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 competive 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′-β-methylenephosphosulfate (3′-mePAPS) from adenosine 2′-phosphate 5′-β-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. © 1991.