Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase

Abstract

Sugars appended to pharmaceutically important natural products influence key pharmacological properties and/or molecular mechanism of action.[1] However, studies designed to systematically understand and/or exploit the role of carbohydrates in drug discovery are often limited by the availability of practical synthetic and/or biosynthetic tools.[2] Among the contemporary options to address this limitation,[3–4] chemoenzymatic glycorandomization utilizes a set of flexible enzymes consisting of an anomeric kinase, sugar-1-phosphate nucleotidylytransferase, and natural product glycosyltransferase (GT).[4–6] While chemoenzymatic glycorandomization has been successfully applied to alter the natural sugar moieties of numerous natural products,[4–8] the process remains primarily restricted by enzyme specificity and availability of suitable GTs for the target of interest. Thus, although there is precedent for improving non-glycosylated therapeutics via glycoconjugation, including colchicine,[9] mitomycin,[10] podophyllotoxin,[11] rapamycin,[12] isophosphoramide mustards,[13] or taxol,[14] such targets remain beyond chemoenzymatic strategies. Recent studies on OleD, the oleandomycin (1) GT from Streptomyces antibioticus (Scheme 1a), revealed an enhanced triple mutant (A242V/S132F/P67T, referred to herein as ‘ASP’) that displayed marked improvement in proficiency and substrate promiscuity.[4] To probe the synthetic utility of this enhanced catalyst and expand upon previous reports of acceptor promiscuity for wild-type (WT) OleD,[15] we report a comparison of the aglycon specificities of the WT and ‘ASP’ OleD variants toward 137 drug-like acceptors. This study highlights the ability of OleD variants to glucosylate a total of 71 diverse acceptors, catalyze iterative glycosylation with numerous substrates, and establishes OleD as the first multifunctional GT capable of generating O-, S- and N-glycosides.