Roddan, Rebecca (2021) Norcoclaurine synthase: structural studies, enzyme engineering and the biocatalytic synthesis of novel alkaloids. PhD thesis, Birkbeck, University of London.
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Abstract
Norcoclaurine synthase (NCS) is a biocatalyst, involved in plant alkaloid biosynthesis. In plants, NCS catalyses a stereoselective Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA), to give (S)-norcoclaurine, the first committed intermediate in benzylisoquinoline alkaloid (BIA) biosynthesis. Previous work has shown that a range of aldehydes and ketones are accepted by NCS, in place of 4-HPAA, to generate single isomer tetrahydroisoquinolines (THIQs). The THIQ moiety is found in many biologically active molecules and NCS provides a facile route towards novel analogues. The aim of this project was to rationalise and extend the substrate scope of NCS, in particular towards a-substituted aldehydes. Despite previous reports, a range of a-methyl substituted aldehydes were shown to be accepted by NCS. A kinetic resolution of the aldehyde was also observed, with the (R)-enantiomer preferentially accepted, leading to THIQ products with two well-defined stereocentres. Single point variants of NCS were shown to improve activities compared with the wild-type enzyme. One variant, M97V was particularly promising and lead to the acceptance of the benzaldehydes as substrates and so the single step syntheses of various (1S)-aryl-THIQs was achieved. To expand the amine substrate scope of NCS, N-methyl-phenylamines were also explored as substrates. Chemoenzymatic cascades were developed to increase the molecular complexity of the NCS-generated THIQs and crystallographic investigations, involving the co-crystallisation of reaction intermediate mimics helped to rationalise NCS activities. Routes towards (R)-THIQs were also explored, involving screening novel variants, NCSs from Nelumbo nucifera and another Pictet-Spenglerase, salsolinol synthase.
Metadata
| Item Type: | Thesis |
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| Copyright Holders: | The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted. |
| Depositing User: | Acquisitions And Metadata |
| Date Deposited: | 06 Oct 2021 08:52 |
| Last Modified: | 01 Nov 2023 14:52 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/46196 |
| DOI: | https://doi.org/10.18743/PUB.00046196 |
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