RESEARCH FRONT CSIRO PUBLISHING Review Aust. J. Chem. 2009, 62, 510–520 www.publish.csiro.au/journals/ajc Recent Developments in Glycoside Synthesis with Glycosynthases and Thioglycoligases Bojana Raki ´ c A and Stephen G. Withers A,B A Centre for High-Throughput Biology and Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver V6T 1Z1, Canada. B Corresponding author. Email: withers@chem.ubc.ca Glycosynthases are hydrolytically incompetent engineered glycosidases that catalyze the high-yielding synthesis of glycoconjugates from glycosyl fluoride donor substrates and appropriate acceptors. Glycosynthases from more than 10 glycoside hydrolase families have now been generated, allowing the synthesis of a wide range of oligosaccharides. Recent examples include glycosynthase-mediated syntheses of xylo-oligosaccharides, xyloglucans, glycolipids, and aryl glycosides. Glycosynthases have also now been generated from inverting glycosidases, increasing the range of enzyme scaffolds. Improvement of glycosynthase activity and broadening of specificity has been achieved through directed evo- lution approaches, and several novel high-throughput screens have been developed to allow this. Finally, metabolically stable glycoside analogues have been generated using another class of mutant glycosidases: thioglycoligases. Recent developments in all these aspects are discussed. Manuscript received: 28 January 2009. Final version: 31 March 2009. Introduction Glycoconjugates, oligo- and polysaccharides are found throughout all living organisms and are involved in critical biological events including cellular differentiation, cellular communication, host–pathogen interactions, and oncogenesis. [1] Some oligo- and polysaccharides are structural and storage biopolymers and have found applications in the biotechnology industry. [2,3] Glycoconjugates, however, present important syn- thetic targets for therapeutics. [4,5] Although the methodologies of synthetic chemistry have advanced greatly, the complexity of glycoconjugates and oligo- and polysaccharides still makes their synthesis very challenging, especially on large scales. Control- ling regio- and stereoselective formation of glycosidic bonds is difficult and requires multiple protecting group manipulations that are laborious and typically result in low yields. [6] Enzymes offer an alternative synthetic approach, in which there is inherent Bojana Raki´ c obtained her B.Sc. in organic synthesis under the supervision of Professor Radomir Saiˇ ci´ c, at the University of Belgrade, Serbia. On receiving an international fellowship, she moved to Canada and obtained her Ph.D. in 2007 at the University of Ottawa/National Research Council of Canada under the supervision of Professor John Pezacki, where she worked on small-molecule effects on the hepatitis C virus. Currently, she is a Postdoctoral Fellow at the University of British Columbia with Professor Stephen Withers, where she works on developing chemical biology approaches to study the function of mammalian sialyltransferases. SteveWithers was trained (B.Sc. and Ph.D.) at the University of Bristol, UK, where he obtained his Ph.D. with Dr Michael Sinnott. He moved to Canada as a postdoctoral fellow, applying heteronuclear NMR to the study of enzymatic catalysis with Drs Brian Sykes and Neil Madsen in the Department of Biochemistry at the University of Alberta. In 1982, he moved to the University of British Columbia (UBC) as Assistant Professor of Chemistry. He now holds the Khorana Chair of Chemistry and Biochemistry at UBC and serves as the Director of CHiBi, the Centre for High-Throughput Biology at UBC. control of regio- and stereoselectivity, and often produce higher yields in fewer steps. [7] There are two main approaches to the enzymatic synthe- sis of glycoconjugates. The first is to use glycosyltransferases, which transfer sugars from nucleotide donors onto glycosyl acceptors. [8,9] One drawback of this approach is that many of these enzymes are membrane-bound, making them difficult to isolate or to express in a recombinant form; thus, syntheses are often restricted to small scales. However, bacterial glyco- syltransferases are proving valuable in helping to overcome this problem. Another problem is that the nucleotide donor sugars are expensive and in some cases unstable. Furthermore, the nucleotide by-product is in many cases a strong inhibitor of the enzyme. Although recycling schemes can alleviate some of these problems, alternative approaches are important. © CSIRO 2009 10.1071/CH09059 0004-9425/09/060510