Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids
Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids
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DOI：
10.1093/glycob/cww084
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发表时间：
2016-11-01
期刊：
影响因子：
4.3
通讯作者：
Biot, Christophe
中科院分区：
文献类型：
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作者：
Gilormini, Pierre-Andre;Lion, Cedric;Biot, Christophe
天然和合成修饰的胞苷一磷酸活化唾液酸（CMP-Sias）是糖生物学领域的重要研究资产：除其他应用外，它们可用于探测聚糖，检测细胞表面的唾液酸化缺陷或进行唾液酸转移酶活性的详细研究。然而，这些化学工具由于水解而非常不稳定，并且获得起来非常耗时和昂贵。它们几乎不可能以令人满意的纯度储存，并且它们的制备需要多个费力的纯化步骤，这通常导致严重的产物损失。利用原位时间分辨P-31磷核磁共振（P-31 NMR），我们精确地建立了一些CMP-Sias（包括CMP-Neu 5Ac、CMP-Neu 5Gc、CMP-SiaNAl和CMP-SiaNAz）在几种实验条件下的形成和降解动力学。P-31 NMR可以在未氘化的溶剂中进行，并且是一种灵敏且非破坏性的技术，其允许直接原位监测和优化涉及含磷物质的化学-酶促合成。因此，我们表明，CMP-唾液酸衍生物可以稳健地获得高产量使用容易获得的脑膜炎奈瑟菌CMP-唾液酸合酶。这种集成的工作流程需要不到一个小时的时间，新鲜制备的CMP-Sias可以直接转移到唾液酸化生物测定中，而无需任何纯化步骤。
Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved P-31 phosphorus nuclear magnetic resonance (P-31 NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. P-31 NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.