Development of Imine Reductases and Reductive Aminases for Chiral Amine Synthesis

296 Pages·2017·13.97 MB·English

by  Godwin Aleku

Development of Imine Reductases and Reductive
Aminases for Chiral Amine Synthesis

A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy (PhD) in the Faculty of Science and Engineering

Godwin Abazho Aleku
2016

School of Chemistry
Manchester Institute of Biotechnology
Contents
List of Tables ..................................................................................................................................... 9
Abstract .......................................................................................................................................... 11
Dedication ....................................................................................................................................... 13
Acknowledgement........................................................................................................................... 14
Abbreviations .................................................................................................................................. 15
Chapter 1 ........................................................................................................................................ 17
1.1. Introduction .......................................................................................................................... 17
1.1 Chiral Amines: significance and sources ................................................................................. 19
1. 2. Biocatalysis: an emerging source of chiral amines ................................................................ 20
1.3. Biocatalytic kinetic/dynamic resolution routes to chiral amines ............................................ 23
1.3.1. Lipases ........................................................................................................................... 23
1.3.2. Monoamine Oxidases (MAOs) ........................................................................................ 24
1.4. Biocatalytic asymmetric synthetic routes to chiral amines ..................................................... 25
1.4.1. ω-Transaminases (TAs) ................................................................................................... 25
1.4.3. Ammonia Lyases (ALs) .................................................................................................... 26
1.5 Engineered reductive aminases.............................................................................................. 27
1.6. Imine reductases................................................................................................................... 28
1.6.1. Reported imine bioreduction using whole cells with no implicated genes ....................... 30
1.6.2. Imine reductases in biosynthesis pathways .................................................................... 32
1.6.3. Known bacterial imine reductases .................................................................................. 36
1.7. Mining of genomic databases reveals a wide distribution of bacterial imine reductases. ....... 38
1.8. Mechanisms of IRED-catalysed reactions .............................................................................. 39
1.8.1. Catalytic mechanism ...................................................................................................... 39
1.8.2. Stereoselectivity of IREDs ............................................................................................... 41
1.8.3. Substrate specificity of IREDs ......................................................................................... 41
1.9. Biocatalytic synthesis of chiral amines: gaps and unmet needs.............................................. 41
1.10. PhD aims ............................................................................................................................. 42
1.11. References .......................................................................................................................... 43
Chapter 2 ........................................................................................................................................ 47
Material and methods ..................................................................................................................... 47
2.1. General information ............................................................................................................. 47
2.2. Chemicals ............................................................................................................................. 48
2
2.2.1. Cyclic imines and amines used as substrates/product standards for the biocalytic imine
reduction ................................................................................................................................. 48
2.2.2. Carbonyl compounds used as substrates for AspRedAm catalysed reductive amination . 49
2.2.3. Amine nucleophiles used as substrates/coupling partners for reductive amination of
carbonyl compounds. .............................................................................................................. 51
2.3. Strains and plasmids ............................................................................................................. 52
2.4. Chemical synthesis ................................................................................................................ 52
2.4.1. General reductive amination procedures for the preparation of amines (58n, 75a, 75d,
75n, 81a, 85a, 88a & 88g). ....................................................................................................... 52
2.5. Bioinformatics....................................................................................................................... 55
2.5.1. Sequence data retrieval and subcellular localization studies ........................................... 55
2.5.2. Phylogenetic analysis ..................................................................................................... 55
2.6. Molecular biology ................................................................................................................. 55
2.6.1. Cloning and expression of AoIRED .................................................................................. 56
2.6.2. Cloning and expression of AspRedAm ............................................................................. 57
2.6.3. Cloning of IREDs from Mesorhizobium sp and S. glauca .................................................. 60
2.7. Protein production and purification ...................................................................................... 60
2.7.1. Production and Purification of of N-terminal His tagged AspRedAm .............................. 60
6
2.7.2. Production and Purification of N-terminal His tagged AoIRED........................................ 61
6
2.7.3. Production and purification of MSMIRED and SgIRED ..................................................... 63
2.8. Biotransformations ............................................................................................................... 63
2.8.1. Whole-cell Biotransformation for imine reduction.......................................................... 63
2.8.2. Biotransformations using isolated enzymes for reduction of imines ............................... 63
2.8.3. Biotransformations using isolated enzymes for reductive amination of carbonyl
