No abstract

For the first time, the thioglycoligase strategy has been successfully applied to alpha-glycosidases. The alpha-thioglycoligases derived from the family 31 glycosidases, alpha-xylosidase from E. coli (YicI) and alpha-glucosidase from Sulfolobus solfataricus, catalyze thioglycoligase reactions using alpha-glycosyl fluorides and deoxythioglycosides as donors and acceptors, respectively, in yields up to 86%. In addition, we describe the Michaelis complex of YicI using one of the thioglycosides as a nonhydrolyzable substrate analogue and discuss the structural insights this yields into the specificity and mechanism of family 31 alpha-glycosidases and the molecular basis of an associated genetic disease.

Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.

Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-beta-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from alpha-xylobiosyl fluoride to either p-nitrophenyl beta-xylobioside or benzylthio beta-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, beta-(1 --> 4) linkages.