Synthetic and Biosynthetic Studies on Antibiotic Natural Products

Abstract

Mupirocin is a mixture of metabolites isolated from Pseudomonas fluorescens with pseudomonic acid A (PA-A 1) as the major bioactive component, which possesses a C17 polyketide-derived monic acid moiety with a functionalised tetrahydropyran ring esterified by an unusual 9-HN acid fatty acid side chain. Details of the timing and mechanism of esterification remain to be proven, but it is proposed to be catalysed by MupB. To investigate the esterification process, a mutasynthesis approach was developed to prepare novel pseudomonic acid analogues 27, desepoxyPA-B with C7 side chain (7-HH). DesepoxyPA-B 26 was isolated from a P. fluorescens mmpE∆OR/∆mupU-pJH2 mutant and converted in a series of transformations involving hydrolysis, protection, esterification, deprotection and selective oxidation to give the target 27.

Feeding the ∆mupW strain of P. fluorescens with 26 converted into PA-C 3, losing the 8-hydroxyl group. With the same mutant, feeding 27 produced the novel metabolite C7-PA-C 74, but no fatty acid chain extension to the 9-HN side-chain occurred, suggesting that chain extension occurs earlier in the biosynthetic pathway before THP formation. In support of this proposal, feeding studies with the linear biosynthetic intermediate mupirocin W5 (21) with a C7 side chain showed some extension to mupirocin W4 7 the C9 side chain.

Further substrates were prepared, including the pantetheine derivatives of acid 53, 58, 61-C9, and 72 C7, and each was upgraded to acyl carrier proteins (ACPs) for assays. Results indicated that MupB functions as an acyltransferase, successfully transferring 53_MmpE_ACP to 53_MacpB during mupirocin biosynthesis. No hydrolysis occurred without MupB, highlighting the specificity of ACP interactions. Experiments involving holo/malonyl_MacpB, MmpB_TE, and MmpB_KS domains were also conducted using the synthetic substrates. Overall, these results support that esterification cannot occur before the formation of the C9 fatty acid side chain.

Chapter 3 describes preliminary studies toward the asymmetric synthesis of intermediates which have potential value in investigations into the mechanism of β-branching in polyketide biosynthesis. The approach involves opening anhydrides such as 101 using protected pantetheine and the catalyst 100.

Date of Award	17 Mar 2026
Original language	English
Awarding Institution	University of Bristol
Supervisor	Chris L Willis (Supervisor) & Matthew P Crump (Supervisor)