water ...................129 6.17. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Acetic Acid ..........130 6.18. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in p-Xylene ..............130 6.19. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Mesitylene ...........131 6.20. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in n-Decane ..............131 6.21. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3-Methyl-3Cyclohexenecarboxylic Acid 40 with Pd/C and Nitrobenzene in Mesitylene .................132 6.22. PdCl2-AMS Catalyzed Dehydrogenation of 4-Methyl-3-cyclohexnecarboxylic Acid 39 .....................................................................132 6.23. Pd/C-AMS Catalyzed Dehydrogenation of 4-Methyl-3-cyclohexnecarboxylic Acid 39 132 6.24. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexnecarboxylic Acid 39 ......................133 6.25. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexnecarboxylic Acid 39 and 3-Methyl-3-cyclohexnecarboxylic Acid 40 ..............................................................133 6.26. Aerobic Oxidative Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39....134 6.27. Vapor Phase Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 .............134 6.28. Vapor Phase Dehydrogenation of a Mixture of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3-Methyl-3-cyclohexnecarboxylic Acid 40 .........................................................135 viii REFERENCES ............................................................................................................................137 CHAPTER FIVE .........................................................................................................................143 Synthesis of Biobased Trimethyl Trimellitate .............................................................................143 1. Introduction ..............................................................................................................................143 2. Trimellitic Acid Production .....................................................................................................144 3. Biobased Trimellitic Acid / Trimethyl Trimellitate .................................................................146 4. New Synthesis Route of Biobased Trimellitic Acid ................................................................147 5. Protocatechuate 3,4-Dioxygenase ............................................................................................149 6. 1,2,4-Butanetricarboxylic Acid ................................................................................................152 6.1. Introduction ........................................................................................................................152 6.2. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid .....................................................152 7. Results ......................................................................................................................................154 7.1. Microbial Synthesis of 2E,4Z-3-Carboxylic Acid .............................................................154 7.1.1. E. coli WN1/pYNB6.009A ..........................................................................................154 7.1.1.1. Cloning Overview ..................................................................................................154 7.1.1.2. Construction of pKD16.179A and pYNB6.009A ..................................................155 7.1.1.3. Host Strain E. coli WN1 Phenotype Confirmation ................................................158 7.1.2. Protocatechuate 3,4-Dioxygenase Activity ..................................................................159 7.1.3. Fed-Batch Fermentor Synthesis of 2E,4Z-3-Carboxymuconic Acid ...........................159 7.1.3.1. Glucose-rich Condition Fermentation....................................................................159 7.1.3.2. Glucose-limited Condition Fermentation ..............................................................161 7.2. Isolation of 2E,4E-3-Carboxymuconic Acid from Fermentation Broth ............................163 7.3. Synthesis of Biobased Trimethyl Trimellitate ...................................................................164 7.3.1. Isomerization of 2E,4E-3-Carboxymuconic Acid 54 to 2Z,4E-3-Carboxymuconic Acid 13 .................................................................................164 7.3.2. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene ............................165 7.3.3. Esterification of 2E,4E-3-Carboxymuconic Acid 54 ...................................................165 7.3.4. Cycloaddition of Trimethyl 3-Carboxymuconate 56 and Ethylene .............................166 7.3.5. Aromatization of Cycloadduct 57 ................................................................................167 7.3.5.1. Dehydrogenation Catalyzed by Pd/C in Solvents ..................................................167 7.3.5.2. Aerobic Oxidative Dehydrogenation of Cycloadduct 57.......................................168 7.3.5.3. Vapor Phase Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/SiO2 ............169 7.4. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid .....................................................170 8. Discussion ................................................................................................................................171 9. Experimental ............................................................................................................................172 9.1 Spectroscopic Measurements ..............................................................................................172 9.2. Gas Chromatography .........................................................................................................172 9.3. Bacterial Strains and Plasmids ...........................................................................................172 9.4. Storage of Bacterial Strains and Plasmids .........................................................................173 9.5. Culture Medium .................................................................................................................173 9.6. Preparation and Transformation of Electrocompetent Cell ...............................................174 9.7. Small Scale Purification of Plasmid DNA .........................................................................175 9.8. Large Scale Purification of Plasmid DNA .........................................................................176 9.9. Isolation of Klebsiella pneumoniae subsp. pneumoniae (ATCC25597) Genomic DNA ..176 9.10. Plasmid pKD16.179A ......................................................................................................177 ix 9.11. Plasmid pYNB6.009B .....................................................................................................178 9.12. Plasmid pYNB6.009A .....................................................................................................180 9.13. E. coli WN1 .....................................................................................................................180 9.14. Protocatechuate 3,4-Dioxygenase Activity ......................................................................181 9.15. Fed-Batch Fermentation General .....................................................................................182 9.16. Glucose-rich Condition Fermentation..............................................................................184 9.17. Glucose-limited Condition Fermentation ........................................................................185 9.18. Removal of Cells and Protein ..........................................................................................187 9.19. Isolation of 2E,4E-3-Carboxymuconic Acid 54 ..............................................................187 9.20. 2E,4E-3-Carboxymuconic Acid 54 isolation Process Investigation ................................188 9.21. Isomerization of 2E,4E-3-Carboxymuconic Acid 54 to 2Z,4E-3-Carboxymuconic Acid 13 ..............................................................................188 9.22. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene ................................189 9.22.1. Reaction in 1,4-Dioxane at 164 °C ............................................................................189 9.22.2. Reaction in m-Xylene at 150 °C ................................................................................189 9.22.3. Reaction in 1,4-Dioxane at 100 °C ............................................................................190 9.22.4. Reaction in m-Xylene at 100 °C ................................................................................190 9.23. Esterification of 2E,4E-3-Carboxymuconic Acid 54 .......................................................190 9.24. Cycloaddition of Trimethyl 3-Carboxymuconate 56 and Ethylene .................................191 9.25. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Decane ...............................192 9.26. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Dichlorobenzene ................193 9.27. Aerobic Oxidative Dehydrogenation of Cycloadduct 57.................................................193 9.28. Vapor Phase Dehydrogenation of Cycloadduct 57 ..........................................................193 9.29. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid 37 ..............................................194 REFERENCES ............................................................................................................................196 x LIST OF TABLES Table 2.1. Dehydration of Shikimic Acid in Various 1-Butyl-3-methylimidazolium Salts 40a-40e ..................................................................34 Table 2.2. Impact of Shikimic Acid Concentration in 1-Butyl-3-methylimidazolium Bromide 40b on Dehydration Yield and Accountability of Shikimic Acid Consumption ....................34 Table 2.3. 1-Butyl-3-methylimidazolium Bromide 40b Recycle ..................................................36 Table 2.4. Dehydration of Shikimic Acid in Various Ionic Liquids 41-45 ...................................37 Table 2.5. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide 40b ...................................................................42 Table 2.6. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Chloride 40a ...................................................................42 Table 2.7. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Iodide 40c .......................................................................43 Table 2.8. Lewis Acid 2 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide 40b ...................................................................43 Table 2.9. m-Hydroxybenzoic Acid Synthesis from Acetylated Shikimic Acid in Various 1-Butyl-3-3methylimidazolium Salts 40a-40e ...................................................45 Table 3.1. Shikimic Acid Partition Coefficient in Various Solvents .............................................76 Table 3.2. Problems Seen During KARR® Column Optimization ...............................................77 Table 3.3. Countercurrent Extraction of Microbe-Synthesized Shikimic Acid .............................79 Table 3.4. % Purity and % recovery of Crystallized Shikimic Acid .............................................80 Table 3.5. Instrument Setting of First Phase ..................................................................................88 Table 3.6. Instrument Setting of Second Phase .............................................................................89 Table 3.7. Auto-antifoam Setting...................................................................................................89 Table 4.1. Cycloaddition of Acrylic Acid and Isoprene ..............................................................108 Table 4.2. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid in Various Solvents..112 xi Table 5.1. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene...........................165 Table 5.2. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Solvents ........................168 Table 5.3. PCR Cycle for pcaHG ................................................................................................177 Table 5.4. PCR Cycle for serA – aroFFBR – ParoF Cassette ...........................................................179 Table 5.5. First Phase of Glucose-rich Fermentation ..................................................................184 Table 5.6. Second Phase of Glucose-rich Fermentation ..............................................................185 Table 5.7. First Phase of Glucose-limited Fermentation .............................................................186 Table 7.8. Second Phase of Glucose-limited Fermentation .........................................................186 xii LIST OF FIGURES Figure 2.1. ortho- and para-intermediate ......................................................................................22 Figure 3.1. A: Centrifugal Extractor, B: Rotor of Centrifugal Extractor .......................................70 Figure 3.2. A: Packed Column, B: Sieve Tray Column, C: RDC Extractor, D: SCHEIBEL® Column ...............................................................................................70 Figure 3.3. KARR® Column .........................................................................................................70 Figure 3.4. KARR® Column Configuration..................................................................................73 Figure 3.5. A: Tangential (Cross) Flow Filtration, B: Dead-End Filtration ..................................74 Figure 3.6. Hollow Fibers ..............................................................................................................74 Figure 3.7. Continuous Liquid-Liquid Extractor ...........................................................................82 Figure 4.1. Vapor Phase Plug Reactor .........................................................................................117 Figure 5.1. Preparation of Plasmid pKD16.179A ........................................................................156 Figure 5.2. Preparation of Plasmid pYNB6.009B .......................................................................157 Figure 5.3. Preparation of Plasmid pYNB6.009A .......................................................................158 Figure 5.4. Glucose-rich/IPTG(+) Fermentation .........................................................................161 Figure 5.5. Glucose-limited/IPTG(+) Fermentation ....................................................................162 xiii LIST OF SCHEMES Scheme 1.1 Methane is an Ozone Precursor ....................................................................................4 Scheme 1.2. Formation of Cyclohexane during Aromatization of Cyclohexene ............................6 Scheme 1.3. Palladium Catalyst Cycle with Anthraquinone-2-sulfonate Sodium Salt ...................8 Scheme 1.4. Proposed Mechanism for Regeneration of Pd(TFA)2 with Oxygen............................9 Scheme 1.5. Proposed Mechanism for PdII Catalyzed Aerobic Dehydrogenation ..........................9 Scheme 1.6. Aromatization of 3,4-Dimethylcyclohex-3-enecarboxylic Acid ...............................10 Scheme 1.7. Aerobic Oxidative Dehydrogenation of Cyclohexenes to Arene Derivatives ..........11 Scheme 1.8. Dehydration of Microbially Synthesized Shikimic Acid ..........................................12 Scheme 1.9. Synthesis of Biobased p-Toluic Acid m-Toluic Acid ...............................................13 Scheme 1.10. Synthesis of Biobased Trimethyl Trimellitate ........................................................13 Scheme 2.1. p-Hydroxybenzoic Acid Production from Benzene ..................................................23 Scheme 2.2. Benzene Metabolism .................................................................................................25 Scheme 2.3. Biocatalytic Routes to p-Hydroxybenzoic Acid........................................................27 Scheme 2.4. Biocatalytic Routes to p-Hydroxybenzoic Acid........................................................29 Scheme 2.5. Shikimic Acid Dehydration in Ionic Liquid ..............................................................33 Scheme 2.6. Ionic Liquids Used in Shikimic Acid Dehydration ...................................................33 Scheme 2.7. Possible Mechanism of Shikimic Acid Dehydration ................................................40 Scheme 2.8. Aromatization of Acetylated Shikimic Acid .............................................................44 Scheme 2.9. Possible Mechanism of Acetylated Shikimic Acid Aromatization ...........................46 Scheme 3.1. The Common Pathway of Aromatic Amino Acid Biosynthesis ...............................67 Scheme 3.2. Biosynthesis of Erythrose-4-phosphate .....................................................................68 xiv Scheme 3.3. Increasing Phosphoenolpyruvic Acid Availability ...................................................68 Scheme 3.4. Dehydration of Shikimic Acid ..................................................................................81 Scheme 4.1. Aerobic Catalytic Oxidation of p-Xylene to Terephthalic Acid .............................101 Scheme 4.2. Catalytic Pathway for Amoco Mid-Century Oxidation of p-Toluic Acid ..............101 Scheme 4.3. Biobased p-Xylene ..................................................................................................103 Scheme 4.4. Xylene-Free Synthesis of Biobased Terephthalic Acid ..........................................104 Scheme 4.5. Alder’s Reaction Sequence .....................................................................................105 Scheme 4.6. Cycloaddition of Acrylic Acid and Isoprene...........................................................107 Scheme 4.7. Aromatization of 4-Methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3cyclohexenecarboxylic acid 40 ...............................................................................110 Scheme 4.8. H2SO4 Oxidation......................................................................................................111 Scheme 4.9. Proposed Reaction Mechanism of H2SO4 Oxidation ..............................................111 Scheme 4.10. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid on Pd/AMS.........113 Scheme 4.11. Stahl’s Reported Reaction .....................................................................................114 Scheme 4.12. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid and 3Methyl-3-cyclohexenecarboxylic Acid ....................................................................114 Scheme 4.13. Aerobic Oxidative Dehydrogenation of Cyclohexenes to Arene Derivatives ......115 Scheme 4.14. Aerobic Oxidative Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid .......................................................................................................................115 Scheme 4.15. Vapor Phase Aromatization of 4-Methyl-3-cyclohexenecarboxylic Acid and 3-Methyl-3-cyclohexenecarboxylic Acid ................................................................118 Scheme 5.1. Plasticizers ...............................................................................................................143 Scheme 5.2. Oxidation of Pseudocumene....................................................................................145 Scheme 5.3. Biobased Trimellitate ..............................................................................................147 Scheme 5.4. New Synthesis Route of Biobased Trimellitic Acid ...............................................148 xv Scheme 5.5. Biosynthesis Pathway of 2E,4Z-3-Carboxymuconic Acid ......................................149 Scheme 5.6. b-Ketoadipate Pathway ...........................................................................................151 Scheme 5.7. First Phase of Aromatic Compound Degradation ...................................................151 Scheme 5.8. Synthesis of 1,2,4-Butanetricarboxylic Acid from 2E,4Z-3-Carboxymuconic Acid ..................................................................................152 Scheme 5.9. Biobased 1,2,4-Butanetricarboxylic Acid Synthesis Route ....................................153 Scheme 5.10. Lysine Biosynthesis Pathway ................................................................................154 Scheme 5.11. Protocatechuate 3,4-Dioxygenase .........................................................................159 Scheme 5.12 Fermentation Products............................................................................................160 Scheme 5.13. Isomerization of 2E,4E-3-Carboxylic Acid 54 .....................................................164 Scheme 5.14. Nuclear Overhauser Effect ....................................................................................164 Scheme 5.15. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene .....................165 Scheme 5.16. Esterification of 2E,4E-3-Carboxymuconic Acid 54 ............................................166 Scheme 5.17. Nuclear Overhauser Effect ....................................................................................166 Scheme 5.18. Cycloaddition of Trimethyl 3-Carboxymuconate 56a,b and Ethylene .................167 Scheme 5.19. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Solvent ....................167 Scheme 5.20. Aerobic Oxidative Dehydrogenation of Cycloadduct 57 ......................................168 Scheme 5.21. Vapor Phase Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/SiO2 ...........169 Scheme 5.22. Synthesis of 1,2,4-Butanetricarboxylic Acid 37 ...................................................170 xvi KEY TO ABBREVIATIONS Ac acetyl AcOH acetic acid AMS anthraquinone-2-sulfonate sodium salt Ap ampicillin ApR ampicillin resistance ASA acetylated shikimic acid ADP adenosine monophosphate ATP adenosine triphosphate BMIm 1-butyl-3-methylimidazolium Bp base pair BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide BTEX benzene, toluene, ethylbenzene and xylenes BTX benzene, toluene and xylenes BuOH butanol CIAP calf intestinal alkaline phosphatase CoA coenzyme A DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate DHQ 3-dehydroquinic acid DHS 3-dehydroshikimic acid DIPEA N,N-diisopropylethylamine DMF dimethylformamide xvii DNA deoxyribonucleic acid dNTP Deoxynucleotide solution mix DO dissolved oxygen E4P D-erythrose E.C. enzyme commission number E. coli Escherichia coli EPA United Sates Environmental Protection Agency EtOAc ethyl acetate EtOH ethanol FBR feed back resistant GC gas chromatography h hour HPLC high-performance liquid chromatography IPTG isopropyl b-D-1-thiogalactopyranoside K. pneumoniae Klebsiella pneumoniae LCP liquid crystalline polymer MCL maximum contaminant limit MeOH methanol MHBA m-hydroxybenzoic acid Min minute MS mass spectrometer NAD nicotinamide adenine dinucleotide NMP N-methyl-2-pyrrolidone 4-phosphate xviii NOE nuclear Overhauser effect spectroscopy mp melting point NMR nuclear magnetic resonance spectroscopy OD optical density OTf trifluoromethanesulfonate OSHA Occupational Safety and Health Administration PCA protocatechuic acid PCR polymerase chain reaction PEP phosphoenolpyruvate PET polyethylene terephthalate PHBA p-hydroxybenzoic acid Ppm parts per million PTS PEP:carbohydrate phosphotransferase PVC polyvinylchloride RDC rotating disc contactor ROS reactive oxygen species rt room temperature SA shikimic acid sec second sp. species subsp. subspecies TCA tricarboxylic acid cycle Temp temperature xix TFA trifluoroacetate THF tetrahydrofuran TLC thin-layer chromatography TsOH p-toluenesulfonic acid TSP sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid xx CHAPTER ONE 1. Introduction As part of the generation that was born and raised during the 1970’s oil crisis, the fear of decreasing petrochemical availability was omnipresent. This fear has declined today due to exponentially increasing production of unconventional gases such as shale gas, tight gas and coalbed methane. Unconventional gas is the gas that can be produced by using hydraulic fracturing, horizontal drilling or other techniques to expose additional reservoirs of hydrocarbons.1 Use of hydraulic fracturing to extract oil and gas from shale/rock/coalbed formations, has advanced rapidly over the last two decades.2 Extraction of shale gas in the United States has increased more than ten times in the last decade alone.3 The United States has become self-sufficient in gas production despite recently being one of the largest importers of natural gas in the world.2 According to the U.S. Department of Energy, the United States had 3.5 ´ 1011 m3 of proven coalbed methane reserves and 5.0 ´ 1012 m3 of proven reserves of shale gas methane.4a They also estimated the United States had 3000 ´ 1012 m3 of methane as methane hydrate.4b Even though there is no commercial production of methane from methane hydrate, Japan recently reported the world’s first offshore methane hydrate production field test.4c Many aromatic chemicals are derived from benzene, toluene and xylenes (BTX) but the challenge of deriving BTX from methane remains. The conversion of methane to BTX via methanol has been suggested to be the most promising route for conversion of methane to BTX.5 Methane is converted to methanol via steam reforming. The catalysts for steam reforming are quartz (silica) or metal exchanged zeolite.6,7 Selective conversion of methanol to aromatics is catalyzed by metal exchanged ZSM-5.8 The highest distribution of products among BTX from this process is xylenes, while the lowest is benzene.8 The selective disproportionation of toluene   1 over zeolite can yield p-xylene and benzene.9 Biobased aromatic chemicals production may appear unnecessary since we can produce BTX from methane. However, other factors need to be considered such as climate change, avoidance of toxic starting materials and/or byproducts, and significant consumer preference for biobased products. The United Nations encourages implementation of carbon pricing systems for greenhouse gas emissions such as carbon taxation and cap-and-trade policies.10 A carbon pricing system has already been implemented or is scheduled for implementation in small number of countries, states and provinces. A cap-and-trade program has been in place in California since 2012.11 Washington and Oregon have been deliberating the implementation of a carbon tax since 2015.10,12 Although the first carbon tax proposal in Washington was rejected by voters in November 2016, the proposed legislation is currently being revised.13 In petrochemical production, significant amounts of carbon are wasted as CO2. As an example, 30-40% of carbon is wasted as CO2 and CO during the production of maleic anhydride from n-butane.14 Synthesis of biobased maleic anhydride from furfural, which is obtained from hydrolysis of biomass, also loses 40% of carbon during oxidation,15,16 although this lost carbon originated from atmospheric CO2 fixed by plants. The U.S. Department of Agriculture (USDA) encourages production and use of biobased products as part of the BioPreferred® program. The USDA reported that the total contribution of the biobased product industry to the U.S. economy in 2013 was $369 billion and employed four million workers.17 One example of a commercial biobased product is the 30% (by weight) biobased polyethylene terephthalate water bottle produced for and marketed by The Coca-Cola Company.18 The radiation from sunlight bounces off the Earth surface and emits longwave radiation. Absorbance of longwave radiation and emission of the energy by CO2 is the basis of its   2 greenhouse effect.19a There is also a report that elevated CO2 concentrations in the atmosphere affects plant physiology, which increases greenhouse effect.19b The elevated CO2 concentrations in the atmosphere reduces the evapotranspiration of plants, which is reducing the concentration of water vapor in the atmosphere.19b Even though water has a greenhouse gas effect,19a clouds near the land keeps temperature low by increasing the planetary albedo and causing less solar radiation to reach the surface. The decreasing the greenhouse gas effect of water cannot compensate the increased solar radiation to reach the surface.19b Methane has more of a greenhouse gas effect than CO2 because methane absorbs shorter wavelength (8 µm) radiation than CO2 (10 µm and 14 µm).19a Degradation of methane produces ozone, which is also a greenhouse gas.20 The ozone layer in the stratosphere protects us by absorbing UV radiation. Ozone in the troposphere leads to a greenhouse effect by absorbing longwave (9 µm) radiation.19a,20a High concentrations of methane along with nitrogen oxides (NOx) increases the concentration of ozone in the troposphere (Scheme 1).20 Hydroxyl radical abstracts a hydrogen from methane, forming •CH3. The •CH3 reacts with molecular oxygen to form •OOCH3. An oxygen atom is abstracted from •OOCH3 by NO, yielding •OCH3. Formaldehyde is produced when •OCH3 reacts with molecular oxygen. Formaldehyde is photolyzed to CO and H2 by sunlight.20e An oxygen atom is released from NO2 upon photolysis by sunlight, and the oxygen atom reacts with molecular oxygen, forming ozone (Scheme 1.1).20   3 Scheme 1.1. Methane is an Ozone Precursor Methane gas leakage at shale gas drilling and production sites is a problem. It is estimated that 2.2 ´ 109 kg/yr of methane is emitted annually in the United States.21 Another problem at shale gas drilling site is a groundwater contamination from the shale gas/oil, chemicals used during hydraulic fracturing and disturbance of surface soil.22 Dichloromethane was detected from private, residential water wells (within one mile) the Barnett shale region in Texas.22a The average dichloromethane concentration was 0.08 mg/L while the maximum contaminant limit (MCL) of dichloromethane suggested by the U.S. Environmental Protection Agency is 0.005 mg/L.22a The source of dichloromethane is thought to be from disturbance of surface soil, which was already contaminated with dichloromethane. Methane contaminates underground water at active shale gas extraction sites. The highest methane level in the underground water from Wyoming, north-east Pennsylvania and New York was 70 mg/L.22b All the samples with high methane concentration was obtained within 1 km from the active shale gas   4 extraction sites.22b Hydraulic fracturing utilizes organic solvents containing benzene, toluene, ethylbenzene and xylenes (BTEX).22c The contaminated water and soil produced from drilling and hydraulic fracturing are injected into a ground. The shallow groundwater near the disposal ground contained 390 µg/L of benzene and 150 µg/L of m/p-xylenes while EPA’s maximum concentration level for benzene was 5 µg/L.22c There was an incident caused by Marcellus shale gas wells in Pennsylvania. Natural gas flooded into initially drinkable groundwater used by several households.22d The groundwater was located to 1-3 km from hydraulic fracturing ares. In addition to methane (47 mg/L), the water was observed to foam.22d Later, the cause of foaming was linked to 2-n-butoxyethanol, which was one component of the fracturing fluid.22d In addition to greenhouse gas emissions and groundwater contamination, earthquakes related to shale gas production is a concern. The magnitude and the area impacted by reported earthquakes related to hydraulic fracturing are relatively small (magnitude 2-3) relative to tectonic earthquake activity.23ab However, there were some significant earthquakes in Oklahoma caused by injection of wastewater produced from hydraulic fracturing. On September 3rd 2016, there was the earthquake with magnitude 5.8, which occurred approximately 15 km to the northwest of the town Pawnee.23c It damaged several buildings in the town. The earthquake was felt across multiple states up to distance over 1500 km.23c Aromatic compounds are ubiquitous in commodity, specialty and pharmaceutical chemicals. Because aromatics are generally toxic to microbes,24 biobased aromatic compounds are often synthesized from nontoxic biobased intermediates. An ideal strategy is a synthesis of biobased hydroaromatics that can be either dehydrated or dehydrogenated to aromatics in a single step. We can perform elimination reactions on biobased cyclohexenes possessing appropriately placed leaving groups. In the absence of leaving groups, dehydrogenation   5 reactions need to be employed. This thesis focuses on biobased cyclohexenes that are aromatized either by dehydration reactions or by dehydrogenation reactions. On its face, selective aromatization of cyclohexenes seems easy, but it has troubled chemists for more than 50 years. The difficulty of this transformation is a formation of cyclohexanes. One theory is that cyclohexenes can disproportionate into one equivalent of benzene and two equivalents of cyclohexane (Scheme 1.2A).25 The other theory could be the reduction timing of H-PdII-H to Pd0-H2 complex (Scheme 1.2B). The H-PdII-H needs to be reduced to Pd0-H2 complex before it hydrogenates other alkene (Scheme 1.2B). Scheme 1.2. Formation of Cyclohexane during Aromatization of Cyclohexene Early research examined the aromatization of cyclohexane and cyclohexene to benzene using a Ni catalyst.28ab In the 1970’s, Pd and Pt became popular catalysts for investigation of the reaction conditions required for cyclohexene aromatization. A catalyst that works well for dehydrogenation of cyclohexene tends to work better for hydrogenation of cyclohexene.27a In order to lead the reaction toward dehydrogenation instead of hydrogenation, high temperatures are required for the reactions.27a For example, the suggested reaction temperature by Johnson Matthey is 5-100 °C for hydrogenation reaction in solvent while 180-250 °C is suggested for dehydrogenation reaction in solvents.27c C-C bond cleavage with attendant formation of coke   6 becomes a problem at high temperatures.27a Hydrogenolysis of ethane to methane with metal catalysts under hydrogen gas at 205°C was used to compare the ease of C-C bond cleavage.27b When the relative reaction rates per unit surface area of metal were compared as C-C bond cleavage relative activity, Ni had 105 higher relative specific activity when compared with Pd and Pt.27b This trend suggested Ni would have more of a coke formation problem than Pd or Pt during dehydrogenation reaction.27ab Trost and Metzner used palladium(II) trifluoroacetate (TFA) and a hydrogen acceptor to prevent the disproportionation of cyclohexene to benzene and cyclohexane in 1980.28 They examined multiple molecules as hydrogen acceptors although most inhibited the reaction. The selectivity towards benzene was slightly improved using three equivalents of maleic acid as the hydrogen acceptor.28 Later, chemists started focusing on a catalytic cycle between PdII and Pd0 such as that seen in the Wacker process. For example, the oxidation of ethylene to acetaldehyde utilizes PdCl2 and CuCl2 as cocatalysts. PdII oxidizes ethylene to acetaldehyde as it is reduced to Pd0. CuII oxidizes Pd0 back to PdII while being reduced to CuI. Oxygen oxidizes CuI back to CuII.29 One issue of the Wacker process is the production of a chlorinated side product.30 In order to avoid the chlorinated side product, an alternative electron-transfer system for the reoxidation of Pd0 was desired. Sheldon and Sobczak proposed anthraquinone-2-sulfonate sodium salt (AMS) as a cocatalyst with either PdCl2 or Pd/C for aromatization of cyclohexene to benzene in 1991. They chose AMS for their catalyst system because it was found to be reoxidized easily by molecular oxygen (Scheme 1.3). They reported quantitative yields of benzene on a Pd/AMS catalytic system with molecular oxygen.31 They did not demonstrate the utility of their system to substituted cyclohexenes.   