Site-selective glycosylation of hemoglobin with variable molecular weight oligosaccharides: potential alternative to PEGylation.
Site-selective glycosylation of hemoglobin with variable molecular weight oligosaccharides: potential alternative to PEGylation.
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DOI：
10.1021/ja300893t
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发表时间：
2012-05-02
影响因子：
15
通讯作者：
Wang, Peng G.
中科院分区：
文献类型：
--
作者：
Styslinger, Thomas J.;Zhang, Ning;Bhatt, Veer S.;Pettit, Nicholas;Palmer, Andre F.;Wang, Peng G.
聚（乙二醇）（PEG）缀合（即PEG化）是增加治疗性蛋白质和胶体的循环半衰期的常用策略，然而，几乎没有可行的替代方案来复制其功能。在这里，我们报告了一种方法，用于快速位点选择性糖基化的蛋白质与各种大小的碳水化合物，高达10,000 Da的分子量（MW），因此，作为一个潜在的替代聚乙二醇化。更重要的是，开发的方法有两个独特的特点。首先，避开了通常伴随碳水化合物片段修饰的传统保护基策略，允许用各种大小的聚糖对所需蛋白质进行容易的位点选择性糖基化。其次，所采用的方法不受寡糖大小的限制;因此，可以采用与治疗性蛋白质的PEG化中使用的PEG的MW相似的聚糖。为了证明该技术的有用性，用一系列增加MW（504至~ 10，000 Da）的碳水化合物对血红蛋白（Hb）进行位点选择性糖基化。基于文献中存在的大量生物化学和生物物理学知识选择Hb，并且因为其用作人工红细胞替代品合成/配制中的前体。在Hb的成功位点选择性糖基化之后，在体外研究增加聚糖MW对Hb的生物物理性质的影响。
Poly(ethylene glycol) (PEG) conjugation (i.e. PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids, however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with various sized carbohydrates, up to a molecular weight (MW) of 10,000 Da, thus, serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with various sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of a similar MW to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (504 to ~10,000 Da). Hb was selected based on the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb’s biophysical properties was investigated in vitro.
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