Synthetic Chain Terminators Off‐Load Intermediates from a Type I Polyketide Synthase

Abstract

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.

Chemical Probes for the Functionalization of Polyketide Intermediates

“…Recent efforts in these directions have highlighted both the innate and engineered chemical flexibility of PKSs in accepting a variety of non-native substrates to generate novel polyketide derivatives in vitro and in vivo, thus opening up the exploitation of novel chemical spaces and bioactivities for this class of natural products. [9] We have recently developed synthetic chain terminators capable of capturing polyketide biosynthetic intermediates in vitro [10] and in vivo. [11] These chemical probes (e.g., the b-ketoacids 1 a,b; Scheme 1; generated in situ from the hydrolysis of the corresponding methyl esters) [11] compete with ACP-bound malonate units for polyketide chain extension.…”

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

“…One promising approach to do this is suggested by our recent demonstration that a non-hydrolysable SNAC thioester analogue of (methyl)malonyl-CoA can successfully offload partially assembled polyketide chains from a modular PKS. [30] Experimental Section Experimental details and synthetic procedures are given in the Supporting Information.…”

Insights into Lasalocid A Ring Formation by Chemical Chain Termination In Vivo

“…[16][17][18] This approach has proved successful in vitro for the isolation of intermediates from iterative [16] and modular [17] recombinant enzymes. This strategy utilizes carba(dethia) mimics of the malonyl units normally recruited for polyketide formation to intercept and off-load truncated biosynthetic species from PKSs (Scheme 2, box).…”

“…This strategy utilizes carba(dethia) mimics of the malonyl units normally recruited for polyketide formation to intercept and off-load truncated biosynthetic species from PKSs (Scheme 2, box). [16][17][18] This approach has proved successful in vitro for the isolation of intermediates from iterative [16] and modular [17] recombinant enzymes. We have recently also shown that this methodology can be employed for in vivo studies: derivatives of intermediates of erythromycin biosynthesis were conveniently isolated from the ethyl acetate extracts of the soil bacterium Saccharopolyspora erythraea grown in the presence of malonyl carba(dethia) N-acetyl cysteamine esters.…”

“…In addition, derivative species Scheme 2. In vivo release of derivatives of intermediates from the lasalocid A polyketide synthase by using the synthetic chain terminators 6 a-c (box), [17][18] generated in situ by the hydrolysis of the corresponding methyl esters 5 a-c [18] (Table 1). The stereochemistry of the derivatives has yet to be established.…”