7 Scheme 1.3. Palladium Catalyst Cycle with Anthraquinone-2-sulfonate Sodium Salt There was no progress in aromatization of cyclohexenes during the 1990’s and 2000’s. However, there was significant progress in elaborating catalytic cycles between PdII and Pd0 suitable for aerobic Pd-catalyzed oxidation of alcohols/alkenes to a ketones/aldehydes,32-34 amminations34 and annulations.33-35 Nitrogen-donor ligands were commonly used in those transformations in order to initiate and/or accelerate the reactions.32-34 Later Stahl explained the role of nitrogen-donor ligands were to form soluble Pd nanoparticles.36 Labinger reported 100% selectivity for aromatization of cyclohexene to benzene using a Pd(TFA)2/O2 system.25 They suggested that insertion of O2 into a Pd-H bond 3 produces a peroxy intermediate 4, which reacts with trifluoroacetic acid to regenerate the catalyst 5 (Scheme 1.4). The shortcoming of their approach was a low conversion.25   8 Scheme 1.4. Proposed Mechanism for Regeneration of Pd(TFA)2 with Oxygen25 Stahl reported a method for Pd(II)-catalyzed aerobic dehydrogenation of cyclohexanones to phenols in 2011.37 His catalyst system consisted of Pd(TFA)2, 2-(N,N-dimethylamino)pyridine and p-toluenesulfonic acid (TsOH). Stahl observed that the electron deficient ligand would benefit C-H activation and b-hydride elimination. TsOH was suggested to protonate the pyridine ligand to afford a more electron deficient ligand. Stahl proposed that the Pd(II) is reduced to Pd(0), which is then oxidized back to Pd(II) by O2 (Scheme 1.5).37,38 Stahl’s research focus was aromatization of cyclohexanones, such as 2-methylcyclohexanone, 3-methylcyclohexanone, 4methylcyclohexanone, and 4-phenylcylohexanone to phenol derivatives.38 However he reported one result with the same catalyst system that afforded a 99% yield for the aromatization of 3,4dimethylcyclohex-3-enecarboxylic acid 6 (Scheme 1.6).37 He did not report another aromatization of a cyclohexene for four years. Scheme 1.5. Proposed Mechanism for PdII Catalyzed Aerobic Dehydrogenation37,38   9 Scheme 1.6. Aromatization of 3,4-Dimethylcyclohex-3-enecarboxylic Acid37 A few years later, Stahl published a tandem oxidative coupling/dehydrogenation reaction. The catalyst system was modified to employ a different nitrogen-donor ligand and a different additive. The most significant change was the additive. TsOH interfered with oxidative coupling reactions but quinones, particularly AMS, were beneficial.39 Stahl’s research in oxidative dehydrogenation continued to focus on the aromatization of cyclohexanone to phenol.39 The aromatization of cyclohexene came back under the spotlight in 2015.40 Stahl reported a catalyst system consisted of Pd(TFA)2, AMS and MgSO4 as a drying agent which could aromatize cyclohexenes.40 The quantitative or near quantitative yields were reported with various substituted cyclohexenes including: aromatic, alkyl, ester, cyclic anhydride, ketone, imide and carboxylic acid groups (Scheme 1.7).40   10 Scheme 1.7. Aerobic Oxidative Dehydrogenation of Cyclohexenes to Arene Derivatives40 However, this reaction is very sensitive to minor changes in the substituents. The reaction yield was nearly quantitative when the substituent was an N-aryl amide bearing chloride 18. The reaction yield was very poor when the substituent was an N-aryl amide bearing bromide 19 (Scheme 1.7).40 One carboxylic acid as a substituent 10 produced a quantitative yield of benzoic acid; two carboxylic acids substituents 11 produce a poor yield of phthalic acid with a significant quantity of benzoic acid from decarboxylation (Scheme 1.7).40 Stahl’s research is a significant development in aromatization of cyclohexenes, but all of his reported successes were limited to substituents in the 4- and/or 5-position of the cyclohexenes without any substituent at other positions.40 This thesis focuses on synthesis and aromatization of biobased cyclohexenes. An   11 elimination reaction was performed on biobased cyclohexene 21 possessing appropriately placed leaving groups. In the absence of leaving groups, dehydrogenation reactions were employed. In Chapter 2, microbially synthesized shikimic acid 21 is aromatized to p-hydroxybenzoic acid 22 and m-hydroxybenzoic acid 23 by a dehydration reactions (Scheme 1.8). 4-Methylcyclohex-3enecarboxylic acid 26 and 3-methylcyclohex-3-enecarboxylic acid 27, which are formed by cycloaddition of acrylic acid 25 with isoprene 24, are aromatized to p-toluic acid 28 and m-toluic acid 29 by dehydrogenation in Chapter 4 (Scheme 1.9). In Chapter 5, (2E,4Z)-hexa-2,4-diene1,3,6-tricarboxylic acid ((2E,4Z)-3-carboxymuconic acid) 30 is synthesized by a recombinant E. coli strain. The biobased cyclohexene 32 is synthesized by cycloaddition of the trimethyl 3carboxymuconate 31 and ethylene and then aromatized by dehydrogenation (Scheme 1.10). A vapor phase plug reactor was developed for the dehydrogenation reaction to aromatize biobased cyclohexenes 26, 27, 32. Stahl’s oxidative aerobic dehydrogenation reaction was also tested to aromatize the biobased cyclohexenes 26 and 32. No aromatization of cyclohexenes 26 or 32 was observed. Scheme 1.8. Dehydration of Microbially Synthesized Shikimic Acid   12 Scheme 1.9. Synthesis of Biobased p-Toluic Acid m-Toluic Acid Scheme 1.10. Synthesis of Biobased Trimethyl Trimellitate   13 REFERENCES   14 REFERENCES 1.   Alberta Energy Regulator. What is Unconventional Oil and Gas? https://www.aer.ca/aboutaer/spotlight-on/unconventional-regulatory-framework/what-is-unconventional-oil-and-gas (accessed Mar 30, 2017). 2.   Stephenson, M. H. Shale gas in North America and Europe. Energy Sci. Eng. 2016, 4, 4-13. DOI: 10.1002/ese3.96. 3.   U.S. Energy Information Administration. U.S. Shale Production. https://www.eia.gov/dnav/ng/hist/res_epg0_r5302_nus_bcfa.htm (accessed Dec. 19, 2016). 4.   (a) U.S. Department of Energy, U.S. Crude Oil and Natural Gas Proved Reserves, Year-end 2015. https://www.eia.gov/naturalgas/crudeoilreserves/pdf/usreserves.pdf (accessed Mar 13, 2017). (b) United States Department of Energy. 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Study for manufacturing methyl ethyl ketone from n-butene by Wacker process (Part 2): Reaction of 1-butene with palladium chloride-cupric chloride aqueous solution at elevated temperature and Pressure Sekiyu Gakkaishi 1983, 26, 8-14 31.  Sheldon, R. A.; Sobczak. J. M. Catalytic oxidative dehydrogenation of cyclohexene. J. Mol. Catal. 1991, 68, 1-6 32.  Nishimura, T.; Uemura, S. Novel palladium catalytic systems for organic transformations. Synlett 2004, 201-216. 33.  Stoltz, B. M. Palladium catalyzed aerobic dehydrogenation: from alcohols to indoles and asymmetric catalysis. Chem. Lett. 2004, 33, 362-367. 34.  Sigman, M. S.; Schultz, M. J. The renaissance of palladium(II)-catalyzed oxidation chemistry. Org. Biomol. Chem. 2004, 2, 2551-2554. 35.  Toyota, M.; Ihara, M. Development of palladium-catalyzed cycloalkenylation and its application to natural product synthesis. Synlett 2002, 1211-1222. 36.  Pun, D.; Diao, T.; Stahl, S. S. Aerobic dehydrogenation of cyclohexanone to phenol catalyzed by Pd(TFA)2 / 2-dimethylaminopyridine: evidence for the role of Pd nanoparticles. J. Am. Chem. Soc. 2013, 135, 8213-8221. DOI: 10.1021/ja403165u. 37.  Izawa, Y.; Pun, D.; Stahl, S. S. Palladium-catalyzed aerobic dehydrogenation of substituted cyclohexanones to phenols. Science 2011, 333, 209-213. DOI: 10.1126/science.1204183.   18 38.  Diao, T.; Stahl, S. S. Synthesis of cyclic enones via direct palladium-catalyzed aerobic dehydrogenation of ketones. J. Am. Chem. Soc. 2011, 133, 14566-14569. DOI: 10.1021/ja206575j. 39.  Izawa, Y.; Zheng, C.; Stahl, S. S. Aerobic oxidative Heck / dehydrogenation reaction of cyclohexanones: efficient access to meta-substituted phenols. Angew. Chem. Int. Ed. 2013, 52, 3672-3675. DOI: 10.1002/anie.201209457. 40.  Iosub, A. V.; Stahl, S. S. Palladium-catalyzed aerobic oxidative dehydrogenation of cycohexenes to substituted arene derivatives. J. Am. Chem. Soc. 2015, 137, 3454-3457. DOI: 10.1021/ja512770u.   19 CHAPTER TWO Synthesis of Biobased p-Hydroxybenzoic Acid 1. Introduction p-Hydroxybenzoic acid is used in the manufacture of liquid crystalline polymers (LCPs). The first LCP developed and commercialized in 1955 was poly(p-hydroxybenzoic acid).1 This polymer was insoluble in standard organic solvents and possessed a high melting temperature (350 °C). These properties allow p-hydroxybenzoic acid to be used for many useful applications but also cause difficulty in processing.1 In order to alleviate processing challenges, various comonomers are employed, such as m-hydroxybenzoic acid, 6-hydroxynaphthoic acid, isophthalic acid, terephthalic acid, hydroquinone, bisphenol, 1,4-naphthalene dicarboxylic acid, 1,5naphthalene dicarboxylic acid and 2,6-naphthalene dicarboxylic acid.2 Though the presence of comonomers in the crystal lattice lowers the melting temperature of LCPs, the polymer as a whole is nonetheless heat resistant. A physical term, coefficient of thermal expansion, describes how much a material expands when it is heated. The expansion coefficient of some LCPs are comparable to the expansion coefficient of glass, steel, ceramic and glass fiber/epoxy materials. LCPs, which are used in the construction of circuit boards,3 have a low expansion coefficient. This is very desirable for electronic device casings, connectors and other parts. LCP use also addresses the demand to reduce chip size and weight. The physical strength and thermostability of LCPs require less thickness to hold components of the circuit board than glass fiber/epoxy resin.4b Therefore, the use of LCPs has increased quickly over time in electronic devices such as circuit boards, semiconductors and liquid crystal displays.4 Applications extend beyond electronic devices. LCPs’ high melting temperature allows them to form nonstick bakewares.5 LCPs are also quite strong and are being used to create cut-resistant yarns, which can be   20 manufactured into a material for lightweight garments that protect against sharps object and knives.6 Esterified p-hydroxybenzoic acids (parabens) are widely used as preservatives in cosmetics, creams, and oral aqueous medicines such as cough syrup and some intravenous medicines. The most widely marketed parabens are: methylparaben, ethylparaben, propylparaben and butylparaben. Different esters of parabens lead to different solubilities and different antimicrobial activities.7 Parabens have also been shown to bind to human estrogen receptors.7b However, the affinities of parabens to the estrogen receptors are 104 to 106 times less than estradiol.7c Methyl and ethylparaben have lower in vitro and in vivo estrogenic activity than butyl or propylparaben.7b Even though there are concerns about the possible role of parabens in decreased sperm viability and incidence of breast cancer, current data does not support these concerns.7c p-Hydroxybenzoic acid ($5.40/kg) is more expensive than other monomers such as terephthalic acid ($0.73/kg).8 Global LCP production was estimated at 58.7 ´ 106 kg/yr with a market size of $950 million in 2016. The market size of LCPs has been increasing every year based on data between 2012 and 2015.9 Grand View Research reported the revenue from parabens in the global cosmetic preservative market was valued at only $33 million in 2015.10 2. p-Hydroxybenzoic Acid Production In 1860, Kolbe discovered that the reaction of sodium phenoxide with carbon dioxide could produce salicylic acid (o-hydroxybenzoic acid).11 He later filed a patent in which the selectivity of the reaction could be altered to p-hydroxybenzoic acid by using potassium hydroxide instead of sodium hydroxide.12 The selectivity of p-hydroxybenzoic acid increases   21 with an increasing ionic radius of the alkali metal (Figure 2.1).13 The carboxylation of metal phenoxide salts was improved by Kolbe himself in 1875 then by Schmitt in 1885.14 This KolbeSchmitt reaction is still the basis for manufacture of hydroxybenzoic acids. Figure 2.1. ortho- and para-intermediate p-Hydroxybenzoic acid 6 is currently manufactured from benzene 1 (Scheme 1). Benzene 1 is transformed to cumene 2 by the acid-catalyzed reaction of benzene 1 and propylene. Currently, zeolites are employed as the acid catalysts.15 In 1944, Hock reported the synthesis of phenol 4 and acetone from cumene 2. Cumene 2 is first oxidized by molecular oxygen to cumene hydroperoxide 3, which is then cleaved to phenol 4 and acetone by a Brønsted acid.16 After World War II, the Hock process became the standard commercial method to manufacture phenol 4 and acetone.17 In the Kolbe-Schmitt reaction, potassium phenolate, which results from deprotonation of phenol 4 using KOH, is reacted at 120 °C with carbon dioxide at 10 atm pressure. The potassium p-hydroxybenzoate 5 is neutralized with H2SO4 which produces a stoichiometric amount of potassium salt byproduct, KHSO4.13   22 Scheme 2.1. p-Hydroxybenzoic Acid Production from Benzene It is not difficult to obtain the starting materials required for the manufacture of phydroxybenzoic acid thanks to many years of process development. Benzene and propylene are produced from petroleum and natural gas/shale gas (Chapter 1). Potassium hydroxide is manufactured by potassium chloride electrolysis. In the United States, potassium chloride is obtained from the Great Salt Lake Desert in Utah and subterranean brines of Searles Lake in California.18 Carbon dioxide is recovered as a byproduct of ammonia and hydrogen production.19 Sulfuric acid is manufactured from either pyrites (FeS2) or elemental sulfur. Combustion of elemental sulfur or heating pyrites produces sulfur dioxide gas which is oxidized to sulfur trioxide gas by a vanadium oxide catalyst (V2O5).20a,b Reaction of sulfur trioxide with water leads to sulfuric acid. In the United States, 80% of sulfuric acid is produced from elemental sulfur combustion which produces electrical power in addition to sulfuric acid.20c Most mining of elemental sulfur in North America is localized near the Gulf of Mexico and spans a region extending through parts of Mexico, Texas and Louisiana.20d,e Therefore, raw materials for phydroxybenzoic acid production are readily available. There are some significant concerns with current manufacture of p-hydroxybenzoic acid, which include occupational hazards from exposure to carcinogenic benzene and toxic phenol.   23 3. Toxicity of Benzene and Phenol Benzene causes acute myeloid leukemia,21a-g hematotoxicity (decrease in blood cells counts)21d,e,g and is suspected of causing non-Hodgkin lymphoma.21b,j,k The permissible occupational exposure limit to benzene in the U.S. is 1 part per million (8h time-weighted average) and actual occupational exposure levels are typically below the limit.21a,e However, benzene still causes hematotoxicity in workers exposed to below 1 part per million.21a,e Some individuals are more susceptible to benzene toxicity than the other.21e Single-nucleotide polymorphisms in the genes encoding cytochrome-P450, myeloperoxidase and quinone oxidoreductase were observed in those more susceptible to benzene toxicity than the other.21e The various metabolites of benzene are thought to be critical factors in benzene toxicity (Scheme 2.2).21a-d,f Benzene 1 is metabolized in the liver and lungs.21a Some of those metabolites are excreted21i while others are metabolized further in the bone marrow.21a Benzene is oxidized to benzene oxide 7 by cytochrome-P450 that exists in equilibrium with its tautomer, oxepin 9.21a The majority of benzene oxide 7 rearranges to phenol 4 spontaneously.21a The remainder is converted to catechol, trans,trans-muconaldehyde 10 or S-phenylmercapturic acid 8.21a Phenol 4 is either excreted21i or metabolized to 1,4-hydroquinone 12.21a 1,4-Hydroquinone 12 is converted to either the reactive metabolite 1,2,4-benzenetriol 18 in the liver / lungs or to another reactive metabolite 1,4-benzoquinone 14.21a Metabolism of oxepin 9 produces the reactive trans,transmuconaldehyde 10.21a,b,d,f Because benzene oxide 7, 1,4-benzoquinone 14 and trans,transmuconaldehyde 10 are electrophiles, they can react with peptides and proteins easily and interfere with cellular function.21a,b,d,f,h The reactive oxygen species (ROS) 19 produced from the metabolites by peroxidases in the bone marrow can bind to cellular macromolecules and also generate oxygen radicals.21a,f The formation of 1,4-benzoquinone 14 in the bone marrow by   24 myeloperoxidase particularly is thought to be a key step in causing benzene carcinogenicity.21a-c Scheme 2.2. Benzene Metabolism 1 a O j HN S excreted in urine O O CO2H 7 8 Liver and Lung HO 18 OH 15 OH ROS 19 excreted in urine OH OH e,f 16 12 OH i h O 17 O e,f O HO CO2H 11 g a i Bone marrow CHO 10 OH 4 HO d 9 OH excreted in urine as e conjugated sufates and glucuronides f OH a a c b HO2C OHC 13 excreted in urine as conjugated sufates h and glucuronides i O O 14 toxicity (a) cytochrome-P450 (b) spontaneous (c) epoxide hydrolase (d) peroxidase (e) uridine diphosphate glucuronosyltransferase (f) phenol sulfotransferase (g) dihydrodiol dehydrogenase (h) quinone oxidoreductase, NAD(P)H (i) myeloperoxidase (j) glutathione-S-transferase ROS: reactive oxygen species (oxygen radicals)   25 The Occupational Safety and Health Administration (OSHA) set the occupational exposure limit to phenol at 5 parts per million (8h time-weighted average).22 Most of the severe health problems from phenol exposure occur due to skin contact with phenol. Phenol is absorbed through the skin rapidly and taken into blood stream. Though most of absorbed phenol will be excreted in the urine, skin contact with large quantity of phenol can be lethal. A case wherein 25% of an individual’s skin was exposed to liquid phenol resulted in a rapid death within 10 minutes.22 This was one of several rapid death cases caused by dermal exposure to phenol. The causes of death were respiratory depression, cardiac arrest and renal failure.22 Chronic dermal exposure to small quantities of phenol also causes health problems, such as cardiac arrhythmias, muscle pain, enlarged and tender liver, kidney damage, emaciation, skin inflammation and necrosis.22 The mechanisms of toxicity of phenol are unknown, although phenol is metabolized via the same pathway responsible for benzene metabolism. Phenol has not been found to cause hematotoxicity or leukemia.22 4. Biocatalytic Routes to p-Hydroxybenzoic Acid Biocatalytic synthesis of p-hydroxybenzoic acid has been attempted as an alternative to the Kolbe-Schmitt synthesis. One approach used the microbial oxidation of toluene 20 to phydroxybenzoic acid 6 (Scheme 2.3). Various Pseudomonas putida hosts have been engineered to heterologously express tmoABCDEF-encoded toluene monooxygenase from Pseudomonas mendocina KR1.23 The toluene 4-monooxygenase catalyzes the conversion of toluene 20 into pcresol 21, which is converted in three steps by pcu genes to p-hydroxybenzoic acid 6 (Scheme 2.3).23 An advantages to this approach is that substrate, toluene 20 is not toxic. However, the rates to convert toluene 20 to p-hydroxybenzoic acid 6 are slow and the titer of p-   26 hydroxybenzoic acid in the culture medium is low (2 g/L).23 Also the low solubility of toluene in water would become a problem in large scale fermentation. Phenylphosphate carboxylase from the strict anaerobe Thauera aromatica has been used to convert phenylphosphate 24 into p-hydroxybenzoic acid 6 (Scheme 2.3).24a-d A partially purified oxygen sensitive enzyme, phenylphosphate carboxylase has been used for this conversion.24a-d This route cannot avoid use of benzene 1 and phenol 4. It also requires phenol 4 to be phosphorylated to phenylphosphate 24 prior to carboxylation. There was one report of in vitro phenol phosphorylation with enzymes isolated from Shigella flexneri.24e Genetic engineering of Thauera aromatica has not progressed to the point where conversion of phenol 4 to phydroxybenzoic acid 6 has been achieved. Scheme 2.3. Biocatalytic Routes to p-Hydroxybenzoic Acid TmoABCDEF: toluene 4-monooxygenase; PcuBXA: p-cresol utilization gene; PcuC: p-hydroxybenzaldehyde dehydrogenase Microbe-catalyzed conversion of glucose into p-hydroxybenzoic acid (Scheme 2.4) has the advantage of using nontoxic, renewable glucose as a starting material.25 Carbon flow is   27 directed into and through the common pathway of aromatic amino acid biosynthesis to chorismic acid (Scheme 4, 26/27 to 34).25 Chorismate lyase encoded by ubiC in E. coli then catalyzes the conversion of chorismic acid 34 into p-hydroxybenzoic acid 6 with pyruvic acid formed as the byproduct (Scheme 2.4).25 Numerous strategies have been developed for directing carbon flow into chorismic acid 34. However, ubiC-encoded chorismate lyase is a problematic enzyme having a very low turnover of about 1 sec-1 and inhibited by the p-hydroxybenzoic acid 6 produced with an inhibition constant (Ki) of 2.1 µM.25c For comparison, the chorismate lyase Km for substrate chorismic acid 34 is 29 µM.25c The toxicity of p-hydroxybenzoic acid synthesized from glucose towards microbes has been reduced with resin-based extraction.26 During microbial synthesis of p-hydroxybenzoic acid under fermentor-controlled conditions, culture broth is continuously passed through an anion exchange resin,26 which is a polystyrene support functionalized with quaternary amine groups.27 Phenolics such as p-hydroxybenzoic acid bind to the resin and are removed from the fermentation broth. Resin-based extraction increases the titers of microbially synthesized, resinbound p-hydroxybenzoic acid by a factor of about fourfold over the concentration of phydroxybenzoic acid synthesized in lieu of resin based extraction.26 The total p-hydroxybenzoic acid afforded was equivalent to a titer of 23 g/L.26   28 Scheme 2.4. Biocatalytic Routes to p-Hydroxybenzoic Acid 26 HO CO2H OPO3H2 + AroFFBR CO2H OH OH O H2O3PO HO CO2H H2O3PO H OH CO2H AroB O AroD O OH CO2H AroE O OH OH OH OH 28 29 30 HO 27 AroK AroL ATP CO2H CO2H CO2H CO2H AroC UbiC OH 6 OH OH 31 O pyruvic acid CO2H AroA H3PO4 OH H2O3PO O OH OH 32 TyrA TyrB NADH CO2 NH3 CO2 CO2H OH C(O)SCoA H O - PAL 35 H2O3PO 33 34 NAD+ PEP CO2H OH NH4 *1 OH 36 CoASH ATP OH 37 acetylCoA CO2H *2 HCHL OH 38 OH 6 PEP: phosphoenolpyruvate; E4P: D-erythrose 4-phosphate; DAHP: 3-deoxy-D-arabino-heptulosonate 7phosphate; DHQ: 3-dehydroquinic acid; DHS: 3-dehydroshikimic acid; AroFFBR: DAHP synthase; AroB: DHQ synthase; AroD: DHQ dehydratase; AroE: shikimate dehydrogenase; AroK: shikimate kinase I; AroL: shikimate kinase II; AroA: 5-enolpyruvylshikimate 3-phosphate synthase; AroC: chrosmate synthase; UbiC: chorismate lyase; TyrA: chorismate mutase-prephenate dehydrogenase; TyrB: tyrosine aminotransferase; PAL: phenylalanine ammonia lyase; *1: p-coumaroyl-CoA synthetase; HCHL: 4-hydroxycinnamoyl-CoA hydratase/lyase; *2: p-hydroxybenzaldehyde dehydrogenase An alternative microbial synthesis of p-hydroxybenzoic acid 5 has been created in Pseudomonas putida S12 where either glucose or glycerol was converted into tyrosine 35 28 followed by phenylalanine ammonia lyase-catalyzed conversion of tyrosine into p-coumaric acid 36 (Scheme 2.4).29 A series of enzymes native to Pseudomonas putida S12 including pcoumaroyl-CoA synthetase, 4-hydroxycinnamoyl-CoA hydratase/lyase and phydroxybenzaldehyde dehydrogenase catalyzed the conversion of p-coumaric acid 36 into phydroxybenzoic acid 6 (Scheme 2.4). Native p-hydroxybenzoate hydroxylase was inactivated to   29 prevent catabolism of product p-hydroxybenzoic acid 6 in Pseudomonas putida S12.29 This combination of anabolism and catabolism avoids the challenges inherent in employing chorismate lyase in microbial synthesis of p-hydroxybenzoic acid 6 from glucose (Scheme 2.4). The yields of p-hydroxybenzoic acid obtained were competitive with the yields of phydroxybenzoic acid microbially synthesized from glucose via chorismate lyase in lieu of resinbased extraction. However, the yield is too low (16 %) to be suitable for commercial production.29b Synthesis of p-hydroxybenzoic acid in transgenic plants has also been accomplished. Expression of either E. coli ubiC-encoded chorismate lyase (Scheme 2.4) and Pseudomonas fluorescens hchl-encoded 4-hydroxycinnamoyl-CoA hydratase/lyase (Scheme 2.4) in tobacco (Nicotiana tabacum)30-32 and sugar cane (Saccharum hybrids)33 has led to formation of glucosylated p-hydroxybenzoic acid. The majority of the glucosylated p-hydroxybenzoic acid is conjugated as an ether through the C-4 phenolic oxygen while the remainder is conjugated as an ester at the C-1 carboxylate.30-32 This glucosylation with subsequent accumulation of the conjugated p-hydroxybenzoic acid in vacuoles is essential to the successful management of phydroxybenzoic acid’s toxicity in plants. An additional critical role of glucosylation is the elimination of p-hydroxybenzoic acid’s severe feedback inhibition of ubiC-encoded chorismate lyase activity. Nuclear transformation with chloroplast-targeted ubiC and hchl has dominated approaches to achieving synthesis of glucosylated p-hydroxybenzoic acid in plants.30,31,33 However, the highest levels of glucosylated p-hydroxybenzoic acid in plant leaves has been achieved with integration of ubiC into the chloroplast genome.32 Nuclear transformation of ubiC led to phenotypically healthy tobacco plants, which accumulated glucosylated p-hydroxybenzoic   30 acid to 0.52 dry wt% in leaves.30 This corresponds to 0.24 dry wt% of p-hydroxybenzoic acid after correcting for the mass of the glucose conjugate. Nuclear transformation of tobacco with hchl gave phenotypically unhealthy tobacco plants accumulating 1.3 dry wt% glucosylated phydroxybenzoic acid in leaves corresponding to approximately 0.61% dry wt% of phydroxybenzoic acid.31 In sugar cane, nuclear transformation with ubiC yielded plants that accumulate a mean value of 0.41 dry wt% glucosylated p-hydroxybenzoic acid (0.19 dry wt% equivalent of p-hydroxybenzoic acid).33 Nuclear transformation of sugarcane with hchl led to plants that accumulate a mean value of 0.7 dry wt% glucosylated p-hydroxybenzoic acid (0.32 dry wt% equivalent of p-hydroxybenzoic acid).33 Integration of ubiC into the tobacco chloroplast genome afforded phenotypically healthy plants accumulating a mean value of 14 dry wt% glucosylated p-hydroxybenzoic acid (6.6 dry wt% equivalent of p-hydroxybenzoic acid).32 In 1892, Eykman reported dehydration of shikimic acid 31 to form p-hydroxybenzoic acid 5.34 This reaction has been revisited recently with a report of the formation of phydroxybenzoic acid and m-hydroxybenzoic acid in 40% and 13% yields, respectively by refluxing in 12 M HCl for 14 h.35 Heating shikimic acid at 120 °C with 1 M H2SO4 in acetic acid solution for 16 h led to a 57% yield of p-hydroxybenzoic acid and a 9% yield of mhydroxybenzoic acid.35 Although all of the shikimic acid was consumed in these reactions, mass accountability was poor.35 Significant quantities of an uncharacterized black precipitate were formed in addition to the p-hydroxybenzoic acid and m-hydroxybenzoic acid.35 An appealing advantage of synthesizing p-hydroxybenzoic acid from shikimic acid follows from commercial sourcing of shikimic acid from select plants or microbial synthesis of shikimic acid from glucose. These sources of shikimic acid were identified during commercialization of the anti-influenza drug Tamiflu®,36 which is synthesized from shikimic   31 acid. Commercially, shikimic acid is obtained from the star fruit and leaves of Illicium verum36 in China and Vietnam,37 and microbial synthesis from glucose under fermentor-controlled conditions.38 A number of plants other than Illicium verum have been reported to produce significant concentrations of shikimic acid.39 The shikimic acid is found in unconjugated, freeacid form in plant tissue.39b In this chapter, various ionic liquids are explored as solvent and co-catalyst for the synthesis of p-hydroxybenzoic acid by acid-catalyzed dehydration of shikimic acid. Chemical dehydration of nontoxic shikimic acid for synthesis of p-hydroxybenzoic acid allows us to avoid use of toxic phenol derived from carcinogenic benzene and the large salt wastes streams from Kolbe-Schmitt synthesis.   32 5. Results 5.1. Conversion of Shikimic Acid to p-Hydroxybenzoic Acid in Ionic Liquid Bromides Unlike earlier reported dehydrations of shikimic acid,34,35 acid-catalyzed dehydration of shikimic acid using a number of different ionic liquid bromides has been discovered to undergo essentially quantitative conversion to a mixture of p-hydroxybenzoic acid and m-hydroxybenzoic acid. The earlier reported acid-catalyzed dehydrations of shikimic acid in acetic acid yielded 57% of p-hydroxybenzoic acid and 9% of m-hydroxybenzoic acid.35 Typical reaction conditions involved heating a 20 weight percent (wt%) solution of shikimic acid in ionic liquid bromide at its melting point under reduced pressure (0.4 mbar) with 20 mol % H2SO4 (Scheme 2.5). The vacuum was employed to remove water byproduct. Scheme 2.5. Shikimic Acid Dehydration in Ionic Liquid CO2H HO 20 mol% H2SO4 OH ionic liquid 90-180 ℃ OH 0.4 mbar SA CO2H CO2H CO2H OH OH PHBA MHBA OH OH PCA Scheme 2.6. Ionic Liquids Used in Shikimic Acid Dehydrations The ionic liquid bromides that were examined included: 1-butyl-3-methylimidazolium bromide 40b (Table 2.1, entry 2), 1-butylpyridinium bromide 41b (Table 2.4, entry 2), tetrabutylphosphonium bromide 42b (Table 2.4, entry 5), 1-butyl-2,3-dimethylimidazolium   33 bromide 43b (Table 2.4, entry 7), 1-butyl-2-methylpyridinium bromide 44b (Table 2.4, entry 8), and 1-butyl-1-methylpyrrolidinium bromide 45b (Table 2.4, entry 9). Table 2.1. Dehydration of Shikimic Acid in Various 1-Butyl-3-methylimidazolium Salts 40a-40e Entry 1 2 3 4 5 Ionic Liquid 40a 40b 40c 40d 40e X=Cl X=Br X=I X=HSO4 X=BF4 Temp Time 90 °C 90 °C 90 °C 90 °C 90 °C 13 h 13 h 13 h 13 h 13 h % Yield PHBA MHBA2 1 0 76 19 1 0 3 0 9 0 1 PCA3 % SA4 0 0 1 0 0 78 1 79 2 38 1 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3PCA=protocatechuic acid % SA=percent remaining shikimic acid. 4 Table 2.2. Impact of Shikimic Acid Concentration in 1-Butyl-3-methylimidazolium bromide 40b on Dehydration Yield and Accountability of Shikimic Acid Consumption 1 Entry IL1 [SA]2 Temp Time 1 2 3 4 40b 40b 40b 40b 20 wt % 40 wt % 50 wt % 60 wt % 90 °C 90 °C 90 °C 90 °C 13 h 13 h 13 h 13 h % Yield PHBA3 MHBA4 76 19 73 17 70 16 62 13 % SA5 1 0 0 6 IL=ionic liquid; 2 [SA]=shikimic acid concentration in IL; 3 PHBA=p-hydroxybenzoic acid; 4 MHBA=m-hydroxybenzoic acid; 5 % SA=percent remaining shikimic acid Acid-catalyzed dehydrations in various ionic liquid bromides afforded differing yields of p-hydroxybenzoic acid and ratios of p-hydroxybenzoic acid to m-hydroxybenzoic acid formed with a range of 2:1 to 4:1. Furthermore, the mass balance and reaction selectivity favoring phydroxybenzoic acid and m-hydroxybenzoic acid varied as a function of the ionic liquid employed. 1-Butyl-3-methylimidazolium bromide 40b (Table 2.1, entry 2) gave the highest yields for acidcatalyzed formation of p-hydroxybenzoic acid. The highest weight percent of shikimic acid that could be dehydrated without a reduction in percent yield of p-hydroxybenzoic acid was also a consideration given the expense of ionic liquids relative to more traditional organic solvents. With 1-butyl-3-methylimidazolium bromide 40b as solvent, the wt% of shikimic acid could be increased from 20 wt% to 40 wt/% without   34 significant diminution in the yield of p-hydroxybenzoic acid, the ratio of p-hydroxybenzoic acid to m-hydroxybenzoic acid formed or the nearly quantitative conversion of shikimic acid to these two dehydration products (Table 2.2, entry 2 vs. entry 1). Increasing the concentration of shikimic acid to 50 wt% relative to 1-butyl-3-methylimidazolium bromide 40b resulted in a 10% reduction in the combined formation of p-hydroxybenzoic acid and m-hydroxybenzoic acid (Table 2.2, entry 1 vs. 3). Increasing the concentration of shikimic acid to 60 wt% relative to 1butyl-3-methylimidazolium bromide 40b resulted in significant reductions in the yield of phydroxybenzoic acid and the mass balance (Table 2.2, entry 1 vs. entry 4). The wt% of shikimic acid was kept at 20 wt% during evaluation of all other ionic liquids as solvents for acid-catalyzed dehydration of shikimic acid. Recycling the ionic liquid helps to avoid costly waste of these relatively expensive solvents. Fresh 1-butyl-3-methylimidazolium bromide 40b was synthesized in situ from 1methylimidazole and 1-bromobutane. Shikimic acid 40 wt% (12 g) in 1-butyl-3methylimidazolium bromide 40b (30 g) was heated at 90 °C under reduced pressure (0.4 mbar) with 20 mol % H2SO4 for 24 h. After addition of water to the reaction crude, p-hydroxybenzoic acid and m-hydroxybenzoic acid were extracted into EtOAc. This initial run gave an 80% yield of p-hydroxybenzoic acid and an 18% yield of m-hydroxybenzoic acid (Table 2.3, entry 1). The aqueous layer was concentrated and dried under reduced pressure and then used as the 1-butyl-3methylimidazolium bromide 40b for a second dehydration of shikimic acid using the same ionic liquid used for the first dehydration. This second dehydration using recycled 1-butyl-3methylimidazolium bromide 40b afforded a 77% yield of p-hydroxybenzoic acid and a 16% yield of m-hydroxybenzoic acid with 2% of unreacted shikimic acid (Table 2.3, entry 2). The viscosity of this reaction solution was significantly higher than first reaction, and the magnetic   35 stir bar was barely stirring the solution. After addition of water to the reaction crude, phydroxybenzoic acid and m-hydroxybenzoic acid were extracted into EtOAc. The aqueous layer was treated with activated carbon, and then concentrated and dried under reduced pressure. This activated carbon-treated 1-butyl-3-methylimidazolium bromide 40b was used for a third dehydration of shikimic acid. This third dehydration afforded a 72% yield of p-hydroxybenzoic acid and a 11% yield of m-hydroxybenzoic acid (Table 2.3, entry 3). In the second reaction, the ionic liquid performed nearly as well as the first reaction. In the third reaction, the ionic liquid showed a significant reduction in the yield of p-hydroxybenzoic acid and m-hydroxybenzoic acid with poor mass balance. Bromide is known to be oxidized by H2SO4 forming Br2 and SO2 when H2SO4 is used as the solvent.40 For the second and third dehydration reactions, 20 mol% H2SO4 was added each time. This suggest a possible redox between bromide and H2SO4 in the ionic liquid. The reduction of the recycled ionic liquid performance could due to a loss of bromide. Table 2.3. 1-Butyl-3-methylimidazolium Bromide 40b Recycle 1 Entry IL1 1 2 3 40b 40b 40b First Dehydration Second Dehydration Third Dehydration % Yield PHBA3 MHBA4 80 18 77 16 72 11 % SA5 0 2 0 IL=ionic liquid; 2 [SA]=shikimic acid concentration in IL; 3 PHBA=p-hydroxybenzoic acid; 4 MHBA=m-hydroxybenzoic acid; 5 % SA=percent remaining shikimic acid Dehydration of shikimic acid in 1-butylpyridinium bromide 41b gave a 62% yield of phydroxybenzoic acid. Of the shikimic acid consumed 77% could be accounted for in the formation of p-hydroxybenzoic acid and m-hydroxybenzoic acid (Table 2.4, entry 2). As with the 1-butyl3-methylimidazolium halides 40a-c, dehydration of shikimic acid in 1-butylpyridinium bromide 41b gave a much larger yield of p-hydroxybenzoic acid relative the corresponding dehydration yields in 1-butylpyridinium chloride 41a and 1-butylpyridinium iodide 41c (Table 2.4, entry 1 and   36 3). 1-Butylpyridinium salts 41 reactions were run at 140 °C, which was the melting temperature of 1-Butylpyridinium chloride 41a. Table 2.4. Dehydration of Shikimic Acid in Various Ionic Liquids 41-45. Entry 1 2 3 4 5 6 7 8 9 Ionic Liquid 41a 41b 41c 42a 42b 42c 43b 44b 45b X=Cl X=Br X=I X=Cl X=Br X=I X=Br X=Br X=Br Temp Time 140 °C 140 °C5 140 °C5 110 °C6 110 °C6 110 °C6 110 °C7 160 °C8 180 °C9 5 13 h 13 h 13 h 13 h 13 h 13 h 13 h 13 h 13 h % Yield PHBA MHBA2 8 7 62 15 15 3 0 0 44 23 0 0 66 19 54 16 35 10 1 PCA 0 0 0 0 0 8 0 0 0 3 % SA4 58 0 29 80 0 82 0 0 0 1 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3PCA=protocatechuic acid; 4 % SA=percent remaining shikimic acid, 5140 °C was the melting temperature of 41a, 6110 °C was the melting temperature of 42c, 7 110 °C was the melting temperature of 43b, 8160 °C was the melting temperature of 44b, 9180 °C was the melting temperature of 45b. Tetrabutylphosphonium salts 42 reactions were run at 110 °C, which was the melting temperature of tetrabutylphosphonium iodide 42c. Tetrabutylphosphonium bromide 42b (Table 2.4, entry 5) afforded a unique result leading to the highest ratio of m-hydroxybenzoic acid to phydroxybenzoic acid of any ionic liquid bromide examined. 1-Butyl-2,3-dimethylimidazolium bromide 43b (Table 2.4, entry 7), 1-butyl-2-methylpyridinium bromide 44b (Table 2.4, entry 8), and 1-butyl-1-methylpyrrolidinium bromide 45b (Table 2.4, entry 9) were also examined as ionic liquid solvents with the reaction temperatures at their melting temperature. Among all of ionic liquid bromides, 1-butyl-1-methylpyrrolidinium bromide 45b (Table 2.4, entry 9) gave the lowest yield of p-hydroxybenzoic acid with the lowest mass balance.   