compounds .............................................................................................................................. 64
2.8.4. Biotransformations using isolated enzymes for oxidative deamination of amines ........... 64
2.7. Enzyme Assays ...................................................................................................................... 65
2.7.1. Measurement of reductive aminase activity ................................................................... 65
2.7.2. Measurement of imine reducing activity ........................................................................ 66
2.7.3. Measurement of deaminase activity .............................................................................. 66
2.8. Protein Engineering .............................................................................................................. 66
2.8.1. Site directed mutagenesis .............................................................................................. 67
2.8.2. Construction of mutant libraries (NNK libraries) ............................................................. 68
2.8.3. Production of mutants ................................................................................................... 68
2.9. Throughput activity screening for saturation library .............................................................. 68
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2.9.1. Primary/initial activity screening .................................................................................... 68
2.9.2. Secondary library throughput screening ......................................................................... 69
2.10. Analysis: Chromatography columns, conditions and retention times for investigated
substrates .................................................................................................................................... 70
2.10.1. Analysis of imines/iminium ions and biotransformation products ................................. 70
2.10.1. Analysis of products of reductive amination of carbonyl compounds ............................ 73
2.11. References .......................................................................................................................... 77
Chapter 3 ........................................................................................................................................ 78
Characterisation of four novel enzymes with NADPH-dependent imine reducing activity ................ 78
3.1. Mining of protein databases highlighted a wide distribution of imine reductases in eukaryotes
and prokaryotes .......................................................................................................................... 80
3.2. Phylogenetic analysis highlights homology and orthology relationship between fungal and
bacteria IREDs. ............................................................................................................................ 82
3.2.1 Branch A- ‘True IREDs’? ................................................................................................... 83
3.2.2. Branches B and C- promiscuous IRED activity? ................................................................ 84
3.2.1 Cloning, expression and purification of putative IREDs ........................................................ 85
3.3. Enzymological properties of AoIRED and AspREDAM ............................................................. 88
3.4. Biotransformation of model imine substrates using whole-cell IRED biocatalyst system ........ 89
3.5. Kinetic parameters ................................................................................................................ 93
3.6. Conclusion ............................................................................................................................ 96
3.7. Supporting information ......................................................................................................... 96
3.8. References ............................................................................................................................ 96
Chapter 4 ........................................................................................................................................ 98
Stereoselectivity and synthetic application of an imine reductase from Amycolatopsis orientalis
(AoIRED) .......................................................................................................................................... 98
4. 1. Background .......................................................................................................................... 98
4.2. Substrate specificity and stereoselectivity ........................................................................... 102
4.2.1. Substrate specificity and stereoselectivity towards substituted pyrrolines, piperideines
and azepines .......................................................................................................................... 102
4.2.2. Substrate specificity towards bulky imines ................................................................... 104
4.3. Inversion of stereoselectivity in the reduction of 1-methyl-3,4-dihydroisoquinoline 48. ...... 105
4.4. Determination kinetic parameters ...................................................................................... 110
4.5. Site directed mutagenesis of AoIRED active site residues .................................................... 111
4.6. Conclusion .......................................................................................................................... 115
4.7. Supporting information ....................................................................................................... 116
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4.8. References .......................................................................................................................... 116
Chapter 5 ...................................................................................................................................... 117
The reductive aminase from Aspergillus oryzae: substrate specificity, mechanism and synthetic
applications ................................................................................................................................... 117
5. 1. Background ........................................................................................................................ 117
5.2. Substrate specificity of AspRedAm in reductive amination .................................................. 118
5.2.1. Carbonyl acceptors....................................................................................................... 120
5.2.2. Amine nucleophiles .......................................................................................................... 122
5.2.3. Reductive amination of carbonyl compounds: predicting ease of reactivity ...................... 124
5.2.4. Amine-ketone equilibrium: what can work at reduced ratios? .......................................... 124
5.3. Comparison of reductive amination activities of AspRedAm , AoIRED and MSMIRED ....... 127
5.4. Kinetic studies ..................................................................................................................... 128
5.4.1. Kinetic parameters ....................................................................................................... 128
