Glycosystems Laboratory, Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
Org Biomol Chem. 2012 Mar 14;10(10):2146-63. doi: 10.1039/c2ob06607f. Epub 2012 Jan 31.
The synthesis of well-defined oligosaccharides is crucial for the establishment of structure-activity relationships for specific sequences of heparin, contributing to the understanding of the biological role of this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal functional group that allows selective conjugation of the probes and expands their use as chemical tools in glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to produce efficiently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps, avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the basic fibroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the amount of sugar required for attachment to the solid support. Using this approach we quantified heparin-protein interactions, and surface dissociation constants for the synthetic heparin derivatives were determined.
具有明确结构的寡糖的合成对于建立肝素特定序列的结构-活性关系至关重要，有助于理解这种多糖的生物学作用。非常方便的是，合成的寡糖含有一个正交官能团，允许探针的选择性缀合，并扩展了它们作为糖生物学中化学工具的用途。我们在这里介绍了使用 n+2 模块化方法合成一系列胺官能化肝素寡糖。仔细优化了糖苷化反应条件，以有效地产生所需的合成中间体，其在还原端具有 N-苄氧羰基保护的氨乙基间隔基。微波加热的使用极大地促进了 O-和 N-磺化步骤，避免了与这些反应相关的实验问题。合成的寡糖被固定在 384 孔微量滴定板上，并与肝素结合蛋白，碱性成纤维细胞生长因子 FGF-2 成功探测。十六烷基三甲基溴化铵的使用将糖与固体载体结合所需的量最小化。使用这种方法，我们定量了肝素-蛋白质相互作用，并确定了合成肝素衍生物的表面离解常数。