37 5.2. Dehydrations of Shikimic Acid in non-Bromide Ionic Liquids While ionic liquid bromides were the best solvents for acid-catalyzed dehydration of shikimic acid, the corresponding ionic liquid chlorides showed low reactivity when employed as solvents. Only a 1% yield of p-hydroxybenzoic acid was obtained when acid-catalyzed dehydration of shikimic acid was run in 1-butyl-3-methylimidazolium chloride 40a (Table 2.1, entry 1). Use of 1-butylpyridinium chloride 41a as the solvent for acid-catalyzed dehydration of shikimic acid led to only an 8% yield of p-hydroxybenzoic acid (Table 2.4, entry 1). No phydroxybenzoic acid was detected when dehydration of shikimic acid was run in tetrabutylphosphonium chloride 42a (Table 2.4, entry 4). Poor results were also encountered when hydrogen sulfate and tetrafluoroborate were used as counteranions (Table 2.1, entry 4-5). A 3 % yield of p-hydroxybenzoic acid was obtained when 1-butyl-3-methylimidazolium hydrogen sulfate 40d was used (Table 2.1, entry 4) as the solvent for acid-catalyzed dehydration of shikimic acid. Of the shikimic acid consumed only this 3% could be accounted for in the formation of p-hydroxybenzoic acid. Use of 1-butyl-3- methylimidazolium tetrafluoroborate 40e as solvent for shikimic acid dehydration also led to a low yield of p-hydroxybenzoic acid with a modest mass balance (Table 2.1, entry 5). Use of ionic liquid iodides as the solvent for acid-catalyzed dehydration of shikimic acid gave yields of p-hydroxybenzoic acid that were uniformly lower relative to the corresponding ionic liquid bromides. An interesting feature of ionic liquid iodides was the formation of protocatechuic acid (3,4-dihydroxybenzoic acid) as a product. Protocatechuic acid formation requires oxidation of the C-3 alcohol of shikimic acid. The resulting dehydroshikimic acid will dehydrate to the observed protocatechuic acid.41 1-Butyl-3-methylimidazolium iodide 40c afforded a 1% yield of p-hydroxybenzoic acid, 1% yield of protocatechuic acid, and no detectable formation of m-   38 hydroxybenzoic acid (Table 2.1, entry 3). Dehydration of shikimic acid in 1-butylpyridinium iodide 41c led to a 15% yield of p-hydroxybenzoic acid and a 3 % yield of m-hydroxybenzoic acid (Table 2.4, entry 3). An 8% yield of protocatechuic acid with no p-hydroxybenzoic acid or mhydroxybenzoic acid was detected when dehydration of shikimic acid was run in tetrabutylphosphonium iodide 42c (Table 2.4, entry 6). 5.3. A Possible Mechanism for Bromide-Catalyzed Dehydration of Shikimic Acid The much higher yields of hydroxybenzoic acids when shikimic acid is dehydrated in ionic liquid bromides relative to ionic liquid chlorides, ionic liquid iodides, or ionic liquids with other counteranions is particularly striking. This suggests that the ionic liquid bromides are playing an essential catalytic role in addition to serving as the solvents for the dehydrations. Protonation by H2SO4 of the C-3 hydroxyl group of shikimic acid (Scheme 2.7) may precede deprotonation of the shikimate carboxylic acid and may result in an exchange of the resulting shikimate carboxylate with the bromide counteranion (Scheme 2.7). Nucleophilic attack by bromide at C-3 would displace water and generate a bromoshikimate intermediate (Scheme 2.7). This bromoshikimate could then partition between elimination reactions that ultimately lead to either p-hydroxybenzoic acid or m-hydroxybenzoic acid (Scheme 2.7). Bromide is a good nucleophile42 and a good leaving group.43 By contrast, chloride is not as good a nucleophile42 or as good of a leaving group43 as bromide. Chloride may consequently be diminished relative to bromide in its ability to nucleophilically displace water from the protonated shikimate intermediate (Scheme 2.7). Subsequently, a chloroshikimate intermediate may undergo elimination to the diolefin intermediates (Scheme 2.7) less readily than a bromoshikimate intermediate. This could explain the poor yields of hydroxybenzoic acid products when shikimic acid dehydration is run in ionic liquid chlorides. The low yields or absence of p-hydroxybenzoic acid formation with ionic liquids   39 with counteranions such as hydrogen sulfate (Table 2.1, entry 4) and tetrafluoroborate (Table 2.1, entry 5) may be explained by the non-nucleophilic nature of these anions. Scheme 2.7. A Possible Mechanism of Shikimic Acid Dehydration [BMIm]+ XCO2H H2SO4 HO OH HSO4 H OH [BMIm]+ CO2- O H [BMIm]+ XCO2H - OH X OH OH [BMIm]+ CO2- OH [BMIm]+ H2SO4 CO2 H2SO4 HX [BMIm]+ = H OH O H N N H2SO4 HX OH OH H 2O OH H 2O [BMIm]+ CO2H2SO4 HX OH [BMIm]+ CO2H2SO4 HX H 2O [BMIm]+ CO2- OH OH H 2O H2SO4 HX OH In contrast to chloride, iodide is a better nucleophile42 and a better leaving group43 relative to bromide. This might lead to the expectation of ionic liquid iodides performing at least as well as ionic liquid bromides in the dehydration of shikimic acid. However, ion exchange considerations may be important with exchange of the carboxylate of shikimic acid with the counteranion of the ionic liquid being an essential step in the overall dehydration. With formation of an intimate ion pair between the shikimate carboxylate and the ionic liquid ammonium cation, the counteranion is released to play its essential role in the subsequent nucleophilic covalent catalysis. Using data from ion exchange resins, the binding affinity of anions with organic ammonium cations is ranked in the order: iodide >> bromide > chloride >> acetate.44 As a consequence, exchange of shikimate with the iodide of ionic liquid iodides may be a slow step.   40 This slower formation of an intimate ion pair between shikimate and the organic ammonium cation component of ionic liquid iodides may explain the lower rates and lower yields for shikimic acid dehydration in ionic liquid iodides. 5.4. Lewis Acid Catalyzed Conversion of Shikimic Acid to p-Hydroxybenzoic Acid in 1Butyl-3-methylimidazolium Salts Lewis acid-catalyzed reactions of shikimic acid dissolved in 1-butyl-3methylimidazolium salts 40 in the absence of sulfuric acid were also explored. The reaction conditions entailed heating a 20 weight percent (wt%) solution of shikimic acid in 1-butyl-3methylimidazolium salts 40 with 20 mol % Lewis acids at 120 °C under reduced pressure (0.4 mbar) except when lower boiling Lewis acids were used. In these cases, the reaction was conducted under N2. Boron tribromide, zirconium(IV) bromide, scandium(III) bromide, hafnium(IV) bromide, germanium(IV) bromide and aluminium bromide were observed to catalyze essentially quantitative dehydration of shikimic acid to a mixture of p-hydroxybenzoic acid and m-hydroxybenzoic acids in 1-butyl-3-methylimidazolium bromide 40b (Table 2.5, entry 1-6). Interestingly CuBr2, FeBr3 and AuBr3 formed almost as much protocatechuic acid as mhydroxybenzoic acid (Table 2.5, entry 8-10). The oxidation state of the metals affected significantly to the reaction. There was no reaction with CuBr or FeBr2 (Table 2.5, entry 18 and 19). The importance of bromide as the counteranion of the ionic liquid was very similar to H2SO4-catalyzed reactions. Boron trichloride, zirconium(IV) chloride, hafnium(IV) chloride, and aluminium chloride catalyzed reactions of shikimic acid dissolved in 1-butyl-3methylimidazolium chloride 40a afforded little to no hydroxybenzoic acid (Table 2.6, entry 1-4).   41 Table 2.5. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide 40b Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 catalyst * BBr3 ZrBr4 ScBr3 HfBr4 * GeBr4 AlBr3 YBr3 CuBr2 FeBr3 AuBr3 VBr3 TiBr4 CrBr3 SnBr4 LaBr3 PdBr2 InBr3 CuBr FeBr2 PHBA 76% 74% 73% 73% 72% 71% 66% 64% 63% 61% 61% 58% 57% 22% 21% 4% 3% 0% 0% 1 % Yield MHBA2 21% 21% 20% 21% 21% 19% 19% 16% 18% 16% 16% 18% 16% 6% 6% 1% 1% 0% 0% PCA3 0% 0% 0% 0% 0% 0% 0% 15% 13% 22% 1% 0% 0% 0% 0% 0% 0% 0% 0% % SA4 0% 0% 0% 3% 0% 7% 8% 0% 0% 0% 0% 1% 0% 57% 69% 91% 96% 99% 99% * rxn = under N2(g); 1 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3 PCA=protocatechuic acid; 4 % SA=percent remaining shikimic acid Table 2.6. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Chloride 40a Entry 1 2 3 4 5 catalyst * BCl3 ZrCl4 HfCl4 AlCl3 * TiCl4 PHBA 26% 0% 0% 0% 12% 1 % Yield MHBA2 10% 0% 0% 0% 6% PCA3 0% 0% 0% 0% 3% % SA4 54% 93% 92% 94% 46% * rxn = under N2(g); 1 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3 PCA=protocatechuic acid;4 % SA=percent remaining shikimic acid Boron triiodide, zirconium(IV) iodide, hafnium(IV) iodide, and aluminium iodide catalyzed reactions of shikimic acid dissolved in 1-butyl-3-methylimidazolium iodide 40c afforded moderate yields of p-hydroxybenzoic acid and m-hydroxybenzoic acid in addition to formation of protocatechuic acid (Table 2.7, entry 1-4). However, the percentage of shikimic acid   42 consumed that could be accounted for in formation of p-hydroxybenzoic acid, m-hydroxybenzoic acid and protocatechuic acid was low. Table 2.7. Lewis Acid 20 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Iodide 40c Entry catalyst 1 2 3 4 BI3 ZrI4 HfI4 AlI3 PHBA 32% 26% 33% 32% 1 % Yield MHBA2 5% 5% 6% 6% PCA3 14% 10% 13% 12% % SA4 23% 2% 1% 2% 1 3 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; PCA=protocatechuic acid; 4 % SA=percent remaining shikimic acid The catalyst loading of boron tribromide, zirconium(IV) bromide, scandium(III) bromide, hafnium(IV) bromide, germanium(IV) bromide and aluminium bromide in the catalyzed dehydration of shikimic acid in 1-butyl-3-methylimidazolium bromide 40b could be lowered to 2 mol % without significant reduction of yield and mass accountability (Table 2.5, entry 1-6 vs. Table 8 entry 1-6). When 2 mol% of H2SO4 was used as the catalyst, the conversion of shikimic acid became noticeably reduced (Table 2.8, Entry 7). This might be due to the redox reaction between H2SO4 and bromide. Table 2.8. Lewis Acid 2 mol% Catalyzed Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide 40b Entry catalyst 1 2 3 4 5 6 7 * BBr3 ZrBr4 ScBr3 HfBr4 * GeBr4 AlBr3 H2SO4 % Yield PHBA1 MHBA2 74% 21% 70% 20% 71% 20% 71% 20% 75% 21% 72% 20% 67% 18% * % SA3 0% 0% 1% 1% 0% 1% 7% rxn = under N2(g); 1 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3 % SA=percent remaining shikimic acid   43 5.5. Conversion of (3R,4S,5R)-Triacetoxy-1-cyclohexenecarboxylic Acid to mHydroxybenzoic Acid in 1-Butyl-3-methylimidazolium Salts Scheme 2.8. Aromatization of Acetylated Shikimic Acid The elimination reaction of acetoxy groups from (3R,4S,5R)-triacetoxy-1cyclohexenecarboxylic acid (acetylated shikimic acid) 46 in various 1-butyl-3methylimidazolium salts 40 were explored. This reaction was very slow and it required higher temperatures and longer reaction times to achieve complete conversion than the dehydration of non-acetylated shikimic acid. m-Acetoxybenzoic acid, p-acetoxybenzoic acid, mhydroxybenzoic acid and p-hydroxybenzoic acid were observed by 1H NMR in the reaction solution during the course of the reaction. In small scale reactions, acetoxybenzoic acids were hydrolyzed by the time acetylated shikimic acid had been completely consumed. Despite the 1butyl-3-methylimidazolium salts were dried before reaction, there must have been enough water to hydrolyze the product because of the hygroscopicity of ionic liquids. The reaction conditions entailed heating a 10 weight percent (wt%) solution of acetylated shikimic acid 46 in 1-butyl-3methylimidazolium salts 40 at 200 °C under active flow of nitrogen (bubbler) with 12 mol % H2SO4 (Scheme 2.8). While the selectivity of shikimic acid dehydration reaction in 1-butyl-3methylimidazolium salts 40 favors p-hydroxybenzoic acid, the elimination reaction of acetoxy groups from acetylated shikimic acid favors the formation of m-hydroxybenzoic acid (Table 2.9, entry 1-4). Selectivity favoring m-hydroxybenzoic acid was highest in 1-butyl-3   44 methylimidazolium chloride 40a to produce a 73% yield of m-hydroxybenzoic acid (Table 2.9, entry 1). Table 2.9. m-Hydroxybenzoic Acid Synthesis from Acetylated Shikimic Acid in Various 1-Butyl-3-methylimidazolium Salts 40a-40e Entry Ionic Liquid 1 2 3 4 5 40a 40b 40c 40d 40e 1 X=Cl X=Br X=I X=HSO4 X=BF4 Temp Time 200 °C 200 °C 200 °C 200 °C 200 °C 1d 1d 1d 1d 1d % Yield PHBA1 MHBA2 0 73 14 48 0 40 0 6 21 15 % ASA3 0 0 0 0 0 PHBA=p-hydroxybenzoic acid; 2 MHBA=m-hydroxybenzoic acid; 3 % ASA=percent remaining acetylated shikimic acid Acetoxy group elimination of acetylated shikimic acid 46 showed a different trend in product selectivity relative to shikimic acid dehydration, which suggests a different mechanism is at play. A stronger base shows more selectivity to the meta product when chloride, bromide and iodide are compared (Table 2.9, entry 1-3). If this is an E2 reaction, the first step is a protonation of pseudo axial acetoxy groups (Scheme 2.9).   45 Scheme 2.9. Possible Mechanism of Acetylated Shikimic Acid Aromatization OAc 5 3 AcO 1 2 4 AcO 46a AcO 6 3 CO2H AcO CO2H 1 6 2 5 OAc 46b CO2H AcO 4 OAc CO2H CO2H OAc 48 OAc 49 AcO 47 OAc CO2H CO2H RO H 2O OR 51 6 R = Ac 50 R = H 39 There are two pseudo axial acetoxy groups in the conformation 46a at C-4 and C-5. Elimination of C-4 acetoxy group would lead to meta product only (Scheme 2.9). Elimination of the C-5 acetoxy yields the intermediate 48, which can lead to either para or meta products. The C-3 proton of the intermediate 48 is more acidic than the C-4 proton and therefore would lead to meta product. There is only one pseudo axial acetoxy group in conformer 46b, and the elimination of this acetoxy group leads to a para product (Scheme 2.9). The conformer 46a possesses two pseudo axial acetoxy groups while the conformer 46b possesses only one pseudo axial acetoxy group. The pseudo axial interaction in cyclohexene is less sterically strained than the diaxial interaction in cyclohexanes.45 There are three possible acetoxy groups for a trans diaxial elimination at the first step (scheme 2.9). Two of them would lead to meta product. Of the two trans diaxial elimination, one is exclusively leads to meta product. The other can lead to either meta or para product. Since the C-3 proton is most acidic, the C-3 proton and the C-4 acetoxy group would more likely eliminated leading meta product. This might be the reason that   46 meta product is predominant in the reaction of chloride, bromide and iodide (Table 2.9, entry 13). 6. Discussion Microbial synthesis of shikimic acid from glucose38 has the advantage of utilizing a starting material that is nontoxic and renewable. This differs from the use of toxic toluene46 and toxic phenol22, which are derived from carcinogenic benzene,21a-g as the starting materials in the microbial oxidation of toluene23 to p-hydroxybenzoic acid and the enzymatic conversion of phenyl phosphate24a-d to p-hydroxybenzoic acid (Scheme 2.3). Microbial synthesis of shikimic acid from glucose followed by chemical dehydration of shikimic acid to p-hydroxybenzoic acid lacks the conciseness of the direct microbial syntheses of p-hydroxybenzoic acid from glucose. One of the direct microbial syntheses of p-hydroxybenzoic acid from glucose involving chorismate lyase has titer of 23 g/L (Scheme 2.4).26 This was achieved by employment of ion exchange resins during fermentation in order to prevent enzyme feedback inhibition.26 Ion exchange resins are relatively expensive and introduce an external loop to the fermentation involving passage of culture broth through a resin bed which increases the risk of contamination. The other direct microbial syntheses of p-hydroxybenzoic acid from glucose involves tyrosine as one of the intermediates. It does not require employment of ion exchange resins during the fermentation, however the yield of p-hydroxybenzoic acid is low (16%).29b Plant-synthesized versus microbe-synthesized shikimic acid is another comparison to consider. Shikimic acid can be synthesized from glucose in 34% yield affording shikimic acid concentrations of 72 g/L (Chapter 3). Plants that synthesize shikimic acid require a growing season before plant tissue containing shikimic acid can be harvested. By contrast, microbial synthesis of   47 shikimic acid only requires 61 h for microbial growth/microbial production with an additional 24 h for fermentor setup, harvesting, and fermentor cleanout. Transgenic plants produce p-hydroxybenzoic acid directly. However, p-hydroxybenzoic acid is conjugated as either a phenolic or carboxylate glucoside.30,31,33 The highest producing transgenic plant produces (after correcting for the mass of the conjugated glucose) the equivalent of 6.6 dry wt% of p-hydroxybenzoic acid.32 There is no report in the literature that describes how p-hydroxybenzoic acid glucosides are extracted from transgenic plants. Also, the process to extract p-hydroxybenzoic acid from the transgenic plant generates a stoichiometric amount of glucose as byproduct, which will need to be captured and utilized. Utilization of fields only for one molecule is not economically efficient. Another advantage of a synthesis with plants is the low turnover. It is not flexible to the market change. The chemical dehydration of shikimic acid in 1-butyl-3-methylimidazolium bromide using H2SO4 can yield 80% of p-hydroxybenzoic acid and 18% m-hydroxybenzoic acid (Table 2.3, entry 1). The concentration of shikimic acid in the reaction solution can be up to 40 wt% of 1-butyl-3methylimidazolium bromide. Employment of recycled 1-butyl-3-methylimidazolium bromide provides comparable yields of p-hydroxybenzoic acid and m-hydroxybenzoic acid to using fresh ionic liquid (Table 2.3). The m-hydroxybenzoic acid can be separated from p-hydroxybenzoic acid by crystallization. m-Hydroxybenzoic acid is also used as one of the copolymers in LCPs.2 Therefore, the mixture of p-hydroxybenzoic acid and m-hydroxybenzoic acid in the mother liquor is a valuable product.   48 7. Experimental 7.1. General 1 H NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to residual non-deuterated solvent in CDCl3 (δ = 7.26 ppm) and CD3OD (δ = 3.34 ppm). Chemical shifts for 1H NMR spectra in D2O is reported (in parts per million) relative to residual non-deuterated sodium salt of 3(trimethylsilyl) propionic-2,2,3,3-d4 acid, TSP (δ = 0.00 ppm). 13 C NMR spectra were recorded at 125 MHz and chemical shifts for these spectra were reported (in parts per million) relative to CDCl3 (δ = 77.4 ppm) and CD3OD (δ = 49.9 ppm). 1-Butyl-3-methylimidazolium iodide, 1-butyl-3-methylimidazolium hydrogen sulfate, 1butyl-3-methylimidazolium tetrafluoroborate, 1-butylpyridinium bromide, 1-butyl-1methylpyrrolidinium bromide, 1-iodobutane, tributylphosphine, 1-methylimidazole, 1chlorobutane, 1-bromobutane, acetic anhydride, pyridine and 3-(trimethylsilyl) propionic2,2,3,3-d4 acid were purchased from Sigma-Aldrich. 1-Butyl-2,3-dimethylimidazolium bromide, 1-butylpyridinium chloride, 1-butylpyridinium iodide, 1-butyl-2-methylpyridinium bromide and tetrabutylphosphonium chloride were purchased from Ionic Liquid Technologies. Tetrabutylphosphonium bromide was purchased from CYTEC. 1-Iodobutane and tributylphosphine were distilled prior to use. Acetic anhydride was dried with P2O5, and then distilled. Pyridine was dried over MS 4 Å. Other chemicals were used without further purification.   49 7.2. Product Analysis HPLC analysis was performed on an Agilent 1100. Shikimic acid was quantified by HPLC using a Grace AlltimaTM amino column (4.6 ´ 250 mm, 5 μm particle size) and isocratic elution with 60/40, (v/v); CH3CN/H2O (140 mM NH4+HCO2−, pH 4.0). The amino column was stored in a solution of 5/95, (v/v); methanol/hexane when it was received from the manufacture. In order to convert the column to weak base ion-exchange mode, the column was washed with 1 L of reagent grade isopropanol (0.2 mL/min) followed by 1 L of HPLC grade acetonitrile (0.2 mL/min) prior to the first use. Shikimic acid molecular standard was obtained from Roche. pHydroxybenzoic acid and m-hydroxybenzoic acid were separated from one another with baseline resolution and quantified by HPLC using an Agilent ZORBAX SB-C18 (4.6 x 150 mm, 5 μm particle size) column and isocratic elution with 15/85, (v/v); CH3CN/H2O (100 mM NH4+HCO2−, pH 2.5). The ZORBAX was stored in a solution of 85/15, (v/v); methanol/H2O when it was received from the manufacture. The column was washed with 1 L of HPLC grade acetonitrile prior to the first use. p-Hydroxybenzoic acid molecular standard was obtained from Spectrum while the m-hydroxybenzoic acid molecular standard was obtained from Sigma-Aldrich. The deionized, distilled water used to make the required buffer solutions was filtered with a DURAPORE® 0.45 µm HV filter (MILLIPORE). The acetonitrile was HPLC grade. Syringe filtration (0.45 µm Whatman filter) was followed by HPLC analysis for all the sample. 7.3. Screening of Ionic Liquid for Dehydration of Shikimic Acid Screening of ionic liquid for dehydration of shikimic acid employed a 25 mL stripping flask fitted with a magnetic stir bar and a gas adaptor. Approximately 5 g of ionic liquid was dried in the flask overnight under reduced pressure at 50 °C. The oil bath temperature was raised   50 to a temperature high enough to melt the ionic liquid under the reduced pressure: 1-butyl-3methylimidazolium salts at 90 °C, 1-butylpyridinium salts at 140 °C, tetrabutylphosphonium salts at 110 °C, 1-butyl-2,3-dimethylimidazolium bromide at 110 °C, 1-butyl-2methylpyridinium bromide at 160 °C, 1-butyl-1-methylpyrrolidinium bromide at 180 °C. The flask containing dry ionic liquid was opened under N2 and weighed. Shikimic acid 20 wt% relative to the ionic liquids’ weight was added. Shikimic acid was dissolved at the melting temperature of the ionic liquid under N2, and then maintained at that temperature for 30 min under vacuum (0.4 mbar). The flask was opened under N2 and H2SO4 (0.2 eq) was added. The reaction solution was heated for 13 h under vacuum (0.4 mbar) and the resulting crude solution was analyzed by HPLC. 7.4. Comparison of Shikimic Acid Concentration in Dehydration of Shikimic Acid Determining the maximum concentration limit of shikimic acid in 1-butyl-3methylimidazolium bromide ([BMIm]Br) for the dehydration of shikimic acid employed a 25 mL stripping flask fitted with a magnetic stir bar and a gas adaptor. Approximately 5 g of [BMIm]Br was dried in the flask overnight under reduced pressure at 50 °C. The flask containing dry [BMIm]Br was weighed and opened under N2. Shikimic acid 20 wt% relative to the [BMIm]Br weight was added. Shikimic acid was dissolved at 90 °C under N2 and heat was maintained for 30 min under vacuum (0.4 mbar). The flask was opened under N2 and H2SO4 (0.2eq) was added. The reaction solution was heated at 90 °C for 13 h under vacuum (0.3 mmHg) and the resulting crude solution was analyzed by HPLC.   51 7.5. Synthesis of 1-Butyl-3-methylimidazolium Bromide47a 1-Bromobutane (101 mL, 0.936 mol) was added to 49.5 mL (0.621 mol) of 1methylimidazole in a 500 mL round bottom flask heated in a 70 °C oil bath fitted with a reflux condenser open to the atmosphere. A vigorous exothermic reaction was observed after heating at 70 °C for approximately 15 min. The flask was removed from the oil bath until the vigorous exothermic reaction ceased and then placed back in the 70 °C oil bath for 13 h. 1-Bromobutane (30.0 mL, 0.279 mol) was added to the flask, and continued heating at 70 °C oil for an additional 6 h. Unreacted 1-bromobutane was removed under reduced pressure at 55 °C. This afforded 1butyl-3-methylimidazolium bromide (135 g, 99%) as a yellow solid. 1H NMR (CDCl3) δ = 10.6 (s, 1H), 7.32 (dd, J = 3.5, 2 Hz, 1H), 7.25 (dd, J = 3.5, 2 Hz, 1H), 4.31 (dd, J1 = J2 = 7.5 Hz, 2H), 4.11 (s, 3H), 1.86-1.92 (m, 2H), 1.37 (m, 2H), 0.950 (dd, J = 7.5, 7.0 Hz, 3H). 13 C NMR (CDCl3) δ = 138.2, 123.6, 122.0, 50.3, 37.1, 32.5, 19.8, 13.8. mp: 72.0-76.0 °C (lit.47b 69-70 °C). 7.6. Recycling 1-Butyl-3-methylimidazolium Bromide In situ synthesized 1-Butyl-3-methylimidazolium bromide (29.9 g, 136 mmol) ([BMIm]Br) was dried in a 200 mL stripping flask overnight at 90 °C under vacuum. The flask containing dry [BMIm]Br was opened under N2 and shikimic acid (11.9 g, 68.5 mmol) was added. After dissolving shikimic acid at 90 °C under N2, the temperature was maintained for 30 min under vacuum (0.4 mbar). The flask was opened under N2 and H2SO4 (0.730 mL, 13.7 mmol) was added. The acidified reaction solution was then heated at 90 °C for 24 h under vacuum (0.4 mbar). After cooling the reaction solution to rt, it was diluted with H2O (30 mL) and extracted with EtOAc (300 mL) for 6 h in a liquid-liquid continuous extractor. Concentrating the organic layer gave a beige-colored solid consisting of 7.52 g of p-   52 hydroxybenzoic acid (80%), and 1.69 g of m-hydroxybenzoic acid (18%) based on HPLC analysis. Water was removed from the aqueous layer on a rotary evaporator under high vacuum. The concentrated brown oil was dried under reduced pressure and solidified overnight giving 28.0 g (128 mmol) of [BMIm]Br as a brown solid. The flask containing this recycled [BMIm]Br was opened under N2 and shikimic acid (11.2 g, 64.1 mmol) was added. After the shikimic acid dissolved by heating at 90 °C under N2, heating was continued under vacuum (0.4 mbar) for 30 min. The flask was opened under N2, and H2SO4 (0.684 mL, 12.8 mmol) was added. The acidified reaction solution was then heated at 90 °C for 24 h under vacuum (0.4 mbar). The reaction solution after cooling was diluted with H2O (30 mL), and extracted with EtOAc (300 mL) for 6 h in a liquid-liquid continuous extractor. Concentrating the organic layer gave browncolored solids consisting of 6.85 g of p-hydroxybenzoic acid (77%) and 1.40 g of mhydroxybenzoic acid (16 %) based on HPLC analysis. In addition to further discoloration, viscosity of the [BMIm]Br had increased during its second use. Activated carbon (DARCO®, KB-B 100 mesh) (4.00 g) was added to the aqueous layer of the extraction from the second shikimic acid dehydration using recycled [BMIm]Br, and the mixture was swirled with an orbital shaker at rt and 200 rpm. The activated carbon was removed by filtration. Concentrating and drying this filtrate gave a yellow-colored oil. Adding a small crystal of [BMIm]Br resulted in solidification of the ionic liquid (17.0 g, 77.6 mmol) as a yellow-colored solid. This twice recycled [BMIm]Br was then used for a third dehydration of shikimic acid. The [BMIm]Br was dried in a 200 mL stripping flask overnight at 90 °C under vacuum. The flask containing dry [BMIm]Br was opened under N2, and shikimic acid (7.06 g, 40.5 mmol) was added. There was shikimic acid (0.20 g, 1.2 mmol) carried over from the   53 second reaction. After the shikimic acid dissolved with heating at 90 °C under N2, heating at 90 °C was continued under vacuum (0.4 mbar) for 30 min. The flask was opened under N2, and H2SO4 (0.432 mL, 8.11 mmol) was added. The acidified reaction solution was then heated at 90 °C for 24 h under vacuum (0.4 mbar). The reaction solution after cooling was diluted with H2O (30 mL), and extracted with EtOAc (300 mL) for 6 h in a liquid-liquid continuous extractor. Concentrating the organic layer gave a beige-colored solid consisting of 4.13 g of phydroxybenzoic acid (72%), and 0.61 g of m-hydroxybenzoic acid (11 %) based on HPLC analysis. 7.7. Lewis Acid Catalysts Screening for Dehydration of Shikimic Acid The ionic liquid was dried in the flask overnight under reduced pressure at 50 °C. The oil bath temperature was raised to 90 °C to melt the ionic liquid under reduced pressure. The melted ionic liquid (5 g) was poured into a 25 mL stripping flask containing a magnetic stir bar. Shikimic acid (1 g, 5.74 mmol) and a Lewis acid catalyst were added. Shikimic acid was dissolved at 90 °C under N2 and then vacuum (0.4 mbar) was applied. BCl3, TiCl4, BBr3 and GeBr4 reactions were run under active flow of N2 due to their low boiling points. The oil bath temperature was raised to 120 °C and held at that temperature for 9 h. The resulting crude solution was analyzed by HPLC. 7.8. Synthesis of Tetra-(n)-butylphosphonium Iodide48 1-Iodobutane (53.6 mL, 471 mmol) was added to tri-(n)-butylphosphine (59 mL, 236 mmol) in a 500 mL round bottom flask fitted with a reflux condenser connected to an active Ar flow using a gas bubbler. The solution was stirred for 1 h at rt then 20 h at 100 °C under an active flow of Ar. Addition of diethyl ether (80 mL) to the reaction solution after cooling the   54 reaction to rt gave a white solid precipitate. Filtration and drying under reduced pressure allowed tetra-(n)-butylphosphonium iodide (91.0 g, 100 %) as a white solid. 1H NMR ((CD3)2CO): δ = 2.47-2.53 (m, 2H), 1.66-1.73 (m, 2H), 1.52 (ddddd, J1 = J2 = J3 = J4 = J5 = 7.34 Hz, 2H), 0.956 (t, J = 7.0 Hz, 3H). 13C NMR ((CD3)2CO) δ = 24.4, 24.2, 23.8, 23.7, 19.0, 18.7, 13.4. mp: 98-100 °C (Lit.48 95-96°C). 7.9. (3R,4S,5R)-Triacetoxy-1-cyclohexenecarboxylic Acid Shikimic acid (32.2 g, 185 mmol) was stirred with acetic anhydride (156 mL, 1.65 mol) and pyridine (156 mL, 1.93 mol) at rt for 24 h in a 1 L stripping flask. The reaction solution was concentrated in vacuo. The residue was diluted with a solution of hexanes/EtOAc (1:2) with 0.5% AcOH and washed with 200 mL of 1% (v/v) H2SO4 (3x). A plug of chromatographic grade silica gel (60 Å pore size, 40-63μm mesh) was prepared with a solution of hexanes/EtOAc with AcOH (3:1, 0.5%). The crude solution was filtered through the plug of silica gel. Concentrating the filtrate in vacuo afforded a colorless oil (42.1 g, 76%). 1H NMR (CDCl3) δ = 6.83-6.85 (m, 1H), 5.72-5.73 (m, 1H), 5.23-5.28 (m, 2H), 2.85 (ddd, J = 19, 4.5, 2 Hz, 1H), 2.41 (ddd, J = 19, 3.5, 2 Hz, 1H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H). 13 C NMR (CDCl3) δ = 170.8, 170.3, 170.2, 135.4, 130.9, 67.7, 67.0, 66.3, 28.3, 21.3, 21.0. 7.10. Screening of Ionic Liquid for m-Hydroxybenzoic Acid Synthesis from (3R,4S,5R)Triacetoxy-1-cyclohexenecarboxylic Acid Screening of ionic liquid for m-hydroxybenzoic acid synthesis from (3R,4S,5R)triacetoxy-1-cyclohexene-1-carboxylic acid employed a 25 mL stripping flask fitted with a magnetic stir bar and a gas adaptor. Ionic liquid (5 g) was added to (3R,4S,5R)-triacetoxy-1cyclohexene-1-carboxylic acid (0.5 g). It was dried overnight under reduced pressure at 50 °C.   55 The flask containing dry ionic liquid and (3R,4S,5R)-triacetoxy-1-cyclohexene-1-carboxylic acid was weighed. The flask was opened under N2, and H2SO4 (0.12eq) was added. It was heated for 24 h under an active flow of N2 using a gas bubbler and the resulting crude solution was analyzed by HPLC. 7.11. 1-Butyl-3-methylimidazolium Chloride49a 1-Chlorobutane (230 mL, 2.20 mol) was added to 122 mL (1.53 mol) of 1methylimidazole in a 500 mL round bottom flask heated in a 95 °C oil bath fitted with a reflux condenser connected to an active flow of N2 using a gas bubbler. The solution was heated at 95 °C for 38 h. Another 40 mL of 1-chlorobutane (0.383 mol) was added, and then continued heating at 95 °C for total 3 days. Removing unreacted substrate with a rotary evaporator and drying under reduced pressure overnight allowed 1-butyl-3-methylimidazolium chloride (267 g, 100 %) as a yellow solid. 1H NMR (CDCl3): δ 11.2 (s, 1H), 7.16 (dd, J = 3, 1.5 Hz, 1H), 7.14 (dd, J = 3.5, 2 Hz, 1H), 4.31 (dd, J1 = J2 = 7.5 Hz, 2H), 4.11 (s, 3H), 1.85-1.92 (m, 2H), 1.38 (m, 2H), 0.958 (dd, J = 7.5, 7.0 Hz, 3H). 13 C NMR (CDCl3) δ = 139.5, 123.1, 121.6, 50.3, 37.0, 32.5, 19.9, 13.8. mp: 60.0-65.0 °C (Lit.49b 65-69 °C). 7.12. m-Hydroxybenzoic Acid Synthesis from (3R, 4S, 5R)-Triacetoxy-1-cyclohexenecarboxylic Acid 1-Butyl-3-methylimidazolium chloride ([BMIm]Cl) was dried and melted in a round bottom flask at 90 °C under vacuum overnight. The [BMIm]Cl (211 g, 1.20 mol) was poured into a 2 L three neck round bottom flask containing (3R,4S,5R)-triacetoxy-1-cyclohexene-1carboxylic acid (20.9 g, 69.5 mmol) and then H2SO4 (0.445 mL, 8.34 mmol) was added. The   56 solution was stirred with an overhead stirrer under an active flow of N2 and heated at 200 °C for 24 h. 0.7 M NaOH (240 mL) was added to the solution and heated at 100 °C for 3 h. The solution was acidified with H2SO4 to pH 3.4 and then extracted with EtOAc (750 mL) for 3 days in a continuous liquid-liquid extractor. The organic extract was concentrated to afford brown oil containing 6.7 g (70%) of m-hydroxybenzoic acid based on HPLC analysis. This brown oil was decolorized using continuous flow conditions. A decolorizing column (D = 5.4 cm) was constructed with a piece of Kimwipe to plug the column outlet below a layer of 6.7 g Celite® 545 topped with a layer consisting of 3.6 g activated charcoal (DARCO® G-60, 100 mesh) dispersed in 15.4 g flash chromatography grade silica gel (60 Å pore size, 40-63μm mesh). After loading, the decolorizing column was washed with an EtOAc/AcOH (98.6:1.4 v/v) solution. Concentrating this solution gave a light brown oil. This light brown oil was diluted with 2 M NaOH (50 mL) and washed with CH2Cl2 (50mL). The remaining aqueous solution was acidified with HCl to pH 0.0, crystallized at 4 °C, filtered, and dried to afford m-hydroxybenzoic acid 5.3 g (54%) as a white solid with a 97.8% purity based on HPLC analysis. 1H NMR (CD3OD): δ = 7.52 (dt, J = 8.0, 1.3 Hz, 1H), 7.45 (dd, J = 2.5, 1.5 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.03 (ddd, J = 8.5, 3.0, 1.0 Hz, 1H). 13 C NMR (CD3OD) δ = 170.8, 159.6, 134.0, 131.3, 122.7, 121.8, 118.1. mp: 196-198 °C (Lit.50 197-200 °C).   57 REFERENCES   58 REFERENCES 1.   Aelony, D.; Renfrew, M. M. Polyester resins. US 2728747, December 27, 1955. 2.   (a) Hanabatake, M. Production method of aromatic polyesters. JP S5657821, May 20, 1981. (b) Layton, R.; Cleay, J. W. Wholly aromatic polyesters with reduced char content. EP 0347228, December 20, 1989. (c) Huspeni, P. J.; Stern, B. A.; Frayer, P. D. 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Plant Cell 2001, 13, 1669-1682. 32.  Viitanen, P. V.; Devine, A. L.; Khan, M. S.; Deuel, D. L.; Van Dyk, D. E.; Daniell, H. Metabolic engineering of the chloroplast genome using the Escherichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiol. 2004, 136, 4048-4060. 33.  McQualter, R. B.; Chong, B. F.; Meyer, K.; Van Dyk, D. E.; O’Shea, M. G.; Walton, N. J.; Viitanen, P. V.; Brumbley, S. M. Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid. Plant Biotechnol. J. 2005, 3, 29-41. 34.  Eykman, J. F. Ueber die shikimisaure. Ber. Dtsch. Chem. Ges. 1891, 24, 1278-1303. 35.  Gibson, J. M.; Thomas, P. S.; Thomas, J. D.; Barker, J. L.; Chandran, S. S.; Harrup, M. K.; Draths, K. M.; Frost, J. W. Benzene-free synthesis of phenol. Angew. Chem. Int. Ed. 2001, 40, 1945-1948. 36.  Abrecht, S.; Harrington, P.; Idling, H.; Karpf, M.; Trussardi, R.; Wirz, B.; Zutter, U. The synthetic development of the anti-influenza neuraminidase inhibitor Oseltamivir phosphate (Tamiflu®): a challenge for synthesis & process research. Chimia 2004, 58, 621-629. 37.  Rapisarda, A. Economic Importance and Market Trends of the Genera Pimpinella, Illicium, and Foeniculum. In Illicium, Pimpinella and Foeniculum. Jodral, M. M., Ed.; CRC: Boca Raton, 2004; p 191-218. 38.  (a) Draths, K. M.; Knop, D. R.; Frost, J. W. Shikimic acid and quinic acid: replacing isolation from plant sources with recombinant microbial biocatalysis. J. Am. Chem. Soc. 1999, 121, 1603-1604. DOI: 10.1021/ja9830243. (b) Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von Daeniken, R.; Weber, W.; Frost, J. W. Hydroaromatic equilibration during biosynthesis of shikimic acid. J. Am. Chem. Soc. 2001, 123, 10173-10182. DOI: 10.1021/ja0109444. (c) Chandran, S. S.; Yi, J.; Draths, K. M.; von Daeniken, R.; Weber, W.; Frost, J. W. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol. Prog. 2003, 19, 808-814. DOI: 10.1021/bp025769p.   63 39.  (a) Avula, B.; Wang, Y.-H.; Smillie, T. J.; Khan, I. A. Determination of shikimic acid in fruits of Illicium species and various other plant samples by LC-UV and LC-ESI-MS. Chromatographia 2009, 69, 307-314. DOI: 10.1365/s10337-008-0884-z. (b) Bohm, B. A. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid). Chem. Rev. 1965, 65, 435-466. 40.  Hirayama, Y. ICL-IP Japan. Global Bromine Industry and Its Outlook. http://www.bromine.chem.yamaguchiu.ac.jp/library/L02_Global%20Bromine%20Industry.pdf (accessed 6/14/2017). 41.  Li, W.; Xie, D.; Frost, J.W. Benzene-free synthesis of catechol: interfacing microbial and chemical catalysis. J. Am. Chem. Soc. 2005, 127, 2874-2882. DOI: 10.1021/ja045148n. 42.  Pearson, R. G.; Songstad, J. Application of the principle of hard and soft acids and bases to organic chemistry. J. Am. Chem. Soc. 1967, 89, 1827-1836. 