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Efficient chemoenzymatic synthesis of terpenes

Huynh, Florence 2020. Efficient chemoenzymatic synthesis of terpenes. PhD Thesis, Cardiff University.
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Abstract

Terpenes and terpenoids are natural products produced by terpene synthases from linear polyprenyl diphosphates. These enzymes catalyse a complex cascade of reactions involving carbocations, modification in bonding and hybridisation in a regio- and stereoselective manner. A great number of them are high-value compounds as they display relevant biological activities and their supply is limited. The terpenoid artemisinin is, for example, the most used treatment against malaria and currently extracted from human cropped Artemisia annua. Recent studies have promoted the in vitro use of natural or unnatural substrates with terpene synthases to generate existing or novel terpenoids. Chemically synthesised farnesyl diphosphates containing heteroatoms, hydroxy, fluoride or alkyl groups have been used with sesquiterpene synthases (STS) to generate new sesquiterpene analogues. This chemoenzymatic approach has allowed the discovery of compounds showing potential application in the agriculture, flavour, or fragrance industry. This thesis focuses on the design and exploration of methodologies to address the challenges of the chemoenzymatic production of natural and novel terpenoids. The first part of the thesis focuses on improving the diphosphorylation procedure of farnesol and its analogues. A one-pot enzymatic diphosphorylation involving undecaprenol kinase and a variant of isopentenyl phosphate kinase was proven to convert farnesol successfully to farnesyl diphosphate (FDP) in 95% yield. In comparison to the traditional method, a chemical disphosphorylation, which gives FDP in 40% yield on average. Several farnesol analogues were also diphosphorylated using this method showing its potential versatility, namely: 6,15-dimethylfarnesol, 14,15-dimethylfarnesol, 10,11-epoxyfarnesol and 8-methoxyfarnesol. The sesquiterpene product arising from the incubation of 10,11-epoxyfarnesyl diphosphate with germacradien-4-ol synthase was isolated and fully characterised for the first time. Ultimately, this work illustrated how the high-performance counter current chromatography (HPCCC), a technique traditionally used for chromatography, could be used as a liquid-liquid reactor for biocatalysis to improve sesquiterpene yields. Several sesquiterpenes were synthesised in a high yield using the efficient mixing created by the centrifugal forces in the HPCCC apparatus. Carrying out the reaction in this counter current chromatography apparatus gave >70% yields while being almost 10 times faster than the segmented flow system and 70 times faster than batch. This methodology also found applications in stereoselective esterification with lipases, cutting the reaction time from one hour in batch to six minutes using the HPCCC apparatus.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 20 January 2021
Last Modified: 22 Jan 2021 14:52
URI: http://orca.cardiff.ac.uk/id/eprint/137791

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