Chemoenzymatic Synthesis of Glycosaminoglycans

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.