43.  Noyce, D. S.; Virgilio, J. A. The synthesis and solvolysis of 1-phenylethyl disubstituted phosphinates. J. Org. Chem. 1972, 37, 2643-2647. 44.  Alexandratos, S. D. ion-exchange resins: a retrospective from industrial and engineering chemistry research. Ind. Eng. Chem. Res. 2009, 48, 388-398. DOI: 10.1021/ie801242v. 45.  Rickborn, B.; Lwo, S. Conformational effects in cyclic olefins. Kinetic and stereochemistry of epoxidation of some alkyl-substituted cyclohexenes. J. Org. Chem. 1965, 30, 2212-2216. 46.  Flowers, L.; Boyes, W.; Foster, S.; Gehlhaus, M.; Hogan, K.; Marcus, A.; McClure, P.; Osier, M.; Ronney, A. Toxycological Review of Toluene; U.S. Environmental Protection Agency; Washington D.C., 2005. 47.  (a) Gruzdev, M. S.; Ramenskaya, L. M.; Chervonova, U. V.; Kumeev, R. S. Preparation of 1butyl-3-methylimidazolium salts and study of their phase behavior and intramolecular intractions. Russ. J. Gen. Chem. 2009, 79, 1720-1727. (b) Golovanov, D. G.; Lyssenko, K. A.; Antipin, M. Y.; Vygodskii, Y. S.; Lozinskaya, E. I.; Shaplov, A. S. Cocrystal of an ionic liquid with organic molecules as a mimic of ionic liquid solution. Cryst. Growth Des. 2005, 5, 337-340. DOI: 10.1021/cg030086i. 48.  Rodriguez-Zubiri, M.; Anguille, S.; Brunet, J. Intermolecular hydriamination of nonactivated alkenes catalyzed by Pt(II) or Pt(IV)-n-Bu4PX (X = Cl, Br, I) systems: key effect of the halide anion. J. Mol. Catal. A-Chem. 2007, 271, 145-150. DOI: 10.1016,/j.molcata.2007.02.011. 49.  (a) Khrizman, A.; Chen, H.; Moyna, G. Synthesis of sequentially deuterated 1-n-butyl-3methylimidazolium ionic liquids. J. Labelled Compd. Rad. 2011, 54, 401-407. DOI: 10.1002/jlcr.1884. (b) Wilkes, J. S.; Levisky, J. A.; Wilson, R. A.; Hussey, C. L. Dialkylimidazolium chloroaluminate melts: a new class of room-temperature ionic liquids for   64 electrochemistry, spectroscopy, and synthesis. Inorg. Chem. 1982, 21, 1263-1264. 50.  Elming, N. Transformation of 2-(b, b-dicarbomethoxyethyl)-furan into m-hydroxybenzoic acid. Acta Chem. Scand. 1956, 10, 1664-1666.   65 CHAPTER THREE Scale-Up of the Synthesis of Biobased p-Hydroxybenzoic Acid 1. Introduction The reaction conditions for the synthesis of biobased p-hydroxybenzoic acid were described in Chapter 2. This chapter focuses on the scale-up of the synthesis of biobased phydroxybenzoic acid. Scale-up includes: (a) the microbial synthesis of shikimic acid from glucose under fermentor-controlled conditions from 1 L to 20 L; (b) elaboration and scale-up of a modified isolation and purification of shikimic acid from fermentation broth; (c) scale-up of the aromatization of shikimic to form p-hydroxybenzoic acid from 1 g reaction to 250 g reaction; (d) scale-up of the purification of biobased p-hydroxybenzoic acid. The starting material, shikimic acid, is microbially synthesized with E. coli SP1.1/pKD15.071B, which was previously genetically engineered in the Frost group.1 Although fed-batch fermentation at 1 L scale with this strain had been performed routinely, scale-up of the microbial synthesis to 20 L scale in the Frost group has not been previously attempted. The current commercial method for isolation of shikimic acid from fermentation broth employs ion exchange columns followed by crystallization from highly flammable solvents. Use of ion exchange columns requires evaporating large quantities of water and generates large salt waste streams associated with regeneration of the ion exchange resins. The aromatization of shikimic to form phydroxybenzoic acid was scaled up from 1 g reaction to 250 g reaction. In addition to biobased p-hydroxybenzoic acid synthesis, microbial synthesis of shikimic acid is also scaled up from a 1 L fermentation to a 20 L fermentation in this chapter. Furthermore, downstream isolation and purification of shikimic acid is developed. Employment of hollow fiber filtration for cell removal and tangential flow filtration systems for protein removal from the 30 L fermentation   66 broth greatly expedited cell removal and broth clarification. Shikimic acid is highly water soluble, which makes isolation from fermentation broth challenging. A continuous countercurrent extraction of shikimic acid was developed to overcome this challenge. 2. E. coli SP1.1/pKD15.071B Scheme 3.1. The Common Pathway of Aromatic Amino Acid Biosynthesis OH O H2O3PO H AroF AroG AroH OH E4P + H2O3PO Pi H2O3PO OH CO2H HO O PEP AroB O Pi OH CO2H HO OH H2O O OH OH DAHP DHQ CO2H CO2H PEP AroA H2O3PO O CO2H H2O3PO Pi OH EPSP S3P HO2C AroC Pi CO2H CO2H O OH OH AroK AroL OH OH CO2H AroD DHS NADPH AroE NADP CO2H ATP HO ADP OH OH shikimic acid L-tyrosine L-phenylalanine OH prephenic acid O CO2H CO2H NH2 OH chorismic acid L-tryptophan 2-aminobenzoic acid E4P: erythrose 4-phosphate; PEP: phosphoenolpyruvate; DAHP: 3-deoxy-D-arabino-heptulsonic acid 7-phosphate; DHQ: 3-dehydroquinic acid; DHS: 3-dehydroshikimic acid; S3P: shikimate 3-phosphate; EPSP: 5-enolpyruvylshikimate 3-phosphate; AroF: DAHP synthase (tyrosine feedback); AroG: DAHP synthase (phenylalanine feedback); AroH: DAHP synthase (tryptophan feedback); AroB: DHQ synthase; aroD: DHQ dehydratase; AroE: shikimate dehydrogenase; AroK: shikimate kinase I; AroL: shikimate kinase II; AroA: EPSP synthase; AroC: chorismate synthase Shikimic acid is a key intermediate in the common pathway of aromatic amino acid biosynthesis. This pathway, which is found in plants, bacteria, fungi and parasites,2 converts phosphoenolpyruvic acid (PEP) and erythrose 4-phosphate (E4P) into chorismic acid via seven enzyme-catalyzed reactions (Scheme 3.1).2a,3   67 Scheme 3.2. Biosynthesis of Erythrose 4-phosphate O OH OH H H2O3PO + H2O3PO TktA OH OH O H2O3PO OH O OH glyceraldehyde 3-phosphate OH H + H O PO 2 3 fructose 6-phosphate OH OH O OH xylulose 5-phosphate E4P TktA: transketolase; E4P: erythrose 4-phosphate Scheme 3.3. Increasing Phosphoenolpyruvic Acid Availability OH OH OH O O PTS OH HO OH OH glycolysis continues OH H2O3PO O OH glucose O PEP H2O3PO OH OH glucose 6-phosphate O pyruvic acid PpsA AMP + Pi ATP PEP: phosphoenolpyruvic acid; PpsA: PEP synthase; PTS: PEP:carbohydrate phosphotransferase Modification of the host genome for microbial synthesis of shikimic acid included the insertion of aroB into the serA locus and disruption of aroL and aroK genes in the genome of E. coli SP1.1.1a,1b,4,5 An extra copy of aroB enables overexpression of 3-dehydroquinic acid synthase, while disruption of shikimate kinase-encoding aroL and aroK prevents phosphorylation of shikimic acid. Disruption of L-serine biosynthesis due to loss of genomic serA-encoded phosphoglycerate dehydrogenase ensures that plasmid pKD15.071B, which encodes serA, aroFFBR, aroE, tktA and ppsA is retained by E. coli SP1.1 during cultivation in minimal salts medium in the absence of L-serine supplementation.1a-c,4ab,6,7a,8a Overexpression of aroFFBR, which encodes a mutant isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase insensitive to feedback inhibition by tyrosine, helps to increase carbon flow into the common pathway.1a,4b Shikimate dehydrogenase, encoded by aroE, was overexpressed to compensate for feedback inhibition of AroE by shikimic acid.1a,4a In vitro experiments have   68 shown that E4P dimerization occurs at high concentrations of E4P in aqueous solutions.7b By balancing the rate of E4P consumption with the rate of E4P synthesis in vivo, the steady state concentration of E4P may be sufficiently low to avoid dimerization.7a The maintenance of a low steady state concentration of E4P in vivo to avoid dimerization may explain why E4P has never been detected in biological systems. In order to increase the availability of E4P to the common pathway, tktA-encoded transketolase was also overexpressed (Scheme 3.2).1b,9 PEP drives PTSmediated transport and phosphorylation of glucose to glucose 6-phosphate. There is a competition for PEP between PTS-mediated glucose transport and biosynthesis of DAHP (Scheme 3.3).8b In order to compensate for the competition with PTS-mediated glucose transport, PEP availability was increased by amplified expression of ppsA-encoded PEP synthase so that pyruvic acid is recycle back to PEP (Scheme 3.3).1c 3. Countercurrent Flow Extraction There are two general methods of liquid-liquid extraction in the chemical industry. One employs a mix/decant tank, which is similar to a huge separatory funnel. The other is countercurrent extraction. Disadvantages to a mix/decant tank include the requirement of multiple solvent additions to achieve the desired number of theoretical plates (one extraction is equivalent to one theoretical plate), recovery and recycling of the large volume of solvent required for each extraction and the requirement that the extraction be performed in batches. Countercurrent extraction, on the other hand, has several advantages over the mix/decant method of extraction. These include a higher number of theoretical extraction plates and the ability to be operated continuously. There are different types of countercurrent extraction equipment such as a centrifugal extractor, packed column, sieve tray column, rotating disc contactor (RDC) extractor, SCHEIBEL® column and KARR® reciprocating column.10a   69 * Configuration example when solvent density is lower than broth Figure 3.1. A: Centrifugal Extractor, B: Rotor of Centrifugal Extractor10 * Configuration example when solvent density is lower than broth Figure 3.2. A: Packed Column, B: Sieve Tray Column, C: RDC Extractor, D: SCHEIBEL® Column * Configuration example when solvent density is lower than broth; broth is dispersed in the solvent. Figure 3.3. KARR® Column In these countercurrent extraction systems, a high density liquid (heavy phase) and a lower density liquid (light phase) flow in opposite directions. In a centrifugal extractor, a heavy phase and a light phase are mixed in a rotor (Figure 3.1A).10 The heavy phase is siphoned off from the outer area of the rotor while the light phase is collected near the center of the rotor (Figure 3.1B).10 Packed columns have a mesh structure in the column which disrupts the flow of one of   70 the phases, breaking it up into droplets. These droplets have a high surface area which facilitates the interaction between the heavy and light phases (Figure 3.2A).10a A sieve tray column use sieves rather than a mesh column to break up one of the phases into droplets (Figure 3.2B).10a Rotating disc contactor (RDC) extractors and SCHEIBEL® columns have rotating impellers for the same reason (Figure 3.2C and 2D).10a KARR® reciprocating columns (KARR® column) have a stack of plates on a shaft, which oscillates up-and-down in order to disrupt one of the phases into droplets (Figure 3.3).10a Each instrument has different characteristics. Centrifugal extractors can handle small density differences between two phases while providing several theoretical plates. However, these extractors are susceptible to fouling and plugging.10a Packed columns and sieve tray columns can process larger volumes per unit time than the other four extraction systems, although the number of theoretical plates is lower than the other four extraction systems. Also, packed columns are not suitable for small scale extractions.10a RDC extractors are suitable for viscous material but have fewer theoretical plates than centrifugal extractors, SCHEIBEL® columns or KARR® columns.10a SCHEIBEL® columns have a high number of theoretical plates and are suitable for a wide range of sample volumes.10a KARR® columns also have a high number of theoretical plates and can be suited to a wide range of sample volumes but they have the additional advantage of having the highest process volume per unit time . 10a In this chapter, a KARR® column was employed to extract shikimic acid from fermentation broth. The fermentation broth and the extraction solvent are fed into the KARR® column, one from the top and the other from the bottom, and flow through the column in opposite directions. The fermentation broth feed and the extraction solvent are assigned for different phases. One phase, called the continuous phase, fills the column chamber and is fed at   71 a high flow rate. The other phase is fed more slowly and therefore disperses into small droplets. (Figure 3.3). This dispersion of one phase into a continuous phase affords a high extraction surface area. Whether the broth feed is used as the continuous or dispersed phase depends on the molecule to be extracted and the characteristics of both broth and solvent. Four configuration choices using light or heavy extraction solvents are illustrated in Figure 3.4.10 A light solvent such as n-butanol or EtOAc will have a lower density than the fermentation broth. A heavy solvent such as dichloromethane or chloroform will have a higher density than the broth. The interface level of each configuration is adjusted so as to not disturb the addition of either feed nor solvent (Figure 3.4).10a The solvent of configuration A and B has a lower density than the broth (Figure 3.4A and B). The column is filled with broth and the solvent is dispersed in the configuration A (Figure 3.4A). In the configuration B, the column is filled with solvent and the broth is dispersed (Figure 3.3 and 4B). The solvent of configuration C and D has a higher density than broth (Figure 3.4C and D). The column is filled with broth and the solvent is dispersed in configuration C (Figure 3.4C). In configuration D, the column is filled with solvent and the broth is dispersed (Figure 3.4D). The configuration of the KARR® column, the flow rate of the solvent and feed, the stroke rate of the plates stack shaft (agitation speed) and the material that the plates stack shaft is made of are experimentally determined.   72 Figure 3.4. KARR® Column Configuration10 4. Tangential (Cross) Flow Filtration Employment of a tangential (cross) flow filtration shortens the time required to remove cells and proteins from fermentation broth. In a tangential flow filtration system, the feed stream flows perpendicularly to pores of the filter (Figure 3.5A).12 The retentate is recirculated as a portion of the feed stream to complete filtration without clogging the filter with cells or proteins. In a dead-end filtration system, the feed stream flows parallel to the pores of the filter resulting in a buildup of cells or proteins on the filter (Figure 3.5B),12 which slows and eventually halts filtration.   73 Figure 3.5. A: Tangential (Cross) flow filtration, B: Dead-End Filtration A hollow fibers cartridge is made of multiple straw-shaped filters (Figure 3.6A). The feed stream enters the one end of straws and the retentate comes out from the other end. Each straw possesses numerous pores that can separate various particle by their sizes (Figure 3.6B). Figure 3.6. Hollow Fibers In this chapter, microbial synthesis of shikimic acid from glucose is scaled up to 20 L scale. Downstream processing of the fermentation broth employs tangential flow filtration to remove cells and proteins followed by a countercurrent extraction of shikimic acid with a KARR® column. 1-Butyl-3-methylimidazolium bromide is employed as both solvent and catalyst in the scaled up synthesis of biobased p-hydroxybenzoic acid from the microbially synthesized shikimic acid.   74 5. Results 5.1. Shikimic Acid Titers and Yield The shikimic acid synthesizing strain,1 E. coli SP1.1/pKD15.071B was cultured at 20 L scale in a 30 L working volume fermentor at pH 7.0 and 33 °C. Glucose-rich culture conditions were employed such that the concentration of glucose in the medium was maintained in the range of 19-43 g/L throughout the run. The stir and airflow rate were allowed to vary in order to maintain dissolved O2 level at 10% of air saturation. The air flow was supplemented with O2 when the stir and airflow rate could not maintain dissolved O2 levels. This 20 L cultivation of E. coli SP1.1/pKD15.071B was run for 61 h and achieved a shikimic acid titer of 72 g/L synthesized in a 34% yield from glucose. The final fermentation broth volume was 30 L after addition of glucose solution, NH4OH and 2N H2SO4 to control pH. Prior to isolation and purification of shikimic acid, cells were removed from crude fermentation broth by filtration through a hollow fiber membrane column (GE Healthcare). Protein was then removed using a tangential flow filtration apparatus fitted with four 10 kD ultrafiltration cassettes (Sartorius). 5.2. Shikimic Acid Extraction Solvent Screening The partition coefficient of shikimic acid in several solvents was measured (Table 3.1). Cell-free, protein-free shikimic acid fermentation broth was concentrated by boiling off water to approximately 1/5 of the original broth volume and then acidified with H2SO4 to pH 2.5. The final volume was adjusted with water to 1/4 of original broth volume. Each extraction was performed in a vial with 0.5 mL concentrated broth and 0.5 mL solvent. The solvent and the broth were mixed by continuously rotating vertically (7 rotations/min) for 12 h using a hybridization incubator. The shikimic acid concentration of extracts and raffinates were   75 determined by HPLC. The partition coefficient of shikimic acid was calculated using Formula 1. Formula 1. Partition Coefficient Partition Coefficient = !"  $%  &'()*+( !"  $%  )*,,$%*(& The total of 14 solvents were tests including nine alcohols, three ketones and two carboxylic acids (Table 3.1). Methanol, ethanol, n-propanol, isopropanol, acetone, acetic acid and propionic acid are miscible with water. Surprisingly, not only could n-propanol, isopropanol and acetone work as extraction solvents, they were the best three solvents in the screening experiments (Table 3.1, entry 6, 7 and 10). These three solvents were miscible with a fermentation broth when it was not concentrated. However, highly concentrated fermentation broth led to a twophase mixture upon addition of the water-miscible solvents. Table 3.1. Shikimic Acid Partition Coefficient in Various Solvents entry solvent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 methanol ethanol n-butanol 2-butanol isobutanol n-propanol isopropanol n-hexanol cyclohexanol acetone 4-methyl-2-pentanone 2-butanone acetic acid propionic acid relative polarity13a 0.762 0.654 0.586 0.506 0.552 0.617 0.546 0.559 0.509 0.355 0.327 0.269 0.648 0.611 solubility in H2O13b (g/L at pH 2) 234 183 48 68 68 100 141 9 44 95 12 47 197 96 partition coefficient no separation no separation 0.192 0.234 0.161 0.466 0.627 0.055 0.181 0.957 0.113 0.100 no separation no separation 5.3. Countercurrent Extraction of Shikimic Acid The configuration of the KARR® column, the flow rate of the solvent and feed, the stroke rate of the plates stack shaft (agitation speed) and the impact of the materials used to   76 construct the plates stack shaft among other valuable were experimentally determined. There are some clues that assist in making these decisions. The guidelines to make the decisions were collected and summarized in Table 2.10,11 If following suggestion of the table 2 does not solve a coalesce, a different solvent should be used (Table 3.2, entry 1). A rag layer is a layer of solids that builds up at the interface. This might be prevented by reversing phases (Table 3.2, entry 2). If the solids are impurities, the rag layer may be filtered and the filtrate is recirculated into the column. When a band of droplets builds up at the interface (emulsion band), this indicates that there is not enough time to let the dispersed phase settle. Reversing phases, slowing down the flow rates or reducing agitation rate will give the dispersed phase more time to settle at the interface (Table 3.2, entry 3). When a phase exits from the wrong exit port, it is called entrainment. Reducing flow rates and/or reducing agitation rate will prevent an entrainment. Alternatively, a mixture of two phases can be separated outside of the column (Table 3.2, entry 4). If a second interface forms in the column (flooding), the flow rate should be increased or agitation rate should be reduced (Table 3.2, entry 5). Table 3.2. Problems Seen During KARR® Column Optimization10,11 entry   Problem Description 1 Coalesce dispersed phase is fused 2 Rag layer solids built up at the interface 3 Emulsion band a band of droplets built up at interface 4 Entrainment a phase exits from the from the wrong exit port 5 Flooding second interface forms in the column 77 Suggestion Increase agitation speed Change flow rate Reverse phases Change solvent Reverse phases Filter the rag layer liquid and recirculate Reverse phases Slow down flow rate Reduce agitation speed Reduce flow rate Reduce agitation speed Separate into two phase outside of column Increase the flow rate Reduce agitation speed The cell-free, protein-free shikimic acid fermentation broth was concentrated by boiling off water to approximately 20% of the original broth volume. Concentrated H2SO4 was then added to adjust the concentrated fermentation broth to pH 2.5. Water was subsequently added so that the final volume of the concentrated, pH-adjusted solution was 25% of the starting fermentation broth volume. A KARR® column (Figure 3.3) was used to extract shikimic acid from the concentrated fermentation broth. Water-miscible solvents (t-butanol, acetone, npropanol, isopropanol) and water-immiscible solvents (n-butanol, 2-butanol) were examined as extracting solvents. Acidified concentrated broth (100 mL) containing shikimic acid (30 g) was subjected to countercurrent extraction using n-butanol, 2-butanol and t-butanol. t-Butanol was melted before use. A broth feed flow rate of 5 mL/min and a solvent flow rate of 40 mL/min were used (Figure 3.3). The % recovery of shikimic acid from the fermentation broth for nbutanol, 2-butanol and t-butanol were 57%, 60% and 73% respectively (Table 3.3). The acidified concentrated broth (100 mL) containing shikimic acid (30 g) was also submitted to countercurrent extraction using n-propanol, acetone and isopropanol. A broth feed flow rate of 5 mL/min and a solvent flow rate of 10 mL/min were used in these instances (Figure 3.3). The % recovery of shikimic acid from the fermentation broth for n-propanol, acetone and isopropanol were 77%, 80% and 80% respectively (Table 3.3). These water-miscible solvents led to a thick, doughy raffinate that clogged the countercurrent extraction column resulting in termination of the extraction during scale-up. The % recovery of the isopropanol and acetone extractions were better than n-propanol. The clogging problem was worse in acetone extraction than isopropanol. Therefore, isopropanol was chosen as the extraction solvent. An isopropanol/water (9:1, v/v) solution was used as the extraction solvent. Addition of water to the isopropanol eliminated the problem of raffinate clogging of the countercurrent extractor. Finally, acidified concentrated   78 broth (1000 mL) containing shikimic acid (272 g) was submitted to countercurrent extraction using isopropanol/water, 9:1, v/v. A broth feed flow rate of 10 mL/min and a solvent flow rate of 20 mL/min were used (Figure 3.3). This extraction with isopropanol/water, 9:1, v/v solution achieved a 95% recovery (Table 3.3). Table 3.3. Countercurrent Extraction of Microbe-Synthesized Shikimic Acid Entry Solvent 1 2 3 4 5 6 n-butanol 2-butanol t-butanol n-propanol acetone isopropanol isopropanol/water, 9:1, v/v 7 Shikimic Acid (g) in Broth 30 30 30 30 30 30 Shikimic Acid (g) Extracted 17 18 22 23 24 24 Shikimic Acid % Recovery 57 60 73 77 80 80 272 259 95 5.4. Comparison of Shikimic Acid Crystallization Solvents n-Butanol, 2-butanol, n-propanol, isopropanol, t-butanol and acetone containing varying contents of shikimic acid (Table 3.3) were concentrated using a rotary evaporator until formation of the first solid was observed, and crystallization subsequently allowed to proceed at rt overnight. The amount of shikimic acid obtained, % recovery, and purity as a function of solvent is summarized in Table 2. n-Butanol resulted in the highest % recovery (75%) and the best purity (94.3%) among these solvents (Table 3.4). No precipitate of shikimic acid was observed after standing at rt overnight for t-butanol and acetone extracts.   79 Table 3.4. % Purity and % Recovery of Crystallized Shikimic Acid Entry Solvent (azeotrope°C, wt% water)14 1 Shikimic Acid in Extract (g) Isolated (g) % Recovery %Purity n-butanol (93 °C, 43%) 17.0 13.6 75 94.3 2 2-butanol (87 °C, 27%) 17.5 9.5 51 93.1 3 n-propanol (88 °C, 29%) 23.3 8.8 34 89.4 4 isopropanol (80 °C, 13%) 24.4 12.2 47 94.1 5.5. Purification of Shikimic Acid The cell-free, protein-free shikimic acid fermentation broth 4 L was aliquoted from 30 L broth. A concentrated, pH-adjusted solution 1 L was prepared as aforementioned. Shikimic acid was extracted from this concentrated, pH-adjusted solution to isopropanol/water, 9:1, v/v with a KARR® column. Isopropanol/water, 9:1, v/v extract (2910 mL) containing shikimic acid (257 g) was decolorized with activated charcoal (10 wt% of shikimic acid). This solution was filtered and then concentrated. The residue was dissolved in boiling water (250 mL) prior to addition of n-butanol (1500 mL). Azeotropic removal of water using a rotary evaporator was conducted until crystallization was first observed and the solution immediately poured into a crystallization dish to afford the first crop of shikimic acid. During the azeotropic removal of water, a dark, brown-colored solid formed gradually at the bottom of the stripping flask. This was kept in the stripping flask when the crystallization solution was poured out. The dark, brown-colored solid remaining in the stripping flask was dissolved in boiling water (100 mL). n-Butanol (550 mL) was then added to the solution. The water was once again removed using a rotary evaporator until crystallization just began. The solution was poured into a crystallization dish to afford a   80 second crop of shikimic acid. The first and second crops of crystallized shikimic acid were collected by filtration after solutions of crystallizing shikimic acid were allowed to stand at rt overnight. For the first crop, 160 g of shikimic acid was obtained (61% recovery) with a purity of 98.4%. For the second crop, 36 g of shikimic acid was obtained (14% recovery) with a purity of 97.9%. This overall process afforded 196 g of shikimic acid (75% recovery). 5.6. Scale Up Synthesis of p-Hydroxybenzoic Acid Scheme 3.4. Dehydration of Shikimic Acid CO2H HO 20 mol% H2SO4 OH OH 1 nBu N Br Me 90 ℃,24 h N CO2H CO2H OH OH 2 3 The dehydration reaction was scaled up to 250 g of shikimic acid 1 in 1-butyl-3methylimidazolium bromide. 1-Butyl-3-methylimidazolium was synthesized in situ from 1methylimidazole and 1-bromobutane. Shikimic acid in 1-butyl-3-methylimidazolium bromide (40 wt%) was heated at 90 °C with 20 mol % H2SO4 for 24 h. The headspace of the reaction vessel was swept with a vigorous flow of N2. This reaction afforded a 74% yield of phydroxybenzoic acid 2 and a 19% yield of m-hydroxybenzoic acid 3. The p-hydroxybenzoic acid 2 and m-hydroxybenzoic acid 3 were extracted into EtOAc with a continuous liquid-liquid extractor (Figure 3.7) followed by decolorization with activated charcoal. Concentration of the decolorized extract followed by crystallization in water afforded 64% p-hydroxybenzoic acid 2 as a white solid with a purity of 98.9%.   81 Figure 3.7. Continuous Liquid-Liquid Extractor 6. Discussion The scale-up of the microbial synthesis of shikimic acid to 20 L scale employed O2supplemented air to afford a shikimic acid concentration in the fermentor broth of 72 g/L synthesized in 34% yield from glucose. Hollow fiber filtration of the 30 L of fermentation broth removed E. coli SP1.1/pKD15.071B cells followed by tangential flow filtration of the cell-free fermentation broth to remove protein resulting from cell lysis. The both filtration processes are suitable for 30 L or higher volume fermentation broth. With respect to reported strategies for isolation of shikimic acid, Royal DSM performed countercurrent extraction of shikimic acid from concentrated fermentation broth residue using 2butanone.15 The DSM process began with the removal of water in the fermentation broth and ended with the crystallization of shikimic acid, achieving a 57% overall recovery of shikimic acid with 96% purity. During their countercurrent extraction, 1.2 L of the extraction solvent, 2butanone, was used to extract only 10 g of shikimic acid.15 This use of a large volume of solvent for isolation of a small quantity of shikimic acid is not practical. Unlike DSM’s shikimic acid   82 extraction method, the countercurrent extraction using isopropanol/water, 9:1, v/v required only 77 mL of the solvent per 10 g of shikimic acid. Starting from concentration of fermentation broth to crystallization of shikimic acid, this novel process achieved a 71% overall recovery of shikimic acid with 98% purity. The dehydration reaction of shikimic acid was successfully scaled up with a nearly quantitative conversion into p-hydroxybenzoic acid (74%) and m-hydroxybenzoic acid (19%). The isolated yield of p-hydroxybenzoic acid was 64% with a purity of 98.9%. The impurity in the isolated product was m-hydroxybenzoic acid (1%). The mother liquor from the crystallization contained p-hydroxybenzoic acid (10%) and m-hydroxybenzoic acid (17%). m-Hydroxybenzoic acid is often used as one of copolymers in LCPs.16 Therefore, the mixture of p-hydroxybenzoic acid and m-hydroxybenzoic acid in the mother liquor can be used in polymerizations to afford melt-processible LCPs.   83 7. Experimental 7.1. General 1 H NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to residual non-deuterated solvent in CDCl3 (δ = 7.26 ppm) and CD3OD (δ = 3.34 ppm). Chemical shifts for 1H NMR spectra in D2O is reported (in parts per million) relative to residual non-deuterated sodium salt of 3(trimethylsilyl) propionic-2,2,3,3-d4 acid, TSP (δ = 0.00 ppm). 13 C NMR spectra were recorded at 125 MHz and chemical shifts for these spectra were reported (in parts per million) relative to CDCl3 (δ = 77.4 ppm) and CD3OD (δ = 49.9 ppm). 1-methylimidazole, 1-bromobutane and 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid were purchased from Sigma-Aldrich. 7.2. Product Analysis HPLC analysis was performed on an Agilent 1100. Shikimic acid was quantified by HPLC using a Grace AlltimaTM amino column (4.6 x 250 mm, 5 μm particle size) and isocratic elution with 60/40, (v/v); CH3CN/H2O (140 mM NH4+HCO2−, pH 4.0). The amino column was stored in a solution of 5/95, (v/v); methanol/hexane when it was received from the manufacture. In order to convert the column to weak base ion-exchange mode, the column was washed with 1 L of reagent grade isopropanol (0.2 mL/min) followed by 1 L of HPLC grade acetonitrile (0.2 mL/min) prior to the first use. Shikimic acid molecular standard was obtained from Roche. pHydroxybenzoic acid and m-hydroxybenzoic acid were separated from one another with baseline resolution and quantified by HPLC using an Agilent ZORBAX SB-C18 (4.6 x 150 mm, 5 μm particle size) column and isocratic elution with 15/85, (v/v); CH3CN/H2O (100 mM NH4+HCO2−,   84 pH 2.5). The ZORBAX was stored in a solution of 85/15, (v/v); methanol/H2O when it was received from the manufacture. The column was washed with 1 L of HPLC grade acetonitrile prior to the first use. p-Hydroxybenzoic acid molecular standard was obtained from Spectrum while the m-hydroxybenzoic acid molecular standard was obtained from Sigma-Aldrich. The deionized, distilled water used to make the required buffer solutions was filtered with a DURAPORE® 0.45 µm HV filter (MILLIPORE). The acetonitrile was HPLC grade. Syringe filtration (0.45 µm Whatman filter) was followed by HPLC analysis for all the sample. 7.3. Culture Medium All solutions were prepared in distilled, deionized water. M9 salts (1 L) contained Na2HPO4 (6 g), KH2PO4(3 g), NH4Cl (1 g), and NaCl (0.5 g). M9 minimal medium (1 L) contained D-glucose (10 g), MgSO4 (0.24 g), thiamine pyrophosphate (0.010 g), L-phenylalanine (0.040 g), L-tyrosine (0.040 g), L-tryptophan (0.040 g), p-hydroxybenzoic acid (0.010 g), paminobenzoic acid (0.010 g), and 2,3-dihydroxybenzoic acid (0.010 g) in 1 L of M9 salts. Solutions of M9 salts, MgSO4, and glucose were autoclaved individually and then mixed. Solutions of amino acids, aromatic vitamins and thiamine pyrophosphate were sterilized through 0.22 μm membranes. Solid medium was prepared by addition of BactoTM agar to a final concentration of 1.5% (w/v) to the liquid medium. The fermentation medium was prepared with water (16 L), K2HPO4 (150 g), ammonium iron (III) citrate (6 g), citric acid monohydrate (42 g), concentrated H2SO4 (24 mL), concentrated NH4OH (82 mL), L-phenylalanine (14 g), L-tyrosine (14 g), and L-tryptophan (7 g). This solution was sterilized in the fermentor using a built-in sterilization function. The solution (2 L) containing following supplements were added immediately prior to initiation of the fermentation: glucose (600 g), MgSO4 (2.4 g), thiamine hydrogenchloride (0.010 g), p-hydroxybenzoic acid   85 (0.100 g), p-aminobenzoic acid (0.100 g), 2,3-dihydroxybenzoic acid (0.100 g) and trace minerals, including (NH4)6(Mo7O24)·∙4H2O (0.037 g), ZnSO4·∙7H2O (0.029 g), B(OH)3 (0.247 g), CuSO4·∙5H2O (0.025 g), and MnCl2·∙4H2O (0.158 g). Glucose and MgSO4 (1 M) were autoclaved separately, while solutions of aromatic vitamins, thiamine pyrophosphate and trace minerals were sterilized through 0.22 μm membranes. 7.4. Bacteria Strain and Plasmid E. coli SP 1.1 was previously engineered in the group.6b,c Plasmid pKD15.071B, which was constructed previously in the group6a was transformed into E. coli SP1.1. 7.5. Fed-Batch Fermentation (General) A B. Braun BIOSTAT® C-DCU fermentor (30 L) connected to a DCU-3 system was used for fed-batch fermentations at 20 L scale. Data acquisition utilized MFCS/win 3.0 software (Sartorius Stedim Systems), which was installed in a personal computer (Digilink) operated by Windows® 7 Professional. Matson Marlow 101U/R peristaltic pump was used for administration of feed glucose solution. Temperature and pH were controlled with PID loop maintaining at 33 °C and pH 7.0±0.1 with 2N H2SO4 and NH4OH. Impeller speed was varied between 100 and 600 rpm to maintain dissolved oxygen (D.O.) levels at 10% air saturation. Airflow was increased from 0.5 L/min to 20 L/min to maintain dissolved oxygen (D.O.) levels at 10% air saturation. The air was supplemented with pure oxygen when the 20 L/min air flow could not provide enough oxygen. DO was measured using a Hamilton OxyFerm FDA 120 (catalogue number 237450) fitted with an Optiflow FDA membrane (catalogue number 237140). pH was measured with Mettler Toledo 405-DPAS-SC-K85/120 (catalogue number 104054479). The concentration of glucose in the medium was maintained at glucose-rich culture conditions   86 defined as a glucose concentration in the range of 19-43 g/L. Glucose levels were maintained throughout the run by addition of feed glucose solution (60% w/v). Glucose concentration was measured with GlucCellΤΜ and glucose test strip (CESCO Bioengineering Co., Ltd.). Antifoam 204 (Sigma-Aldrich) was added as needed to mitigate foam accumulation. Inoculant was started by introduction of a single colony picked from an agar plate into 5 mL of M9 medium. The culture was grown at 37 °C with agitation at 250 rpm until turbid (24h) and subsequently transferred to 100 mL of M9 medium. This 100 mL culture was grown at 37 °C and 250 rpm until the OD600 reached 2.0-3.0 (12 h). Two flasks containing 1 L of M9 media were inoculated with a portion of this 100 mL culture such that the initial OD600 = 0.05. These two 1 L cultures were grown at 37 °C and 250 rpm until the OD600 reached 1.0-2.0 (9-11 h). The fermentation vessel was inoculated with these two 1 L of cultures thus initiating the fermentation run (t = 0 h). 