Department of Chemistry, The Ohio State University, Columbus, Ohio, USA.
Nat Protoc. 2010 Apr;5(4):636-46. doi: 10.1038/nprot.2010.3. Epub 2010 Mar 11.
Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP-GlcNAc/GalNAc. Other UDP-GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from L-fucose, GDP-fucose is constructed by one bifunctional enzyme L-fucose pyrophosphorylase (FKP) via two reactions.
酶法合成利用糖基转移酶是一种高效、高选择性地构建多糖的有力方法。糖核苷酸是 Leloir 型糖基转移酶催化的酶糖基化反应中基本的供体分子。这些供体的应用受到其有限可用性的限制。在本方案中，N-乙酰葡萄糖胺（GlcNAc）/N-乙酰半乳糖胺（GalNAc）由 N-乙酰己糖胺激酶（NahK）磷酸化，然后由 N-乙酰葡萄糖胺尿苷酰转移酶（GlmU）焦磷酸化生成 UDP-GlcNAc/GalNAc。根据这些酶对修饰糖底物的耐受性，还可以制备其他 UDP-GlcNAc/GalNAc 类似物。以 L-岩藻糖为起始原料，通过两步反应，由双功能酶 L-岩藻糖焦磷酸化酶（FKP）构建 GDP-岩藻糖。