5.4.2. Steady state mechanism .............................................................................................. 130
5.2.3. Product inhibition ........................................................................................................ 131
5.5. Synthetic applications of AspRedAm ................................................................................... 133
5.5.1. Reductive amination .................................................................................................... 133
5.5.2. Enantioselective reduction of imines catalysed by AspRedAm ...................................... 136
5.4. Structure guided mutagenesis studies ................................................................................. 138
5.5 AspRedAm versus IREDs: evidence for AspRedAm role in imine formation. .......................... 140
5.6. Conclusion .......................................................................................................................... 144
5.8. Supporting information ....................................................................................................... 144
5.9. References .......................................................................................................................... 145
Chapter 6 ...................................................................................................................................... 146
Structure guided engineering of a reductive aminase and an imine reductase ............................... 146
6.1. Background ......................................................................................................................... 146
6.1 Addressing protein aggregation problem of AoIRED wild type enzyme through rational protein
engineering ............................................................................................................................... 147
6.1.1 The effect of N241X substitutions on stereoselectivity upon reduction of imine substrates
.............................................................................................................................................. 149
6.1.2. Effect of N241 substitution towards improving solution behaviour of AoIRED .............. 152
6.2 Determination of kinetic constants of AoIRED Variants ........................................................ 153
6.3 Engineering enantioselectivity in the Aspergillus oryzae reductive aminase (AspRedAm) ..... 155
6.3.1. Improving the (R)-selectivity of wild-type AspRedAm ................................................... 156
6.3.2 Inversion of stereoselectivity by single point mutant AspRedAm W210A ....................... 159
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6.4 Kinetic constants and implication of substrate binding on catalysis and stereoselectivity. .... 164
6.5. Conclusion .......................................................................................................................... 168
6.6. Supporting information ....................................................................................................... 168
6.7. References .......................................................................................................................... 168
Chapter 7 ...................................................................................................................................... 170
Oxidative deamination of primary and secondary amines towards kinetic resolution of racemic
amines .......................................................................................................................................... 170
7.1. Background ......................................................................................................................... 170
7.2. Substrate specificity of AspRedAm in the oxidative deamination of racemic amines............ 172
7.3 AspRedAm-catalysed kinetic resolution of racemic amines employing an NADPH oxidase
cofactor recycling system .......................................................................................................... 174
7.4. An AspRedAm mutant Q240A with improved deaminase selectivity.................................... 178
7.5. Kinetic resolution system using coupled imine co-substrate ................................................ 178
7.6. Kinetic resolution of racemic amines using a "sacrificial amine" as a co-substrate. .............. 179
7.7. AspRedAm catalysed reductive amination using cyclic amine as a co-susbtrate ................... 182
7.9. AspRedAm-NOX-ammonia borane cascade enables the efficient deracemisation of racemic
amines....................................................................................................................................... 186
7.8. Conclusion .......................................................................................................................... 188
7.9. References .......................................................................................................................... 189
Chapter 8 ...................................................................................................................................... 190
General discussion and conclusion ................................................................................................ 190
8.1. Discovery and characterisation of novel imine reductases (IREDs) ....................................... 191
8.2. Discovery and characterisation of novel multifunctional biocatalyst.................................... 192
8.3. Substrate specificity/reactivity model/chart ........................................................................ 193
8.4. Mechanistic insights ............................................................................................................ 193
8.5. Steroeselectivity ................................................................................................................. 195
8.6. Engineering ......................................................................................................................... 195
8.6.1. Engineering AoIRED ...................................................................................................... 195
8.6.1 Engineering AspRedAm ................................................................................................. 196
8.7. Synthetic applications ......................................................................................................... 196
8.8. Future direction .................................................................................................................. 197
8.8.1. Searching for highly active broad spectrum reductive aminases ................................... 197
8.8.2. AspRedAm in potential cascade reactions .................................................................... 199
8.9. Projected impacts of this work ............................................................................................ 199
8.10. References ........................................................................................................................ 200
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Appendix 1 .................................................................................................................................... 202
