Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases
Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases
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DOI：
10.1002/adsc.201100375
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发表时间：
2011-09
影响因子：
5.4
通讯作者：
Claudia Rech;R. R. Rosencrantz-R.;K. Křenek;H. Pelantová;P. Bojarová;C. Römer;F. Hanisch;V. Křen;L. Elling
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文献类型：
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作者：
Claudia Rech;R. R. Rosencrantz-R.;K. Křenek;H. Pelantová;P. Bojarová;C. Römer;F. Hanisch;V. Křen;L. Elling
聚-N-乙酰乳糖胺(Poly-LacNAc，[3Galβ1，4GlcNAcβ1]n)多糖在糖-蛋白质相互作用中起着重要作用。Poly-LacNAc的合成，无论是化学合成还是酶促合成，通常的特点是由于去保护和/或纯化步骤，在连续合成过程中产物损失很大。在这项工作中，我们提出了一种结合重组糖基转移酶的一锅法合成聚-LacNAc寡糖。通过对聚乳酸混合物的分级，我们能够分离出最多含有六个N-乙酰乳糖胺(LacNAc)单元的多糖。所涉及的重组β1，4-半乳糖基转移酶-1(β4GalT-1)和β1，3-N-乙酰氨基葡萄糖转移酶(β3GlcNAcT)与多达8个糖基单位的分离的糖底物的活性测定表明，β3GlcNAcT对四糖有偏好，而β4GalT-1对特定的糖链长度没有偏好。这些发现引导我们通过改变底物和酶的比例来优化组合一锅法合成，以及开始合成不同链长的聚-LacNAc。因此，我们提出了一种优化的聚-LacNAc的合成方法，通过结合两种糖基转移酶和尿苷-二磷酸葡萄糖/N-乙酰氨基葡萄糖4‘-差向异构酶的一锅策略，在最大限度地减少反应时间和产物损失的同时，以较高的产率合成了长达六个LacNAc单元的长多聚-LacNAc糖链。所得产物是生物材料表面生物功能化和构建用于组织工程的人工细胞外基质的重要配体。
Poly-N-acetyllactosamine (Poly-LacNAc, [3Galβ1,4GlcNAcβ1]n) glycans play an essential role in carbohydrate-protein interactions. The synthesis of poly-LacNAc, both chemical and enzymatic, is typically characterized by high losses of product during sequential synthesis, due to deprotection and/or purification steps. In this work we present a one-pot synthesis of poly-LacNAc oligosaccharides by combining recombinant glycosyltransferases. By fractionation of the poly-LacNAc glycan mixture we were able to isolate glycans with up to six N-acetyllactosamine (LacNAc) units. Activity measurements of the involved recombinant β1,4-galactosyltransferase-1 (β4GalT-1) and β1,3-N-acetylglucosaminyltransferase (β3GlcNAcT) with isolated glycan substrates of up to eight sugar units revealed a preference of β3GlcNAcT for the tetrasaccharide and no preference of β4GalT-1 for a specific glycan length. These findings led us to the optimization of combinatorial one-pot synthesis by variation of substrate and enzyme ratios, as well as starting the synthesis with various poly-LacNAc chain lengths. Consequently, we present here an optimized poly-LacNAc synthesis by the combination of two glycosyltransferases and a uridine-diphospho-glucose/N-acetylglucosamine 4′-epimerase as one-pot strategy resulting in long poly-LacNAc glycans with up to six LacNAc units in high yields while minimizing reaction time and product loss. The obtained products are important ligands for the biofunctionalization of biomaterial surfaces and the construction of an artificial extracellular matrix for tissue engineering.