The Development of Novel Biocatalysts for the Asymmetric Hydroamination of Alkenes A Thesis Submitted to the University of Manchester for the Degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2014 Sarah Louise Lovelock School of Chemistry 1 Contents List of Figures .............................................................................................................................. 5 List of Schemes ........................................................................................................................... 8 List of Tables ............................................................................................................................. 10 Abstract ..................................................................................................................................... 12 Declaration ................................................................................................................................ 13 Copyright Statement ................................................................................................................. 14 Acknowledgements ................................................................................................................... 15 Abbreviations ............................................................................................................................ 16 Chapter 1: Introduction .......................................................................................................... 18 1.1 Introduction to Directed Evolution ............................................................................. 19 1.2 Biocatalysis in Amine Synthesis................................................................................ 20 1.3 Optically Active α-Amino Acids ................................................................................. 24 1.3.1 Methods of α-Amino Acid Synthesis ................................................................. 25 1.4 Ammonia Lyases ....................................................................................................... 29 1.4.1 Phenylalanine Ammonia Lyases ....................................................................... 32 1.4.2 Catalytic Mechanism ......................................................................................... 41 1.4.3 Key Active Site Residues .................................................................................. 47 1.4.4 Previous Mutagenesis Work ............................................................................. 49 1.5 Objectives .................................................................................................................. 49 Chapter 2: Identifying a Candidate for Directed Evolution ................................................. 51 2.1 Introduction ................................................................................................................ 52 2.2 Analytical Methods for the Determination of PAL Catalyzed Activity ........................ 52 2.2.1 HPLC Analysis .................................................................................................. 54 2.2.2 Spectrophotometric Analysis ............................................................................. 56 2.2.3 L-Amino Acid Oxidase Coupled Assay .............................................................. 58 2.3 Optimization of PAL Expression................................................................................ 59 2.4 Optimization of PAL Catalyzed Reaction Conditions ................................................ 63 2.4.1 Amination Reaction Conditions ......................................................................... 63 2.4.2 Deamination Reaction Conditions ..................................................................... 64 2.5 Determining the Kinetic Constants for PAL Catalyzed Reactions ............................ 65 2.5.1 Deamination Kinetics ........................................................................................ 65 2.5.2 Amination Kinetics ............................................................................................. 67 2.6 Comparison of PcPAL, RgPAL and AvPAL as Whole Cell Biocatalysts for the Production of Non-Proteinogenic Amino Acids ..................................................................... 69 2.6.1 Analytical Scale Biotransformations .................................................................. 70 2.6.2 Large Scale Reactions and Product Isolation ................................................... 74 2.6.3 D-Amino Acid Synthesis .................................................................................... 74 2.7 Discussion and Conclusion ....................................................................................... 75 2.7.1 Chosen Target for Evolution ............................................................................. 77 Chapter 3: Mechanistic Studies of PAL Catalyzed D-Amino Acid Formation ................... 79 3.1 Introduction ................................................................................................................ 79 2 3.2 Time Dependence on e.e. ......................................................................................... 80 3.3 Wild-type AvPAL Catalyzed Deamination of 4-Nitrophenylalanine ........................... 82 3.4 Thermodynamic Position of Equilibrium .................................................................... 83 3.5 Investigating the Activity of 4-Nitro-cis-Cinnamic Acid .............................................. 85 3.6 Site-Directed Mutagenesis of Key Active Site Residues .......................................... 85 3.6.1 Kinetic Constants for S168A Catalyzed Reactions ........................................... 88 3.6.2 NaBH4 Reduced AvPAL .................................................................................... 89 3.7 Isotope Labelling Studies to Determine the Stereoselectivity of AvPAL Catalyzed Amination and Deamination Reactions ................................................................................. 91 3.7.1 Synthesis of Deuterated Standards .................................................................. 92 3.7.2 NMR Study of the Amination Reaction of Labelled Substrates ........................ 94 3.7.3 NMR Study of the Deamination Reaction of Labelled Substrates .................... 96 3.8 Docking Study ........................................................................................................... 99 3.9 Conclusions ............................................................................................................. 101 Chapter 4: Testing the Substrate Promiscuity of Wild-type PALs ................................... 104 4.1 Introduction .............................................................................................................. 104 4.2 Chemical Synthesis of Amine Substrates ............................................................... 106 4.3 Chemical Synthesis of Alkene Substrates .............................................................. 108 4.4 Analytical Methods .................................................................................................. 110 4.5 PAL Activity Towards Novel Substrates .................................................................. 111 4.6 Nucleophile Promiscuity .......................................................................................... 116 4.7 Conclusion ............................................................................................................... 118 Chapter 5: Assay Development and Screening ................................................................. 119 5.1 Introduction .............................................................................................................. 120 5.2 Assay Development ................................................................................................ 120 5.2.1 Measuring the Absorbance of Alkene Products .............................................. 120 5.2.2 Derivatisation Methods .................................................................................... 122 5.2.3 Enzyme Coupled Assays ................................................................................ 123 5.2.4 Summary of Assays ........................................................................................ 132 5.3 Screening ................................................................................................................ 134 5.3.1 Screening Variants for Enhanced D-Selectivity ............................................... 134 5.3.2 Screening Variants for Activity Towards Amine Substrates ............................ 141 5.4 Conclusions and Future Work ................................................................................. 143 Chapter 6: Materials and Methods ...................................................................................... 145 6.1 Chemicals and Reagents ........................................................................................ 146 6.2 Genes and Enzymes ............................................................................................... 146 6.3 Instruments .............................................................................................................. 148 6.4 Buffers and Solutions .............................................................................................. 148 6.5 Protein Production and Purification