Efficient Synthesis of Glycoconjugates by means of Automated Glycosynthesizer

研制了一种可用于高效、实用的糖缀合物合成的“酶基自动糖合成仪”。第一个版本的合成器命名为'高尔基'已经在这个项目中完成。固定化糖基转移酶催化糖分子从糖核苷酸到水溶性引物聚合物的转移反应，其中糖结合在柔性聚合物上。近年来，由于糖基转移酶克隆研究的进展，各种重组糖基转移酶变得可行。其中5种重组糖基转移酶（B-1，4-GalT、a-2，3-SiaT、a-2，6-SiaT、B-1，3-GlcNacT和a-1，3-FucT）已被成功固定化并在我们的合成仪中实现。由来自Ia型无乳链球菌的B-1，3-N-乙酰基-D-葡糖胺基转移酶（B-1，3-GlcNAcT）和麦芽糖结合蛋白（MBP）组成的融合蛋白已在大肠杆菌中以可溶性和高活性形式产生。水溶性引物聚合物是具有简单糖侧链作为糖基受体的聚丙烯酰胺基材料，其显著增强重组糖基转移酶与引物聚合物之间的亲和力。此外，引物聚合物可以通过使用简单的凝胶渗透色谱法在连续的糖延伸反应之后容易地从反应混合物中分离。酶的糖延伸过程可以通过NMR光谱和基于荧光共振能量转移（FRET）的分析来监测。虽然GalT的糖延伸反应可以定量进行，但目前SiaT和FucT的糖基化效率较低。我们将研究这些糖基转移酶的糖基化条件，以提高自动合成仪实际使用的转移反应的效率。

An 'enzyme-based automated glycosynthesizer' which can be applied for efficient and practical synthesis of glycoconjugates has been developed. The first version of the synthesizer named 'Golgi' has been accomplished in this project. Immobilized glycosyltransferases catalyze transfer reactions of sugar molecules from sugar nucleotides to water-soluble primer polymers in which sugars bound on the flexible polymers. Recently, a variety of recombinant glycosyltransferases are becoming practicable owing to advances in cloning studies of glycosyltransferases. Five of these recombinant glycosyltransferases, b-1,4-GalT, a-2,3-SiaT, a-2,6-SiaT, b-1,3-GlcNacT and a-1,3-FucT, have been immobilized successfully and is implemented in our synthesizer. A fusion protein composed of b-1,3-N-acetyl-D- glucosaminyltransferase (b-1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) have been produced in Escherichia coli as a soluble and highly active form. Water-soluble primer polymers, which are polyacrylamide-based materials with simple sugar side-chains as glycosyl acceptors, drastically enhance the affinity between recombinant glycosyltransferases and the primer polymers. In addition, the primer polymers can be easily isolated from the reaction mixture after the sequential sugar-elongation reactions by employing simple gel- permeation chromatography. The sugar-elongation processes by enzyme can be monitored by both NMR spectroscopy and fluorescence resonance energy transfer (FRET)-based analysis.Although sugar-elongation reactions by GalT proceed quantitatively, the efficiency of glycosylation by SiaT and FucT are low at the present time. We will examine the condition of glycosylation by these glycosyltransferases to improve the efficiency of the transfer reactions for practical usage of the automated synthesizer.