Step-Economic Synthesis of Biomimetic β-Ketopolyene Thioesters and Demonstration of Their Usefulness in Enzymatic Biosynthesis Studies

Abstract

Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic β-ketopolyene thioesters that is based on an Ir-catalyzed reductive Horner−Wadsworth−Emmons olefination. New β-ketotriene and pentaenethioates of pantetheine and N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.

In vitro studies of maleidride-forming enzymes

“…In order to probe the selectivity of the BfL1 hydrolase we synthesised SNAC, pantetheine, CoA and ACP-bound potential substrates (Scheme 2). 22,23 In our previous work we assumed that the acyl group should be unsaturated (e.g. an E-hex-2-enoyl substrate) but this was unproven, 10 so we made both E-hex-2enoyl 14a-d and hexanoyl 15a-d substrates.…”

Studying a Bottleneck of Multimodular Polyketide Synthase Processing: the Polyketide Structure-Dependent Performance of Ketoreductase Domains

“…The stereoselectivity of isolated KRs is reproduced in vitro if the structure of the acyl part of SNAC thioesters matches that of the biosynthetic precursor but can be entirely altered in response to minimal structural changes. [20][21][22][23]27 Several studies, for example, showed that TylKR1 from the first elongation module of the tylactone-forming PKS reduces the racemic pentane thioate 21 to the expected 2-D,3-D-configured product (B1type reduction) by si face hydride addition to 2-D-21 that is continuously formed by spontaneous C-2 racemization. In contrast, a 2-L,3-L-configured main product (A2-type) was obtained with butanethioate 20, resulting from the re face reduction of 2-L-20.…”

“…21−23 In the case of MycKRB from the mycolactone PKS, the expected B-type selectivity was observed with polyene 29, mimicking polyunsaturated precursors like 50, and not with 21. 19,27 The structure of the polyketide chain thus has a relevant influence on the stereoselectivity of KR domains, making structure−function relationships crucial for a better understanding of natural PKS and for successful PKS engineering.…”

“…31 MycKRB is a general B-type KR that acts as B0-or B1-type depending on the biosynthetic precursor. 19,27 The KR domains were produced as N-terminal His 6 -tagged fusion proteins by heterologous expression in Escherichia coli and purified (Figure S1). As previous studies had found a tagdependent binding mode switch in reductase active sites, Cterminally His 6 and Pro 5 -tagged derivatives of EryKR1 and TylKR1 were included to evaluate this effect.…”

Cross-linking of a polyketide synthase domain leads to a recyclable biocatalyst for chiral oxygen heterocycle synthesis

“…[5][6][7] On the other hand, PKS domains frequently show relaxed substrate specicity and, particularly in the case of domains that catalyse synthetically attractive reactions, an application in chemoenzymatic synthesis is thus conceivable. 6,[8][9][10][11][12][13] Various recent reports have highlighted the existing potential of PKS enzymes for this purpose. [14][15][16][17][18][19] Immobilisation is an effective way to increase the practical value of enzymes by providing them with higher stability and recyclability.…”