Thioester Mediated Biocatalytic Amide Bond Synthesis with In Situ Thiol Recycling

Abstract

The conversion of carboxylic acids to thioesters is a key step in the biosynthesis of natural products, resulting in activation of the acyl groups for subsequent reactions, e.g. acylation of nucleophiles including carbon-carbon bond formation. For example, thioesters of Coenzyme A (CoA-SH; e.g. acetyl-S-CoA) are intermediates in many metabolic pathways, and are increasingly recognised as important cofactors for epigenetic post-translational modifications, such as N-, O- and S-acylations of proteins. However, the limited availability of a broad range of structurally diverse thioesters has limited their wider exploitation in biochemistry, cell biology and biotechnology. Furthermore, the high cost of CoA-SH impairs its use in stoichiometric quantities. To address these challenges we show that the adenylation (A-) domain of the carboxylic acid reductase (CAR) from Segniliparus rugosus (CARsr-A) can function as a broad spectrum acyl-S-CoA synthetase, to generate acyl-S-CoA intermediates from a wide range of carboxylic acids. In addition, CARsr-A was able to generate thioesters from structurally simpler thiols such as pantetheine. The resulting thioesters were then used as substrates for acyltransferases to synthesise a wide range of amides, including the more difficult to prepare, but pharmaceutically relevant aryl amides. Importantly, CoA-SH is recycled during the reaction and can be used in sub-stoichiometric quantities. This approach has also been applied to acylate a histone peptide H4-20 with a range of carboxylic acids, including non-natural chemical labels, by employing a lysine acetyltransferase (HATp300). Overall, this combination of a broad spectrum biocatalyst for thioester synthesis, together with in-situ CoA-SH recycling, provides a generic platform for thioester-dependent cell-free synthesis, with potential applications beyond amide bond formation.

Efficient Enzymatic Synthesis of Carbamates in Water Using Promiscuous Esterases/Acyltransferases

“…[8] Although this enzyme-catalyzed coupling of free carboxylic acids and amines is appealing, a major challenge for broader application has been the dependency on stoichiometric amounts of co-factors like ATP, which requires additional recycling systems. [9] In the past, the promiscuous aminolysis of esters and carbonates mediated by lipases was used to synthesize amides and carbamates, respectively. [10] Candida antarctica lipase B (CAL-B), for example, catalyzed this biotransformation in pure organic solvents to achieve product formation and to avoid the undesired hydrolysis of the target products.…”