9.1. List of Publications .................................................................................................................. 202
Appendix 2 .................................................................................................................................... 204
9.2. Additional results for AoIRED mutants .................................................................................... 204
9.2.1. Biotransformations data AoIRED variants at N171. ....................................................... 204
9.2.2 Kinetic parameters of AoIRED variants at N171. ............................................................ 204
9.2.3. Biotransformations data AoIRED variants at Y179......................................................... 205
9.2.4. Kinetic parameters of AoIRED variants at N171. ........................................................... 205
Appendix 3 .................................................................................................................................... 207
9.3. Plasmids Details ...................................................................................................................... 207
9.3.1. Plasmid details for Aspergillus oryzae IRED (AspRedAm) .................................................. 207
9.3.1.1. Sequence: pET28a-His -AspRedAm ............................................................................ 207
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9.3.1.2. AspRedAm Insert Gene Details .................................................................................. 209
9.3.1.3 AspRedAm Protein sequence ..................................................................................... 209
9.3.2. Plasmid details for Amycolatopsis orientalis IRED (AoIRED) .......................................... 209
9.3.2.1. AoIRED plasmid Sequence: pET28a-His -AoIRED ....................................................... 209
6
9.3.2.2. AoIRED Insert Gene Details ........................................................................................ 211
9.3.2.3. AoIRED protein sequence .......................................................................................... 211
9.3.3 Plasmid details for Mesorhizobium IRED (MSMIRED) ............................................................. 211
9.3.3.1. MSMIRED Sequence: pET28a-His -MSMIRED ............................................................. 211
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9.3.3.2. MSMIRED Insert Gene Details ................................................................................... 213
9.3.3.3. MSMIRED Protein sequence ...................................................................................... 213
9.3.4. Plasmid details for S. glauca (SgIRED)................................................................................... 213
9.3.4.1 SgIRED Insert Gene Details ......................................................................................... 213
9.3.4.2 SgIRED Protein sequence............................................................................................ 214
Appendix 4: Pimers........................................................................................................................ 214
9.4. Lists of mutagenic primers ...................................................................................................... 214
9.4. Lists of mutagenic primers for AspRedAm ....................................................................... 214
9.4.2 Lists of mutagenic primers for AoIRED ........................................................................... 215
Appendix 5 .................................................................................................................................... 217
Supporting information for steady state kinetic studies of AspRedAm ........................................... 217
9.5.1. Steady state kinetic mechanism ................................................................................... 223
9.5.3. Product inhibition studies ............................................................................................ 224
9.5.4. Rate equation for competitive inhibition ...................................................................... 226
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Appendix 6 .................................................................................................................................... 227
9.6. Chromatograms ...................................................................................................................... 227
9.6.1. Chromatograms for the analysis of AoIRED catalysed reactions described in Chapter 4. ... 227
9.6.2. Chromatograms for AspRedAm catalysed reactions described in Chapter 5 & 6. .............. 236
9.6.3 Chromatograms for AspRedAm-catalysed Kinetic Resolution of racemic amines ............... 266
9.6.4 pH and buffer optimization for the AspRedAm-NADPH oxidase (NOX) kinetic resolution
system ....................................................................................................................................... 268
9.6.5. Analysis of biotransformation results kinetic resolution of racemic amines substrates using
the AspRedAm-Nox kinetic resolution system ............................................................................ 274
9.6.6 Chromatograms for the kinetic resolution of racemic amines using a cyclic imine or cyclic
amine to recycle the cofactor .................................................................................................... 285
9.6.7. Analysis of biotransformation results for the deracemisation of racemic amines using
AspRedAm-Nox –ammonia borane cascade. .............................................................................. 291

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List of Tables
Table 1.1. Different species of imine reductases .............................................................................. 30
Table 2.2. Carbonyl compounds used as substrates for AspRedAm catalysed reductive amination. . 50
Table 2.3. Amine nucleophiles used as substrates/coupling partners for reductive amination of
carbonyl compounds ....................................................................................................................... 51
Table 2.4. Summary of yields and sizes of purified IREDs ................................................................. 63
Table 2.5. Typical PCR reaction set up. ............................................................................................. 67
Table 2.6. PCR conditions ................................................................................................................ 67
Table 2.7. Reaction set up for one pot phosphorylation and ligation and transformation of E.coli with
mutant plasmid. .............................................................................................................................. 68
Table 2.8. HPLC analysis: Methods and retention times for imine substrates and biotransformation
products. ......................................................................................................................................... 71
Table 2.9. GC analysis: Methods and retention times of imine substrates and products from
biotransformation run on CP-ChiraSil-DEX CB column using derivatised samples. ............................ 72
Table 2.10. HPLC analysis: methods and retention times for carbonyl acceptors and amine products.
