A simple synthesis of sugar nucleoside diphosphates by chemical coupling in water.

The use of glycosyltransferases in chemoenzymatic oligosaccharide synthesis is attractive, provided that the enzymes are available, since it eliminates the tedious multistep protectiondeprotection and chemical glycosylation procedure that characterizes classic chemical synthesis. The sugar nucleoside diphosphates (sugar-NDPs) that most glycosyltransferases require as glycosyl donors (e.g. UDP-Glc, UDP-Gal, ADP-Glc etc.) can be prepared either chemically or through enzymatic methods including recycling systems. The enzymatic preparation of sugar-NDPs has the advantages of being simple and compatible with glycosyltransferase reactions but requires access to the necessary enzymes. In contrast, chemical synthesis is more complex but is especially powerful for the preparation of analogues that cannot easily be prepared using enzymes owing to a low tolerance of the enzyme for substrate modifications. Chemical methods for the synthesis of sugar-NDPs have recently been comprehensively reviewed and eloquently discussed. The most commonly used procedures involve the formation of the pyrophosphate linkage by coupling of an activated nucleoside-5’-monophosphate (NMP, typically a morpholidate or imidazolide) with the sugar-1-phosphate; in these reactions the addition of catalysts like nitrogencontaining heterocycles can effect substantial improvements in both rate and yield. Newer methods include the “cycloSal” phosphotriester coupling and mixed phosphite– phosphate formation followed by oxidation. Direct coupling of anomerically activated monosaccharides with nucleoside diphosphates, such as uridine 5’-diphosphate, represents a useful alternative approach (reviewed in reference [3]). These chemical procedures are normally performed in anhydrous organic solvents and usually employ per-O-acetylated sugars and phosphates, such as their tetraalkylammonium salts, that are soluble in organic solvents. The syntheses are notoriously difficult to reproduce from laboratory to laboratory, especially when researchers have little previous experience with chemical sugar nucleotide synthesis. We report herein a simple method for the chemical synthesis of sugar-NDPs in a one-pot reaction in water. The procedure involves only the sequential addition of commercially available compounds, and the crude product solution can be used directly as a glycosyl donor source in glycosyltransferase-mediated oligosaccharide synthesis. We found that the reaction of 2-chloro-1,3-dimethylimidazolinium chloride (DMC, 1) with imidazole (2) in D2O at room temperature gave 2-imidazolyl-1,3-dimethylimidazolinium chloride (ImIm, 3), within five minutes, along with imidazole hydrochloride (Im-HCl; Scheme 1). The formation of 3 was confirmed by H NMR spectroscopy and ESI–MS. We then examined whether 3 would convert phosphate groups into reactive phosphorimidazolide groups in aqueous solution. Phosphorimidazolides have been extensively used for pyrophosphate bond formation in anhydrous organic solvents.