Automated Solid Phase Synthesis of oligosaccharides as tools for structural and functional analysis

Abstract

Automated Glycan Assembly (AGA) is introduced in chapther 1. AGA is a powerful technique for the solid-phase synthesis of oligosaccharides. These glycans have potential as analytical standards and as substrates for structural and functional analysis of different carbohydrate-degrading enzymes. AGA is based on the addition of different building blocks (BB) to a functionalized solid support; oligosaccharides of different lengths and branching patterns can be produced after iterative cycles of AGA. In chapter 2 AGA was employed to prepare the core structure of arabinomannosides (AM) from M. tuberculosis, containing α-(1,6)-Man, α-(1,5)-Ara and α-(1,2)-Man linkages. The introduction of a capping step after each glycosylation and further optimized reaction conditions (time and temperature) allowed for the synthesis of a series of oligosaccharides, ranging from hexa- to branched dodecasaccharides. These improvements towards a more robust AGA platform ensure high coupling efficiencies over long sequences. In a collaboration work with Dr. Abragam, the limits of AGA were surpassed, granting access to a 100-mer α-(1,6) polymannoside. The flexibility of AGA in the synthesis of long structures was demonstrated by the convergent block coupling. A set of oligosaccharide fragments prepared by AGA gave a multiple-branched 151-mer polymannoside, the largest polysaccharide prepared by any synthetic method to date. This collection of arabinomannosides was used as standards for the developing of a new analytical technique, namely, direct imaging of single glycan molecules with sub-nanometer resolution using scanning tunneling microscopy (STM). In collaboration with Dr. Xu Wu, direct visualization of mannosides at sub-nanometer resolution permitted the differentiation of α-(1,2) and α-(1,6) linkages together with the localization of the branching point at a single-molecule scale. This technique is expected to be useful for the identification of recurrent structural features of glycans with biological importance. In Chapter 3 AGA was employed to synthesize a collection of six linear α-(1,6)-mannosides and seven β-(1,3)-glucans with specific β-(1,6) and β-(1,4) substitution patterns containing a free reducing end, using two different traceless photolabile linkers. These compounds were used to characterize carbohydrate-degrading enzymes obtained from marine sources. Synthetic α-(1,6)-mannosides permitted the characterization of the putative mannanase GH76A from Salegentibacter sp.. In collaboration with Dr. Solanki, a detailed 3D structure of the active site of GH76A was obtained after the co-crystallization of synthetic mannose tetramer with mutant mannanase GH76. Incubation of these synthetic α-(1,6)-mannosides with GH76A generated hydrolyzed glycans, this suggested that the enzyme GH76A functions as an endo α-(1,6)- mannanase