Dibasic amines as competitive ions improve the resolution between polyanionic nucleotides.

Aliphatic diamines when used as single ion pairing reagents were capable of resolving 3'-,5'- and 2'-,5'- nucleotidyl diphosphates from one another while conventional ion pairing reagents did not separate these positional isomers. The use of 1,2-diamines resulted in the greatest resolution while increasing spacing between the amino groups progressively reduced the resolution while increasing the retention volume. A competitive ion pairing system was also developed using triethylamine as an additional ion pairing reagent. Using this system ethylenediamine, 1,2- and 1,3-diaminopropane were nearly equivalent in their ability to resolve adenosine 3'-phosphate 5'-phosphate, from adenosine 2'-phosphate 5'-phosphate, and adenosine 3'-phosphate 5'-beta-methylenephosphosulfate (3'-mePAPS) from adenosine 2'-phosphate 5'-beta-methylenephosphosulfate (2'-mePAPS), respectively. The ability to easily resolve these positional isomers allows the use of a more simplified synthetic procedure that does not involve the use selective protecting groups to specifically phosphorylate the 2' or 3' hydroxyl group. We have used this procedure on a semipreparative scale to obtain small quantities of both mePAPS and 2'-mePAPS for use in enzymatic studies.