Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.
Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.
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DOI：
10.1039/c5sc02025e
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发表时间：
2015-10-01
期刊：
影响因子：
8.4
通讯作者：
Wang PG
中科院分区：
文献类型：
--
作者：
Li L;Liu Y;Ma C;Qu J;Calderon AD;Wu B;Wei N;Wang X;Guo Y;Xiao Z;Song J;Sugiarto G;Li Y;Yu H;Chen X;Wang PG
高效的化学酶合成策略和基于hilic的纯化方法能够快速获得n -聚糖异构体库。由于n -聚糖的复杂性、多样性和低丰度，蛋白质上n -聚糖的定量、表征和生物功能研究仍然是具有挑战性的任务。结构上定义的n -聚糖（特别是同分异构体）库的可用性对于帮助解决这些任务至关重要。我们在此报告了一种高效的化学酶策略，即核心合成/酶延伸（CSEE），用于快速生产各种n -聚糖。从5个化学制备的构建块开始，通过在聚合片段偶联策略中一致使用低聚糖硫醚作为糖基化供体，化学合成了8个含有一个或两个末端n -乙酰基-d-氨基葡萄糖（GlcNAc）残基的n -聚糖核心结构。然后通过4种强大的糖基转移酶催化的酶反应，将每个核心结构扩展到5到15个n -聚糖序列。利用Neu5Gc和核聚焦成功合成n -聚糖进一步扩展了酶的延伸能力。同时，采用酰胺柱高效液相色谱法对n -聚糖进行快速、高效的纯化（纯度约为98%）。共成功制备了73个n -聚糖（63个异构体），并通过MS2和NMR对其进行了表征。综上所述，CSEE策略为“批量生产”结构明确的n -聚糖提供了一种实用的方法，n -聚糖是糖科学的重要标准和探针。
An efficient chemoenzymatic synthesis strategy and a HILIC-based purification approach enabled rapid access to an N-glycan isomer library. Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to the complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycan (especially isomer) libraries is essential to help solve these tasks. We report herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-d-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of the enzymatic extension. Meanwhile, high performance liquid chromatography with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS2 and NMR. In summary, the CSEE strategy provides a practical approach for “mass production” of structurally defined N-glycans, which are important standards and probes for glycoscience.