7.6. Fed-Batch Fermentation of Shikimic Acid The supplement solution (2 L) was added to the fermentation medium (16 L) followed by inoculation with the two 1 L inoculants. The initial volume of fermentation broth was 20 L with 30 g/L glucose concentration. The concentration of glucose in the medium was maintained in the range of 19-43 g/L throughout the run by addition of feed glucose solution (60% w/v). Oxygen supplementation was required at t = 16-22 h in order to maintain dissolved oxygen (D.O.) levels at 10% air saturation. The cell growth reached stationary phase at t = 30 h. A total of 6.6 kg of glucose was consumed after 61 h based on 1H NMR. The broth volume increased gradually due to cell growth, product, addition of glucose solution and NH4OH (1600 mL), which was used to adjust pH. Final broth volume was approximately 30 L. For 1H NMR   87 quantification of shikimic acid, the broth was concentrated to dryness under reduced pressure, concentrated to dryness one additional time from D2O, and then dissolved in D2O containing a known concentration of the sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid. The concentration of shikimic acid was determined by comparison of integral corresponding to shikimic acid (2.75 ppm, ddt, J = 18.1, 5.3, 1.6 Hz, 1H) with integral corresponding to TSP (δ = 0.00 ppm, s, 9H). The instrument setting was split into two phase. During first phase, DO was maintained at 10 % by impeller speed primarily until the impeller speed reached to the maximum (600 rpm). And then airflow was increased until it reached to the maximum (20.0 L/min). The pO2 control loop setting for first phase was following (Table 3.5.) : Table 3.5. Instrument Setting of First Phase DEADB 0.5% sat CASCADE 1 STIR 2 AIRF Cascade Minimum Maximum XP TI STIR 5.0% 30.0% 150.0% 100.0 s AIRF 1.2% 40.0% 90.0% 50.0 s SUBS 0.0% 100.0% 90.0% 50.0 s O2EN 0.0% 100.0% 90.0% 50.0 s STIR: 5.0% = 100 rpm, 30.0% = 600 rpm AIRF: 0.2% = 1.00 L/min, 40.0% = 20.00 L/min SUBS setting did not matter since it was off. TD 0.0 s 0.0 s 0.0 s 0.0 s Hysterese 01:00 m:s 01:00 m:s 05:00 m:s 05:00 m:s Mode auto auto off auto The cascade setting was altered to second phase when both impeller speed and airflow reached to their maximum. During second phase, DO was maintained at 10 % by impeller speed while the airflow was held at 20.0 L/min. The airflow was supplemented with O2 automatically when the impeller speed was not sufficient to maintain DO at 10%. The pO2 control loop setting for second phase was following (Table 3.6.) :   88 Table 3.6. Instrument Setting of Second Phase DEADB 0.5% sat CASCADE 1 AIRF 2 STIR Cascade Minimum Maximum XP STIR 5.0% 30.0% 150.0% AIRF 40.0% 40.0% 90.0% SUBS 0.0% 100.0% 90.0% O2EN 0.0% 100.0% 90.0% STIR: 5.0% = 100 rpm, 30.0% = 600 rpm AIRF: 40.0% = 20.00 L/min SUBS setting did not matter since it was off. TI 100.0 s 50.0 s 50.0 s 50.0 s TD 0.0 s 0.0 s 0.0 s 0.0 s Hysterese 01:00 m:s 01:00 m:s 05:00 m:s 05:00 m:s Mode auto auto off auto The antifoam was added automatically and manually as needed. The position of foam sensor was gradually raised while total broth volume was increasing. The auto-antifoam setting was following (Table 3.7.) : Table 3.7. Auto-antifoam Setting SENSI CYCLE PULSE Med-High 15:00 00:01 7.7 Cell Removal Raw fermentation broth was filtered with a hollow fiber cartridge (GE Healthcare xFP-xx-55), equipped with a portable utility/sprinkler pump (Simer 2825ss) to remove cells. The hollow fiber cartridge passes cell free broth through 0.1 µm cutoff membrane and recirculates the retentate through 1 mm diameter hollow fibers. When the retentate became approximately 2 L, it was not enough volume to sustain the broth flow. The retentate was diluted with 2 L of water to increase the volume and reduce the viscosity. The diluted retentate was filtered until the flow ceased. This filtration afforded 30.1 L of cell free broth containing 2,160 g of shikimic acid.   89 7.8. Protein Removal The protein in the cell free broth was removed with four Sartocon® ECO Hydrostart® membrane cut off 10 kD cassettes equipped with SartoJet® Pump 4-Piston Diaphragm Pump. This tangential flow filtration of cell free broth was performed with the pressure toward the membrane of 25±5 psi and a back pressure of 6±1 psi. When the retentate became approximately 1 L, it was not enough volume to sustain the broth flow. The retentate was diluted with 2 L of water to increase the volume and reduce the viscosity. The diluted retentate was filtered until the flow ceased. 7.9. Shikimic Acid Extraction Solvent Screening in Vials Cell-free, protein-free shikimic acid fermentation broth was concentrated by boiling off water to approximately 1/5 of the original broth volume and then acidified with H2SO4 to pH 2.5. The final volume was adjusted with water to 1/4 of original broth volume. The extraction was performed in a vial (8 ´ 25 mm). Each vial contained 0.5 mL concentrated broth and 0.5 mL solvent. The solvent and the broth were mixed by continuously rotating vertically (7 rotations/min) for 12 h using a hybridization incubator. The shikimic acid concentration of extracts and raffinates were determined by HPLC. 7.10. KARR® Column General The bench-top KARR® column (220 mL) was manufactured by Koch Modular Process Systems, KMPS). A Teflon and a stainless-steel plate stack assembly built on stainless steel shafts were supplied with the KARR® column. All the KARR® column extraction experiment utilized the Teflon plates stack shaft. The solvent, broth feed and raffinate flow rates were regulated with QD High Speed-High Flows Metering Pump (Fluid Metering Inc.).   90 7.11. Comparison of Shikimic Acid Extraction Solvents in KARR® Column Cell-free, protein-free shikimic acid fermentation broth was concentrated by boiling off water to approximately 1/5 of the original broth volume, and then acidified with H2SO4 to pH 2.5. The final volume was adjusted with water to 1/4 of original broth volume. This concentrated broth (100 mL) containing shikimic acid (30 g) was subjected to countercurrent extraction using n-butanol, 2-butanol and t-butanol. t-Butanol was melted before use. A broth feed flow rate of 5 mL/min and a solvent flow rate of 40 mL/min were used. The amount of extracted shikimic acid from the fermentation broth to n-butanol, 2-butanol and t-butanol were 17 g (57%), 18 g (60%) and 22 g (73%) respectively based on HPLC. The concentrated broth (100 mL) containing shikimic acid (30 g) was additionally submitted to countercurrent extraction using n-propanol, acetone and isopropanol. A broth feed flow rate of 5 mL/min and a solvent flow rate of 10 mL/min were used. The amount of extracted shikimic acid from the fermentation broth to n-propanol, acetone and isopropanol were 23 g (77%), 24 g (80) and 24 g (80%) respectively based on HPLC. Finally, the concentrated broth (1000 mL) containing shikimic acid (272 g) was submitted to countercurrent extraction using isopropanol/water, 9:1, v/v. A broth feed flow rate of 10 mL/min and a solvent flow rate of 20 mL/min were used. The amount of extracted shikimic acid from the fermentation broth to isopropanol/water, 9:1, v/v solution was 259 g (95%) based on HPLC. 7.12. Comparison of Shikimic Acid Crystallization Solvents Extracts contained shikimic acid as a function of solvent were as follows: n-butanol 17.0 g, 2-butanol 17.5 g, n-propanol 23.3 g, isopropanol 24.4 g, t-butanol 22.1 g and acetone 23.6 g. The extract was vacuum filtered through Whatman® #1 filter paper to remove particulate. The   91 extracts were concentrated using a rotary evaporator until formation of the first solid was observed, and crystallization subsequently allowed to proceed at rt overnight. The crystallized solid was collected by filtration and washed with the extraction solvent followed by acetone. After drying under vacuum, the amount of shikimic acid obtained, % recovery, and purity as a function of solvent were: n-butanol 13.6 g (75% recovery, 94.3% purity), 2-butanol 9.5 g (51% recovery, 93.1% purity), n-propanol 8.8 g (34% recovery, 89.4 % purity), isopropanol 12.2 g (47% recovery, 94.1 % purity). No precipitate of shikimic acid was observed after standing at rt overnight for t-butanol and acetone extracts. 7.13. Decolorizing Isopropanol/Water (9:1 v/v) Extract and Evaporative Crystallization from n-Butanol of Shikimic Acid Isopropanol/water. 9:1, v/v extract (2910 mL) containing shikimic acid (257 g) was separated into three 2 L Erlenmyer flasks. Activated charcoal (DARCOTM G-60, decolorizing, ACROS OrganicsTM, 8.6 g) was added to each flask. The flasks were shaken on an orbital shaker at 150 rpm at rt for 1 h. The activated charcoal was removed by vacuum filtration through 64 g of Celite 545 in an OD 17 cm Buchner funnel fitted with Whatman #1 filter paper. This filtrate was concentrated using a rotary evaporator. The residue was dissolved in boiling water (250 mL), and then n-butanol (1500 mL) was added. The water was removed azeotropically with a rotary evaporator until crystallization began. The solution was poured into a crystallization dish to afford a first crop of shikimic acid. A dark, brown-colored solid remained in the stripping flask, which was dissolved in boiling water (100 mL). n-Butanol (550 mL) was added to the solution. The water was again removed azeotropically with a rotary evaporator until crystallization began. The solution was poured into a crystallization dish to afford a second crop   92 of shikimic acid. After overnight crystallization, the crystallized solid was collected by filtration and washed with cold n-butanol followed by acetone. The solid was dried under vacuum to afford shikimic acid, which was analyzed with HPLC. For the first crop, 160 g of off-white colored shikimic acid was obtained (61% recovery) with a purity of 98.4%. For the second crop, 35.6 g of off-white shikimic acid was obtained (14% recovery) with a purity of 97.9%. This overall process afforded 196 g of off-white shikimic acid (75% recovery, 98.1% purity). 1H NMR (D2O, TSP δ = 0.00) δ = 6.80 (dt, J = 3.8, 1.8 Hz, 1H), 4.45 (td, J = 4.0, 1.9 Hz, 1H), 4.04 (ddd, J = 8.3, 6.5, 5.2 Hz, 1H), 3.78 (dd, J = 8.2, 4.3 Hz, 1H), 2.75 (ddt, J = 18.1, 5.3, 1.6 Hz, 1H), 2.23 (ddt, J = 18.2, 6.6, 1.5 Hz, 1H). 13 C NMR (D2O, TSP δ = 0.00) δ = 175.0, 137.9, 134.9, 74.3, 69.6, 68.9, 34.1. mp: 1st crop 182-184 °C, 2nd crop 184-185 °C (lit.17 184-185 °C). 7.14. Scale-up Dehydration of Shikimic Acid in 1-Butyl-3-methylimidazolium Bromide Ionic liquid 1-butyl-3-methylimidazolium bromide was synthesized in situ. 1Bromobutane (491 mL, 4.57 mol) was added to 228 mL (2.86 mol) of 1-methylimidazole in a 2 L three-neck round bottom flask heated in a 70 °C oil bath fitted with two reflux condensers open to the atmosphere. A vigorous exothermic reaction was observed after heating at 70 °C for approximately 15 min. The flask was removed from the oil bath until the vigorous exothermic reaction ceased and then placed back in the 70 °C oil bath for 20 h. Unreacted 1-bromobutane and water were removed by distillation under reduced pressure at 55 °C. This afforded 1-butyl3-methylimidazolium bromide (619 g, 99%) as a yellow oil. 1H NMR (CDCl3) δ = 10.6 (s, 1H), 7.32 (dd, J = 3.5, 2 Hz, 1H), 7.25 (dd, J = 3.5, 2 Hz, 1H), 4.31 (dd, J1 = J2 = 7.5 Hz, 2H), 4.11 (s, 3H), 1.86-1.92 (m, 2H), 1.37 (m, 2H), 0.950 (dd, J = 7.5, 7.0 Hz, 3H). 138.2, 123.6, 122.0, 50.3, 37.1, 32.5, 19.8, 13.8.   93 13 C NMR (CDCl3) δ = In situ synthesized 1-Butyl-3-methylimidazolium bromide ([BMIm]Br) was first dried at 55 °C under vacuum in the 2 L three-neck round bottom flask in which this ionic liquid was generated. The flask containing dry [BMIm]Br (618 g, 3.23 mol) was opened under N2 and shikimic acid (purity 94%, 262 g, 1.42 mol) was added. The flask was fitted with an overhead stirrer and two gas adaptors. The reaction vessel was swept with an active N2 flow. The reaction solution temperature was maintained at 90 °C. After the shikimic acid dissolved, H2SO4 (15.1 mL, 283 mmol) was added. The reaction solution was heated at 90 °C for 24 h under an active flow of N2 to afford 145 g (74%) of p-hydroxybenzoic acid and 37.2 g (19%) of mhydroxybenzoic acid based on HPLC analysis. The reaction solution was diluted with H2O (1000 mL) and separated into two portions. Each portion was extracted with EtOAc (1400 mL) for 18 h in a continuous liquid-liquid extractor. The brown EtOAc extracts were then combined and it contained 140 g of p-hydroxybenzoic acid (71%), and 36.7 g of m-hydroxybenzoic acid (19%) based on HPLC analysis. The brown organic extract was concentrated to approximately 1.5 L. This solution was decolorized using continuous flow conditions. A decolorizing column (D = 8.0 cm) was constructed with a piece of Kimwipe to plug the column outlet below a layer of 75 g Celite® 545 topped with a layer consisting of 75 g activated charcoal (DARCO® G-60, 100 mesh) dispersed in 375 g flash chromatography grade silica gel (60 Å pore size, 40-63μm mesh). After loading, the decolorizing column was washed with an EtOAc/AcOH, 98.6:1.4, v/v solution. Concentrating this solution gave a gray solid. This gray solid was dissolved in 1000 mL boiling H2O, crystallized at rt, filtered, and dried to afford p-hydroxybenzoic acid 126 g (64%) as a white solid with purity 98.9% based on HPLC analysis. 1H NMR (CD3OD): δ = 7.897.92 (m, 2H), 6.83-6.86 (m, 2H). 13 C NMR (CD3OD) δ = 170.9, 164.2, 133.9, 123.6, 116.9. mp: 208-212 °C (Lit.18 210-211 °C).   94 REFERENCES   95 REFERENCES 1.   Draths, K. M.; Knop, D. R.; Frost, J. W. Shikimic acid and quinic acid: replacing isolation from plant sources with recombinant microbial biocatalysis. J. Am. Chem. Soc. 1999, 121, 1603-1604. (b) Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von Daeniken, R.; Weber, W.; Frost, J. W. Hydroaromatic equilibration during biosynthesis of shikimic acid. J. Am. Chem. Soc. 2001, 123, 10173-10182. (c) Chandran, S. S.; Yi, J.; Draths, K. M.; von Daeniken, R.; Weber, W.; Frost, J. W. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol. Prog. 2003, 19, 808-814. 2.   (a) Herrmann, K. M. The Common Aromatic Biosynthetic Pathway. In Amino Acids: Biosynthesis and Genetic Regulation. Herrmann, K. M., Ed.; Somerville, R. L., Ed.; Addison-Wesley: Massachusetts, 1983; p301-322. (b) Roberts, F.; Roberts, C. W.; Johnson, J. J.; Kyle, D. E.; Krell, T.; Coggins, J. R.; Coombs, G. H.; Milhous, W. K.; Tzipori, S.; Ferguson, D. J. P.; Chakrabarti, D.; McLeod, R. Evidence for the shikimate pathway in apicomplexan parasites. Nature 1998, 393, 801-805. (c) Bentley, R. The shikimate pathwayA metabolic tree with many branches. Crit. Rev. Biochem. Molbiol. 1990, 25, 307-384. 3.   (a) Garner, C.; Herrmann, K. M. Biosynthesis of Phenylalanine. In Amino Acids: Biosynthesis and Genetic Regulation. Herrmann, K. M., Ed.; Somerville, R. L., Ed.; Addison-Wesley: Massachusetts, 1983; p323-338. (b) Camakaris, H.; Pittard, J. Tyrosine Biosynthesis. In Amino Acids: Biosynthesis and Genetic Regulation. Herrmann, K. M., Ed.; Somerville, R. L., Ed.; Addison-Wesley: Massachusetts, 1983; p339-350. (c) Somerville, R. L. Tryptophan: Biosynthesis, Regulation, and Large-Scale Production. In Amino Acids: Biosynthesis and Genetic Regulation. Herrmann, K. M., Ed.; Somerville, R. L., Ed.; Addison-Wesley: Massachusetts, 1983; p351-378. 4.   (a) Dell, K. A.; Frost, J. W. Identification and removal of impediments to biocatalytic synthesis of aromatics from D-glucose: Rate-limiting enzymes in the common pathway of aromatic amino acid biosynthesis. J. Am. Chem. Soc. 1993, 115, 11581-11589. (b) Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. (c) Frost, J. W.; Bender, J. L. Kadonaga, J. T.; Knowles, J. R.Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme. Biochemistry 1984, 23, 4470-4475. (d) Takeda, Y.; Nishimura, A.; Nishimura, Y.; Yamada, M.; Yasuda, S.; Suziki, H.; Hirota, Y. Synthetic ColE1 plasmids carrying genes for oenicillin-binding proteins in Escherichia coli. Plasmid 1981, 6, 86-98. 5.   Lobner-Olesen, A.; Marinus, M. G. Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli. J. Biotechnol. 1992, 174, 525-529. 6.   (a) Anton, I. A.; Coggins, J. R. Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase. Biochem. J. 1988, 249, 319-326. (b) Anton, J. A.; Coggins, J. R. Subcloning of the Escherichia coli shikimate dehydrogenase gene (aroE) from the transducting bacteriophage spc1. Biochem. Soc. T. 1984, 12, 275-276. (c) Jaskunas, S.   96 R.; Lindahl, L.; Nomura, M. Specialized transducing phages for ribosomal protein genes of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 6-10. 7.   (a) Draths, K. M.; Pompliano, D. L. Conley, D. L.; Frost, J. W.; Berry, A.; Disbrow, G. L.; Staversky, R. J.; Lievense, J. C. Biocatalytic synthesis of aromatics from D-glucose: The role of transketolase. J. Am. Chem. Soc. 1992, 114, 3956-3962. (b) Blackmore, P. F.; Williams, J. F.; MacLeod, J. K. Dimerization of erythrose 4-phosphate. Febs Lett. 1976, 64, 222-226. 8.   (a) Li, K. Kai Li Laboratory Notebook KL I p018-113 1995. (b) Schaub, J.; Mauch, K.; Reuss, M. Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data. Biotechnol. Bioeng. 2008, 99, 1170-1185. 9.   Hecquet, L.; Fessner, W.-D.; Helanine, V.; Charmantray, F. New Applications of Transketolase: Cascade Reactions for Assay Development. In Cascade Biocatalysis; Riva, S.; Fessner, W.-D., Ed.; Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim: Germany, 2014; pp315-337. 10.  (a) Koch Modular Process System, LLC. Liquid Extraction Overview. https://modularprocess.com/liquid-liquid-extraction/ (accessed Jan 04, 2017). (b) Chemical Engineering, College of Engineering, University of Michigan. Extractor. http://encyclopedia.che.engin.umich.edu/Pages/SeparationsChemical/Extractors/Extractors.ht ml#GJ5VCUA23E0CST2FYB5C (accessed Apr 06, 2017). 11.  Suggestion from my own experience. 12.  Spectrum Labs. Competitive Edge. http://spectrumlabs.com/filtration/Edge.html (accessed Mar 25, 2017) 13.  (a) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry, 3rd ed.; Wiley-Vch: Weinheim, 2003; p 418-424. (b) SciFinder, Predicted properties. Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 (©1994-2017 ACD/Labs) 14.  Smallwood, I. M. Solvent Recovery Handbook, 2nd ed.; CRC Press: Boca Raton, 2002. 15.  Van der Does, T.; Booij, J.; Kers, E. E.; Leenderts, E. J. A. M.; Sibeijn, M.; Agayn, V. Process for the recovery of shikimic acid. WO 0206203, Jannuary 24, 2002. 16.  (a) Hanabatake, M. Production method of aromatic polyesters. JP S5657821, May 20, 1981. (b) Layton, R.; Cleay, J. W. Wholly aromatic polyesters with reduced char content. EP 0347228, December 20, 1989. (c) Huspeni, P. J.; Stern, B. A.; Frayer, P. D. Blends of liquid crystalline polymers of hydroquinone poly(iso-terephthalates) p-hydroxybenzoic acid polymers and another LCP containing oxybisbenzene and naphthalene derivatives. WO 9004002, April 04, 1990. (d) Waggoner, M. Liquid crystalline polymer composition. WO 9606888, July 03, 1996. (e) Shepherd, J. P.; Linstid, C. H. III. Stretchable liquid crystal polymer composition. WO 02064661, August 22, 2002. 17.  Payne, R.; Edmonds, M. Isolation of shikimic acid from star aniseed. J. Chem. Educ. 2005,   97 82, 599-600. 18.  Kikuchi, M.; Sato, K.; Shiraishi, Y.; Nakayama, R.; Watanabe, R.; Sugiyama, M. Constituents of Berchemia racemosa Sieb. et Zucc. Yakugaku Zasshi 1990, 110, 354-357.   98 CHAPTER FOUR Synthesis of Biobased Terephthalic Acid 1. Introduction 1,4-Benzenedicarboxylic acid (terephthalic acid) and 1,3-benzenedicarboxylic acid (isophthalic acid) are used in polymers, particularly polyethylene terephthalate (PET). In 2012, the world PET consumption was over 19.8 million tons.1 The global consumption of terephthalic acid was 47 million tons with a market value of $60 billion in 2012.2 Other significant uses of terephthalic acid are in synthetic fibers3 and liquid crystalline polymers (LCPs)4. In PET, 5-7% of isophthalic acid is introduced into the polymer in order to disrupt the crystallization of the molten resin as it cools. This affords a transparent polymer and lowers the melt process temperature.5 Isophthalic acid is also used for alkyd resins, high-temperature-resistant nylon, plasticizers5 and LCPs4. Terephthalic acid and isophthalic acid are currently produced in industry via catalytic oxidation of p-xylene and m-xylene, respectively.6a,b     99 2. Terephthalic Acid and Isophthalic Acid Production Terephthalic acid and isophthalic acid are produced from para- and meta-xylene by the Amoco Mid-Century process, which is an aerobic catalytic oxidation in acetic acid. The reaction is catalyzed by cobalt, manganese and bromide compounds.6a,7 It is a radical process, which is depicted in Scheme 1.6a, 8 In the first step, a bromine radical abstracts a hydrogen from the methyl group of p-xylene 1 producing 4-methylbenzyl radical 2 and HBr. The 4-methylbenzyl radical 2 reacts with oxygen to form the peroxide 3, which becomes 4-methylbenzaldehyde 4 upon oxidizing CoII to CoIII. A bromine radical abstracts another hydrogen from 4methylbenzaldehyde 4 producing radical intermediate 5, which reacts with oxygen to form the peracid 7. This peracid 7 reacts with 4-methylbezylaldehyde 4 producing two molecules of ptoluic acid 10 in a Baeyer-Villiger type reaction via intermediates 8 and 9.8 Repetition of this process upon the other methyl group produces terephthalic acid 11 (Scheme 4.1).6a,8 The role of manganese is to reduce CoIII back to CoII and oxidize bromide back to bromine radical (Scheme 4.2).6a     100 Scheme 4.1. Aerobic Catalytic Oxidation of p-Xylene to Terephthalic Acid Scheme 4.2. Catalytic Pathway for Amoco Mid-Century Oxidation of p-Toluic Acid Commercial production of terephthalic acid utilizes the oxidation of non-renewable p-xylene. A significant shortcoming of this process is the emission of greenhouse gases, CO2 and methane.   101 Overoxidation of p-xylene to CO2 and acetic acid oxidation to both CO2 and methane creates a large carbon footprint.6a Methane has a 25-fold greater impact on climate change relative to CO2 on a wt/wt basis.6c During production of 1000 kg terephthalic acid from p-xylene, 165 kg CO2 is generated. Of this CO2, 40% comes from overoxidation of p-xylene and 60% comes from oxidation of acetic acid.9 Switching to biobased terephthalic acid production, which being with starting materials that have one or both carboxylic acids are already in the place, can reduce the overall emission of greenhouse gas. Several routes of biobased terephthalic acid production have been proposed. 3. Biobased Terephthalic Acid There are two broad routes in the syntheses of biobased terephthalic acid. One involves pxylene intermediacy (Scheme 4.3) while the other does not (Scheme 4.4). An example of a route using p-xylene as an intermediate proceeds through biobased isobutanol 13, which is microbially synthesized from glucose.10 It can then be dehydrated to isobutene11,12 14 or oxidized to isobutanal13 16 (Scheme 4.3). Dimerization of isobutene 14 produces 2,4,4-trimethylpentene 15,11 which undergoes catalytic dehydrocyclization to p-xylene.12 Isobutanol 13 can also be reacted with isobutanal13 16 producing 2,5-dimethylhexadiene 17, which is dehydrocyclized to pxylene.13 p-Xylene also can be synthesized from cycloaddition of 2,5-dimethylfuran 18 with ethylene followed by dehydration of the cycloaddition product.14 Cycloaddition of 2,5dimethylfuran 18 with acrolein 19 followed by an oxidation, dehydration, and decarboxylation also leads to p-xylene.15 Biobased 2,5-dimethylfuran 18 can be synthesized by hydrogenation16 of 5-hydroxymethylfurfural 22, which is chemically derived from glucose or fructose.17 Catalytic reforming of glucose-derived sorbitol18 23 followed by hydrogenation and dehydration   102 affords a mixture of aromatics.19 Scheme 4.3. Biobased p-Xylene (a) γ-Al2O3, 325 °C, 95%. (b) CuO, 325 °C, 95%. (c) ZSM-5, 160 °C, 69%. (d) CrOx/Al2O3, 550 °C, 42%. (e) NbxOy•H2O, 225 °C, 35%. (f) CrOx/Al2O3, 450 °C, 68%. (g) CuRu/C, n-BuOH, 220 °C, 6.8 bar H2, 71%. (h) C, 200-335 °C, 35-41 bar, 30%. (i) i) Sc(OTf)3, MS (4Å), CDCl3, 55 °C ii) NaClO2, H2O2, NaH2PO4, H2O, CH3CN, 0 °C, 77%. (j) H2SO4, 0 °C, 48%. (k) Cu2O, bathophenanthroline, NMP/quinoline, 210 °C, 91%. (l) i) Pt-Re/C, H2, 230 °C, 27 bar; ii) Ru/C, H2, 160 °C, 55 bar; iii) H-ZSM-5, 400 °C. One method of synthesizing biobased terephthalic without p-xylene intermediacy (Scheme 4.4) starts with 5-hydroxymethylfurfural 22. Oxidation of 5-hydroxymethylfurfural 22 to 2,5-furandicarboxylic acid 2420 followed by cycloaddition with ethylene leads to trace levels of biobased terephthalic acid 11a.21 Another route involves dimerization of dimethyl succinate 25b,22 which is derived from esterification of microbially synthesized23 succinic acid 25a, affords succinylcuccinate 26. Hydrogenation of succinylcuccinate 26 to 27 followed by dehydration and dehydrogenation gives dimethyl terephthalate 11b.24 This can then be hydrolyzed to terephthalic   103 acid. A third route to terephthalic acid that avoids p-xylene utilizes dimerization of microbially synthesized26 malic acid 28 followed by esterification affording methyl coumalate 29b.25 Cycloaddition of 29b with methyl pyruvate leads to dimethyl terephthalate 11b.27 Microbially synthesized28 cis,cis-muconic acid 30 can also be used to make terephthalic acid.29 cis,cisMuconic acid 30 is isomerized to cis,trans-muconic acid 31.29 In situ isomerization of cis,transmuconic acid 31 to trans,trans-muconic acid followed by cycloaddition with ethylene affords cycloadduct 32.30 Dehydrogenation of cycloadduct 32 leads to terephthalic acid 11a.30 Finally, conversion of biobased limonene 33 to p-cymene 34 is followed by oxidation of the side chains to give terephthalic acid 11a.31 Scheme 4.4. Xylene-Free Synthesis of Biobased Terephthalic Acid (a) Pt/C, air, 95%, rt. (b) H2O, 200 °C, 13.8 bar, 0.1%. (c) NaOCH3, MeOH,100 °C, 86%. (d) i) Ru/C, H2, MeOH, 120 °C, 68.9 bar; ii) Ru/C,NaOH, MeOH, 195 °C, 68.9 bar, 24%. (e) SO3/H2SO4, 75 °C, 65%. (f) DIPEA, NMP, (CH3O)2SO2, rt, 64%. (g) 200 °C, 59%. (h) pH 4, 60 °C in fermentation broth, 100%. (i) I2, THF, 160 °C, 17.4 bar, 75%. (j) Pt/C, 150 °C, H2O, 55%. (k) H2N(CH2)2NH2, FeCl3, Na, 50 °C to 100°C, 99%. (k) H2N(CH2)2NH2, FeCl3, Na, 50 °C to 100 °C (l) i) HNO3, H2O, reflux; ii) KMnO4, H2O, NaOH, reflux, 85%.   104 In 1952, Kurt Alder synthesized terephthalic acid 11 from acrylic acid 38 and isoprene 37.32 His intention was to prove that isoprene formed upon ring opening of methylenecyclobutane 35 (Scheme 4.5) by trapping the isoprene formed upon ring opening with acrylic acid, thereby affording 4-methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3cyclohexenecarboxylic acid 40. The cycloadduct 4-methyl-3-cyclohexenecarboxylic acid 39 was then aromatized by H2SO4 producing p-toluic acid 41, which was then oxidized with KMnO4 to terephthalic acid 11.32 Scheme 4.5. Alder’s Reaction Sequence This reaction sequence developed by Alder can produce biobased terephthalic acid if microbially synthesized isoprene33 37 and biobased acrylic acid 38 are used.34,35 The realization of commercial biobased terephthalic acid production with the Alder reaction sequence requires improvement of each step. Clery performed the cycloaddition of isoprene and acrylic acid in a high pressure reactor at 95 °C for 2 h and reported a “quantitative yield” of 4-methyl-3cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40 as a 3:1 mixture.36a PET contains a 20:1 mixture of terephthalic acid 11 and isophthalic acid 43.36b The selectivity of Clery’s reaction was inadequate for commercial terephthalic acid production. Tong and Wang modified the Alder reaction sequence to synthesize biobased terephthalic acid by improving cycloaddition selectivity with the use of BH3 catalysis.37 However, they used Alder’s conditions for the next two steps: a stoichiometric H2SO4 oxidation of 4-methyl-3cyclohexenecarboxylic acid 39 to toluic acid 41 and stoichiometric KMnO4 oxidation of toluic   105 acid 41 to terephthalic acid 11.32,37 In this chapter, methods for aromatization of 4-methyl-3cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40 to p-toluic acid 41 and m-toluic acid 42, respectively, are improved. p-Toluic acid 41 synthesized by aromatization of 4-methyl-3-cyclohexenecarboxylic acid 39 with H2SO4 requires a vapor phase filtration in order to utilize the Amoco Mid-Century oxidation process to yield terephthalic acid 11. The purification method is established in this chapter. A vapor phase plug reactor was developed for dehydrogenation reaction to aromatize both 4-methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40 to p-toluic acids 41 and m-toluic acid 42. Also heterogeneous catalysts for cycloaddition of isoprene and acrylic acid was explored.     106 4. Results 4.1. Cycloaddition of Acrylic Acid and Isoprene Scheme 4.6. Cycloaddition of Acrylic Acid and Isoprene Cycloaddition of acrylic acid 38 and isoprene 37 (Scheme 4.6) were performed using at various reaction conditions (Table 4.1). Uncatalyzed neat cycloaddition of acrylic acid 38 and isoprene 37 in a sealed tube at rt yielded 7% of 4-methyl-3-cyclohexenecarboxylic acid (para cycloadduct) 39 and 2% of 3-methyl-3-cyclohexenecarboxylic acid (meta cycloadduct) 40 (Table 4.1, entry 1). The uncatalyzed neat reaction performed in a high pressure reactor (6.9 bar) at 95 °C afforded 60% of para cycloadduct and 19% of meta cycloadduct (Table 4.1, entry 2). Since PET contains a 20:1 mixture of terephthalic acid 11 and isophthalic acid 43,36b the selectivity of this reaction would be inadequate for commercial terephthalic acid production. Heterogeneous catalysts for cycloaddition of acrylic acid 38 and isoprene 37 were explored. Smit reported the use of silica gel as a heterogeneous catalyst in various cycloadditions.38 This reaction uniquely featured a “dry state adsorption condition.” In this dry state adsorption condition, both the diene and dienophile were adsorbed and reacted on the silica gel in the absence of solvent.38 Smit did not report any cycloaddition of dienophile with carboxylic acid moieties. However, he reported that the Diels-Alder reaction of isoprene 37 and methyl vinyl ketone or acrolein yielded 70% and 56 % cycloadducts, respectively, with 95-97% selectivity toward para.38 This dry state adsorption condition was tested on the cycloaddition of acrylic acid 38 and isoprene 37. A mixture of acrylic acid 38 and isoprene 37 were adsorbed onto pre-dried   107 chromatographic grade SiO2 (230-400 mesh, pore size 60 Å) while the weight of the solution and SiO2 was kept at a ratio of 1:15. Table 4.1. Cycloaddition of Acrylic Acid and Isoprene equivalent entry Yield catalyst solvent temp, time pressure 38 37 1 1.0 1.1 - neat rt, 24 h < 1.6 barb 2 1.0 1.1 - neat 95 °C, 2 h 8.3 barc 3 1.0 1.1 SiO2 neat 25 °C, 2 h < 1.6 barc 4 1.0 1.1 SiO2 neat 50 °C, 2h < 1.6 barc 5 1.0 1.5 SiO2 neat 95 °C, 2 h < 1.6 barc 6 1.0 5.0 hexanes rt, 24 h 1 bard 7 1.0 5.0 hexanes rt, 24 h 1 bard 8 1.0 1.1 hexanes rt, 24 h 1 bard 9 1.0 1.1 hexanes rt, 24 h 1 bard 39 40 (mol ratio) zeolite b 8 wt%a zeolite b 36 wt%a zeolite b 8 wt%a zeolite b 16 wt%a 7%e 2%e (3.5 : 1.0) 60%e 19%e (3.1 : 1.0) 6%f 2%f (3.0 : 1.0) 8%f 3%f (2.7 : 1.0) 10%f 2%f (5.0 : 1.0) f 27% 2%f (14 : 1.0) 35%f 4%f (9.0 : 1.0) 23%f 2%f (12 : 1.0) f 27% 2%f (14 : 1.0) a: Zeolite b wt% of hexanes’ weight. b: apparatus = sealed tube c: apparatus = high pressure reactor. d: apparatus = flask e: Both yields were quantified with GC. f: Yield based on 1HNMR integration. After acrylic acid 38 and isoprene 37 were adsorbed onto the SiO2, it was allowed to react at 25, 50, 95 °C in a high-pressure reactor (Table 4.1, entry 3, 4 and 5). A high pressure reactor was employed in order to prevent evaporation of isoprene 37. The cycloadducts were collected by rinsing the SiO2 with EtOAc. The total yield of cycloadducts from the reactions at 25, 50, 95 °C were 8%, 11% and 12% respectively (Table 4.1, entry 3, 4 and 5). Although, a slight improvement in the selectivity toward para cycloadduct was observed in the reaction at 95 °C (5.0:1.0), it was still far below the ideal selectivity (20:1). Carlson employed zeolites in DielsAlder reactions of isoprene and various dienophiles.39 Carlson did not report any cycloaddition   108 of a dienophile with a carboxylic acid. In his report, zeolite b showed 94-99% selectivity toward para cycloadduct with a reasonable yield when zeolite b was used as suspension in the reaction solution.39 Carlson’s reaction conditions were used for the cycloaddition of acrylic acid 38 and isoprene 37. Flame activated zeolite b was suspended in the hexanes solution of acrylic acid 38 and isoprene 37 for 24 h at rt. Two reaction variables were compared in this reaction: the ratio of acrylic acid 38 to isoprene 37 and wt% of zeolite b (Table 4.1, entry 6,7,8 and 9). The crude products were collected by filtration and concentrated. An improvement in the selectivity toward to para cycloadduct was observed in all four reactions with better yields than the uncatalyzed reaction at rt (Table 4.1, entry 1 vs 6, 7, 8 and 9). However, the crude products after filtration to remove the zeolite b and concentration solidified to an intractable solid mass upon standing at room temperature overnight. Investigation into improvement of the yield and para selectivity of the cycloaddition of acrylic acid 38 and isoprene 37 was continued by a co-worker, Kelly K. Miller.40 He discovered that this cycloaddition catalyzed by 2 mol% of TiCl4 could afford a 94% yield of cycloadducts with 97% selectivity (23:1) favoring the para cycloadduct.40 The pure para cycloadduct 39 was provided by Kelly to me to examine aromatization of para cycloadduct 39.40 Isolation of meta cycloadduct 40 was unsuccessful by column chromatography or distillation. The mixture of para and meta cycloadducts (ratio 1.00:1.26) was obtained from the uncatalyzed neat reaction performed in a high-pressure reactor. A crystallization of para cycloadduct 39 in hexanes gave meta cycloadduct 40 enhanced mother liquor. The crystallization of para cycloadduct 39 from the mother liquor gave more meta cycloadduct 40 enhanced mother liquor.   109 4.2. Aromatization of 4-Methyl-3-cyclohexenecarboxylic Acid and 3-Methyl-3cyclohexenecarboxylic Acid 4-Methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40 were aromatized to p-toluic acid 41 and m-toluic acid 42 (Scheme 4.7) by three approaches: (1) oxidation using H2SO4; (2) solution phase dehydrogenation using Pd(0) or Pd(II); (3) vapor phase dehydrogenation using Pd(0). Scheme 4.7. Aromatization of 4-Methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40 Alder reported32 the aromatization of para cycloadduct 39 by oxidation using H2SO4, which was utilized by Tong37 recently. The problem of this reaction was that 21% of the consumed paracycloadduct 39 could not be accounted. 4.2.1. H2SO4 Oxidation As reported by Alder32 and Tong,37 para cycloadduct 39 was heated in concentrated H2SO4 at 100 °C until vigorous SO2 gas evolution ceased. Quenching the reaction solution with ice followed by filtration afforded 79% of p-toluic acid 41 (Scheme 4.8A). The proposed mechanism is depicted in Scheme 4.9.   110 Scheme 4.8. H2SO4 Oxidation A: reaction with 39 only B: reaction with mixture of 39 and 40 (41 yield from 39; 42 yield from 40) Scheme 4.9. Proposed Reaction mechanism of H2SO4 Oxidation The p-toluic acid 41 from this reaction contained impurities, which inhibit the subsequent Amoco Mid-Century oxidation of p-toluic acid 41. This problem was solved by purification of the p-toluic acid by distillation through a plug of chromatographic grade silica gel. A   111 disadvantage of this reaction was a loss of 21% of the p-toluic acid originally in the crude oxidation product. The use of H2SO4 became more problematic when the 1.0:1.3 mixture of para 39 : meta 40 cycloadducts were aromatized to p-toluic acid 41 and m-toluic acid 42, respectively. This reaction afforded 60% of p-toluic acid 41 from the para cycloadduct 39 and 9% of m-toluic acid 42 from the meta cycloadduct 40, respectively (Scheme 4.8B). 4.2.2. Dehydrogenation Catalyzed by Pd/C in Solvents Due to the problem in H2SO4 oxidation reaction, aromatization of para- and metacycloadducts was redirected towards dehydrogenation. The para cycloadduct 39 was aromatized by dehydrogenation with 1.0 mol% Pd/C in various solvents at their boiling points (Table 4.2). Table 4.2. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid in Various Solvents entry solvent temp (°C) 1 2 3 4 5 water acetic acid p-xylene mesitylene n-decane 100 117 138 165 175 yielda 41 44 34% 63% 36% 60% 55% 45% 39% 59% 39% 63% a: yields were quantified by GC Unlike H2SO4 oxidation, almost all of the consumed para-cycloadduct 39 could be accounted for in product para-toluic acid 41 and side products cis-4-methyl-3-cyclohexanecarboxylic acid 44a and trans-4-methyl-3-cyclohexanecarboxylic acid 44b (Table 4.2). Separate dehydrogenative aromatizations of para-cycloadduct 39 with 1.0 mol% Pd/C in water, acetic acid, n-decane and mesitylene resulted in higher yields of cyclohexane side products 44a and 44b than desired product p-toluic acid (Table 4.2, entry 1, 2, 4 and 5). The reaction in p-xylene showed slightly   112 better selectivity: 55% of p-toluic acid and 45% of cyclohexanes (Table 4.2, entry 3). Nitrobenzene is a hydrogen acceptor reported by Johnson Matthey41 to improve the selectivity of dehydrogenations. Theoretically, transfer of hydrogen from a substrate to nitrobenzene results in formation of aniline and water. Nitrobenzene (17 mol%) was examined as the hydrogen acceptor in dehydrogenation of para cycloadduct 39 with 1 mol% Pd/C in mesitylene. It appeared that the reaction was inhibited by the nitrobenzene as evidenced by 69% of unreacted starting material after 6 days with 31% yield of p-toluic acid. This phenomenon was also reported by Trost and Metzner42 during aromatization of cyclohexene. 