Study the specificity of UDP-Sugar pyrophosphorylase using galactose and glucose analogues

• Main Authors: Wen-Cheng Chen, 陳文成
• Other Authors: Chi-Huey Wong
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/42815979583560682180

碩士 === 國立臺灣大學 === 化學研究所 === 100 === Glycans and glycoconjugates are important biomolecules, which participate in various cellular processes, e.g. cancer growth, infiltration, and metastasis. Although many chemical synthetic methods have been developed during last decade, the synthesis of glycoconjugates, especially the stereoselective glycosylation, remains to be problematic. In contrast, enzymatic synthesis using glycosyltransferases is advantageous in terms of not only chemical selectivities but also green chemistry. In this study, we looked into arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP), an enzyme that catalyses the conversion of monosaccharide-1-phosphate to the respective UDP-sugars with broad specificity, and its application for the synthesis of glycoconjugate containing non-natural sugar. We attempted to use kinase to transfer non-nature monosacharides to monosacharide-1-phosphate followed by AtUSP-catalyzed transfermation of these monosacharides-1-phosphates to the respective UDP-sugars. To examine the substrate tolerance of AtUSP, we synthesized glucose and galactose analogues containing azido, fluorous, methyl, vinyl, and alkynyl groups at the C-6 position. The azido and fluorous ananlogues were prepared form C-6 OH free derivatives by SN2 reaction and fluorination, respectively, using DAST. Alkenyl and alkynyl mimics were synthesized from the corresponding aldehydes via Wittig olefination and Bestman-ohira reaction. Finally, methyl analogues were obtained by hydrogenation of corresponding alkenyl derivatives. We also synthesized GlcNAc and GalNAc analogues with different azidoalkylcarbonyl groups and pentynoyl groups. We applied amide bond formation to synthesize these compounds. This methodology is straightforward and may be applicable for the preparation of other sugar analogues. The evaluation of the synthesized analogues as substrates is under way. However, when we used kinase to catalyze the formation of monosaccharide-1-phosphates, the reaction rate is too slow to obtain any kinetic data. The problem may be solved by using more kinases or chemical method to synthesize these monosaccharide-1-phosphates to test AtUSP more quickly.