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Title: Synthetic and enzymatic studies related to branched-chain amino acid metabolism
Author: Rathbone, Daniel Lee
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1987
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Several sytheses of a-acetolactate analogues (2-hydroxy-3-oxo carboxylic esters) and related compounds were developed. The action of the enzyme acetolactate decarboxylase upon these compounds was studied. A synthesis of 3-bromo-2-oxo carboxylic acids, esters and amides was developed. Inhibition studies using methyl 3-bromo-3-methyl-2-oxo butanoate with pig liver esterase indicated that the esters were not suitable mechanism-based inhibitors of the enzyme. A synthesis of 3-hydroxy-2-oxo carboxylic esters was developed. These compounds were found to isomerise, via alkyl group migration, to the corresponding 2-hydroxy-3-oxo carboxylic esters upon treatment with catalytic quantities of dibutyl tin oxide. Cyclic substrates gave access to 6,7 and 8-membered ring-expanded products. A one-step synthesis of 2-hydroxy-3-oxo carboxylic esters, in high yield, from the corresponding c43-unsaturated esters was developed. This involved the use of acidic manganate (VII) in aqueous acetone and was found to be superior to any synthesis of acyclic 2- hydroxy-3-oxo carboxylic esters published to date. The enzyme acetolactate decarboxylase (ADC) was found to decarboxylate the (S)-isomers of CY-acetolactate and its analogues to give the corresponding (R)-(-hydroxyketones in high optical purity. The (R)-substrates were decarboxylated to (R)-ß. 1'-hydroxyketones via prior isomerisation to (S)-(-acetolactate analogues by a tertiary ketol rearrangement involving carboxylate group migration. As a result (-acetolactate analogues with non-identical substituents at the 2- and 3-positions gave structurally different OF-hydroxyketone products upon decarboxylation with ADC. In the case of C-acetolactate, a substrate with identical substituents at the 2- and 3-positions, both enantiomers were converted into (R)-(-)-acetoin with an enantiomeric excess greater than 98%. This represents an unusual example of the enzymic conversion of both enantiomers of a racemic substrate into a single enantiomer of product. The behaviour of the enzyme towards a range of substrates gave some insight into the nature of the enzyme active site. ADC was found to catalyse, stereospecifically, the incorporation of deuterium into the (R)-isomer of racemic acetoin at the methine position. The chirality of the deuterated and non-deuterated components of the partially deuterated mixture was analysed in situ by vibrational circular dichroism measurements. Acetolactate synthase isozyme II (ALS II) was used to generate QE-acetolactate and its homologues from simple 2-oxo carboxylic acids. These were decarboxylated in situ by ADC to give the corresponding a-hydroxyketones of high optical purity. In one example 01-acetohydroxybutyrate was generated by the action of ALS II upon 2- oxobutanoate. The structure of the single c-hydroxyketone prepared by ADC-catalysed decarboxylation of the 06-acetohydroxybutyrate indicated that the (S)-isomer of Qf-acetohydroxybutyrate had been generated. Model studies were carried out on 2-hydroxycyclohexanone for the stereospecific reduction of the enzymatically produced Ck-hydroxyketones to 1,2-diols. No signifiant chiral induction was observed with sodium borohydride in a range of solvents or with lithium aluminium hydride.
Supervisor: Not available Sponsor: Science and Engineering Research Council (Great Britain) (SERC) ; Imperial Chemical Industries plc
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry Chemistry, Organic Biochemistry