Chemoenzymatic Synthesis of Glycosaminoglycans.,Accounts of Chemical Research

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Chemoenzymatic Synthesis of Glycosaminoglycans.
Accounts of Chemical Research ( IF 16.4 ) Pub Date : 2019-11-12 , DOI: 10.1021/acs.accounts.9b00420
Xing Zhang ₁ , Lei Lin ₂ , He Huang ₁ , Robert J Linhardt _{3,

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Affiliation

Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides composed of alternating hexosamine and uronic acid or galatose residue that include hyaluronan, chondroitin sulfate and dermatan sulfate, heparin and heparan sulfate, and keratan sulfate. GAGs display a range of critical biological functions, including regulating cell-cell interactions and cell proliferation, inhibiting enzymes, and activating growth factor receptors during various metabolic processes. Indeed, heparin is a widely used GAG-based anticoagulant drug. Unfortunately, naturally derived GAGs are highly heterogeneous, limiting studies of their structure-activity relationships and even resulting in safety concerns. For example, the heparin contamination crisis in 2007 reportedly killed more than a hundred people in the United States. Unfortunately, the chemical synthesis of GAGs, or their oligosaccharides, based on repetitive steps of protection, activation, coupling, and deprotection, is incredibly challenging. Recent advances in chemoenzymatic synthesis integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions, mimicking the biosynthetic pathway of GAGs, and represent a promising strategy to solve many of these synthetic challenges. In this critical Account, we examine the recent progress made, in our laboratory and by others, in the chemoenzymatic synthesis of GAGs, focusing on heparan sulfate and heparin, a class of GAGs with profound physiological and pharmacological importance. A major challenge for the penetration of the heparin market by homogeneous heparin products is their cost-effective large-scale synthesis. In the past decade, we and our collaborators have systematically explored the key factors that impact this process, including better enzyme expression, improved biocatalysts using protein engineering and immobilization, low cost production of enzyme cofactors, optimization of the order of enzymatic transformations, as well as development of efficient technologies, such as using ultraviolet absorbing or fluorous tags, to detect and purify synthetic intermediates. These improvements have successfully resulted in multigram-scale synthesis of low-molecular-weight heparins (LMWHs), with some showing excellent anticoagulant activity and even resulting in more effective protamine reversal than commercial, animal-sourced LMWH drugs. Sophisticated structural analysis is another challenge for marketing heparins, since impurities and contaminants can be present that are difficult to distinguish from heparin drug products. The availability of the diverse library of structurally defined heparin oligosaccharides has facilitated the systematic analytical studies undertaken by our group, resulting in important information for characterizing diverse heparin products, safeguarding their quality. Recently, a series of chemically modified nucleotide sugars have been investigated in our laboratory and have been accepted by synthases to obtain novel GAGs and GAG oligosaccharides. These include fluoride and azido regioselectively functionalized sugars and stable isotope-enriched GAGs and GAG oligosaccharides, critical for better understanding the biological roles of these important biopolymers. We speculate that the repertoire of unnatural acceptors and nucleotide sugar donors will soon be expanded to afford many new GAG analogues with new biological and pharmacological properties including improved specificity and metabolic stability.

中文翻译：

化学酶法合成糖胺聚糖。

糖胺聚糖（GAG）是一类结构复杂的杂多糖，由交替的六胺和糖醛酸或半乳糖残基组成，包括透明质酸，硫酸软骨素和硫酸皮肤素，肝素和硫酸乙酰肝素和硫酸角质素。GAG显示出一系列关键的生物学功能，包括在各种代谢过程中调节细胞与细胞之间的相互作用和细胞增殖，抑制酶和激活生长因子受体。确实，肝素是一种广泛使用的基于GAG的抗凝药物。不幸的是，天然来源的GAG具有高度的异质性，限制了其结构-活性关系的研究，甚至导致安全隐患。例如，据报道，2007年发生的肝素污染危机使美国一百多人丧生。很遗憾，基于保护，活化，偶联和脱保护的重复步骤，GAG或其寡糖的化学合成极具挑战性。化学酶法合成的最新进展将化学衍生化的灵活性与酶催化的反应相结合，模仿了GAG的生物合成途径，代表了解决许多此类合成挑战的有前途的策略。在这个重要的报告中，我们检查了在我们的实验室和其他方面在化学合成GAG中取得的最新进展，重点是硫酸乙酰肝素和肝素，这是一类具有深远的生理和药理学意义的GAG。均质肝素产品对肝素市场渗透的主要挑战是它们具有成本效益的大规模合成方法。在过去的十年中，我们和我们的合作者已经系统地探索了影响这一过程的关键因素，包括更好的酶表达，使用蛋白质工程和固定化改进的生物催化剂，酶辅因子的低成本生产，酶促转化顺序的优化以及有效技术的开发。，例如使用紫外线吸收标签或荧光标签来检测和纯化合成中间体。这些改进已成功地导致了低分子量肝素（LMWH）的多克级合成，其中一些具有出色的抗凝活性，甚至比商业来源的动物源LMWH药物更有效地逆转了鱼精蛋白。复杂的结构分析是肝素营销的另一个挑战，因为可能存在难以与肝素药物产品区分开的杂质和污染物。结构明确的肝素寡糖多样性文库的可用性促进了我们小组进行的系统分析研究，从而为表征各种肝素产品，维护其质量提供了重要信息。最近，在我们的实验室中研究了一系列化学修饰的核苷酸糖，并已被合成酶所接受，从而获得了新颖的GAG和GAG寡糖。其中包括氟化物和叠氮基区域选择性官能化糖以及稳定的富含同位素的GAG和GAG寡糖，对于更好地了解这些重要生物聚合物的生物学作用至关重要。

更新日期：2019-11-13

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