4.2.3. Catalytic Oxidative Dehydrogenation Sheldon and Sobczak utilized anthraquinone-2-sulfonate sodium salt (AMS) 45 as a cocatalyst with either PdCl2 or Pd/C for aromatization of cyclohexene to benzene.43 They reported quantitative yields of benzene on a Pd/AMS catalytic system with molecular oxygen. We examined their reaction conditions on the aromatization of para cycloadduct 39 (Scheme 4.10). There was no reaction in either PdCl2 with AMS or Pd/C with AMS. Scheme 4.10. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid on Pd/AMS CO2H A 39 DMF/H2O O2 (1 atm) 50 ℃, 13h 3 mol% Pd/C CO2H 10 mol% AMS B 39   3 mol% PdCl2 10 mol% AMS DMF/H2O O2 (1 atm) 70 ℃, 13h no reaction O SO3Na AMS = no reaction 113 O 45 4.2.4. Aerobic Dehydrogenation Stahl reported44 a quantitative yield of aromatization of 3,4-dimethyl-3cyclohexenecarboxylic acid 47 with Pd-catalyzed aerobic dehydrogenation (Scheme 4.11). Scheme 4.11.Stahl’s Reported Reaction Despite the only difference between his substrate and the cycloadducts 39, 40 is possession of one extra methyl at either C-3 or C-4, aromatization of both para 39 and meta 40 cycloadducts showed poor yields (Scheme 4.12). There was no unreacted substrate remaining and the 1H NMR spectrum showed multiple unidentified products. Scheme 4.12. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid and 3-Methyl-3-cyclohexenecarboxylic Acid 5 mol% Pd(TFA)2 10 mol% 2NMe2py CO2H 20 mol% TsOH A mesitylene O2 (1 atm) 100 ℃, 24h 39 CO2H 41 16% CO2H CO2H 5 mol% Pd(TFA)2 10 mol% 2NMe2py 20 mol% TsOH 39 B 40 CO2H 2NMe2py N = 46 NMe2 41 21% mesitylene O2 (1 atm) 100 ℃, 24h 42 16% CO2H 4.2.5. Aerobic Oxidative Dehydrogenation Recently, Stahl reported a modified catalyst system which could aromatize cyclohexenes. It consisted of Pd(TFA)2, AMS 45 and MgSO4 as a drying agent.45 The quantitative or near quantitative yields were reported with various substituted cyclohexenes (Scheme 4.13).   114 However, all of his reported successes were limited to substituents in the 4- and/or 5-position of the cyclohexenes without any substituent at other positions. Scheme 4.13. Aerobic Oxidative Dehydrogenation of Cyclohexenes to Arene Derivatives45 His aerobic oxidative dehydrogenation reaction conditions were tested on aromatization of para cycloadduct 39 (Scheme 4.14). The methyl substituent evidently has a significant impact on the function of the catalyst. No reaction was observed using para 39 as a substrate. Scheme 4.14. Aerobic Oxidative Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid   115 4.2.6. Vapor Phase Dehydrogenation H2SO4 oxidation reaction gave the highest yield (79%) of p-toluic acid 41 in the liquid phase aromatization of para cycloadduct 39. However, the biggest disadvantage of this reaction is the inability to structurally account for 21% of the consumed para-cycloadduct. Also, we would like to avoid the use of hot, concentrated H2SO4 as a stoichiometric oxidant and solvent. Fortunately, the cycloadducts 39 and 40 possess reasonable vapor pressures and therefore vapor phase dehydrogenative aromatization was realizable. A vapor phase dehydrogenation reactor was developed with common laboratory glassware and instruments (Figure 4.1). The reaction substrate placed in a round bottom flask was distilled through Pd/C dispersed in macroporous silica gel (200-425 mesh, pore size 150 Å). The flask containing substrate and a glass tube containing the catalyst were inserted in an oven. The reaction product was collected in collection bulbs at ambient temperature and a U-shaped tube cooled to -78 °C. The entire glassware was rocked by an oscillating pneumatic motor (Sigma) to ensure even heating of the substrate and the catalyst. This apparatus was connected to a water recirculating aspirator pump. In order to avoid deactivation of the catalyst from moisture, a trap filled with a drying agent was placed between the reactor and the aspirator pump (Figure 4.1).   116 Figure 4.1. Vapor Phase Plug Reactor Para cycloadduct 39 was distilled at 0.13 bar and 240 °C through Pd/C dispersed in macroporous silica gel. p-Toluic acid was obtained in 82% yield along with trans-4methylcyclohexanecarboxylic acid 44a and cis-4-methylcyclohexanecarboxylic acid 44b in 5% and 3% yields, respectively (Scheme 4.15A). Not only this yield was comparable with the H2SO4 oxidation reaction but also 90% of the mass of consumed para-cycloadduct could be structurally accounted for in p-toluic acid product 82% and side products cis- and trans- 8%. The aromatization of meta cycloadduct 40 with H2SO4 gave a poor yield (9%) of m-toluic acid 42 (Scheme 4.8B). The vapor phase dehydrogenation of the 3.1:1.0 mixture of para 39:meta 40 resulting from uncatalyzed cycloaddition led to a 69% yield of m-toluic acid 42 from meta cycloadduct 40 along with trans-3-methylcyclohexanecarboxylic acid 49b, cis-3methylcyclohexanecarboxylic acid 49a and unreacted meta cycloadduct 40 in 10%, 13% and 17% yields, respectively (Scheme 4.15B).     117 Scheme 4.15. Vapor Phase Aromatization of 4-Methyl-3-cyclohexenecarboxylic Acid and 3-Methyl-3-cyclohexenecarboxylic Acid A: reaction with 39 only. B: reaction with mixture of 39 and 40 (41 and 44 yields from 39; 42 and 47 yields from 40) unreacted para cycloadduct 39 28%, unreacted meta cycloadduct 40 17%.     118 5. Discussion Alder’s and Tong’s reaction conditions involve a stoichiometric H2SO4 oxidation of 4methyl-3-cyclohexenecarboxylic acid 39 to toluic acid 41 and stoichiometric KMnO4 oxidation of toluic acid 41 to terephthalic acid 11.32,37 p-Toluic acids 41 synthesized by aromatization of 4methyl-3-cyclohexenecarboxylic acid 39 with H2SO4 oxidation required a vapor phase filtration in order to use an Amoco Mid-Century oxidation to produce terephthalic acid 11. Distillation of p-toluic acid 41 synthesized by H2SO4 oxidation through a plug of chromatographic grade silica gel enabled use of an Amoco Mid-Century oxidation of p-toluic acid to obtain. Even though this purification method avoided the use of stoichiometric KMnO4 oxidation of toluic acid 41 to terephthalic acid 11, there were still problems with the H2SO4 oxidation reaction. Those problems included 21% of missing mass and the use of hot, concentrated H2SO4 as a stoichiometric oxidant and solvent. The development of the vapor phase plug reactor for dehydrogenative aromatization solved both of these issues.     119 6. Experimental 6.1. General 1 H NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to CDCl3 (δ = 7.26 ppm). 13 C NMR spectra were recorded at 125 MHz and the shifts for these spectra are reported (in parts per million) relative to CDCl3 (δ = 77.0 ppm). GC spectra were recorded on an Agilent 6890N chromatograph equipped with an autosampler. Zeolite b ammonium and 5 wt% Pd on C (A503023-5) were purchased from Alfa Aesar. All other reagents were purchased from SigmaAldrich. Chemicals were used as received without further purification. 6.2. Cycloaddition Products 39 and 40 Analysis The total concentration of para cycloadduct 39 and meta cycloadduct 40 was quantified using an Agilent DB-FFAP nitroterephthalic acid modified polyethylene glycol coated capillary column (30 m ´ 0.320 mm i.d., 0.25 µm film thickness). Isolated cycloadduct 39 was used as the standard, which was kindly provided by Kelly Miller.40 The series of standard solutions contained 0.464-23.86 mg/mL of cycloadduct 39, 50.3 mg/mL of 3-methylcyclohexanone as an internal standard and toluene as the solvent. The samples from cycloaddition were prepared in the same way as the standard solution. Syringe filtration (0.45 µm Whatman filter) was followed by chromatographic analysis to determine the total concentration of para 39 and meta 40 cycloadducts. The ratio of para cycloadduct 39 and meta cycloadduct 40 was determined using an Agilent HP-5 (5%-Phenyl)-methylpolysiloxane coated capillary column (30 m ´ 0.320 mm i.d., 0.25 µm film thickness). A weighed quantity of the cycloaddition reaction products (12.6 mg,   120 0.0899 mmol) was added to toluene (2 mL) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (376 µL, 1.42 mmol) were then added. Derivatization was complete upon mixing. Syringe filtration (0.45 µm Whatman filter) was followed by chromatographic analysis to determine the ratio of para 39 and meta 40 cycloadducts. 6.3. Analysis of Aromatization Products: p-Toluic Acid 41 and 4-Methyl-3-cyclohexanecarboxylic Acid 44 The concentrations of p-toluic acid 41 and 4-methylcyclohexanecarboxylic acid 44 were quantified using an Agilent DB-FFAP nitroterephthalic acid modified polyethylene glycol coated capillary column (30 m ´ 0.320 mm i.d., 0.25µL film thickness). p-Toluic acid 41 and 4methylcyclohexanecarboxylic acid 44 obtained from Sigma-Aldrich were used as the standards. The series of standard solutions contained 0.51-5.04 mg/mL of p-toluic acid 41 and 1.14-5.02 mg/mL of 4-methylcyclohexanecarboxylic acid 44 with 3.20 mg/mL of 3-methylcyclohexanone as an internal standard and EtOH as the solvent. The samples from aromatization reaction were prepared in the same way as the standard solution. Syringe filtration (0.45 µm Whatman filter) was followed by chromatographic analysis to determine the concentration of p-toluic acid 41 and 4-methylcyclohexanecarboxylic acid 44.     121 6.4. Analysis of Aromatization Products: p-Toluic Acid 41, m-Toluic Acid 42, 4Methylcyclohexanecarboxylic Acid 44, 3-Methylcyclohexanecarboxylic Acid 47, Unreacted 4-Methyl-3-cyclohexenecarboxylic Acid 39 and Unreacted 3-Methyl-3cyclohexenecarboxylic Acid 40 p-Toluic 41 acid and m-toluic acid 42 were separated from one another with baseline resolution and quantified by HPLC using an Agilent ZORBAX SB-C18 (4.6 ´ 150 mm, 5 µm particle size) column and isocratic elution with 20/80, (v/v); CH3CN/H2O (100 mM NH4+HCO2−, pH 2.5). A series of standard solutions in EtOH of p-Toluic 41 acid (0.508-5.01 mg/mL) and mtoluic acid 42 (1.02-5.32 mg/mL) were prepared with p-toluic 41 and m-toluic acid 42 obtained from Sigma-Aldrich. The samples from aromatization reaction were prepared in the same way as the standard solution. Syringe filtration (0.45 µm Whatman filter) was followed by chromatographic analysis to determine the concentration of p-toluic acid 41 and m-toluic acid 42. The concentration of [4-methylcyclohexanecarboxylic acid 44 + 3methylcyclohexanecarboxylic acid 47] and [unreacted 4-methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3-cyclohexenecarboxylic acid 40] were quantified using an Agilent DB-FFAP nitroterephthalic acid modified polyethylene glycol coated capillary column (30 m ´ 0.320 mm i.d., 0.25 µm film thickness). 4-methylcyclohexanecarboxylic acid 44 obtained from SigmaAldrich were used as the standards. Isolated cycloadduct 39 was used as the standard, which was provided by Kelly Miller.40 The series of standard solutions contained 0.49-5.10 mg/mL of 4methylcyclohexanecarboxylic acid 44 and 0.97-4.94 mg/mL of cycloadduct 39 with 3.20 mg/mL of 3-methylcyclohexanone as an internal standard and EtOH as the solvent. The samples from aromatization reaction were prepared in the same way as the standard solution. Syringe filtration (0.45 µm Whatman filter) was followed by chromatographic analysis.   122 The ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b: trans-3-methylcyclohexanecarboxylic acid 47a: cis-3methylcyclohexanecarboxylic acid 47b and unreacted 4-methyl-3-cyclohexenecarboxylic acid 39 to 3-methyl-3-cyclohexenecarboxylic acid 40 were determined using an Agilent HP-5 (5%Phenyl)-methylpolysiloxane coated capillary column (30 m ´ 0.320 mm i.d., 0.25 µm film thickness). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (600 µL, 1.42 mmol) and acetonitrile (400 µL, 2.26 mmol) were added to a reaction crude solid (20.6 mg). Derivatization was complete upon mixing. Syringe filtration (0.45 µm Whatman filter) was followed by analysis to determine the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b: trans-3-methylcyclohexanecarboxylic acid 47a: cis-3methylcyclohexanecarboxylic acid 47b and unreacted 4-methyl-3-cyclohexenecarboxylic acid 39 to 3-methyl-3-cyclohexenecarboxylic acid 40. 6.5. Uncatalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at rt A 100 mL glass pressure vessel (10.3 bar max) equipped with a magnetic stir bar was charged with acrylic acid 38 (8.36g, 116 mmol) and isoprene 37 (8.32g, 122 mmol). This solution was stirred for 24 h at rt. Concentrating this reaction solution afforded 7% of para cycloadduct 39 and 2% of meta cycloadduct 40 based on GC.     123 6.6. Uncatalyzed Cycloaddition of Acrylic Acid and Isoprene in a High Pressure Reactor Isoprene (77.5 g, 1.14 mol) 37 was added to acrylic acid (71.4 g, 0.991 mol) 38 in a Parr Series 4575 high pressure reactor interfaced with a Series 4842 temperature controller. The reactor was flushed with N2 and then pressurized to 8.3 bar with N2. Heating the reactor at 95 °C with stirring (100 rpm) for 2 h led to an initial increase in pressure to 13.8 bar followed by a decline in pressure to 9.7 bar. After allowing the reactor to cool, a yellow heterogeneous reaction crude was obtained containing a 79% yield of a 3.1:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (83.3 g of para cycloadduct 39 and 26.4 g of meta cycloadduct 40). 6.7. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 25 °C Chromatographic grade silica gel was dried in a vacuum oven at 55 °C overnight. A solution containing isoprene 37 (2.70 g, 39.6 mmol) and acrylic acid 38 (2.70 g, 37.5 mmol) was added to the dry silica gel (79.1 g) in a 1 L stripping flask. This flask was rotated for 3 min in an iced-bath. This reaction solution adsorbed on the silica gel was transferred into a Parr Series 4575 high pressure reactor interfaced with a Series 4842 temperature controller in order to prevent evaporation of isoprene 37. The reactor was maintained at 25 °C for 2 h. The reaction product was extracted into EtOAc from the silica gel. Concentrating this extract afforded 8% yield of a 3.0:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (0.42g of para cycloadduct 39 and 0.11 g of meta cycloadduct 40) based product mass and 1H NMR integration.     124 6.8. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 50 °C Chromatographic grade silica gel was dried in a vacuum oven at 55 °C overnight. A solution containing isoprene 37 (2.70 g, 39.6 mmol) and acrylic acid 38 (2.70 g, 37.5 mmol) was added to the dry silica gel (70.8 g) in a 1 L stripping flask. This flask was rotated for 3 min in an ice-bath. This reaction solution adsorbed on the silica gel was transferred into a Parr Series 4575 high pressure reactor interfaced with a Series 4842 temperature controller in order to prevent evaporation of isoprene 37. The reactor was maintained at 50 °C for 2 h. The reaction product was extracted into EtOAc from the silica gel. Concentrating this extract afforded 11% yield of a 2.7:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (0.42g of para cycloadduct 39 and 0.16 g of meta cycloadduct 40) based on product mass and 1H NMR integration. 6.9. Silica Gel Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 at 95 °C Chromatographic grade silica gel was dried in a vacuum oven at 55 °C overnight. A solution containing isoprene 37 (3.83 g, 56.2 mmol) and acrylic acid 38 (2.70 g, 37.5 mmol) was added to the dry silica gel (83.2 g) in a 1 L stripping flask. This flask was rotated 3 min in an ice-bath. This reaction solution adsorbed on silica gel was transferred into a Parr Series 4575 high pressure reactor interfaced with a Series 4842 temperature controller in order to prevent evaporation of isoprene 37. The reactor was maintained at 95 °C for 2 h. The reaction product was extracted into EtOAc from the silica gel. Concentrating this extract afforded 12% yield of a 5.0:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (0.56 g of para cycloadduct 39 and 0.11 g of meta cycloadduct 40) based on product mass and 1H NMR integration.   125 6.10. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 6) Zeolite b (5.29 g) was flame activated under vacuum. Hexanes (67.2 g) and acrylic acid 38 (3.75 g, 52.0 mmol) were added to the flame activated zeolite b under N2. The mixture was stirred for 30 min at rt prior to addition of isoprene 37 (17.7 g, 260 mmol). This reaction mixture was stirred for 24 h at rt under N2. Filtration of the reaction mixture followed by concentrating the filtrate afforded 29% yield of a 14:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (2.00 g of para cycloadduct 39 and 0.14 g of meta cycloadduct 40) based on product mass and 1 H NMR integration. This crude product was a yellow oil initially however, it became an undissolvable solid after standing at rt overnight. 6.11. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 7) Zeolite b (5.28 g) was flame activated under vacuum. Hexanes (14.5 g) and acrylic acid 38 (3.75 g, 52.0 mmol) were added to the flame activated zeolite b under N2. The mixture was stirred for 30 min at rt prior to addition of isoprene 37 (17.7 g, 260 mmol). This reaction mixture was stirred for 24 h at rt under N2. Filtration of the reaction mixture followed by concentrating the filtrate afforded 39% yield of a 9.0:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (2.57 g pf para cycloadduct 39 and 0.29 g of meta cycloadduct 40) based on the mass of product and 1H NMR integration. This crude product was a yellow oil initially however, it became an undissolvable solid after standing at rt overnight.   126 6.12. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 8) Zeolite b (5.24 g) was flame activated under vacuum. Hexanes (67.2 g) and acrylic acid 38 (3.75 g, 52.0 mmol) were added to the flame activated zeolite b under N2. The mixture was stirred for 30 min at rt prior to addition of isoprene 37 (3.90 g, 57.3 mmol). This reaction mixture was stirred for 24 h at rt under N2. Filtration of the reaction mixture followed by concentrating the filtrate afforded 25% yield of a 12:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (1.65 g of para cycloadduct 39 and 0.14 g of meta cycloadduct 40) based on the mass of product and 1H NMR integration. This crude product was a yellow oil initially however, it became an undissolvable solid after standing at rt overnight. 6.13. Zeolite b Catalyzed Cycloaddition of Acrylic Acid 38 and Isoprene 37 (Table 4.1, entry 9) Zeolite b (5.25 g) was flame activated under vacuum. Hexanes (33.6 g) and acrylic acid 38 (3.75 g, 52.0 mmol) were added to the flame activated zeolite b under N2. The mixture was stirred for 1 h at rt prior to addition of isoprene 37 (3.90 g, 57.3 mmol). This reaction mixture was stirred for 24 h at rt under N2. Filtration of the reaction mixture followed by concentrating the filtrate afforded 29% yield of a 14:1.0 ratio of para cycloadduct 39: meta cycloadduct 40 (1.97 g of para cycloadduct 39 and 0.14 g of meta cycloadduct 40) based on the mass of product and 1H NMR integration. This crude product was a yellow oil initially however, it became an undissolvable solid after standing at rt overnight.   127 6.14. H2SO4 Oxidation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 to p-Toluic Acid 41 Concentrated H2SO4 (80.0 mL, 1.50 mol) previously chilled in an ice bath was added dropwise to (20.1 g, 143 mmol) of para cycloadduct 39 (from a TiCl4-catalyzed cycloaddition40) in a 250 mL round bottom flask chilled in an ice bath and fitted with a reflux condenser open to the atmosphere. The solution was heated to 100 °C. Vigorous gas formation was first observed when the reaction mixture temperature reached 60 °C. Heating of the reaction mixture at 100 °C was continued until gas formation ceased. This gas was captured into a vacuumed Erlenmeyer flask and submitted to a GC/MS analysis confirming SO2. Sulfur dioxide: MS (EI) m/z = calculated [M]•+ 64.0, measured 63.9. The reaction mixture was heated for a total of 50 min from the time gas evolution was first observed at 60 °C. The reaction mixture was poured into ice (400 g) immediately after gas formation ceased, and 17.7 g of a tan-colored solid precipitated from solution. This solid was collected by filtration to afford 15.4 g (79%) of p-toluic acid 41 based HPLC. Kugelrohr distillation (50 mL bulbs, 14/20 joint size) of this solid through a plug (9 cm x 1.7 cm) of chromatographic grade silica gel (60 Å pore size, 40-63 µm mesh) under vacuum afforded 15.2 g (78%) of p-toluic acid 5 as a white solid. p-Toluic acid 41: 1H NMR (CDCl3) d = 8.00-8.02 (m, 2H), 7.27-7.29 (m, 2H), 2.44 (s, 3H). 13 C NMR (CDCl3) d = 127.5, 145.0, 130.6, 129.6, 126.9, 22.1. mp: 178-180 °C (Lit.46 178-180 °C). 6.15. H2SO4 Oxidation of a Mixture of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3Methyl-3-cyclohexenecarboxylic Acid 40 to p-Toluic Acid 41 and m-Toluic Acid 42 The mixture of 4-methyl-3-cyclohexenecarboxylic acid 39 and 3-methyl-3cyclohexenecarboxylic acid 40 were prepared from the uncatalyzed cycloaddition of acrylic acid 38 and isoprene 37 in a high pressure reactor. The concentration of meta cycloadduct 40 was   128 increased by removal of para cycloadduct 39 with multiple crystallization in hexanes. Concentrated H2SO4 (3.99 mL, 74.9 mmol) previously chilled in an ice bath was added dropwise to a cycloadducts mixture consisting of para cycloadduct 39 (0.446 g, 3.18 mmol) and meta cycloadduct 40 (0.562 g, 4.01 mmol) in a 15 mL round bottom flask chilled in an ice bath and fitted with a reflux condenser open to the atmosphere. The solution was heated to 100 °C. Vigorous gas formation was first observed when the reaction mixture temperature reached 60 °C. Heating of the reaction mixture at 100 °C was continued until gas formation ceased. The reaction mixture was heated for a total of 13 min from the time gas evolution was first observed at 60 °C. The reaction mixture was poured into ice (20 g) immediately after gas formation ceased, and 0.630 g of a tan-colored solid precipitated from solution. This solid was collected by filtration to afford 0.286 g (66%) of p-toluic acid 39 and 0.0477g (9%) of m-toluic acid 40 based on HPLC analysis. 6.16. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Water Pd (5 wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. Water (6.6 mL) followed by para cycloadduct 39 (1.32g, 9.42 mmol) were added to the 50 mL round bottom flask with three neck (14/20) containing dry Pd/C (0.20g, 0.094 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The reaction mixture was refluxed at 100 °C for 1 h. This reaction mixture was diluted with EtOH to 200 mL. Based on GC analysis, the reaction yield was 34% (0.441 g) of ptoluic acid 41 and 63% (0.849 g) of 4-methylcyclohexanecarboxylic acid 44. 1H NMR integration indicated the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b was 1.3:1.0.   129 6.17. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Acetic Acid Pd (5 wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. Acetic acid (5.3 mL) followed by para cycloadduct 39 (1.12g, 7.99 mmol) were added to the 50 mL round bottom flask with three neck (14/20) containing dry Pd/C (0.17 g, 0.080 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The reaction mixture was refluxed at 117 °C for 1 h. This reaction mixture was diluted with EtOH to 200 mL. Based on GC analysis, the reaction yield was 36% (0.391 g) of ptoluic acid 41 and 60% (0.686 g) of 4-methylcyclohexanecarboxylic acid 44. 1H NMR integration indicated the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b was 1.4:1.0. 6.18. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in p-Xylene Pd (5 wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. p-Xylene (7.7 mL) followed by para cycloadduct 39 (1.32g, 9.42 mmol) were added to the 50 mL round bottom flask with three neck (14/20) containing dry Pd/C (0.20 g, 0.094 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The reaction mixture was refluxed at 138 °C for 1 h. This reaction mixture was diluted with EtOH to 200 mL. Based on GC analysis, the reaction yield was 55% (0.703 g) of ptoluic acid 41 and 45% (0.606 g) of 4-methylcyclohexanecarboxylic acid 44. 1H NMR integration indicated the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b was 1.5:1.0.   130 6.19. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in Mesitylene Pd (5 wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. Mesitylene (6.5 mL) followed by para cycloadduct 39 (1.12g, 7.99 mmol) were added to the 50 mL round bottom flask with three neck (14/20) containing dry Pd/C (0.17 g, 0.080 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The reaction mixture was refluxed at 165 °C for 1 h. This reaction mixture was diluted with EtOH to 200 mL. Based on GC analysis, the reaction yield was 39% (0.428 g) of ptoluic acid 41 and 59% (0.672 g) of 4-methylcyclohexanecarboxylic acid 44. 1H NMR integration indicated the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b was 1.7:1.0. 6.20. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 in n-Decane 5% Pd on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. n-Decane (8.2 mL) followed by para cycloadduct 39 (1.19g, 8.49 mmol) were added to the 50 mL round bottom flask with three neck (14/20) containing dry 5 wt% Pd/C (0.18 g, 0.085 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The reaction mixture was refluxed at 175 °C for 1 h. This reaction mixture was diluted with EtOH to 200 mL. Based on GC analysis, the reaction yield was 39% (0.458 g) of ptoluic acid 41 and 63% (0.765 g) of 4-methylcyclohexanecarboxylic acid 44. 1H NMR integration indicated the ratio of trans-4-methylcyclohexanecarboxylic acid 44a: cis-4methylcyclohexanecarboxylic acid 44b was 1.5:1.0.   131 6.21. Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3-Methyl-3cyclohexenecarboxylic Acid 40 with Pd/C and Nitrobenzene in Mesitylene To a solid containing para cycloadduct 39 (0.478g, 3.41 mmol) and meta cycloadduct 40 (0.0305 g, 0.218 mmol) in a 50 mL round bottom flask with three neck (14/20), are added 5 wt% Pd on C containing approximately 50 wt% water (0.184 g, 0.040 mmol), mesitylene (2.9 mL), AcOH 145 µL and nitrobenzene (77.5 mg, 0.630 mmol). The flask was fitted with a thermometer and reflux condenser open to the atmosphere. The mixture was refluxed at 166 °C for 6 days. The mixture was gravity hot filtered, and the filtrate was concentrated under vacuum. The residue was dissolved in EtOH (100 mL as the final volume) and analyzed with HPLC. This afforded 0.145 g (31 %) p-toluic acid 41 and 8.14 mg (28%) of m-toluic acid 42. 6.22. PdCl2-AMS Catalyzed Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 3943 PdCl2 (0.07 g, 0.4 mmol) and 0.32 g (1.0 mmol) of antoraquinone-2-sulfonate sodium salt (AMS) was added to a 100 mL round bottom flask (14/20) fitted with a magnetic stir bar. DMF (3.0 mL) and water (1.0 mL) were added to the flask. The flask was purged with O2 followed by addition of para cycloadduct 39. The flask was connected to a O2 balloon through an air condenser after purging with O2. The reaction mixture was heated at 50 °C for 13 h. Based on 1 H NMR observation, there was no reaction. 6.23. Pd/C-AMS Catalyzed Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 3943 Pd (5 wt%) on C containing approximately 50 wt% water (1.28 g, 0.30 mmol) and AMS (0.31 g, 1.0 mmol) was added to a 100 mL round bottom flask (14/20) fitted with a magnetic stir   132 bar. DMF (3.0 mL) and water (1.0 mL) were added to the flask. The flask was purged with O2 followed by addition of para cycloadduct 39. The flask was connected to an O2 balloon through an air condenser after purging with O2. The reaction mixture was heated at 70 °C for 13 h. Based on 1H NMR observation, there was no reaction. 6.24. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 44 To a glass test tube (20 ´ 150 mm) fitted with a magnetic stir bar was added Pd(TFA)2 (32.7 mg, 0.0984 mmol), p-toluenesulfonic acid (76.0 mg, 0.400 mmol), 2-(N,Ndimethylamino)pyridine (24.5 mg, 0.201 mmol), para cycloadduct 39 (274 mg, 1.96 mmol) and mesitylene (0.90 mL). The test tube was sealed with a rubber septum and O2 was sparged into the mixture for 10 min. The test tube was fitted with O2 balloon and heated at 100 °C for 24 h. Water 10 mL was added to the reaction mixture, and then extracted in EtOAc (10 mL ´ 3). The organic layer was dried over MgSO4 and concentrated to afford 43.5 mg (16%) of p-toluic acid based on HPLC analysis. 6.25. Aerobic Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3Methyl-3-cyclohexenecarboxylic Acid 4044 To a glass test tube (20 ´ 150 mm) fitted with a magnetic stir bar was added Pd(TFA)2 (37.8 mg, 0.114 mmol), p-toluenesulfonic acid (84.1 mg, 0.442 mmol), 2-(N,Ndimethylamino)pyridine (34.5 mg, 0.282 mmol), a mixture of para cycloadduct 39 (138 mg, 0.984 mmol) and meta cycloadduct 40 (174 mg, 1.24 mmol), and mesitylene (0.94 mL). The test tube was sealed with a rubber septum and O2 was sparged into the mixture for 10 min. The test tube was fitted with O2 balloon and heated at 100 °C for 24 h. Water 10 mL was added to the   133 reaction mixture, and then extracted in EtOAc (10 mL ´ 3). The organic layer was dried over MgSO4 and concentrated to afford 27.1 mg (21%) of p-toluic acid and 27.1 mg (16%) of mtoluic acid based on HPLC analysis. 6.26. Aerobic Oxidative Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 3945 To a glass test tube (20 ´ 150 mm) fitted with a magnetic stir bar was added para cycloadduct 39 (139 mg, 0.994 mmol), Pd(TFA)2 (16.8 mg, 0.0505 mmol), AMS (62.5 mg, 0.201 mmol), MgSO4 (99.8 mg) and chlorobenzene (1.0 mL). The test tube was sealed with a rubber septum and O2 was sparged into the mixture for 10 min while the mixture was stirred. The test tube was fitted with O2 balloon and heated at 110 °C for 24 h. Water 10 mL was added to the reaction mixture, and then extracted in EtOAc (10 mL ´ 3). There was no reaction based on GC analysis and 1H NMR observation. 6.27. Vapor Phase Dehydrogenation of 4-Methyl-3-cyclohexenecarboxylic Acid 39 Davisil Grade 643 silica gel (150Å pore size, 35-70 µm mesh) was dried in a vacuum oven (150 °C, 15 mbar) overnight. Pd (5wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70°C. The dried silica gel (1.67 g) and dried Pd (5wt%) on C (0.18g, 0.085mmol) were thoroughly mixed and then packed into a 9 cm ´ 1.7cm glass tube using glass wool to immobilize the plug reactor material. 4-Methyl-3-cyclohexenecarboxylic acid 39 (10.1 g, 72.0 mmol) obtained from a TiCl4-catalyzed cycloaddition was placed in a 50 mL, 14/20 round bottom flask. Vaporization/dehydrogenation of para cycloadduct 39 employed a Kugelrohr apparatus assembled as follows: the 50 mL flask containing 39, the 9 cm ´ 1.7 cm glass tube containing the plug reactor, three 50 mL collection bulbs in series, a U-shaped tube   134 and finally a straight gas adaptor connected to a water recirculating aspirator pump (Figure 1). The flask containing 39 and plug reactor were inserted into the Kugelrohr oven. The second 50 mL collection bulb and U-shaped tube were cooled to -78°C. Vaporization/dehydrogenation proceeded at 240 °C under vacuum (0.13 bar) with reciprocal oscillating agitation. The white solid that accumulated in the collection bulbs and the U-shaped tube was collected with EtOH washes. The plug reactor contents were suspended in EtOH followed by filtration to remove the Pd on C and silica gel. All of the EtOH washes were combined, concentrated and dried to afford 8.03 g (82 %) of p-toluic acid 41, 0.495 g (5%) of trans-4-methylcyclohexanecarboxylic acid 44a, and 0.405 g (3%) of cis-4-methylcyclohexanecarboxylic acid 44b. 6.28. Vapor Phase Dehydrogenation of a Mixture of 4-Methyl-3-cyclohexenecarboxylic Acid 39 and 3-Methyl-3-cyclohexenecarboxylic Acid 40 Davisil Grade 643 silica gel (150Å pore size, 35-70µm mesh) was dried in a vacuum oven (150 °C, 15 mbar) overnight. Pd (5wt%) on C containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70°C. The dried silica gel (1.73 g) and dried 5wt% Pd on C (0.20 g, 0.094mmol) were thoroughly mixed and then packed into a 9 cm ´ 1.7cm glass tube using glass wool to immobilize the plug reactor material. A mixture of para cycloadduct 39 (8.00 g, 57.1 mmol) and meta cycloadduct 40 (2.61 g, 18.6 mmol) resulting from an uncatalyzed cycloaddition was placed in a 50 mL, 14/20 round bottom flask. Vaporization/dehydrogenation of the mixture of para cycloadduct 39 and meta cycloadduct 40 employed a Kugelrohr apparatus assembled as follows: the 50 mL flask containing 39 and 40, the 9 cm x 1.7 cm glass tube containing the plug reactor, three 50 mL collection bulbs in series, a U-shaped tube and finally a straight gas adaptor connected to a water recirculating aspirator pump (Figure 1). The flask   135 containing cycloadducts 39 and 40 and plug reactor were inserted into the Kugelrohr oven. The second 50 mL collection bulb and U-shaped tube were cooled to -78 °C. Vaporization/dehydrogenation proceeded at 240 °C under vacuum (0.11 bar) with reciprocal oscillating agitation. The white solid that accumulated in the collection bulbs and the U-shaped tube was collected with EtOH washes. The plug reactor contents were suspended in EtOH followed by filtration to remove the Pd on C and silica gel. All of the EtOH washes were combined, concentrated and dried to afford 4.08 g (53 %) of p-toluic acid 41, 0.17 g (2%) of trans-4-methylcyclohexanecarboxylic acid 44a, 0.05 g (1%) of cis-4methylcyclohexanecarboxylic acid 44b, 2.23 g (28%) of unreacted 4-methyl-3cyclohexenecarboxylic acid 39, 1.74 g (69%) of m-toluic acid 42, 0.34 g (13%) of cis-3methylcyclohexanecarboxylic acid 47a, 0.27 g (10%) of trans-3-methylcyclohexanecarboxylic acid 47b and 0.44 g (17%) of unreacted 3-methyl-3-cyclohexenecarboxylic acid 40.       136 REFERENCES       137 REFERENCES 1.   Merchant Research & Consulting Itd. Global PET Supply to Exceed 24.39Mln Tonnes in 2015. https://mcgroup.co.uk/news/20140117/global-pet-supply-exceed-2439-mln-tonnes2015.html (accessed Jan 06, 2017). 2.   Collias, D. I., Harris, A. M., Nagpal, V., Cottrell, I. W., Schultheis, M. W. Biobased terephthalic acid technologies: a literature review. Ind. Biotechnol. 2014, 10, 91-105. DOI: 10.1089/ind.2014.0002. 3.   Sheehan, R. J. 