Chemical and biological tools for nvestigating the catalytic mechanism of 7-epizingiberene synthase

Pople, Huw 2023. Chemical and biological tools for nvestigating the catalytic mechanism of 7-epizingiberene synthase. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

7-epizingiberene synthase (EZS) is a terpene synthase found in the wild tomato plant Solanum habrochites produces 7-epizingiberene, a whitefly repellent, from the less common terpene precursor (2Z,6Z)-farnesyl diphosphate. The hydride shifts that occur during the catalytic mechanism of EZS from Solanum habrochites are currently unknown. The synthesis of deuterated analogues of its natural substrate (2Z, 6Z)-farnesyl diphosphate (zzFPP) is the first step toward solving this problem. The current synthesis of zzFPP from readily available nerol, involves a six-step process including a nonselective synthesis of the 2Z double bond resulting in at least half of the product being the incorrect isomer, followed by a low yielding chemical phosphorylation. Previous work showed that this route was not fit for the purpose of developing substrate analogues. Alterations to the chemical route were tried, including attempted improvements to the chemical phosphorylation step, isomerisation of the incorrect isomer to reclaim yield lost as byproduct and several iterations on a route starting from directed synthesis of the double bond were attempted, all of which were unsuccessful. The development of a chemoenzymatic route to all-Z sesquiterpenes required mutation of the zzFPP synthase (zFPS) from S. habrochites. zFPS L145W, was shown to form neryl diphosphate preferentially when compared to the wildtype and as such, allows for stepwise build-up of zzFPP analogues by first making a neryl diphosphate analogue followed by chain extending to zzFPP without the need for yield damaging steps between extensions. The complete chemoenzymatic route was demonstrated to be viable using IPP, 1d2-IPP and DMAPP although the hydride shifts of the catalytic mechanism of EZS are still unclear.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	12 April 2024
Last Modified:	12 Apr 2024 08:13
URI:	https://orca.cardiff.ac.uk/id/eprint/167918

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