........................................................................................................................................................ 75
Table 2.11. GC-analysis: methods and retention times of carbonyl acceptor substrates and amine
products from biotransformation run on CP-ChiraSil-DEX CB column using underivatised samples .. 76
Table 3.12. Biotransformation results using purified MSMIRED and SgIRED. .................................... 92
Table 3.13. Comparison of the kinetic parameters of AoIRED, AspRedAm, MSMIRED and SgIRED for
selected substrates 48 & 50. ............................................................................................................ 93
Table 4.14. Monocyclic prochiral imines reduced by purified AoIRED. ............................................ 103
Table 4.15. Bulky prochiral imines reduced by purified AoIRED. ..................................................... 105
Table 4.16. Steady-state kinetic parameters for selected AoIRED substrates. ................................. 111
Table 4.17. Biotransformation data for AoIRED mutants at N171, Y179 and N241. ........................ 113
Table 4.18. Kinetic parameters of active site mutants of AoIRED against selected substrates. ....... 114
Table 5.19. Relative activity of carbonyl acceptors in AspRedAm catalysed reductive amination. ... 119
Table 5.20. Relative activity of amines in AspRedAm catalysed reductive amination. ..................... 120
Table 5.21. AspRedAm-catalysed reductive amination of carbonyl compounds at low molar ratios.
...................................................................................................................................................... 126
Table 5.22. Comparison of reductive amination activities of AspRedAm , AoIRED and MSMIRED. .. 128
Table 5.23. AspRedAm Kinetic constants for selected substrates. .................................................. 129
Table 5.24. Product inhibition patterns of AspRedAm catalysed synthesis and oxidation of N-
methylcylohexylamine. .................................................................................................................. 132
9
Table 5.25. Biotransformation results of AspRedAm catalysed reductive amination of cyclic ketones
...................................................................................................................................................... 133
Table 5.26. Biotransformation results of AspRedAm catalysed reductive amination of acyclic carbonyl
compounds. .................................................................................................................................. 135
Table 5.27. Biotransformation results of AspRedAm catalysed reduction cyclic and preformed imines.
...................................................................................................................................................... 137
Table 5.28. Kinetic constants of AspRedAm on selected imine substrates ...................................... 138
Table 5.29. Biotransformation and kinetic data of AspRedAm WT and mutants D169A, D169N &
Y177A. ........................................................................................................................................... 139
Table 6.30. Whole cell biotransformation for the reduction of MDQ (48) and 2-MPN (42a) catalysed
by AoIRED mutants at N241. .......................................................................................................... 151
Table 6.31. Kinetic constants of AoIRED variants at N241 for MDQ 48. .......................................... 154
Table 6.32. Comparison of biotransformation results afforded by AspRedAm WT and its variant
Q240A. .......................................................................................................................................... 158
Table 6.33. Biotransformation results for the reductive amination catalysed by AspRedAm W210A
variant. .......................................................................................................................................... 163
Table 6.34. kinetic constants of AspRedAm variants at Q240 and W210. ....................................... 165
Table 6.35. Comparison of AspRedAm kinetic constants for ketones and amines. ......................... 166
Table 7.36. AspRedAm catalysed kinetic resolution or racemic amine employing an NADPH oxidase
for co-factor recycling.................................................................................................................... 177
Table 7.37. AspRedAm-catalysed kinetic resolution employing different imine co-substrates. ....... 179
Table 7.38. AspRedAm catalysed reductive amination of carbonyl compounds employing MTQ as an
amine co-substrate for co-factor recycling. .................................................................................... 185
Table 7.39. Biotramsformation results for the deracemisation of racemic amines using the
AspRedAm-NOX-ammonia borane cascade.................................................................................... 187
Table 9.40. Biotransformations of imine substrates using WT AoIRED and variants at N171........... 204
Table 9.41. Kinetic parameters of WT AoIRED and variants at N171. .............................................. 204
Table 9.42. Biotransformations of imine substrates using WT AoIRED and variants at Y179. .......... 205
Table 9.43. Kinetic parameters of WT AoIRED and variants at Y179. .............................................. 205
Table 9.44. Biotransformation of imine substrates catalyzed by WT AoIRED and its N241A mutant.
...................................................................................................................................................... 206
Table 9.45. List of mutagenic primers- AspRedAm ......................................................................... 214
Table 9.46. Lists of mutagenic primers for AspRedAm (continued). ................................................ 214
Table 9.47. List of muatanic primers for AoIRED. ........................................................................... 215
10