Phosphoric Acid → Guanosine-3'-diphosphate

A mixture of 0.35 millimoles of the guanosine-3'-monophosphoramidate and 1.5 millimoles of inorganic phosphoric acid was dissolved in 2.0 ml of dried pyridine, the solution was left to stand at room temperature for a period of 3 or 4 days, the undissolved matter was filtered off, and the filtrate was concentrated under vacuum to remove the pyridine. The residue was dissolved in 25 ml of an aqueous solution that contained 1 g of sodium acetate, the solution was adjusted to a pH of 7.0 with sodium hydroxide, and washed five times with ether twice the volume of the solution to remove the guanidium salt. The solution was then passed through a column of Dowex 2 (Cl- type: manufactured by Dow Chemical Co.) to remove the unreacted guanosine-3'-monophosphoramidate. The solution was treated with lithium hydroxide to bring its pH to 6.5, concentrated under vacuum, the residue was dissolved in 25 ml of methanol, to which was added 200 ml of acetone to precipitate the crystal of a lithium salt of guanosine-3'-diphosphate. The crystal was passed through a column of Dowex 50 (H+ type) for desalting. The eluate was collected, concentrated under vacuum, and the residue was lyophilized to give guanosine-3'-diphosphate.