Abstract
In this thesis, we employed IMPACTTM-CN system to overexpress L-fucokinase/GDP-fucose pyrophosphorylase (FKP), α-1,3-fucosyl- transferase (FucT), and β-1,3-N-acetylgalactosaminyltransferase/ β-1,3-galactosyltransferase (LgtD). FKP was used to synthesize GDP-Fucose. The synthesis was performed on preparative scales (230 mg) and the reaction was monitor by high performance liquid chromatography. After quenching the reaction with ethanol, the crude products were purified by gel filtration using BioGel P-2 resin (Bio-Rad). In combination with α-1,3-fucosyltransferase and other glycosyltransferases, Lewis X and sialyl Lewis X have been synthesized successfully. To synthesize P1 antigen, other enzymes such as galactokinase (GalK), β-1,3-N-acetylglucosaminyltransferase (HpGnT) and α-1,4-galactosyltransferase (LgtC) were expressed. After optimization of enzymatic reaction conditions, P1 antigen was synthesized successfully. Furthermore, an fluorous assisted separation was developed to facilitate the purification of enzymatic reaction products. By using a fluorous tag at the reducing end of saccharide, optimized conditions of the enzyme, and fluorous solide phase extraction, the separation time was remarkably reduced to 30 min for each seperation. In addition, α-1,3-fucosyltransferase was found to tolerate a wide range of substrates appended with a fluorous tag such as LacNAc, sialyl LAcNAc, and LacdiNAc in this study. After fucosylation, these substrates were transformed to fluorous tagged Lewis X, sialyl Lewis X and dimeric Lewis X.