Kinetic Preference for the 3‘−5‘-Linked Dimer in the Reaction of Guanosine 5‘-Phosphorylmorpholinamide with Deoxyguanosine 5‘-Phosphoryl-2-methylimidazolide as a Function of Poly(C) Concentration

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Abstract
The formation of the internucleotide bond in diguanylate synthesis was studied in aqueous solution at pH 8 and 0.2 M Mg2+ in the presence and absence of polycytidylate, poly(C). The investigation was simplified by using guanosine 5‘-phosphorylmorpholinamide, mor-pG, which can act only as a nucleophile, and deoxyguanosine 5‘-phosphoryl-2-methylimidazolide, 2-MeImpdG, which can act only as an electrophile. The time-dependent product distribution was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS). In the absence of poly(C) the reaction between mor-pG and 2-MeImpdG yielded small amounts of the dimer mor-pGpdG with a regioselectivity of 2‘−5‘:3‘−5‘ = 3.5. In the presence of poly(C) dimer yields increased and a reversal in regioselectivity occurred; both effects were in proportion to the concentration of the polymer. The results can be quantitatively explained with the proposition that poly(C), acting as the template, catalyzes the reaction between template-bound monomers by about a factor of 4−5 over the reaction in solution and yields dimers with a regioselectivity of 2‘−5‘:3‘−5‘ ≈ 0.33. These findings illustrate the intrinsic preference of guanosine monomers to correctly self-assemble on the appropriate template.