Use of click chemistry to define the substrate specificity of Leishmania β-1,2-mannosyltransferases

Leishmania spp. are human pathogens that utilize a novel beta-1,2-mannon as their major carbohydrate reserve material. We describe a new approach that combines traditional substrate-modification methods and "click chemistry" to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania beta-1,2-mannosyltransferases responsible for beta-1,2-mannon biosynthesis. The library was assembled by using the highly selective copper(I)-catalysed cycloaddition reaction of azides and olkynes to couple an assortment of azide- and alkyne-functionalized small molecules with complementary alkyne- and ozide-functionalized mannose derivatives. All mannose derivatives with a-orientated substituents on the anomeric carbon were found to act as substrates when incubated with a Leishmania mexicana particulate fraction containing GDP-mannose. In contrast, 6-substituted mannose derivatives were not substrates. Representative products formed from the library compounds were analysed by moss spectrometry, methylation linkage analysis and beta-mannosidase digestions and showed extension with up to four beta-1,2-linked mannosyl residues. This work provides insights into the substrate specificity of this new class of glycosyltransferases that can be applied to the development of highly specific tools and inhibitors for their study.