Synthetic Methods for Biologically Active Molecules: Exploring the Potential of Bioreductions

This ready reference focuses on the currently available toolbox of biocatalysed reductions of C=O, C=C and formal C=N double bonds to show which transformations are reliable for use in manufacturing processes and which still require improvements and investigation. Following an introductory chapter, chapters 2-4 present the synthetic strategies which are currently available for the reduction of C=C, C=O and for reductive amination, by means of whole cell catalysts and isolated enzymes. Chapters 5-7 go on to describe the improvements achieved thus far, illustrating the versatility that is currently possible in adapting the enzymes to the requests of organic synthesis. Chapters 8-12 present the improvements brought about by the optimization of reaction conditions, and the use of particular synthetic sequences. The final two chapters describe the actual practical applications of bioreductions for the synthesis of active pharmaceutical ingredients, and for flavors and fragrances. With its excellent and comprehensive overview, this will be of great interest for those working in academia and industry.

Table of Contents:
Preface XIII List of Contributors XV 1 Development of Sustainable Biocatalytic Reduction Processes for Organic Chemists 1 Roland Wohlgemuth 1.1 Introduction 1 1.2 Biocatalytic Reductions of CO Double Bonds 3 1.2.1 Biocatalytic Reductions of Ketones to Alcohols 3 1.2.2 Biocatalytic Reductions of Aldehydes to Alcohols 6 1.2.3 Biocatalytic Reductions of Carboxylic Acids to Aldehydes 8 1.2.4 Biocatalytic Reductions of Carboxylic Acids to Alcohols 8 1.3 Biocatalytic Reductions of CC Double Bonds 8 1.4 Biocatalytic Reductions of Imines to Amines 10 1.5 Biocatalytic Reductions of Nitriles to Amines 12 1.6 Biocatalytic Deoxygenation Reactions 12 1.7 Emerging Reductive Biocatalytic Reactions 14 1.8 Reaction Engineering for Biocatalytic Reduction Processes 16 1.9 Summary and Outlook 17 References 18 2 Reductases: From Natural Diversity to Established Biocatalysis and to Emerging Enzymatic Activities 27 Elena Fernandez-Alvaro and Pablo Dominguez de Maria 2.1 Reductases: Natural Occurrence and Context for Biocatalysis 27 2.2 Emerging Cases of Reductases in Biocatalysis 36 2.2.1 Motivation: The Quest for Novel Enzymes and Reactivities 36 2.2.2 Imine Reductases 36 2.2.3 Nitrile Reductases: The Next Member in the Portfolio of Reductases? 38 2.2.4 Other Emerging N-Based Enzymatic Reductions: Nitroalkenes and Oximes 41 2.2.5 From Carboxylic Acids to Alcohols: Biocatalysis 42 2.3 Concluding Remarks 44 References 44 3 Synthetic Strategies Based on CC Bioreductions for the Preparation of Biologically Active Molecules 49 Francesco G. Gatti, Fabio Parmeggiani, and Alessandro Sacchetti 3.1 Introduction 49 3.2 Bioreduction of a,b-Unsaturated Carbonyl Compounds 53 3.2.1 Aldehydes 54 3.2.2 Ketones 61 3.3 Bioreduction of Nitroolefins 65 3.4 Bioreduction of a,b-Unsaturated Carboxylic Acids and Derivatives 68 3.4.1 Monoesters and Lactones 68 3.4.2 Diesters 71 3.4.3 Carboxylic Acids 73 3.4.4 Anhydrides and Imides 73 3.5 Bioreduction of a,b-Unsaturated Nitriles 74 3.6 Concluding Remarks 76 References 77 4 Synthetic Strategies Based on CO Bioreductions for the Preparation of Biologically Active Molecules 85 Anibal Cuetos, Alba Diaz-Rodriguez, and Ivan Lavandera 4.1 Introduction 85 4.2 Synthesis of Biologically Active Compounds through CO Bioreduction 87 4.2.1 Keto Esters 87 4.2.1.1 a-Keto Esters 87 4.2.1.2 b-Keto Esters 89 4.2.1.3 Other Keto Esters 89 4.2.2 Diketones 90 4.2.3 a-Halo Ketones 91 4.2.4 (Hetero)Cyclic Ketones 94 4.2.5 "Bulky-Bulky" Ketones 96 4.2.6 Miscellaneous 98 4.3 Other Strategies to Construct Biologically Active Compounds 99 4.4 Summary and Outlook 106 References 107 5 Protein Engineering: Development of Novel Enzymes for the Improved Reduction of CC Double Bonds 113 Sabrina Kille and Manfred T. Reetz 5.1 