Chemical Synthesis of Fructose 2,6-Bisphosphate

This chapter describes the chemical synthesis of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase. The method of chemical synthesis of fructose 2,6-bisphosphate is based on the synthesis of fructose 2-phosphate. The treatment of fructose 1-phosphate with dicyclohexylcarbodiimide (DCC) in aqueous pyridine leads to the formation of fructose cyclic 1,2-phosphate. Alkaline hydrolysis of that compound yields fructose 2-phosphate as one product and fructose 1-phosphate as the other. Base-catalyzed ring opening of fructose 1,2-cyclic 6-bisphosphate (II) yields both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate. Fructose 1,6-bisphosphate is destroyed by heating in alkali, whereas the fructose 2,6-bisphosphate is stable and is purified by anion-exchange chromatography. 6-Phosphofructo-1- kinase activity and the activation of 6-phosphofructo-1-kinase by fructose 2,6- bisphosphate are determined with the aldolase-coupled assay. Purified rat liver 6-phosphofructo-1-kinase is used in the activation assay. The overall yield of this method is limited primarily by the base-catalyzed ring opening reaction, but the key to purification is heating in alkali.