Abstract

Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. The biosynthesis of these natural product glycosides involves the action of sugar biosynthetic enzymes and glycosyltransferases (GTs), where many of these enzymes show relaxed substrate specificities. This can make them valuable biocatalytic tools for altering glycosylation as part of a strategy called “glycodiversification.”
The success of glycodiversification greatly depends on the screening method, many of which have limitations with respect to their throughput and ease of use. To address this, we have developed a screening tool for assaying GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. Using our novel high-throughput assay, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. A number of these enzymes showed novel acceptor and donor specificities. Interestingly, three GTs catalyzed the synthesis of novel anthracycline glycosides. Our findings are particularly important towards the glycodiversification of therapeutics in this class, given the clinical value of anthracycline glycosides, and the importance of the sugar residue on the biological activity of these therapeutics.
Epirubicin is a high value anticancer anthracycline drug, as it is reported to have a lower cardiotoxicity than the parental compound, doxorubicin. The sugar moiety, L-acosamine and L-daunosamine is the only difference between epirubicin and doxorubicin. The corresponding nucleotide activated sugar donors are TDP-L-acosamine and TDP-L-daunosamine. Despite the importance of TDP-L-acosamine, a natural biosynthetic pathway for this precursor has not been described. Chemists have synthesized this donor via organic synthetic methods, however, it is a tedious process with multiple chemical conversions. Furthermore, the in vivo production of TDP-L-daunosamine is reported to be low. We have addressed this by engineering an in vitro enzymatic pathway for the synthesis of TDP-L-acosamine and TDP-L-daunosamine based on the substrate flexibility of sugar biosynthetic enzymes. Starting from a common precursor, we have synthesized these nucleotide deoxysugars and a few intermediates of the enzymatic pathway and used one of the produced intermediates in enzymatic characterization. Moreover, these could be used in in vitro glycodiversification and as the starting material for the in vitro enzymatic synthesis of some other valuable deoxysugars.