Introduction 113 5.2 The Protein Engineering Process and Employed Mutagenesis Methods 114 5.3 Examples of Rational Design of Old Yellow Enzymes 117 5.4 Evolving Old Yellow Enzymes (OYEs) 117 5.4.1 Evolving OYE1 as a Catalyst in the Stereoselective Reduction of 3-Alkyl-2-cyclohexenone Derivatives and Baylis-Hillman Adducts 119 5.4.2 Evolving the Pentaerythritol Tetranitrate (PETN) Reductase as a Catalyst in the Reduction of a,b-Unsaturated Carbonyl Compounds and E-Nitroolefins 123 5.4.3 Evolving Nicotinamide-Dependent 2-Cyclohexenone Reductase (NCR) from Zymomonas mobilis for the Reduction of a,b-Unsaturated Ketones 129 5.4.4 Evolving the YqjM from Bacillus subtilis for Enhanced Activity, Substrate Scope, and Stereoselectivity in the Reduction of a,b-Unsaturated Ketones 129 5.5 Conclusions and Perspectives 134 References 134 6 Protein Engineering: Development of Novel Enzymes for the Improved Reduction of CO Double Bonds 139 Nobuya Itoh and Yoshihide Makino 6.1 Introduction 139 6.2 Detailed Characterization of PAR 140 6.2.1 Location of PAR in Styrene Metabolic Pathway 140 6.2.2 Physicochemical Properties of PAR 142 6.2.3 Enzymatic Properties of PAR 147 6.2.4 Docking Model Construction of PAR 151 6.3 Detailed Characterization of LSADH 151 6.3.1 Screening of LSADH from Styrene-Assimilating Soil Microorganisms 151 6.3.2 Physicochemical Properties of LSADH 153 6.3.3 Enzymatic Properties of LSADH 153 6.4 Engineering of PAR for Increasing Activity in 2-Propanol/Water Medium 157 6.4.1 Construction of Sar268 Mutant 157 6.4.2 Construction of HAR1 Mutant 160 6.4.3 Characterization of Sar268 and HAR1 161 6.5 Application of Whole-Cell Biocatalysts Possessing Mutant PARs and LSADH 165 6.5.1 E. coli Whole-Cell Biocatalysts Possessing Mutant PARs and LSADH 165 6.5.2 Application of Immobilized E. coli Whole-Cell Catalysts to Continuous Production of Chiral Alcohol 168 6.5.3 Application of Immobilized E. coli Whole-Cell Catalysts (LASDH) for Regenerating NADH with IPA 171 6.6 Engineering of b-Keto Ester Reductase (KER) for Raising Thermal Stability and Stereoselectivity 172 6.6.1 Enzymatic Properties of KER 172 6.6.2 Engineering of KER and Characterization of Mutant Enzymes 175 6.7 New Approach for Engineering or Obtaining Useful ADHs/Reductases 177 6.7.1 Engineering the Coenzyme Dependency of Ketol-Acid Reductoisomerase (KARI) 177 6.7.2 Engineering Substrate- and Stereospecificity of Reductases by Structure-Guided Method 178 6.7.3 Engineering Database: Systematic Information of Sequence-Structure-Function 179 6.7.4 Metagenomics 180 References 181 7 Synthetic Applications of Aminotransferases for the Preparation of Biologically Active Molecules 187 Sachin Pannuri, Sanjay Kamat, and Abraham R. Martin-Garcia 7.1 Introduction 187 7.1.1 Aminotransferases 187 7.1.2 Transamination Reaction 188 7.1.3 Stereoselectivity of Aminotransferases 189 7.2 Applications 192 7.2.1 Biotransformation Process 192 7.2.2 Biologically Active Molecules 195 7.2.3 Process Economy 196 7.3 Challenges 196 7.3.1 Substrate Specificity 197 7.3.2 Improving Reaction Yield 197 7.3.3 Process Scale-Up 200 7.4 Future Research Needs 203 7.5 Conclusions 203 References 204 8 Strategies for Cofactor Regeneration in Biocatalyzed Reductions 209 Selin Kara, Joerg H. Schrittwieser, and Frank Hollmann 8.1 Introduction: NAD(P)H as the Universal Reductant in Reduction Biocatalysis 209 8.2 The Most Relevant Cofactor Regeneration Approaches - and How to Choose the Most Suitable One 210 8.2.1 Electrochemical Regeneration of NAD(P)H 212 8.2.2 H2 as Reducing Agent 213 8.2.3 Formates as Reducing Agents 215 8.2.4 Phosphites as Stoichiometric Reductants 218 8.2.5 Alcohols as Stoichiometric Reductants 218 8.2.6 Glucose as Stoichiometric Reductant 223 8.3 Coupling the Reduction Reaction to a Regeneration Reaction Producing a Valuable Compound 225 8.4 Avoiding NAD(P)H: Does It Also Mean Avoiding the Challenge? 