DOI: 10.1002/cbic.200600159 Use of Click Chemistry to Define the Substrate Specificity of Leishmania b-1,2- Mannosyltransferases Phillip van der Peet, [a] Carlie T. Gannon, [a] Ian Walker, [a] Zoran Dinev, [a] Marcus Angelin, [a] Shanna Tam, [a] Julie E. Ralton, [b] Malcolm J. McConville,* [b] and Spencer J. Williams* [a] Introduction Leishmaniasis threatens 350 million people across almost 100 countries, with some countries experiencing infection rates in the hundreds of thousands per year. [1] Untreated, visceral forms of leishmaniasis can lead to severe haemorrhaging, sep- ticaemia, intercurrent infection and, eventually, death. [2] There are currently no well-defined vaccines against Leishmania and existing drug treatments are severely limited by low efficacy, severe side affects, high cost in endemic areas and the emer- gence of parasite strains that are resistant to front-line drugs. Leishmania spp. are transmitted by sandfly vectors (belonging to the genus Phlebotomus or Lutzomya) that carry the infec- tious flagellated promastigote stage. Promastigotes injected into the skin of the mammalian host during a sandfly blood- meal are rapidly internalized by phagocytic cells including macrophages, dendritic cells and neutrophils. Internalized pro- mastigotes subsequently differentiate into aflagellate amasti- gotes in the mature phagolysosome of these cells and contin- ue to proliferate primarily in macrophages. The obligate intra- cellular amastigote stages can induce active disease or remain latent for decades and are the major target for new anti-leish- manial therapies. Leishmania amastigotes accumulate a novel class of intracel- lular oligosaccharides that function as the major carbohydrate reserve material of these parasites. [3] These oligosaccharides are composed exclusively of b-1,2-linked mannose with a degree of polymerization of 4–40. [3] Targeted deletion of the genes involved in the biosynthesis of b-1,2-mannan and/or mannose-containing surface glycoconjugates suggests that b- 1,2-mannan might be required for intracellular survival in mac- rophages. [4–6] Recent studies have shown that the initial steps in b-1,2-mannan biosynthesis are complex and involve a novel mannose–cyclic-phosphate primer. [7] Short mannose oligosac- charides, with or without the cyclic phosphate, are subse- quently elongated with b-1,2-linked mannosyl residues by non- processive mannosyltransferase(s). [7] Initial studies have surpris- ingly shown that the elongation reactions can be primed by mannose or methyl a- and b-mannosides (unpublished data). Based on these preliminary results, we sought to develop a rapid approach to determine the substrate tolerance of the Leishmania b-1,2-mannosyltransferases in greater detail. This [a] P. van der Peet, C. T. Gannon, Dr. I. Walker, Dr. Z. Dinev, M. Angelin, S. Tam, Dr. S. J. Williams School of Chemistry, Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville, Victoria 3010 (Australia) Fax: (+ 61)3-9347-8124 E-mail: sjwill@unimelb.edu.au [b] Dr. J. E. Ralton, Prof. M. J. McConville Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville, Victoria 3010 (Australia) Fax: (+ 61)3-9348-1421 Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Leishmania spp. are human pathogens that utilize a novel b-1,2- mannan as their major carbohydrate reserve material. We de- scribe a new approach that combines traditional substrate-modi- fication methods and “click chemistry” to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania b-1,2-mannosyltransferas- es responsible for b-1,2-mannan biosynthesis. The library was as- sembled by using the highly selective copper(I)-catalysed cycload- dition reaction of azides and alkynes to couple an assortment of azide- and alkyne-functionalized small molecules with comple- mentary alkyne- and azide-functionalized mannose derivatives. All mannose derivatives with a-orientated substituents on the anomeric carbon were found to act as substrates when incubat- ed with a Leishmania mexicana particulate fraction containing GDP-mannose. In contrast, 6-substituted mannose derivatives were not substrates. Representative products formed from the li- brary compounds were analysed by mass spectrometry, methyla- tion linkage analysis and b-mannosidase digestions and showed extension with up to four b-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 develop- ment of highly specific tools and inhibitors for their study. 1384 # 2006 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim ChemBioChem 2006, 7, 1384–1391