Engineering of a thermostable alcohol dehydrogenase towards imine reduction
The enantioselective reduction of carbon-nitrogen double bonds is a powerful tool for accessing chiral secondary amines. These are important building blocks in the production of many high-added value chemicals, with an estimated 40-45 % of all pharmaceuticals and 20 % of agrochemicals containing a c...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2018
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| Online Access: | https://eprints.nottingham.ac.uk/51890/ |
| _version_ | 1848798597448990720 |
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| author | Nethercott, Alanna Mary |
| author_facet | Nethercott, Alanna Mary |
| author_sort | Nethercott, Alanna Mary |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | The enantioselective reduction of carbon-nitrogen double bonds is a powerful tool for accessing chiral secondary amines. These are important building blocks in the production of many high-added value chemicals, with an estimated 40-45 % of all pharmaceuticals and 20 % of agrochemicals containing a chiral amine intermediate. However, most of their current methods of manufacture are inefficient and wasteful. As a consequence, there is an interest to develop better synthetic routes to chiral amines based on the use of catalytic technologies and of atom efficient reagents. One approach is with the use of enzymes, due to their high activities and selectivities, mild reaction conditions and their renewable origin. The enzymatic reduction of imines is a relatively recent area of research, and the known imine reductases (IREDs) have low activities and limited substrate scope. The research presented here investigates new routes to make imine reduction biocatalysts, whereby an alcohol dehydrogenase is used as a starting point to engineer reaction promiscuity. These enzymes catalyse the reduction of a similar functionality, the carbon-oxygen double bond. The mode of substrate activation differs between the two enzymes: a Brønsted acid is used in imine reductases, while a zinc(II) Lewis acid is used in alcohol dehydrogenases.
The well-characterised thermostable alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was selected, due to its broad substrate range and stability. Docking studies with selected imines suggested that these substrates could be accommodated in the hydrophobic binding pocket. Both imines and amines were shown to act as mixed inhibitors of the native TADH activity, and it was suggested that inhibition occurred due to binding elsewhere in the active site. Three approaches were investigated towards the engineering the TADH active site to avoid inhibition and promote imine activation, based on both genetic and chemical modifications.
First, sixteen zinc-devoid TADH mutants containing aspartic acid or tyrosine at selected positions within the active site were created, in an effort to mimic the key features found in existing imine IREDs. Whilst ketone reduction activity was abolished in these mutants, no imine reduction activity could be restored. Second, a chemical modification approach was proposed, involving the replacement of the catalytic zinc(II) ion by a rhodium(I), which is known to activate imines in synthetic catalysts. Zinc was successfully removed from the active site to yield apo-TADH, as shown by the absence of catalytic activity. Rhodium(I) was observed to non-selectively bind to apo-TADH and to restore a small degree of native activity, however no imine reduction activity was observed. Finally, a novel iridium(III) complex containing a nicotinamide-functionalised N-heterocyclic carbene ligand was designed and synthesised. This complex was capable of performing transfer hydrogenation of an aromatic ketone and an aromatic imine, in both organic and aqueous conditions. An increased activity was observed for the imine compared to the ketone in aqueous media employing sodium formate as the hydride source. Inhibition studies showed that the Ir-catalyst acted as a mixed inhibitor for TADH, suggesting that interactions with the protein do not occur at the co-factor binding site. |
| first_indexed | 2025-11-14T20:22:18Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-51890 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:22:18Z |
| publishDate | 2018 |
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| repository_type | Digital Repository |
| spelling | nottingham-518902025-02-28T12:04:53Z https://eprints.nottingham.ac.uk/51890/ Engineering of a thermostable alcohol dehydrogenase towards imine reduction Nethercott, Alanna Mary The enantioselective reduction of carbon-nitrogen double bonds is a powerful tool for accessing chiral secondary amines. These are important building blocks in the production of many high-added value chemicals, with an estimated 40-45 % of all pharmaceuticals and 20 % of agrochemicals containing a chiral amine intermediate. However, most of their current methods of manufacture are inefficient and wasteful. As a consequence, there is an interest to develop better synthetic routes to chiral amines based on the use of catalytic technologies and of atom efficient reagents. One approach is with the use of enzymes, due to their high activities and selectivities, mild reaction conditions and their renewable origin. The enzymatic reduction of imines is a relatively recent area of research, and the known imine reductases (IREDs) have low activities and limited substrate scope. The research presented here investigates new routes to make imine reduction biocatalysts, whereby an alcohol dehydrogenase is used as a starting point to engineer reaction promiscuity. These enzymes catalyse the reduction of a similar functionality, the carbon-oxygen double bond. The mode of substrate activation differs between the two enzymes: a Brønsted acid is used in imine reductases, while a zinc(II) Lewis acid is used in alcohol dehydrogenases. The well-characterised thermostable alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was selected, due to its broad substrate range and stability. Docking studies with selected imines suggested that these substrates could be accommodated in the hydrophobic binding pocket. Both imines and amines were shown to act as mixed inhibitors of the native TADH activity, and it was suggested that inhibition occurred due to binding elsewhere in the active site. Three approaches were investigated towards the engineering the TADH active site to avoid inhibition and promote imine activation, based on both genetic and chemical modifications. First, sixteen zinc-devoid TADH mutants containing aspartic acid or tyrosine at selected positions within the active site were created, in an effort to mimic the key features found in existing imine IREDs. Whilst ketone reduction activity was abolished in these mutants, no imine reduction activity could be restored. Second, a chemical modification approach was proposed, involving the replacement of the catalytic zinc(II) ion by a rhodium(I), which is known to activate imines in synthetic catalysts. Zinc was successfully removed from the active site to yield apo-TADH, as shown by the absence of catalytic activity. Rhodium(I) was observed to non-selectively bind to apo-TADH and to restore a small degree of native activity, however no imine reduction activity was observed. Finally, a novel iridium(III) complex containing a nicotinamide-functionalised N-heterocyclic carbene ligand was designed and synthesised. This complex was capable of performing transfer hydrogenation of an aromatic ketone and an aromatic imine, in both organic and aqueous conditions. An increased activity was observed for the imine compared to the ketone in aqueous media employing sodium formate as the hydride source. Inhibition studies showed that the Ir-catalyst acted as a mixed inhibitor for TADH, suggesting that interactions with the protein do not occur at the co-factor binding site. 2018-07-13 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/51890/1/PhD%20Thesis%20Alanna%20M%20Nethercott_corrected%202018.pdf Nethercott, Alanna Mary (2018) Engineering of a thermostable alcohol dehydrogenase towards imine reduction. PhD thesis, University of Nottingham. Chiral secondary amines; Imines; Imine reductases; Biocatalysis; Alcohol dehydrogenase |
| spellingShingle | Chiral secondary amines; Imines; Imine reductases; Biocatalysis; Alcohol dehydrogenase Nethercott, Alanna Mary Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title | Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title_full | Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title_fullStr | Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title_full_unstemmed | Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title_short | Engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| title_sort | engineering of a thermostable alcohol dehydrogenase towards imine reduction |
| topic | Chiral secondary amines; Imines; Imine reductases; Biocatalysis; Alcohol dehydrogenase |
| url | https://eprints.nottingham.ac.uk/51890/ |