Glyco (randomization/diversification) is a term that encompasses strategies to diversify a core drug scaffold via enzymatic glycosylation to provide sets of analogs wherein the sole diversity element is a carbohydrate. This review covers the influence of glycosylation upon various drug properties, the classes of glycosyl-conjugating enzymes amenable to glyco(randomization/diversification) schemes, approaches to the synthesis of required substrates and specific examples of glycorandomized libraries utilizing both wild-type and engineered enzymes.

We report the ability of simple glycoside donors to drastically shift the equilibria of glycosyltransferase-catalyzed reactions, transforming NDP-sugar formation from an endo- to an exothermic process. To demonstrate the utility of this thermodynamic adaptability, we highlight the glycosyltransferase-catalyzed synthesis of 22 sugar nucleotides from simple aromatic sugar donors as well as the corresponding in situ formation of sugar nucleotides as a driving force in context of glycosyltransferase-catalyzed reactions for small molecule glycodiversification. These simple aromatic donors also enabled the first general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering, and other fundamental sugar nucleotide-dependent investigations. This study directly challenges the general notion that NDP-sugars are ‘high-energy’ sugar donors when taken out of their traditional biological context.

A sweet library: Two variants (wild‐type (WT) and a triple mutant) of glycosyltransferase (GT) OleD have been shown to catalyze glycosylation of over 70 substrates, formation of O‐, S‐ and N‐glycosidic bonds, and iterative glycosylation (see scheme). Identified substrates include nucleophiles not previously known to act in GT reactions and span numerous natural product and therapeutic drug classes.

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.carbohydrate | enzyme | glycobiology | protein engineering T he lack of accessibility and availability of uncommon and uniquely functionalized sugar nucleotides (NDP-sugars) continues to restrict research focused upon understanding the regulation, biosynthesis, and/or role of glycosylated macromolecules and glycosylated small molecules in biology or therapeutic development (1-7). Although there are many reported chemical, enzymatic, and chemoenzymatic strategies for NDP-sugar synthesis, those that extend beyond the reach of common biological sugars (e.g., Dglucose, D-galactose, etc.) nearly all suffer from long reaction times (>16 h), relatively low yields, and difficulties associated with product purification and/or stability (3,4,8,9). Thus, the development of robust methods for sugar nucleotide synthesis directly compatible to the downstream biological processes to be studied may be advantageous.From a traditional viewpoint, NDP-sugars are used as donors by Leloir glycosyltransferases (sugar nucleotide-dependent enzymes) for formation of glycosidic bonds. However, many glycosyltransferase (GT)-catalyzed reactions are known to be readily reversible, enabling the "pirating" of unique sugars from natural products or alternative donors (resulting in generation of the respective sugar nucleotide) and one-pot sugar exchange reactions between unique natural products (4, 10-13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14-18) and simple donors designed to fundamentally alter the reaction thermodynamics, recently enabled a unique platform for NDP-sugar synthesis and a high throughput colorimetric screen for NDP-sugar formation and utilization (19). While the prior platform proof-of-concept study highlighted the syntheses of 22 natural and nonnatural TDP/UDP-sugars from 11 distinct 2-chloro-4-nitrophenyl glycoside donors using a single GT catalyst (Fig. 1A) (19), the substrate specificity of the glycosyltransferase used ...

Glycodiversification of natural products is an effective strategy for small molecule drug development. Recently, improved methods for chemo-enzymatic synthesis of glycosyl donors has spurred the characterization of natural product glycosyltransferases (GTs), revealing that the substrate specificity of many naturally occurring GTs as too stringent for use in glycodiversification. Protein engineering of natural product GTs has emerged as an attractive approach to overcome this limitation. This review highlights recent progress in the engineering/evolution of enzymes relevant to natural product glycodiversification with a particular focus upon GTs.