228 8.5 Conclusions 230 References 231 9 Solvent Effects in Bioreductions 239 Yan Ni, Hui-Lei Yu, and Jian-He Xu 9.1 Introduction 239 9.2 Solvent Systems for Biocatalytic Reductions 240 9.2.1 Bioreduction in Aqueous Systems 240 9.2.2 Bioreduction in Monophasic Aqueous-Organic Systems 241 9.2.3 Bioreduction in Biphasic Aqueous-Organic Systems 243 9.2.4 Bioreduction in Micro- or Nonaqueous Systems 245 9.2.5 Bioreduction in Nonconventional Media 247 9.2.5.1 Ionic Liquids 247 9.2.5.2 Supercritical Fluids 250 9.2.5.3 Combining ILs and SFs 251 9.2.5.4 Gas-Phase Media 252 9.2.5.5 Reverse Micelles 254 9.3 Solvent Control of Enzyme Selectivity 255 9.4 Concluding Remarks 257 References 258 10 Application of In situ Product Removal (ISPR) Technologies for Implementation and Scale-Up of Biocatalytic Reductions 263 John M. Woodley 10.1 Introduction 263 10.2 Process Requirements for Scale-Up 263 10.3 Bioreduction Process Engineering 265 10.4 In situ Product Removal 267 10.5 Biocatalyst Format 269 10.5.1 Whole-Cell Processes 271 10.5.2 Isolated Enzyme Processes 272 10.6 Selected Examples 273 10.6.1 ISPR with Resins 273 10.6.2 ISPR with Solvent Extraction 274 10.6.3 ISPR with Crystallization 274 10.6.4 Removal of Acetone 275 10.7 Future Outlook 276 10.7.1 Protein Engineering 276 10.7.2 Choice of Methods 277 10.7.3 Process Integration 278 10.8 Conclusions 280 References 280 11 Bioreductions in Multienzymatic One-Pot and Cascade Processes 285 Daniela Monti and Erica E. Ferrandi 11.1 Introduction 285 11.2 Coupled Oxidation and Reduction Reactions 287 11.3 Consecutive and Cascade One-Pot Reductions 292 11.4 Cascade Processes, Including Biocatalyzed Reductive Amination Steps 296 11.5 Other Examples of Multienzymatic Cascade Processes, Including Bioreductive Reactions 302 References 304 12 Dynamic Kinetic Resolutions Based on Reduction Processes 307 Dimitris Kalaitzakis and Ioulia Smonou 12.1 Introduction 307 12.2 Cyclic Compounds 309 12.3 Acyclic a-Substituted-b-Keto Esters and 2-Substituted-1,3-Diketones 313 12.4 Acyclic Ketones and Aldehydes 322 12.5 Conclusions 323 References 324 13 Relevant Practical Applications of Bioreduction Processes in the Synthesis of Active Pharmaceutical Ingredients 329 Gabor Tasnadi and Melanie Hall 13.1 Introduction 329 13.2 Ketoreductases 337 13.2.1 Ethyl 4-chloro-3-hydroxybutanoate 337 13.2.2 Atorvastatin 338 13.2.3 Montelukast 339 13.2.4 Ramatroban 340 13.2.5 Ezetimibe 341 13.2.6 Profens 342 13.2.7 Atazanavir 343 13.2.8 Chemokine Receptor Inhibitor 343 13.2.9 Duloxetin 344 13.2.10 6-Hydroxybuspirone 345 13.2.11 LY 300164 346 13.2.12 Paclitaxel 346 13.3 Ene Reductases 347 13.3.1 Levodione 347 13.3.2 (p)-Dihydrocarvone 348 13.3.3 Butyrolactone - Jasplakinolide and Amphidinolides 348 13.3.4 (R )-Flurbiprofen 349 13.3.5 Ethyl (S )-2-ethoxy-3-(4-methoxyphenyl)propanoate - Tesaglitazar 350 13.3.6 Methyl (Z)-2-bromocrotonate - Antidiabetic Drug Candidates 350 13.3.7 Roche Ester 351 13.3.8 Human Neurokinin-1 Receptor Antagonists 352 13.3.9 Asymmetric Synthesis of Amino Acid Derivatives 353 13.4 Others 355 13.4.1 Amino Acid Dehydrogenase-Catalyzed Processes 355 13.4.1.1 Saxagliptin 355 13.4.1.2 Omapatrilat 356 13.4.1.3 Inogatran 357 13.4.1.4 Corticotropin-releasing Factor-1 (CRF-1) Receptor Antagonist 357 13.4.1.5 AG7088 358 13.4.2 Pyrrolo[2,1-c][1,4]benzodiazepines (Antitumor Agents) 358 13.4.3 Dihydrofolate Reductase 359 13.4.4 b-Carbolines 359 13.5 Bioreduction-Supported Processes 361 13.6 Outlook 363 References 365 Index 375

About the Author :
Elisabetta Brenna received her laurea (1989) and PhD (1993) in chemistry from the University of Milan. In 1996, she became Assistant Professor at Politecnico di Milano, where she is now Associate Professor. Her main scientific interests are the enzyme-mediated synthesis of the single enantiomers of chiral biologically active compounds, and the use of SNIF NMR technique for tracing back the synthetic history of active pharmaceutical ingredients and flavors. She is author of more than 120 publications on international journals.