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In 2010, 6.0 ´ 109 kg of plasticizers were consumed globally.1a Flexible polyvinylchloride (PVC) constitutes 80-90% of global plasticizer production.1b Classes of plasticizers include phthalates, terephthalates, epoxies, aliphatics (adipates and hydrogenated phthalates), trimellitates, polymerics, and phosphates (Scheme 5.1).2 In 2005, phthalates accounted for 88% of world plasticizer consumption. Phthalate usage declined to 70% of plasticizer consumption in 2014 and is projected to continue declining to 65% of plasticizer consumption in 2019.1b This decline is attributed to a demand for higher performance plasticizers and concerns regarding the impact of phthalates on human health and the environment. Scheme 5.1. Plasticizers O O O O OR O phthalate O OR O O OR OR O n n O n n CO2R CO2R hydrogenated phthalate 143 O RO CO2R O adipates RO epoxies OR O O n n terephthalate O RO O O O n n O OR OR   O trimellitate P OR OR phosphate Testicular atrophy in adult male rats exposed to di-n-butyl phthalate was reported more than 40 years ago.3,4 More recent studies have shown that exposing pregnant female rats to di-nbutyl or diethyl phthalate can lead to deformities in reproductive organs of male fetuses. These deformities caused structural and functional problems that persisted throughout the rats’ lifetime.5-7 Other reports have correlated elevated concentrations of phthalate diester or phthalate monoester, a metabolite of phthalate, in urine and semen of human males with various abnormalities. Takahashi examined the urine and blood of workers exposed to high levels of din-butyl and diethyl phthalates.8 He reported a relationship between a high concentration of phthalate monoester in the urine with a significantly lower concentration of free-testosterone in the blood.8 Calafat related a high concentration of monoethyl phthalate ester in urine with a high incidence of DNA damage in male test subjects sperm cells.9 Saxena found that men who have a high concentration of diethyl / di-n-butyl / diethylhexyl phthalates in their semen tend to suffer from infertility, low sperm count, abnormal sperm cells or DNA fragmentation within sperm cells.10 Since the toxicity of phthalate varies with the carbon chain of esters, each phthalate needs to be evaluated separately.11 Of the non-phthalate plasticizers, trimellitates and diisononyl cyclohexane-1,2dicarboxylate (DINCH) have limited evidence of antiandrogenicity11 and are resistant to high temperatures because they are very structurally stable and have extremely low volatility. These characteristics make trimellitates suitable for use in automobile interiors, high-specification electrical cable insulation and sheathing, construction materials, toys and medical devices. The demand for trimellitates is expected to grow in Asia Pacific, Europe and North America.12   144 2. Trimellitic Acid Production Trimellitic acid 2 is commercially produced by oxidation of pseudocumene 1 (Scheme 5.2),13,14 which is produced from catalytic reforming of crude oil fractions such as naphtha.15,16 The oxidation of pseudocumene 1 employs a modified Amoco oxidation process.14 Amoco oxidation (Co2+, Mn2+, Br- in AcOH with air) produced a 53% yield of trimellitic acid17 2 along with undesired partial oxidation products 3 (Scheme 5.2).13,18 Byproducts are attributed to incomplete aryl methyl group oxidations. Subsequent methyl group oxidations have higher energy barriers than initial oxidations. Thus, the last methyl substituent at C-4 is more difficult to oxidize than the first two. Even though increasing reaction temperature could theoretically help the oxidation of the last methyl substituent, it instead induced decarboxylation at C-4 producing a higher concentration of phthalic acid than trimellitic acid 2.17 Another significant problem during oxidation of pseudocumene is precipitation of the catalysts.18 Higher yields of trimellitic acid were obtained with three metals (Co2+, Mn2+, Ce3+, Br- in AcOH with air)13,14,19 oxidation schemes. Amoco Corporation patented a revised Amoco process to produce trimellitic acid 2 from oxidation of pseudocumene 1 with a Ce3+, ZrO2, Co2+, Mn2+, Br- in AcOH with air system (Scheme 5.2).14 The main role of ZrO2 is to prevent the precipitation of the catalysts and increase the catalysts activity.18 Scheme 5.2. Oxidation of Pseudocumene Co2+, Mn2+, BrAmoco Oxidation air (13.8-27.6 bar) AcOH, 198℃ 1 CO2H CO2H CO2H CO2H CO2H 2 R 3 Yield 53 % Co2+, Mn2+, BrZrO2, Ce3+ Revised Amoco Oxidation air (19.3 bar) AcOH, 204℃ 1   145 CO2H CO2H CO2H 2 Yield 90 % R = CH3, CH2OH, CHO 3. Biobased Trimellitic Acid / Trimethyl Trimellitate Two general routes toward the synthesis of biobased trimellitate 2 have been published in the literature. One route involves a pseudocumene 1 intermediate while the other route does not (Scheme 5.3). Disproportionation of xylenes with shape-selective zeolite ZSM-5 produces pseudocumene 1 as a major product.15 Synthesis of biobased p-xylene 4 was discussed in Chapter 4. Ethanol 7 can be reformed to pseudocumene 1, along with fifteen other hydrocarbon products, using Cu-ZSM- 5.20 Catalytic reforming of glycerol 5 with methanol 6 over H-ZSM-5 affords pseudocumene 1 in addition to xylenes, toluene and benzene.21 Glycerol and methanol are byproducts of biodiesel and wood pulp production, respectively.22,23 One route to trimethyl trimellitate that does not involve a pseudocumene intermediate utilizes microbially synthesized24 cis,cis-muconic acid 8, isomerized stepwise to trans,transmuconic acid 10a25,26 prior to esterification to dimethyl trans,trans-muconate 10b (Scheme 5.3). Cycloaddition of dimethyl trans,trans-muconate 10b with methyl acrylate, followed by dehydrogenation affords trimethyl trimellitate 2b.26 Biobased methyl acrylate can be synthesized by esterification of biobased acrylic acid.27-29   146 Scheme 5.3. Biobased Trimellitate (a) ZSM-5, 427-538 °C (b) Cu-ZSM-5, 400 °C (c) HZSM-5, 400 °C, 16 % (d) Co2+, Mn2+, Ce3+, ZrO2, Br-, AcOH, air, 19.3 bar, 204 °C, 90% (e) pH 4, 60 °C in fermentation broth, 100% (f) I2, THF, rt, 84% (g) H2SO4, MeOH, reflux, 95% (h) i) neutral Al2O3, 4-t-butylcatechol, diglyme, 150 °C ii) Pd/C, diglyme, reflux, 46% 4. New Synthetic Route to Biobased Trimellic Acid 3-Carboxy-2E,4Z-muconic acid 12, which is microbially synthesized from glucose can lead to another route for synthesis of biobased trimellitic acid 2 (Scheme 5.4). Isomerization of 3- carboxy-2E,4Z-muconic acid 12 to 3-carboxy-2Z,4E-muconic acid 13 followed by DielsAlder reaction with bioethanol 7-derived ethylene30 15 and subsequent aromatization would lead to trimellitic acid (Scheme 5.4). Additionally, hydrogenation of the cycloadduct 14 would lead to a new aliphatic plasticizer core. This proposed microbial synthesis of 3-carboxy-2E,4Zmuconic acid 12 uses the previous microbial synthesis of 2Z,4Z-muconic acid as its biosynthetic foundation.   147 Scheme 5.4. New Synthetic Route to Biobased Trimellitic Acid 2Z,4Z-Muconic acid is microbially synthesized with E. coli WN1/pWN2.248, which was previously engineered in Frost group (Scheme 5.5).24 Modification of the host genome for microbial synthesis of 2Z,4Z-muconic acid included the insertion of an aroBaroZ cassette into the serA locus31, insertion of a tktAaroZ cassette into the lacZ locus24 and mutation of the aroE gene32,33 in the genome of E. coli WN1. The extra genomic copy of aroB eliminates accumulation of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), while mutation of aroE prevents carbon flow in the common pathway from proceeding beyond 3-dehydroshikimic acid (DHS).24 In order to increase the availability of erythrose 4-phosphate (E4P), the genomic tktA insert results in overexpresssion of transketolase.24 Two genomic copies of the Klebsiella pneumoniae aroZ gene, which encodes DHS dehydratase, enables the conversion of DHS to protocatechuic acid (PCA).24 Disruption of L-serine biosynthesis is due to loss of genomic serAencoded phosphoglycerate dehydrogenase. This ensures that WN1 retains plasmid pWN2.248, which encodes serA, aroY, catA, and aroFFBR. The K. pneumoniae aroY gene encoding PCA decarboxylase enables the conversion of PCA to catechol, and the Acinetobacter calcoaceticus catA gene encoding catechol 1,2-dioxygenase leads to production of 2Z,4Z-muconic acid.24 Plasmid-localized aroFFBR encodes a mutant isozyme of 3-deoxy-D-arabino-heptulosonic acid 7phosphate synthase, AroFFBR, which is insensitive to feedback inhibition by tyrosine increases   148 carbon flow into the common pathway of aromatic amino acid biosynthesis.24 Scheme 5.5. Biosynthetic Pathway of 2E,4Z-3-carboxymuconic Acid E4P: erythrose 4-phosphate; PEP: phosphoenolpyruvate; DAHP: 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DHQ: 3-dehydroquinic acid; DHS: 3-dehydroshikimic acid; PCA: protocatechuic acid AroFFBR: DAHP synthase (tyrosine insensitive); AroB: DHQ synthase; AroD: DHQ dehydratase; AroE: shikimate dehydrogenase; AroZ: DHS dehydratase from Klebsiella pneumoniae; AroY: PCA decarboxylase from K. pneumoniae; CatA: catechol 1,2-dioxygenase from Acinetobacter calcoaceticus; PcaG PcaH: protocatechuate 3,4-dicarboxylase 3-Carboxy-2E,4Z-muconic acid synthesizing E. coli can be constructed with the same host strain WN1,24 which already has the pathway to produce PCA from E4P and PEP (Scheme 5.5). The only new catalytic activity needed is pcaGpcaH-encoded protocatechuate 3,4-dioxygenase. 5. Protocatechuate 3,4-Dioxygenase Protocatechuate 3,4-dioxygenase is endogenous to many bacteria.34-39 A crystal structure of protocatechuate 3,4-dioxygenase has been determined.34 Protocatechuate 3,4-dioxygenase contains sets of a nonheme ferric ion, an a-subunit and a bsubunit ([abFe3+]x).35-39 The number of [abFe3+] depends on the species. Protocatechuate 3,4-dioxygenase of Pseudomonas cepacia   149 is [abFe3+]4; Geobacillus sp. is [abFe3+]5, Pseudomonas putida is [abFe3+]12.35-39 The a-subunit is encoded by pcaG and the b-subunit is encoded by pcaH.35,40 The pcaGpcaH genes encoding protocatechuate 3,4-dioxygenase have been cloned and sequenced35,40 from a variety of different bacterial sources and heterologously expressed in catalytically active form in Escherichia coli.39,41,42 Protochatechuate 3,4-dioxygenase is found in the b-ketoadipate pathway. This aromatic degradation pathway has two branches. One branch degrades protocatechuic acid 15. The other branch degrades catechol 23 (Scheme 5.6).43 Various aromatic compounds are first degraded to protocatechuic acid 15 (Scheme 5.7).43 Protocatechuic acid 15 is then converted by enzymes of the b-ketoadipate pathway to succinyl-CoA 21 and acetyl-CoA 22 via intermediacy of bketoadipate 19 (Scheme 5.6).43 The compounds converted to protocatechuic acid depend on the microbial species, which are frequently isolated from soil.43 Coniferyl alcohol 26, vanillic acid 27, ferulic acid 28 and 4-coumaric acid 29 (Scheme 5.7) are lignin-related monomers from decaying plant materials.44 Shikimic acid 30 and quinic acid 31 (Scheme 5.7) are also from decaying plant materials.43 Benzoic acid 32, p-cresol 34 and 4-chlorobenzoic acid 35 (Scheme 5.7) are pollutants found in soil, which result from human activity.45-47   150 Scheme 5.6. b-Ketoadipate Pathway (a) protocatechuate 3,4-dioxygenase (b) β-carboxy-cis,cis-muconate lactonizing enzyme E.C.5.5.1.2 (c) γ-carboxymuconolactone decarboxylase E.C.4.1.44 (d) catechol 1,2-dioxygenase (e) cis,cis-muconate lactonizing enzyme (f) muconolactone isomerase (g) enollactone hydrolase (h) β-ketoadipate:succinyl-CoA transferase (i) β-ketoadipyl-CoA thiolase Scheme 5.7. First Phase of Aromatic Compound Degradation   151 6. 1,2,4-Butanetricarboxylic Acid 6.1. Introduction Another valuable biobased molecule that can be derived from 3-carboxy-2E,4Z-muconic acid 16 is 1,2,4-butanetricarboxylic acid 37 (Scheme 5.8). Scheme 5.8. Synthesis of 1,2,4-Butanetricarboxylic Acid from 2E,4Z-3-Carboxymuconic Acid 1,2,4-Butanetricarboxylic acid is used as a cross-linker in polyurethane coating formulations. These speciality coatings can tolerate 50 °C water, acidic water and prolonged exposure to sunlight.48 1,2,4-Butanetricarboxylic acid is also used in inkjet cartridges to prevent pigment bleeding.49 Other applications include its utilization as a raw material for gellant in cosmetics,50 a binder in electrochemical cells,51 and used as a synthetic elastomer for cardiac tissue.52 This synthetic elastomer will act as scaffolding for engineered tissue implants.52 6.2. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid There are some reports in the literature suggesting 1,2,4-butanetricarboxylic acid can be made renewable by employing biobased starting materials (Scheme 5.9). Dimerization of methyl acrylate 38, synthesized by esterification of biobased acrylic acid,27 yields molecule 39.53 Hydrocyanation of 39 followed by hydrolysis affords 1,2,4-butanetricarboxylic acid 37.53 Two routes employ microbially synthesized54 butadiene 41 as the starting material. Cycloaddition of butadiene 41 with biobased acrylic acid28,29 42 forms 3-cyclohexenecarbocylic acid 43,55 which is oxidized to 1,2,4-butanetricarboxylic acid 37.56 Dimerization of butadiene57 41 followed by oxidation of the dimer58 44 also affords 1,2,4-butanetricarboxylic acid. Hydrogenolysis of   152 homocitric acid lactone 45 produced by genetically engineered Issatchenkia orientalis can also afford 1,2,4-butanetricarboxylic acid 37.59 Homocitric acid 47 is an intermediate of lysine biosynthesis pathway in Issatchenkia orientalis (Scheme 5.9).59 The homocitric acid 47 lactonizes to homocitric acid lactone 45 in the acidic fermentation broth.59 Scheme 5.9. Biobased 1,2,4-Butanetricarboxylic Acid Synthesis Route (a) tricyclohexylphosphine, chlorobenzene, 60 ℃, 40% (b) HCN, CH3CN, 75 ℃, 100% (c) 12N HCl, 110 ℃, 80% (d) tetraacetyl diborate, -78 ℃→rt, 99% (e) HNO3, NH4VO3, Cu0, 50-60 ℃, 85%. (f) (C4H9)4N+[Fe(CO)3NO]-, FeCl2, 1.5 bar, 100 ℃, 96% (g) HNO3, NH4VO3, 1-methylpicolinium chloride, 170 ℃, 100%. (h) Pt/Pd on C, H2O, 150 ℃, quantitative yield.   153 Scheme 5.10. Lysine Biosynthesis Pathway (a) homocitrate synthase (b) homocitrate dehydratase (c) homoaconitase (d) homoisocitrate dehydrogenase (e) 2-aminoadipate aminotransferase (f) alpha-aminoadipate reductase (g) saccharophine dehydrogenase 1.5.1.10 (h) saccharophine dehydrogenase 1.5.1.7 7. Results 7.1 Microbial Synthesis of 2E,4Z-3-Carboxymuconic Acid 7.1.1. E. coli WN1/pYNB6.009A 7.1.1.1. Cloning Overview 2Z,4Z-Muconic acid is microbially synthesized with E. coli WN1/pWN2.248, which was previously engineered in Frost group (Scheme 5.5).24 Modification of the host genome for microbial synthesis of 2Z,4Z-muconic acid included the insertion of an aroBaroZ cassette into the serA locus31, insertion of a tktAaroZ cassette into the lacZ locus24 and mutation of the aroE gene32,33 in the genome of E. coli WN1. The extra genomic copy of aroB eliminates accumulation of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), while mutation of aroE prevents carbon flow in the common pathway from proceeding beyond 3-dehydroshikimic acid (DHS).24 In order to increase the availability of erythrose 4-phosphate (E4P), the genomic   154 tktA insert results in overexpresssion of transketolase.24 Two genomic copies of the Klebsiella pneumoniae aroZ gene, which encodes DHS dehydratase, enables the conversion of DHS to protocatechuic acid (PCA).24 Disruption of L-serine biosynthesis is due to loss of genomic serAencoded phosphoglycerate dehydrogenase. This ensures that WN1 retains the plasmid pYNB6.009A, which contains pcaHG, serA, aroFFBR genes and ParoF DNA sequence. The pcaHG genes encode protocatechuate 3,4-dioxygenase, which converts protocatechuic acid into 2E,4Z-3-carboxymuconic acid.35,40 The aroFFBR encodes a mutant isozyme of DAHP synthase whose expression is insensitive to feedback inhibition by tyrosine.24 This allows elevated DAHP synthase activity when cells were cultured in medium containing aromatic amino acids. Transcription of aroF is repressed by the TyrR repressor-tyrosine complex.60a There are three sites (TYR R boxes) in the promoter of aroF where the TyrR repressor-tyrosine complex can bind.60a,b Even though, the TYR R boxes of aroFFBR possess weaker affinity to TyrR repressortyrosine complex than wild type, addition of tyrosine to the culture medium still causes some repression of aroF gene.60a The sequence of unmodified native promoter of aroF (ParoF) can titrate away the TyrR repressor-tyrosine complex.60c 7.1.1.2. Construction of pKD16.179A and pYNB6.009A A 1.5 kb DNA fragment encoding pcaHG with its native promoter was amplified from Klebsiella pneumoniae subsp. pneumoniae (ATCC25597) and subsequently digested by EcoRI. Ligation of this fragment into the EcoRI digested pJF118EH resulted in pKD16.179A (Figure 5.1). The resulting pcaHG genes were transcribed in the same direction as the tac promoter located on pJF118EH.   155 Figure 5.1. Preparation of Plasmid pKD16.179A A 3.3 kb DNA fragment containing serA gene, aroFFBR gene and ParoF DNA sequence was amplified from pWN1.162A24 and subsequently digested by SmaI. Ligation of this fragment into the SmaI digested pKD16.179A resulted in pYNB6.009B (Figure 5.2). The resulting serA and aroFFBR genes were transcribed in the opposite direction to the tac promoter located on pKD16.179A.   156 Figure 5.2. Preparation of Plasmid pYNB6.009B The 3.3 kb DNA fragment containing serA gene, aroFFBR gene and ParoF DNA sequence was digested from pYNB6.009B by SmaI. Ligation of this fragment into the SmaI digested pKD16.179A resulted in pYNB6.009A (Figure 5.2). The resulting serA and aroFFBR genes were transcribed in the same direction as the tac promoter located on pKD16.179A.   157 Figure 5.3. Preparation of Plasmid pYNB6.009A 7.1.1.3. Host Strain E. coli WN1 Phenotype Confirmation The host strain E. coli WN1 was previously engineered by Wei Niu in the Frost group.24 The modifications of the host genome included the insertion of an aroBaroZ cassette into the serA locus31, insertion of tktAaroZ cassette into the lacZ locus24 and mutation of the aroE gene32,33 in the genome of E. coli WN1. Therefore, this strain requires supplementation of aromatic amino acids and serine. An insertion of tktAaroZ cassette into the lacZ locus results in a loss of lactose metabolism. A glycerol freeze E. coli WN1 was streaked on a LB Plate. Six isolated colonies were tested on multiple plates to confirm their phenotype. The growth   158 characteristics of the six colonies were: growth as a white colony on MacConkey agar containing lactose; no growth on M9 containing aromatic amino acids and aromatic vitamins; no growth on M9 containing serine, growth on M9 containing aromatic amino acids, aromatic vitamin and serine; growth on LB; and no growth on LB containing ampicillin (Ap). 7.1.2. Protocatechuate 3,4-Dioxygenase Activity In order to measure the protocatechuate 3,4-dioxygenase activity, pKD16.179A was transformed into E. coli DH5a. E. coli DH5a/pKD16.179A was harvested from LB/Ap/1 mM IPTG culture. The specific activity of protocatechuate 3,4-dioxygenase was assayed with crude cell-free lysate. The consumption of protocatechuic acid (PCA) was measured by the slope of absorption at 290 nm over 30 seconds (Scheme 5.11). The specific activity of protocatechuate 3,4-dioxygenase of the cell-free lysate was 1.0 µmol min-1 mg-1. Scheme 5.11. Protocatechuate 3,4-Dioxygenase PcaHG: protocatechuate 3,4-dioxygenase 7.1.3. Fed-Batch Fermentor Synthesis of 2E,4Z-3-Carboxymuconic Acid 7.1.3.1. Glucose-rich Condition Fermentation The 2E,4Z-3-carboxymuconic acid 16 synthesizing strain, E. coli WN1/pYNB6.009A was cultured at 1 L scale in a 2 L working volume fermentor at pH 7.0 and 33 °C. The impeller speed and airflow were allowed to vary in order to maintain dissolved oxygen (DO) level at 10% of air saturation. Antifoam was added manually as needed. Glucose-rich culture conditions   159 were employed such that the concentration of glucose in the medium was maintained in the range of 19-45 g/L (average 28 g/L) throughout the run. Inoculant was initially grown in 5 mL M9 medium in an incubator shaker (37 °C, 250 rpm) for 20 h. This culture was then transferred to 100 mL of M9 medium, cultured in an incubator shaker (37 °C, 250 rpm) for an additional 9 h. The fermentation was initiated when this culture was transferred to the fermentor (t = 0). The initial glucose concentration in the fermentation medium was 30 g/L. For glucose-rich/IPTG(+) experiments, sterile 100 mM IPTG stock solution (12 mg IPTG / each addition) was added at 19 h, 27 h, 38 h, 45 h and 51h. Some of the microbially-synthesized 2E,4Z-3-carboxymuconic acid 16 isomerized to 2E,4E-3carboxymuconic acid 54 and 2-(carboxymethyl)-5-oxo-2,5-dihydrofuran-3-carboxylic acid (lactone) 55 (Scheme 5.12). Scheme 5.12. Fermentation Products The total products 16 and 54 concentration, PCA concentration, lactone 55, calculated dry cell mass and protocatechuate 3,4-dioxygenase activity were observed during fermentation. In glucose-rich/IPTG(+) fermentation, steady accumulation of products 16/54 continued during the entire fermentation (Figure 4). After 68 h of cultivation, 23.7 g/L of 3carboxymuconic acid was synthesized in a 12% (mol/mol) yield from glucose.   160 t= 24 h 45 h PCA 3,4-dioxygenase activity 29 U/mg 29 U/mg Figure 5.4. Glucose-rich/IPTG(+) Fermentation 7.1.3.2. Glucose-limited Condition Fermentation The 2E,4Z-3-carboxymuconic acid 16 synthesizing strain, E. coli WN1/pYNB6.009A was cultured at 1 L scale in a 2 L working volume fermentor at pH 7.0 and 33 °C. The instrument setting was split into two phases. During first phase, the impeller speed (50-750 rpm) and airflow (0.06-1.00 L/min) were allowed to vary in order to maintain DO level at 10% of air saturation. When the airflow reached to 1.00 L/min with impeller speed 750 rpm, both airflow and impeller speed were kept constant. DO level started increasing when all the initial glucose was consumed. Here, the instrument settings were switched to the second phase. During the second phase, airflow and impeller speed remained constant at 1.00 lpm and 750 rpm respectively. Automatic oxygen-sensor-controlled glucose feeding was used to maintain a glucose-limited condition. The impeller speed was lowered as needed. Antifoam was added manually as needed. Inoculant was initially grown in 5 mL M9 medium in an incubator shaker (37 °C, 250   161 rpm) for 20 h. This culture was then transferred to 100 mL of M9 medium and cultured in an incubator shaker (37 °C, 250 rpm) for an additional 7 h. The fermentation was initiated when this culture was transferred to the fermentor (t = 0). The initial glucose concentration in the fermentation medium was 20 g/L. The sterile 100 mM IPTG stock solution (12 mg IPTG / each addition) was added at 19 h, 25 h, 31 h, 37 h, 43 h and 49 h. The total products 16 and 54 concentration, PCA concentration, calculated dry cell mass and protocatechuate 3,4-dioxygenase activity were determined over the course of the fermentation. The glucose-limited/IPTG(+) fermentation condition showed a poor accumulation of products 16/54 during the entire fermentation (Figure 5.5). After 63 h of cultivation, only 1.2 g/L of 3-carboxymuconic acid was synthesized in a 2% (mol/mol) yield from glucose. The synthesis of 2E,4Z-3-carboxymuconic acid 16 with E. coli WN1/pYNB6.009A preferred glucose-rich conditions. t= 24 h 48 h PCA 3,4-dioxygenase activity 27 U/mg 50 U/mg Figure 5.5. Glucose-limited/IPTG(+) Fermentation   162 7.2. Isolation of 2E,4E-3-Carboxymuconic Acid from Fermentation Broth Prior to isolating 2E,4E-3-carboxymuconic acid from the glucose-rich fermentation broth, cells were removed by centrifugation. Protein was then removed using a tangential flow apparatus fitted with four 10 kD ultrafiltration cassettes (Sartorius). A portion of the cellfree/protein-free fermentation broth containing 5.4 g of 2E,4Z-3-carboxymuconic acid 16, 2.9 g of 2E,4E-3-carboxymuconic acid 54 and 2.9 g of lactone 55 was treated with activated charcoal twice. This charcoal-treated broth was concentrated to 1/5 of the original volume with a rotary evaporator. The concentrated broth was chilled in an ice-bath followed by acidification in an ice-bath with H2SO4 to pH 2. A white solid precipitated as the acidified broth was stirred at rt for 2 h. Filtration afforded 4.9 g of a white solid containing 4.3 g of 2E,4E-3-carboxymuconic acid 54 (52% recovery of the 54 in the clarified fermentation broth). In order to investigate the fate of the half of the product not recovered during the isolation process, the same isolation procedure was performed with a another cell-free/protein-free fermentation broth containing 2.1 g of 2E,4Z-3-carboxymuconic acid 16, 2.7 g of 2E,4E-3carboxymuconic acid 54 and 1.4 g of lactone 55. The isolated white solid contained 2.4 g of 2E,4E-3-carboxymuconic acid 54 (50% recovery of the 54 in the clarified fermentation broth). The mother liquor contained 1.3 g of 2E,4E-3-carboxymuconic acid 54 (27% of the 54 in the clarified fermentation broth) and 1.0 g of lactone 55 (21% of the 54 in the clarified fermentation broth). Extraction of the product from this mother liquor afforded a residue containing both 2E,4E-3-carboxymuconic acid 54 and lactone 55. Separation of the two molecules was unsuccessful.   163 7.3. Synthesis of Biobased Trimethyl Trimellitate 7.3.1. Isomerization of 2E,4E-3-Carboxymuconic Acid 54 to 2Z,4E-3-Carboxymuconic Acid 13 2E,4E-3-Carboxymuconic acid 54 was isomerized to 2Z,4E-3-carboxymuconic acid 13 with 22 mol% of iodine in acetonitrile (Scheme 5.13). 2E,4E-3-carboxymuconic acid 54 isomerized to 12% of 2Z,4E-3-carboxymuconic acid 13 and 5% of lactone 55 but 83% of 2E,4E3-carboxymuconic acid 54 remained unreacted. 2Z,4E-3-Carboxymuconic acid 13 was distinguished from 2E,4E-3-carboxymuconic acid 54 by 1H NMR and nuclear Overhauser effect spectroscopy (NOE). The magnetic interactions between the protons on C-2 and C-4 was observed using NOE experiments (Scheme 5.14). The peak of the proton on C-2 was observed after the proton on C-4 was magnetically excited. Scheme 5.13. Isomerization of 2E,4E-3-Carboxymuconic Acid 54 Scheme 5.14. Nuclear Overhauser Effect   164 7.3.2. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene Cycloaddition of 2E,4E-3-carboxymuconic acid 54 and ethylene was attempted in a high pressure reactor (Scheme 5.15). Scheme 5.15. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene Even though the reaction of 2E,4E-3-carboxymuconic acid 54 and ethylene in 1,4-dioxane at 164 °C consumed all the starting material 54, this reaction did not yield the cycloadduct 14 (Table 5.1, entry 1). 1H NMR of this reaction crude and extract showed many unidentifiable resonances. The reaction in m-xylene at 150 °C also consumed all of the starting material 54 but did not yield any cycloadduct 14 (Table 5.1, entry 2). The reaction in 1,4-dioxane at 100 °C yielded no product (Table 5.1, entry 3). 1H NMR observation showed mostly unreacted 2E,4E3-carboxymuconic acid 54 with some unidentifiable resonances. The reaction in m-xylene at 100 °C yielded no product (Table 5.1, entry 4). 1H NMR observation showed mostly unreacted 2E,4E-3-carboxymuconic acid 54 with some unidentifiable resonances. Table 5.1. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and ethylene entry 1 2 3 4 solvent 1,4-dioxane m-xylene 1,4-dioxane m-xylene temperature 164 °C 150 °C 100 °C 100 °C ethylene 34.5 bar 34.5 bar 34.5 bar 34.5 bar rxn time 24 h 12 h 12 h 12 h yield 14 0% 0% 0% 0% 7.3.3 Esterification of 2E,4E-3-Carboxymuconic Acid 54 Esterification of 2E,4E-3-carboxymuconic acid 54 was attempted with dry methanol and H2SO4 as a catalyst. The Fischer esterification reaction at either rt or reflux (65 °C) yielded   165 partially esterified products but no triester. 2E,4E-3-Carboxymuconic acid 54 was successfully fully esterified using oxalyl chloride with catalytic DMF (Scheme 5.16). Purification by flush chromatography afforded a 32% yield of isolated trimethyl 2E,4E-3-carboxymuconate 56a and a 25% yield of trimethyl 2Z,4E-3carboxymuconate 56b. Trimethyl 2E,4E-3-carboxymuconatre 56a was distinguished from 2Z,4E-3-carboxymuconic acid 56b by 1H NMR and nuclear Overhauser effect spectroscopy (NOE). The magnetic interactions between the protons on C-2 and C-4 was observed in NOE experiments (Scheme 5.17). The peak of the proton on C-2 was observed after the proton on C-4 was magnetically excited. Scheme 5.16. Esterification of 2E,4E-3-Carboxymuconic Acid 54 Scheme 5.17. Nuclear Overhauser Effect 7.3.4. Cycloaddition of Trimethyl 3-Carboxymuconate 56 and Ethylene Cycloaddition of trimethyl 3-carboxymuconate 56 was conducted with ethylene (17.2 bar) in m-xylene at 150 °C for 9 h (Scheme 5.18).   166 Scheme 5.18. Cycloaddition of Trimethyl 3-Carboxymuconates 56a,b and ethylene This reaction yielded total 85% of trans-cycloadduct 57a and cis-cycloadduct 57b. The concentrated reaction crude product was subsequently aromatized without purification. A unique feature of this cycloaddition was that trimethyl 2E,4E-3-carboxymuconate 56a could react with ethylene while dimethyl cis,trans-muconate required an isomerization to dimethyl trans,transmuconate61 in order to react with ethylene. 7.3.5. Aromatization of Cycloadduct 57 Cycloadduct 57 was aromatized to trimethyl trimellitate 2b by three strategies: dehydrogenation catalyzed by Pd/C in solvents, aerobic oxidative dehydrogenation,62 and vapor phase dehydrogenation catalyzed by Pd/SiO2. 7.3.5.1 Dehydrogenation Catalyzed by Pd/C in Solvents Cycloadduct 57 was aromatized to trimethyl trimellitate 2b with dehydrogenation catalyzed by Pd/C in solvents (Scheme 5.19). Scheme 5.19. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Solvents   167 A commercially available Pd (5 wt%) on C contained approximately 50 wt% water. It was dried for 3 h under reduced pressure at 70 °C. The Dry 2 mol% Pd on C and decane were added to cycloadduct 57 and refluxed at 174 °C for 3 days. Decane and Pd/C were removed with a silica gel column. A dark brown material remained at the leading edge of the column. The collected fraction was concentrated affording 17% yield of trimethyl trimellitate 2b with no unreacted cycloadduct 57 (Table 5.2, entry 1). Dry 2 mol% Pd/C and dichlorobenzene were added to cycloadduct 57 and heated at 172 °C for 2 days. Pd/C was removed by filtration. The filtrate was concentrated affording 19% of trimethyl trimellitate 2b with no unreacted cycloadduct 57 (Table 5.2, entry 2). Table 5.2. Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/C in Solvents entry solvent temp 1 2 decane dichlorobenzene 174 °C 172 °C rxn time 3d 2d Yield 2b 17% 19% 7.3.5.2. Aerobic Oxidative Dehydrogenation of Cycloadduct 57 Aromatization of cycloadduct 57 was attempted by Stahl’s aerobic oxidative dehydrogenation reaction conditions.62 These consisted of 6 mol% of Pd(TFA)2, 21 mol% anthraquinone-2-sulfonate sodium salt (AMS) 58, 82 mol% MgSO4 and oxygen (1 atm) in chlorobenzene (Scheme 5.20). Scheme 5.20. Aerobic Oxidative Dehydrogenation of Cycloadduct 57 All of Stahl’s reported successful aromatization of cyclohexenes were limited to substituents in   168 the 4- and/or 5-position of the cyclohexene. Other substitution patterns for mono- and disubstituted cyclohexenes were not examined for aerobic oxidative dehydrogenation. Also, no 1,3,6-trisubstituted cyclohexenes were examined as substrates for oxidative dehydrogenation. The aerobic oxidative dehydrogenation gave no reaction to cyclohexene 57 (Scheme 5.20). 7.3.5.3. Vapor Phase Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/SiO2 A vapor phase dehydrogenation reactor was employed for aromatization of cycloadduct 57 (Chapter 4, Figure 4.1). The cycloadduct 57 was distilled at 0.07 bar and 250 °C through 11 mol% Pd/SiO2 dispersed in macroporous silica gel. Trimethyl trimellitate 2b was obtained in 57% yield. In addition, methyl benzoate 59, dimethyl phthalate 60, dimethyl isophthalate 61 and dimethyl terephthalate 62 were obtained as side products in 3%, 4%, 2% and 2% yields, respectively (Scheme 5.21). Scheme 5.21. Vapor Phase Dehydrogenation of Cycloadduct 57 Catalyzed by Pd/SiO2   169 7.4. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid The synthesis of 1,2,4-butanetricarboxylic acid 37 employed a high pressure reactor (34.5 bar). 2E,4E-3-carboxymuconic acid 54 was hydrogenated with 2 mol% Pd/C at rt in water (Scheme 5.22). Scheme 5.22. Synthesis of 1,2,4-Butanetricarboxylic acid 37 This reaction afforded 99% yield of 1,2,4-butanetricarboxylic acid 37. The reaction mixture was filtered and then concentrated to 1/10 of the filtrate volume. The filtrate was acidified with H2SO4 to pH 2 followed by extraction with EtOAc. The extract was dried with MgSO4 subsequently concentrated affording a white residue. This residue was crystallized in acetonitrile at 4 °C. This afforded a 79 % isolated yield of 1,2,4-butanetricarboxylic acid 37. The 1H NMR, 13 C NMR, and mp of biobased 1,2,4-butanetricarboxylic acid 37 synthesized from glucose was identical with 1,2,4-butanetricarboxylic acid purchased from Sigma.   170 8. Discussion A recombinant strain of Escherichia coli capable of synthesizing 2E,4Z-3carboxymuconic acid 16 from glucose was engineered and called WN1/pYNB6.009A. This strain synthesized 23.7 g/L of 3-carboxymuconic acids 16 and 54 in a 12% (mol/mol) yield from glucose under fermentor-controlled conditions. The isolated product, 2E,4E-3-carboxymuconic acid 54 was used to synthesize biobased trimethyl trimellitate 2b and 1,2,4-butanetricarboxylic acid 37. A quantitative yield of biobased 1,2,4-butanetricarboxylic acid 37 was synthesized from 2E,4E-3-carboxymuconic acid 54 with only one step process. The cycloaddition of trimethyl 2E,4E-3-carboxymuconate 56a with ethylene was achieved without an isomerization to trimethyl 2Z,4E-3-carboxymuconate 56b. Cycloadduct 57 was aromatized to trimethyl trimellitate 2b with three methods: dehydrogenation catalyzed by Pd/C in solvents, Stahl’s aerobic oxidative dehydrogenation reaction,62 and vapor phase dehydrogenation catalyzed by Pd/SiO2. Neither of the solution phase aromatization of cycloadduct 57 was successful. The vapor phase dehydrogenation catalyzed by Pd/SiO2 gave the highest yield of trimethyl trimellitate among the three methods tested.   171 9. Experimental 9.1. Spectroscopic Measurements 1 H NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to CDCl3 (δ = 7.26 ppm) or sodium 3(trimethylsilyl)propionate-2,2,3,3-d4 (TSP, δ = 0.00 ppm) with D2O as the solvent. 13 C NMR spectra were recorded at 125 MHz and the shifts for these spectra are reported (in parts per million) relative to CDCl3 (δ = 77.0 ppm). UV and visible measurements were recorded on an Agilent 8453 UV-visible Spectroscopy System with UV-visible ChemStation as the operating software in Dell Dimention 8100. 9.2. Gas Chromatography GC spectra were recorded on an Agilent 6890N chromatograph equipped with an autosampler. The trimethyl trimellitate 2b concentration was determined using an Agilent HP-5 (5%-Phenyl)-methylpolysiloxane coated capillary column (30 m ´ 0.320 mm i.d., 0.25 µm film thickness). The reaction crude product was diluted with acetonitrile in a volumetric flask. Syringe filtration (0.45 µm Whatman filter) was followed by analysis to determine the concentration of trimethyl trimellitate 2b. 9.3. Bacterial Strains and Plasmids E. coli DH5a [F- f80lacZDM15 D(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-,mk+) PhoA supE44 l- thi-1 gyrA96 relA1] was obtained previously from Invitrogen. E. coli WN1 [tsx352 supE42 l- aroE353 malA352(lR) serA::aroBaroZ lacZ::tktAaroZ] was constructed by Wei   172 Niu in the Frost group.24 Plasmid pJF118EH was previously obtained from professor M. Bagdasarian (Michigan State University). Plasmid pWN1.162A was constructed by Wei Niu in the Frost group.24 9.4. Storage of Bacterial Strains and Plasmids All E. coli was stored at -78 °C in glycerol. Plasmids were transformed into E. coli DH5a for a long-term storage. Preparation of a bacterial glycerol freeze started from an inoculation of 5 mL medium with a single colony of the desired strain. E. coli strains were cultured in 5 mL LB medium (with antibiotic when it was appropriate) at 37 °C with agitation for 14 h. Glycerol freeze samples were prepared by addition of bacterial culture 0.75 mL to sterile 80% (v/v) aqueous glycerol solution 0.25 mL). The solutions were gently mixed, allowed to stand at room temp. for 6 h, and then stored at -78 °C. 9.5. Culture Medium BactoTM tryptone, DifcoTM yeast extract and BactoTM agar were obtained from BD. Ampicillin, thiamine hydrogen chloride, 2,3-dihydroxybenzoic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid was obtained from Spectrum. Isopropyl-b-D-thiogalactoside was obtained from Gold Biotechnology. Ammonium heptamolybdate tetrahydrate was obtained from Mallinckrodt. All solutions were prepared in deionized distilled water. LB medium (1 L) contained BactoTM tryptone (10 g), DifcoTM yeast extract (5 g) and NaCl (10 g).63 2´Yeast-tryptone (1 L) contained DifcoTM yeast extract (10 g), BactoTM tryptone (16 g) and NaCl (5 g).63 SOB medium (1 L) contained BactoTM tryptone (20 g), DifcoTM yeast extract (5 g) and NaCl (5 g).64 SOC medium was prepared with SOB medium (1 L), 250 mM   173 KCl (10 mL), 2M MgCl2(5 mL), 1M glucose (20 mL).64 Modified M9 salts (1 L) contained Na2HPO4 (6 g), KH2PO4 (3 g), NH4Cl (1 g) and NaCl (0.5 g).63 M9 minimal medium contained D-glucose (10 g), MgSO4 (0.24 g), and thiamine hydrogen chloride (0.001 g) in 1 L of M9 salts.63 M9 medium (1 L) was supplemented where appropriate with L-phenylalanine (0.040 g), Ltyrosine (0.040 g), L-tryptophan (0.040 g), p-hydroxybenzoic acid (0.010 g), p-aminobenzoic acid (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g). L-Serine was added to a final concentration of 33 mg/L where indicated. Ampicillin was added where appropriate to a final concentration of 50 μg/mL where indicated. Solutions of LB medium, M9 salts, MgSO4, D-glucose, SOB medium, KCl, MgCl2 were autoclaved. Solutions of aromatic vitamins (containing phydroxybenzoic acid, p-aminobenzoic acid, and 2,3-dihydroxybenzoic acid), aromatic amino acids (containing L-phenylalanine, L-tyrosine, L-tryptophan), L-serine, ampicillin and isopropylb-D-thiogalactoside (IPTG) were sterilized through 0.22 μm membrane. 