The synthesis of glucose-6-phosphate and 6-phosphogluconate.

In studies of the enzymatic oxidation of 6-phosphogluconate, relatively large quantities of this substance in high purity were required. This led to a search for a convenient preparation of glucoseB-phosphate. Available methods for the synthesis of glucose-6-phosphate, reviewed by Lardy et al. (1, 2), are laborious and not well suited to the preparation of large quantities of this substance. A procedure has now been developed, based on the phosphorylation of glucose with polyphosphoric acid. Pyrophosphoric acid has been reported to act as a phosphorylating agent for high molecular weight alcohols (3). More recently polyphosphoric or pyrophosphoric acid has been used by Cherbuliez and Weniger (4) in the preparation of phosphoenolpyruvic acid, phosphocholine, phosphoethanolamine, and glycerophosphoric acid. In the direct phosphorylation of glucose with polyphosphoric acid, esterification occurs not only at the 6 position but at other positions as well. Since glucose-g-phosphate is resistant to acid hydrolysis (5), other esters formed could be removed by this means with little loss of glucose-6-phosphate. Selective hydrolysis with acid has been used by LePage and Umbreit (6) for the preparation of glucoseB-phosphate from Embden ester’ The oxidation of glucoseS-phosphate to 6-phosphogluconate with excess bromine for 48 hours at room temperature according to Robison and King (7) resulted in a product which, by enzymatic assay, was only 50 to 70 per cent pure and contained much inactive esterified phosphate. The yield of 6-phosphogluconate was correspondingly low. A study of the rate of oxidation of glucose-6-phosphate by bromine confirmed the finding of Warburg and Christian (8) that glucose-g-phosphate was completely oxidized in 1 to 2 hours at room temperature. With the enzymatic assay described above, the quantitative formation of 6-phosphogluconate could be demonstrated and a pure product obtained in high yield.