School of Chemistry - Theses
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ItemSubstrates, substrate mimics and inhibitors of bacterial and fungal mannanases and transmannosidases( 2016)Glycoside hydrolases are a diverse group of enzymes that have been classified into more than 130 sequenced based families. Glycoside hydrolase family 76 (GH76) is poorly characterized and is populated exclusively by bacterial and fungal members. The only known biochemical function for members of this family has been described for bacterial enzymes, termed α-1,6-mannanases, which hydrolytically cleave α-1,6-mannans, such as those found in the fungal cell wall. Separately, it has been hypothesized that certain fungal members, termed DCW1 and DFG5, are transmannosidases, with the ability to cleave an α-mannosidic linkage within the glycosylphosphatidylinositol (GPI) glycan of cell wall proteins, and then catalyze the formation of a new glycosidic linkage to the cell wall glycan, resulting in covalent attachment of mannoproteins to the fungal cell wall. The first part of this thesis details part of a large collaborative effort dedicated to establishing a detailed chemical mechanism for the foundation member of glycoside hydrolase family 76, Aman6 α-1,6-mannanase from Bacillus circulans. In an effort to obtain non-hydrolyzable substrate mimics, a series of S-linked α-1,6-oligomannosides were synthesized using alternative [2+2+2] approaches that involve assembly from either the reducing end or the non-reducing end. In the first approach, involving construction from the reducing end, coupling of a disaccharide thioacetate with a 6’-iodo reducing end disaccharide, followed by activation of the resulting tetrasaccharide to a 6’’’-iodide, and iterative coupling with the same disaccharide thioacetate afforded an S-linked hexasaccharide, as well as the intermediate di- and tetrasaccharides. In the second approach, involving construction from the non-reducing end, coupling of the above disaccharide thioacetate with an anomeric S-trityl protected 6’-iodo disaccharide, afforded an S-trityl tetrasaccharide, which was converted was converted to a tetrasaccharide thioacetate, which was in turn coupled with the same anomeric S-trityl protected 6’-iodo disaccharide to afford the hexasaccharide, which was finally elaborated to the methyl thioglycoside. X-ray crystallographic studies with the catalytic domain of Aman6 (BcGH76) and the synthetic S-linked oligomannosides provided evidence only for binding of sugar residues in the -3/-2 subsites, and no active site spanning complexes could be obtained for the wildtype enzyme. However, mechanistically informative complexes were obtained with α-1,6-mannosylisofagomine (ManIFG) and an active-site spanning complex with α-1,6-mannopentaose and a catalytically disabled mutant. In tandem with combined quantum mechanics/molecular mechanics calculations, a conformational itinerary for the enzyme-catalyzed reaction was proposed. The second part of this thesis details efforts to develop a non-hydrolyzable trisaccharide homologue of ManIFG that contained only S-linkages. Building upon the methodology and key intermediates developed in the previous part, S-linked di- and trisaccharide isofagomines were readily assembled. Crystallographic studies with BcGH76 revealed the disaccharide to bind unexpectedly in the -3/-2 subsites; the trisaccharide bound in a more orthodox fashon in the -3/-2/-1 subsities, but even so bound without proper engagement with the catalytic machinery, and in particular the catalytic nucleophile. We conclude that the introduction of one or more S-linkages substantially perturbs the structure of the S-linked oligomannosides such that they are poor mimics of substrate. The final chapter of this thesis discloses the synthesis of a GPI anchor fragment. Two tetrasaccharides, α-Man-1,2-α-Man-1,6-α-Man-1,4-α-GlcN-OMe, and the corresponding acetamide, were prepared using a novel procedure inspired by previous work of F. Kong. The route is notable for use of only ester, acetal and amide protecting groups, and unlike previous work, the tetrasaccharides were deprotected. It is anticipated that these tetrasaccharides may prove of use in obtaining direct experimental evidence in support of mannoprotein cross-linking into th fungal cell wall catalyzed by the family GH76 enzymes DCW1/DFG5.