9.6. Preparation and Transformation of Electrocompetent Cells Electrocompetent cells were prepared using procedures modified from Sambrook and Russell.65 Preparation of electrocompetent cells started with inoculation of LB (5 mL) from a single colony followed by incubation in an incubator shaker (37 °C, 250 rpm) for 14 h. The seed culture (2 mL) was transferred to a 2 L Erlenmeyer flask containing 500 mL 2´YT. The cells were cultured in an incubator shaker (37 °C, 250 rpm) until they reached the mid-log phase of growth (OD600 = 0.5-0.7). The flask containing the culture was chilled in an ice-bath for 10 min. The culture was transferred to a sterile centrifuge bottle. The cells were harvested by centrifugation (2000 g, 5 min, 4 °C) and the supernatant was discarded. The cells were kept cold in an ice-bath as much as possible until the end of the procedure. The cells were resuspended in   174 400 mL of ice-cold sterile water, and harvested by centrifugation (2000 g, 10 min, 4 °C). The cells were resuspended in 200 mL of ice-cold sterile water, and harvested by centrifugation (2000 g, 10 min, 4 °C). Once again, the cells were resuspended in 200 mL of ice-cold sterile water, and harvested by centrifugation (2000 g, 10 min, 4 °C). The cells were suspended in 100 mL of sterile ice-cold 10 %(v/v) glycerol aqueous solution. The cells were harvested by centrifugation (2000 g, 10 min, 4 °C). The cells were suspended in 1.5 mL of sterile ice-cold 10%(v/v) glycerol aqueous solution. Aliquots (50 μL) were dispended into ice-cold 1.5 mL sterile microfuge tubes and immediately frozen in liquid nitrogen. The competent cells were stored at -78 °C. Electroporation was performed by Bio-Rad Gene Pulser® II with Gene Pulser® Cuvette (catalogue # 165-2086, 0.2 cm electrode gap). The cuvettes were chilled on ice for 5 min prior to use. Plasmid DNA (dissolved in sterile water 1-5 μL) or purified DNA ligation reaction solution was mixed with 50 μL of electrocompetent cells. The mixture of cells and DNA was transferred into a cuvette. The instrument was set at 2.5 kV, 25 μF and 200 W. A single pulse was applied to a sample which typically resulted in a time constant of 5.2 ms. SOC medium (1 mL) was added into the cuvette and the mixture was transferred into a sterile test tube (20 ´ 150 mm). It was incubated in an incubator shaker (37 °C, 250 rpm) for 1 h, and plated on the appropriate medium. 9.7. Small Scale Purification of Plasmid DNA A single colony of a strain possibly containing the desired plasmid was inoculated into LB/Ap (5 mL). A master plate was created for each colony. After incubation in an incubator shaker (37 °C, 250 rpm) for 14 h, cells were harvested from 3 mL of the culture in a 1.5 mL   175 microfuge tube by centrifugation (5000 g, 1 min). Qiagen ® QIAprep® Spin Miniprep Kit (catalogue number 27104) was used for purification of the plasmid DNA by following the manufacturer’s instructions. The plasmid DNA was eluted from the spin column with 50 μL of sterile water. 9.8. Large Scale Purification of Plasmid DNA A colony from the master plate created during small scale purification of plasmid DNA was used to inoculate 500 mL LB/Ap in a 2 L Erlenmeyer flask. After incubation in an incubator shaker (37 °C, 250 rpm) for 14 h, cells were harvested from approximately 400 mL of the culture in a centrifuge tube by centrifugation (3200 g, 5 min). Qiagen ® Plasmid Maxi Kit (catalogue number 12163) was used for purification of the plasmid DNA by following manufacturer’s instructions. The purified plasmid DNA pellet was dissolved with 500 μL of sterile water. In order to determine the concentration and purity of DNA, the absorbance at 260 nm and 280 nm were measured relative to water. The DNA concentration was calculated based on the absorbance at 260 nm of 50 ng μL-1 cm-1 is 1.0.65 The purity of DNA was assessed with the ratio of absorbance 260 nm / 280 nm.66 A pure DNA solution should have the ratio of 1.8 or above. The ratio becomes low when it is contaminated with protein.66 A sample with the ratio above 1.2 was stored as stock plasmid DNA solution. 9.9. Isolation of Klebsiella pneumoniae subsp. pneumoniae (ATCC 25597) Genomic DNA Klebsiella pneumoniae subsp. pneumoniae (ATCC 25597) was streaked on a LB plate with a glycerol freeze from the group strain collection. LB 5 mL was inoculated with single colony. After incubation in an incubator shaker (37 °C, 250 rpm) for 14 h, cells were harvested   176 from 1 mL of the culture in a 1.5 mL microfuge tube by centrifugation (10,000 g, 2 min). Promega Wizard® Genomic DNA Purification Kit (catalogue number A1120) was used to isolate the genomic DNA by following the manufacturer’s instructions. The DNA concentration was 617 ng/μL based on the absorbance at 260 nm. The ratio of absorbance 260 nm / 280 nm was 1.6. 9.10. Plasmid pKD16.179A The 1.5 kb open reading frame of pcaHG was amplified from Klebsiella pneumoniae subsp. pneumoniae (ATCC 25597) genomic DNA using the following primers containing EcoRI restriction sequences: 5’-AGGAATTCCAGTGCGCAAAACATAACCCA and 5’AGGAATTCGTCACCGCGACGGCTAAATA. The primers were synthesized by Integrated DNA Technologies. Bio-Rad DNA Engine® Peltier Thermal Cycler was used for polymerase chain reaction (PCR). Each reaction (50 μL) contained 0.2 mM dNTPs (Promega), 0.5 μM of each primer, template DNA 250 ng, 1´Phusion buffer and 1 unit of Phusion DNA polymerase (New England BioLabs). PCR cycles were set as follows (Table 5.3.) : Table 5.3. PCR Cycle for pcaHG number of cycle step temperature time length 1 initial denaturation 98 C 2 min denaturation 98 C 30 sec 30 annealing 60-68 C 30 sec extension 72 C 1 min 1 final extension 72 C 5 min 1 storing 4 C hold The PCR product (1.5 kb) was purified with Zymo Research DNA Clean & ConcentratorTM-25 (catalogue number D4006) by following the manufacturer’s protocol.   177 The PCR product (insert) and pJF118EH (vector) were digested with EcoRI-HF (New England BioLabs) at 37 °C for 1 h. The linearized vector was dephosphorylated at 5’ and 3’ end of DNA with New England BioLabs Calf Intestinal Alkaline Phosphate (catalogue number M0290) by following the manufacturer’s protocol. The digested insert and dephosphorylated vector were purified from 0.7 % agarose gel with Zymo Research ZymocleanTM Gel DNA Recovery Kit (catalogue number D4001) by following the manufacturer’s protocol. The insert and vector were ligated with New England BioLabs T4 DNA Ligase (catalogue number M0202S) by following the manufacturer’s protocol. The ligation product was purified with Zymo Research DNA Clean & ConcentratorTM-25. The ligation product was transformed into electrocompetent E. coli DH5α and plated on LB/Ap. Small scale purification of plasmid DNA was performed for six colonies from the LB/Ap plate followed by enzyme digestion with EcoRI-HF at 37 C for 1 h. Further enzyme digestions were performed on two possible candidate plasmids with BamHI-HF, PvuI-HF, SalIHF and SalI/NdeI, confirming both plasmids were pKD16.179A. Large scale purification of plasmid DNA was performed on those two plasmids and stored as pKD16.179A stock solution after confirmation of the products by enzyme digestion with EcoRI-HF, BamHI-HF, PvuI-HF, SalI-HF and SalI/NdeI. 9.11. Plasmid pYNB6.009B pWN1.162A24 was linearized by enzyme digestion with HindIII-HF at 37 °C for 1 h followed by purification with Zymo Research DNA Clean & ConcentratorTM-25. The 3.3 kb DNA fragment containing serA gene, aroFFBR gene and ParoF DNA sequence was amplified from the linearized pWN1.162A using the following primers containing SmaI restriction sequences:   178 5’-TCCCCCGGGTAAATAGTGCAAGG and 5’-TCCCCCGGGATGACGTAACGATAA. The primers were synthesized by Integrated DNA Technologies. Each reaction (50 μL) contained 0.2 mM dNTPs (Promega), 0.2 μM of each primer, template DNA 20 ng, 1.0 mM MgCl2, 1´ thermopol buffer and 2.5 units of Taq DNA polymerase (New England BioLabs). PCR cycles were set as follows (Table 5.4.) : Table 5.4. PCR Cycle for serA - aroFFBR - ParoF Cassette number of cycle step temperature time length 1 initial denaturation 94 C 3 min denaturation 94 C 1 min 30 annealing 56 C 1 min extension 72 C 3 min 18 sec 1 final extension 72 C 4 min 1 storing 4 C hold The PCR product (1.5 kb) was isolated from 0.7 % agarose gel with Zymo Research ZymocleanTM Gel DNA Recovery Kit. The resulting DNA (insert) and pKD16.179A (vector) were digested with SmaI (New England BioLabs) at 37 °C for 2 h. The linearized vector was dephosphorylated at 5’ and 3’ end of DNA with New England BioLabs Calf Intestinal Alkaline Phosphate. The digested insert was purified from 0.7 % agarose gel with Zymo Research ZymocleanTM Gel DNA Recovery Kit. The dephosphorylated vector was purified with Zymo Research DNA Clean & ConcentratorTM-25. The insert and vector were ligated with New England BioLabs T4 DNA. The ligation product was purified with Zymo Research DNA Clean & ConcentratorTM-25. The ligation product was transformed into electrocompetent E. coli DH5α and plated on LB/Ap. Small scale purification of plasmid DNA was performed for 30 colonies from the LB/Ap plate followed by enzyme digestion with SmaI at 37 °C for 1 h. One plasmid contained the insert fragment. Further enzyme digestion was performed on the plasmids with NdeI,   179 confirming the plasmid as pYNB6.009B. Large scale purification of plasmid DNA was performed on this plasmid and stored as pYNB6.009B stock solution after confirmation of the product by enzyme digestion with SmaI and NdeI. 9.12. Plasmid pYNB6.009A pKD16.179A (vector) was digested with SmaI at 37 °C for 2 h. The linearized vector was dephosphorylated at 5’ and 3’ end of DNA with New England BioLabs Calf Intestinal Alkaline Phosphate. The dephosphorylated vector was purified with Zymo Research DNA Clean & ConcentratorTM-25. pYNB6.009B was digested with SmaI at 37 °C for 2 h. The 3.3 kb DNA fragment containing serA gene, aroFFBR gene and ParoF DNA sequence was isolated from 0.7 % agarose gel with Zymo Research ZymocleanTM Gel DNA Recovery Kit. The insert and vector were ligated with New England BioLabs T4 DNA. The ligation product was purified with Zymo Research DNA Clean & ConcentratorTM-25. The ligation product was transformed into electrocompetent E. coli DH5α and plated on LB/Ap. Small scale purification of plasmid DNA was performed for 10 colonies from the LB/Ap plate followed by enzyme digestion with SmaI at 37 °C for 1 h. Nine plasmids contained the insert fragment. Further enzyme digestion was performed on those plasmids with NdeI, confirming six plasmids were pYNB6.009A. Large scale purification of plasmid DNA was performed on one of the plasmids and stored as pYNB6.009A stock solution after confirmation of the product by enzyme digestion with SmaI and NdeI. 9.13. E. coli WN1 The host strain E. coli WN1 was previously engineered by Wei Niu in the Frost group.24   180 A glycerol freeze E. coli WN1 was streaked on a LB Plate and incubated at 37 °C for 14 h. Six isolated colonies were inoculated on multiple plates and incubated at 30 °C for 17 h. The growth characteristics of the six colonies were: growth as a white colony on MacConkey agar containing lactose; no growth on M9 containing aromatic amino acids and aromatic vitamins; no growth on M9 containing serine, growth on M9 containing aromatic amino acids, aromatic vitamins and serine; growth on LB; and no growth on LB containing ampicillin (Ap). 9.14. Protocatechuate 3,4-Dioxygenase Activity pKD16.179A was transformed into E. coli DH5α, and plated on LB/Ap. A single colony was inoculated into 100 mL LB/Ap in 500 mL Erlenmeyer flask followed by incubation in an incubator shaker (37 °C, 250 rpm) for 15 h. This culture was used to inoculate 500 mL LB/Ap in 2 L Erlenmeyer flask, and incubated in an incubator shaker (37 °C, 250 rpm) until OD600 became 0.6. This culture was incubated further for 4 h after addition of 1mM IPTG. The cells were harvested by centrifugation (3200 g, 5 min, 4 °C) and the supernatant was discarded. The mass of wet cells was 2.6 g. The cells were kept cold in an iced-bath as much as possible until the end of the procedure. The cells were resuspended in 250 mL of ice-cold 20 mM tris-acetate pH 7.5, and harvested by centrifugation (3200 g, 5 min, 4 °C). The cells were resuspended in 6 mL of ice-cold 50 mM tris-acetate pH 7.5. The cells were lysed with French Pressure Cell Press (SLM instrument). The instrument setting was following: piston diameter 1 inch, medium ratio, 1100 psig = cell pressure 18000 psig. The cell debris was removed by centrifugation (29000 g, 30 min, 4 °C). The crude cell-free lysate was kept cold in an ice-bath as much as possible until the end of the procedure. Bio-Rad Quick StartTM Bradford 1´ dye Reagent (catalogue number 500-0205) and Quick   181 StartTM Bovine Serum Albumin Standard (catalogue number 500-0206) were used to quantify the concentration of protein by following manufacture procedure. The specific activity of protocatechuate 3,4-dioxygenase was assayed with crude cell-free lysate. The consumption of protocatechuic acid was measured by the slope of absorption at 290 nm (e = 3890 L mol-1 cm-1).67a Each reaction solution 1 mL contained 320 μM protocatechuic acid, 50 mM tris-acetate pH 7.5 and 110 μL of diluted cell lysate.67b The specific activity of protocatechuate 3,4-dioxygenase of the cell-free lysate was 1.0 μmol min-1 mg-1. 9.15. Fed-Batch Fermentation General The fermentation medium was prepared with water (800 mL), K2HPO4 (7.5 g), ammonium iron (III) citrate (0.3 g), citric acid monohydrate (2.1 g), concentrated H2SO4 (1.2 mL), concentrated NH4OH (4.1 mL), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), and Ltryptophan (0.35 g) in a 2 L fermentation vessel. This vessel containing the medium with antifoam (5 drops) was autoclaved. The solution (100 mL) containing following supplements were added immediately prior to initiation of the fermentation: glucose (30 g), MgSO4 (0.12 g), thiamine hydrogen chloride (0.001 g), p-hydroxybenzoic acid (0.010 g), p-aminobenzoic acid (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g) and trace minerals, including (NH4)6(Mo7O24)·∙4H2O (3.7 mg), ZnSO4·∙7H2O (2.9 mg), B(OH)3 (24.7 mg), CuSO4·∙5H2O (2.5 mg), and MnCl2·∙4H2O (15.8 mg). The initial glucose solution, feed glucose solution and MgSO4 solutions were autoclaved. Solutions of aromatic vitamins (containing p-hydroxybenzoic acid, p-aminobenzoic acid, and 2,3-dihydroxybenzoic acid), trace minerals solution and IPTG solution were sterilized through 0.22 μm membrane. Antifoam (Sigma 204) was added to the fermentation broth as needed.   182 Fermentations employed BIOSTAT® B-DCU system controlled by DCU Tower with a 2 L working volume fermentor vessel. Data acquisition utilized MFCS/win 3.0 software (Sartorius Stedim Systems), which was installed in a personal computer (Digilink) operated by Windows® 7 Professional. Matson Marlow 101U/R peristaltic pump was used for administration of feed glucose solution. Temperature and pH were controlled with PID loop maintaining at 33 °C and pH 7.0±0.1 with 2N H2SO4 and NH4OH. Impeller speed was varied between 50 and 1800 rpm to maintain dissolved oxygen (DO) levels at 10% air saturation. Airflow was increased from 0.06 L/min to 1.0 L/min to maintain DO levels at 10% air saturation. DO was measured using a Hamilton OxyFerm FDA 225 (catalogue number 237452) fitted with an Optiflow FDA membrane (catalogue number 237140). pH was measured with Hamilton EasyFerm Plus K8 200 (catalogue number 238627). Glucose concentration was measured with GlucCellΤΜ and glucose test strip (CESCO Bioengineering Co., Ltd.). Antifoam 204 (Sigma-Aldrich) was added as needed to mitigate foam accumulation. For 1H NMR quantification of product concentration, the broth was concentrated to dryness under reduced pressure, concentrated to dryness one additional time from D2O, and then dissolved in D2O containing a known concentration of the sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid. The concentration was determined by the integral corresponding to 2E,4Z-3-carboxymuconic acid 16 (5.99 ppm, d, J = 12 Hz, 1H); 2E,4E-3-carboxymuconic acid 54 (6.14 ppm, d, J = 16 Hz, 1H); protocatechuic acid 15 (6.94 ppm, d, J =8.5 Hz, 1 H); lactone 55 (5.60 ppm, ddd, J = 2.5, 3.5, 8.5 Hz, 1H) with integral corresponding to TSP (δ = 0.00 ppm, s, 9H). Cell densities were determined by dilution of fermentation broth with M9 salts followed by measurement of absorption at 600 nm (OD600). Dry cell weight of E. coli cells (g/L) was calculated using a conversion coefficient of 0.39 g L-1 OD600-1.68   183 9.16. Glucose-rich Condition Fermentation Glucose-rich culture condition was employed such that the concentration of glucose in the medium was maintained in the range of 19-45 g/L (average 28 g/L) throughout the run. Inoculant was initially grown in 5 mL M9 medium in an incubator shaker (37 °C, 250 rpm) for 20 h and subsequently transferred to 100 mL of M9 medium. This 100 mL culture was grown at 37 °C and 250 rpm until the OD600 reached 1.0-2.0 (9 h). Baffles were installed in the 2 L fermentor vessel for glucose-rich experiments. The fermentation was initiated when this culture was transferred to the fermentor (t = 0). The initial glucose concentration in the fermentation medium was 30 g/L. For glucose-rich/IPTG(+) experiment, sterile 100 mM IPTG stock solution (12 mg IPTG / each addition) was added at 19 h, 27 h, 38 h, 45 h and 51h. The instrument settings were split into two phases. During the first phase, DO was maintained at 10 % by increasing impeller speed until the speed reached the set maximum (750 rpm). Airflow was then increased until it reached the set maximum (1.00 L/min). The pO2 control loop setting for first phase was as follows (Table 5.5) : Table 5.5. First Phase of Glucose-rich Fermentation DEADB 0.5% sat CASCADE 1 STIR 2 AIRF Cascade Minimum Maximum XP TI TD Hysterese Mode STIR 2.5% 37.5% 150.0% 100.0 s 0.0 s 01:00 m:s auto AIRF 2.0% 33.3% 90.0% 50.0 s 0.0 s 01:00 m:s auto O2EN 0.0% 100.0% 90.0% 50.0 s 0.0 s 05:00 m:s off STIR: 2.5% = 50 rpm, 37.5% = 750 rpm AIRF: 2.0% = 0.06 L/min, 33.3% = 1.00 L/min O2EN setting did not matter since it was off. The cascade settings were switched to the second phase when both impeller speed and airflow reached their set maximum values. During the second phase, DO was maintained at 10 % by   184 changing the impeller speed while the airflow was held at 1.00 L/min. The pO2 control loop setting for second phase was as follows (Table 5.6.) : Table 5.6. Second Phase of Glucose-rich Fermentation DEADB 0.5% sat CASCADE 1 AIRF 2 STIR Cascade Minimum Maximum XP TI TD Hysterese Mode STIR 37.5% 90.0% 150.0% 100.0 s 0.0 s 01:00 m:s auto AIRF 33.3% 33.3% 90.0% 50.0 s 0.0 s 01:00 m:s auto O2EN 0.0% 100.0% 90.0% 50.0 s 0.0 s 05:00 m:s off STIR: 37.5% = 750 rpm, 90.0% = 1800 rpm AIRF: 33.3% = 1.00 L/min O2EN setting did not matter since it was off. The impeller speed (STIR) minimum was changed to 400 rpm (20%) at t = 24 h. 9.17. Glucose-limited Condition Fermentation Glucose limited conditions consisted of two phases. The preparation of inoculant and the first phase of fermentation were essentially the same as described for glucose rich conditions. Inoculant was initially grown in 5 mL M9 medium in an incubator shaker (37 °C, 250 rpm) for 20 h and subsequently transferred to 100 mL of M9 medium. This 100 mL culture was grown at 37 °C and 250 rpm until the OD600 reached 1.0-2.0 (7 h). The baffles for the 2 L fermentor vessel were removed for these experiments. The fermentation was initiated when the 100 mL culture was transferred to the fermentor (t = 0). The initial glucose concentration in the fermentation medium was 20 g/L. The sterile 100 mM IPTG stock solution (12 mg IPTG / each addition) was added at 19 h, 25 h, 31 h, 37 h, 43 h and 49 h. The instrument settings were split into two phases. During the first phase, DO was maintained at 10 % by increasing the impeller speed until the impeller speed reached the set maximum (750 rpm). Airflow was then increased   185 increased until it reached the set maximum (1.00 L/min). The pO2 control loop settings for the first phase was as follows (Table 5.7.) : Table 5.7. First Phase of Glucose-limited Fermentation DEADB 0.5% sat CASCADE 1 STIR 2 AIRF Cascade Minimum Maximum XP TI TD Hysterese Mode STIR 2.5% 37.5% 150.0% 100.0 s 0.0 s 01:00 m:s auto AIRF 2.0% 33.3% 90.0% 50.0 s 0.0 s 01:00 m:s auto O2EN 0.0% 100.0% 90.0% 50.0 s 0.0 s 05:00 m:s off STIR: 2.5% = 50 rpm, 37.5% = 750 rpm AIRF: 2.0% = 0.06 L/min, 33.3% = 1.00 L/min O2EN setting did not matter since it was off. The pO2 control loop was turned off when both impeller speed and airflow reached their maximum values. The airflow was held at 1.00 L/min and the impeller speed was held at 750 rpm. DO level started increasing when all the initial glucose was consumed. The instrument setting was switched to pO2 Nutrient control loop while the airflow was held at 1.00 L/min and the impeller speed was held at 750 rpm. The pO2 Nutrient control loop setting for second phase was as follows (Table 5.8.) : Table 5.8. Second Phase of Glucose-limited Fermentation MIN 0.0% MAX DEADB 0.5% sat XP TI 999.9 s TD impeller speed = 750 rpm airflow = 1.0 L/min 100.0% 950.0% 0.0 s These settings createdthe glucose-limited conditions by restricting the addition of feed glucose to only when DO roseabove 10%. The impeller speed was lowered to 600 rpm at 31 h and then 400 rpm at 52 h. Antifoam was added manually as needed.   186 9.18. Removal of Cells and Protein The cells were removed by centrifugation (4600 g, 30 min, 4 °C). The protein in the cell free broth was removed with four Sartocon® ECO Hydrostart® membrane cut off 10 kD cassettes equipped with a SartoJet® Pump 4-Piston Diaphragm Pump. This tangential flow filtration of cell free broth was performed with the pressure toward the membrane of 25±5 psi and a back pressure of 6±1 psi. When the retentate became approximately 200 mL, it was not enough volume to sustain the broth flow. The retentate was diluted with 200 mL of water to increase the volume. The diluted retentate was filtered until the flow ceased. 9.19. Isolation of 2E,4E-3-carboxymuconic acid 54 The cell-free/protein-free broth 627 mL contained 5.4 g of 2E,4Z-3-carboxymuconic acid 16, 2.9 g of 2E,4E-3-carboxymuconic acid 54 and 2.9 g of lactone 55. An activated charcoal (Sigma 242276, Darco G-60, mesh 100-325) 10 g was added to a 2 L Erlenmeyer flask containing 627 mL of the broth. It was swirled for 1h in an incubator shaker (250 rpm, rt). The activated charcoal was removed by filtration with a filter paper. The filtrate was treated with 10 g of the activated charcoal one more time followed by filtration. The filtrate was concentrated to 125 mL with a rotary evaporator. The concentrated broth was chilled in an ice-bath and acidified with H2SO4 to pH 2 while it was kept in an ice-bath. This acidified broth was stirred at rt for 2 h to allow precipitation of the product. The white solid was collected by filtration with a filter paper and dried under reduced pressure overnight. Filtration afforded 4.9 g of white solid containing 4.3 g of 2E,4E-3-carboxymuconic acid 54 (52% recovery) based on 1H NMR analysis. 1H NMR (D2O, TSP δ = 0.00) δ = 7.94 (d, J = 16 Hz, 1H), 6.48 (s, 1H), 6.33 (d, J = 16 Hz, 1H).   13 C NMR (D2O, TSP δ = 0.00) δ = 175.8, 174.1, 173.6, 145.0, 140.2, 132.0, 128.5. 187 mp: decomposed at 169 °C (lit.69 155-159 °C). 9.20. 2E,4E-3-Carboxymuconic Acid 54 Isolation Process Investigation The cell-free/protein-free broth 500 mL containing 2.1 g of 2E,4Z-3-carboxymuconic acid 16, 2.7 g of 2E,4E-3-carboxymuconic acid 54 and 1.4 g of lactone 55 was processed as aforementioned. The isolated white solid and the mother liquor were analyzed with 1H NMR. The isolated white solid (2.7 g) contained 2.4 g of 2E,4E-3-carboxymuconic acid 54 (50% recovery). The mother liquor contained 1.3 g of 2E,4E-3-carboxymuconic acid 54 (27%) and 2.4 g of lactone 55. Since the original cell-free/protein-free broth contained 1.4 g of lactone 55, 1.0 g of the lactone 55 (21%) was produced during this isolation process. 9.21. Isomerization of 2E,4E-3-Carboxymuconic Acid 54 to 2Z,4E-3-Carboxymuconic Acid 13 Iodine (0.03 g, 0.12 mmol) and acetonitrile (10 mL) were added to a 25 mL stripping flask (14/20 neck size) containing a magnetic stir bar and 2E,4E-3-carboxylic acid 54. This flask was connected to a reflux condenser, which was opened to air. This mixture was refluxed at 82 °C for 24 h. The reaction mixture was concentrated and dried under reduced pressure. The brown residue was analyzed by 1H NMR and NOE. For 1H NMR quantification of products, the residue was dissolved in D2O. 1H NMR (D2O, TSP δ = 0.00) 2E,4E-3-carboxymuconic acid 54 δ = 7.94 (d, J = 16 Hz, 1H), 6.48 (s, 1H), 6.33 (d, J = 16 Hz, 1H); 2Z,4E-3-carboxymuconic acid 13 δ = 7.27 (d, J = 16 Hz, 1H), 6.15 (d, J = 16 Hz, 1H), 6.01 (s, 1H); lactone 55 δ = 6.43 (d, J = 2Hz, 1H), 5.62 (ddd, J = 2.5, 3.5, 8.5 Hz, 1H), 3.21 (dd, J = 3.5, 16.5 Hz, 1H), 2.73 (dd, J = 8.5, 16.5 Hz, 1H). The ratio of three products was determined by the integral corresponding to 2E,4E-3-carboxymuconic acid 54 at 7.94 ppm, 2Z,4E-3-carboxymuconic acid 13 at 7.27 ppm   188 and lactone 55 at 5.60 ppm. This reaction afforded 12% of 2Z,4E-3-carboxymuconic acid 13, 5% of lactone 55 and 83% of 2E,4E-3-carboxymuconic acid 54. The magnetic interactions between the protons on C-2 and C-4 was observed using NOE technique (Scheme 13). There was no interaction between protons on C-2 (6.48 ppm) and C-4 (7.94 ppm) of 2E,4E-3carboxymuconic acid 54. There was an interaction between the protons on C-2 (6.01 ppm) and C-4 (7.27 ppm) of 2Z,4E-3-carboxymuconic acid 13. 9.22. Cycloaddition of 2E,4E-3-Carboxymuconic Acid 54 and Ethylene 2E,4E-3-Carboxymuconic acid 54 and a solvent were added in a Parr Series 4590 Micro Bench Top Reactor (25 mL) interfaced with 4848 Reactor Controller. The reactor was flushed with N2 five times, and then flushed with ethylene five times. The reactor was charged with ethylene (34.5 bar). 9.22.1. Reaction in 1,4-Dioxane at 164 °C Heating the reactor at 164 °C with stirring (100 rpm) for 24 h led to a maximum pressure to 72.4 bar. After allowing the reactor to cool, a dark brown reaction crude was obtained. The entire reaction crude was concentrated and an aliquot of the residue was dissolved in DMSO-d6. This reaction did not yield cycloadduct 14 based on 1H NMR. 9.22.2. Reaction in m-Xylene at 150 °C Heating the reactor at 150 °C with stirring (200 rpm) for 12 h led to a maximum pressure to 79.3 bar. After allowing the reactor to cool, a dark brown reaction crude was obtained. The entire reaction crude was concentrated and an aliquot of the residue was dissolved in DMSO-d6. This reaction did not yield cycloadduct 14 based on 1H NMR.   189 9.22.3. Reaction in 1,4-Dioxane at 100 °C Heating the reactor at 100 °C with stirring (300 rpm) for 12 h led to a maximum pressure to 56.9 bar. After allowing the reactor to cool, a white reaction crude was obtained. The reactor was rinsed with water. The water solution was acidified with H2SO4 to pH 1 followed by extraction with Et2O 10 mL ´ 6. The extracted was dried over MgSO4 and concentrated. The residue was dissolved in DMSO-d6. This reaction did not yield cycloadduct 14 with mostly unreacted 2E,4E-3-carboxymuconic acid 54 and some unidentifiable peaks in 1H NMR. 9.22.4. Reaction in m-Xylene at 100 °C Heating the reactor at 100 °C with stirring (300 rpm) for 12 h led to a maximum pressure to 58.6 bar. After allowing the reactor to cool, a white reaction crude was obtained. The reactor was rinsed with water. The water solution was acidified with H2SO4 to pH 1 followed by extraction with EtOAc 10 mL ´ 6. The extracted was dried over MgSO4 and concentrated. The residue was dissolved in DMSO-d6. This reaction did not yield cycloadduct 14 with mostly unreacted 2E,4E-3-carboxymuconic acid 54 and some unidentifiable peaks in 1H NMR. 9.23. Esterification of 2E,4E-3-Carboxymuconic Acid 54 Tetrahydrofuran (20 mL) was added to a 100 mL RBF (2 ´ 24/40 necks) containing 2E,3E-3-carboxymuconic acid (1.02 g, 5.50 mmol) and a magnetic stir bar. The flask was purged with N2, and connected to a N2 bubbler line while the side neck was closed with a rubber septum. The flask was chilled in an iced-bath followed by addition of oxalyl chloride (1.50 mL, 17.7 mmol). The reaction mixture was stirred in an iced-bath for 30 min after addition of dimethylformamide (10 μL, 0.1 mmol). The reaction mixture was continued to stir for 3 h at rt. The reaction solution became dark brown gradually during this 3 h. The flask was chilled in an   190 ice-bath prior to addition of dry cold methanol (20 mL, 494 mmol). The reaction solution was stirred in an ice-bath for 30 min and then continued stirring at rt for 19 h. The reaction solution was concentrated under reduced pressure. The residue was loaded on a silica column. Chromatographic purification (EtOAc/hexanes, 1:11 to 1:5, v/v) gave 746 mg of light white oil containing Trimethyl 2E,4E-3-carboxymuconate 56a and Trimethyl 2Z,4E-3-carboxymuconate 56b. The magnetic interactions between the protons on C-2 and C-4 was observed using NOE technique (Scheme 16). There was no interaction between protons on C-2 (6.70 ppm) and C-4 (8.22 ppm) of trimethyl 2E,4E-3-carboxymuconate 56a. There was an interaction between the protons on C-2 (6.14 ppm) and C-4 (7.23 ppm) of trimethyl 2Z,4E-3-carboxymuconate 56b. The products were quantified by the integral corresponding to trimethyl 2E,4E-3-carboxymuconate 56a (6.61 ppm, d, J = 16 Hz, 1H); trimethyl 2Z,4E-3-carboxymuconate 56b (6.07 ppm, d, J = 16 Hz, 1H) with integral corresponding to maleic anhydride (δ = 7.03 ppm, s, 2H). Trimethyl 2E,4E-3-carboxymuconate 56a (32%) and trimethyl 2Z,4E-3-carboxymuconate 57 (25%) were isolated as a mixture. 1H NMR for trimethyl 2E,4E-3-carboxymuconate 56a: d = 8.22 (dd, J = 1, 16 Hz, 1H), 6.70 (d, J = 1Hz, 1H), 6.61 (d, J = 16 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H). 1H NMR for trimethyl 2Z,4E-3-carboxymuconate 56b: d = 7.23 (d, J = 16 Hz, 1H), 6.14 (s, 1H), 6.07 (d, J = 16 Hz, 1H), 3.87 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H). 9.24. Cycloaddition of Trimethyl 3-Carboxymuconates 56 and Ethylene A solution of trimethyl 3-carboxymuconic acid 56 (0.520 g, 2.28 mmol) in m-xylene was added in a Parr Series 4590 Micro Bench Top Reactor (25 mL) interfaced with 4848 Reactor Controller. The reactor was flushed with N2 five times and then flushed with ethylene five times. The reactor was charged with ethylene (17.2 bar). Heating the reactor at 150 °C with stirring   191 (300 rpm) for 9 h led to a maximum pressure to 34.5 bar. After allowing the reactor to cool, a colorless reaction crude was obtained. A sticky solid remained in the reactor that did not dissolved in common solvents. The crude solution was concentrated under reduced pressure affording a total 85% yield of trans-cycloadduct 57a and cis-cycloadduct 57b. The products were quantified by the integral corresponding to cycloadduct-1 (3.74 ppm, s, 3H); cycloadduct-2 (3.72 ppm, s, 3H) with integral corresponding to tert-butyl methyl ether (δ = 3.21 ppm, s, 3H). The ratio of cycloadduct-1 : cycloadduct-2 were 1.0:1.1. 1H NMR (CDCl3) for mixture of transcycloadduct 57a and cis-cycloadduct 57b: δ = 7.21 (d, J = 3 Hz, 1H), 7.18 (dd, J = 1.5, 4 Hz, 1 H), 3.750 (s, 3H), 3.746 (s, 3H), 3.736 (s, 3H), 3.722 (s, 3H), 3.693 (s, 3H), 3.684 (s, 3H), 3.493.51 (m, overlap 2H), 3.31-3.34 (m, 1H), 3.24-3.28 (m, 1H), 2.13-2.18 (m, 1H), 2.01-2.08 (m, 1H), 1.89-1.97 (m, overlap 4H), 1.81-1.88 (m, overlap 2 H). 9.25. Dehydrogenation of Cycloadducts 57 Catalyzed by Pd/C in Decane 5 wt% Pd/C (Alfa Aesar A102023-5) containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. Dry Pd/C (17.3 mg, 0.008 mmol) and decane (3.0 mL) were added to a 25 mL stripping flask (14/20) containing cycloadduct 57 (77.0 mg, 0.300 mmol). The flask was connected to a reflux condenser opening to air. The mixture was refluxed at 174 °C while reaction progress was observed by TLC (EtOAc : hexanes = 1:2, UV). After 3 days, the reaction mixture was loaded on a silica gel column in order to remove decane. Dark brown material remained at beginning of the column. The collected fraction was concentrated affording 41.7 mg of yellow oil containing 12.9 mg of trimethyl trimellitate 2b (17%) based on GC analysis.   192 9.26. Dehydrogenation of Cycloadducts 57 Catalyzed by Pd/C in Dichlorobenzene 5 wt% Pd/C (Alfa Aesar A102023-5) containing approximately 50 wt% water was dried for 3 h under reduced pressure at 70 °C. Dry Pd/C (38.3 mg, 0.018 mmol) and dichlorobenzene (3.0 mL) were added to a 25 mL stripping flask (neck size 14/20) containing cycloadduct 57 (230 mg, 0.897 mmol). The flask was connected to a reflux condenser open to air. The mixture was heated at 172 °C while reaction progress was monitored by TLC (EtOAc : hexanes = 1:2, UV). After 2 days, the reaction mixture was filtered in order to remove Pd/C. The filtrate was concentrated affording 273 mg of light brown oil containing 43.0 mg of trimethyl trimellitate 2b (19%) based on GC analysis. 9.27. Aerobic Oxidative Dehydrogenation of Cycloadduct 5761 To a glass test tube (20 ´ 150 mm) fitted with a magnetic stir bar was added Pd(TFA)2 (18.8 mg, 0.0565 mmol), AMS (64.9 mg, 0.209 mmol), MgSO4 (100 mg, 0.831 mmol) and cycloadduct 57 (259 mg, 1.01 mmol) dissolved in chlorobenzene (1.0 mL). The test tube was sealed with a rubber septum and O2 was sparged into the mixture for 10 min while the mixture was stirred. The test tube was fitted with O2 balloon and heated at 110 °C for 24 h. 1H NMR spectrum showed nothing but starting material, cycloadduct 57. 9.28. Vapor Phase Dehydrogenation of Cycloadduct 57 Pd/SiO2 (2.4 wt%) was prepared by Kelly Miller and Sebastien Thueillon. Davisil Grade 643 silica gel (150 Å pore size, 35-70 µm mesh) and Pd/SiO2 (2.4 wt%) were dried under reduced pressure (0.3 mbar) at 100 °C for 3h. The dried silica gel (1.00 g) and dried Pd/SiO2 (0.50 g, 0.113 mmol) were thoroughly mixed and then packed into a 9 cm ´ 1.7cm glass tube   193 using glass wool to immobilize the plug reactor material. Vaporization/dehydrogenation of cycloadduct 57 employed a Kugelrohr apparatus assembled as follows: the 50 mL flask containing 57, the 9 cm ´ 1.7 cm glass tube containing the plug reactor, three 5 mL collection bulbs in series, a U-shaped tube and finally a straight gas adaptor connected to two water recirculating aspirator pumps through an oscillating motor and a trap (Chapter 4, Figure 1). The flask containing 57 and plug reactor were inserted into an oven. The U-shaped tube was cooled to -78 °C. Prior to the reaction, the glassware was assembled without the flask containing the substrate. The glass tube containing the catalyst was closed with a glass cap. The catalyst was preheated at 250 °C for 10 min at 0.07 bar and the vacuum was gently released from the system. The 50 mL flask containing cycloadduct 57 was connected to the catalyst tube. Vaporization/dehydrogenation proceeded at 250 °C under vacuum (0.07 bar) with reciprocal oscillating agitation. The colorless clear liquid that accumulated in the collection bulbs and the U-shaped tube was collected with acetonitrile washes into a 10 mL volumetric flask. The plug reactor contents were suspended in acetonitrile followed by filtration to remove the Pd/SiO2. The filtrate was collected in a 5 mL volumetric flask. The acetonitrile solutions contained 148 mg of trimethyl trimellitate 2b (57%), 4.04 mg of methyl benzoate 59 (3%), 8.24 mg of dimethyl phthalate 60 (4%), 4.52 mg of dimethyl isophthalate 61 (2%) and 3.10 mg of dimethyl terephthalate 62 (2%). 9.29. Synthesis of Biobased 1,2,4-Butanetricarboxylic Acid 37 2E,4E-3-Carboxymuconic acid (875 mg, 4.70 mmol) 54 and 5 wt% Pd/C (Alfa Aesar A102023-5) containing approximately 50 wt% (0.46 g, 0.11 mmol) were added to water (130 mL) in a Parr Series 4575 high pressure reactor (500 mL) interfaced with a Series 4842   194 temperature controller. The reactor was flushed with H2 and then pressurized to 34.5 bar with H2. It was kept stirring (200 rpm) at rt for 4 h. Pd/C was removed by filtration affording clear colorless filtrate containing 892 mg of 1,2,4-butanetricarboxylic acid 37 (99%). The product 37 was quantified by the calibration curve generated from integral corresponding to 1,2,4butanetricarboxylic acid 37 (1.87 ppm, dtd, 2H) with integral corresponding to TSP (δ = 0.00 ppm, s, 9H). This filtrate was concentrated with a rotary evaporator down to 20 mL and then acidified with H2SO4 to pH 1.8. The product was extracted by hand with EtOAc 20 mL 6. 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