Use of click chemistry to define the substrate specificity of Leishmania beta-1,2-mannosyltransferases.
Leishmania spp. are human pathogens that utilize a novel beta-1,2-mannan 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-mannan biosynthesis. The library was assembled by using the highly selective copper(I)-catalysed cycloaddition reaction of azides and alkynes to couple an assortment of azide- and alkyne-functionalized small molecules with complementary alkyne- and azide-functionalized mannose derivatives. All mannose derivatives with alpha-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 mass 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.
van der Peet
Phillip
P
School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Gannon
Carlie T
CT
Walker
Ian
I
Dinev
Zoran
Z
Angelin
Marcus
M
Tam
Shanna
S
Ralton
Julie E
JE
McConville
Malcolm J
MJ
Williams
Spencer J
SJ
D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry; D002474:Cell-Free System / Q000378:metabolism; D002855:Chromatography, Thin Layer; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D007894:Leishmania mexicana / Q000201:enzymology*; D008358:Mannose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D008362:Mannosides / Q000138:chemical synthesis / Q000737:chemistry; D008364:Mannosyltransferases / Q000378:metabolism*; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D000480:Alkynes; D001386:Azides; D008362:Mannosides; D008364:Mannosyltransferases; D008358:Mannose