[17] Aldose 1,6-diphosphates

Publisher Summary This chapter describes the method for the preparation of aldose 1,6-diphosphates. A microanalytical procedure is developed to measure the effect of time of incubation and temperature on the rate and extent of the phosphorylation reaction. In this assay, the sugar diphosphate product is partially purified and the rate of formation of acid-labile phosphate is measured. The fully acetylated derivative of D-glucose-6-P is prepared by a modification of the procedure used for the synthesis of completely acetylated derivatives of free hexoses with acetic anhydride in pyridine at 3%. Characterization of a-D-glucose 1,6-diphosphate is discussed. The procedure described in the chapter, with slight modifications has been used to prepare α -D-ribose 1,5-diphosphate, α -D-galactose 1,6-diphosphate, α -D-mannose 1,6-diphosphate, and N-acetyl- α -D-glucosamine 1,6-diphosphate. All the sugar diphosphates prepared by this procedure were active when they were assayed for enzymic activity with rabbit muscle phosphoglucomutase. Some precautions must be taken when preparing sugar diphosphates other than α -D-glucose-l,6-P 2 . Several sugar monophosphate samples contain varing amounts of D-glucose-6-P. To remove this impurity, 5-g samples of D-galactose-6-P and D-mannose-6-P were treated with D-glucose- 6-P dehydrogenase and NADP + to convert traces of D-glucose 6-P to 6-P-gluconic acid. One other precaution that must be taken in the preparation of α -D-mannose-l,6-P 2 and α -D-ribose-l,5-P 2 is to reduce the concentration of HC1 from 10 to 5 m M, during the removal of the residual sugar monophosphate. These sugar diphosphates are considerably more labile to acid hydrolysis than α -D-glucose-l,6-P 2 .