Kinetics of template-directed pyrophosphate-linked dideoxyguanylate synthesis as a function of 2-MeImpdG and poly(C) concentration: Insights into themechanism

Aqueous solutions of deoxyguanosine 5'-monophosphate 8-methylimidazolide, 8 -MeImpdG, yield primarily deoxyguanosine 5'-monophosphate, 5'dGMP, and pyro phosphate-linked dideoxyguanylate, determined at 23 degrees C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid dG(5')ppdG, abbrevia ted G(2)(P) (see Chart 1). The initial rate of G(2)(P) formation, d[G(2)(P) ]/dt in M h(-1), chromatography (HPLC) analysis, exhibits a second-order de pendence on 2-MeImpdG concentration, [G](o), indicating a bimolecular mecha nism of dimerization in the range 0.02 M less than or equal to [G](o) less than or equal to 0.09 M. In the presence of polycytidylate, poly(C), G(2)(P ) synthesis is accelerated and oligodeoxyguanylate products are formed by i ncorporation of 2-MeImpdG molecules. The kinetics of G(2)(P) formation as a function of both monomer and polymer concentration, expressed in C equival ents, were also determined under the above conditions and exhibited a compl ex behavior. Specifically, at a constant [poly(C)], values of d[G(2)(P)]/dt typically increased with [G], with a parabolic upward curvature. At a cons tant [G](o), values of d[G(2)(P)]/dt increase with [poly(C)], but level off at the higher poly(C) concentrations. As [G](o) increases this saturation occurs at a higher poly(C) concentration, a result opposite to expectation for a simple complexation of two reacting monomers with the catalyst prior to reaction. Nevertheless, these results are shown to be quantitatively con sistent with a template-directed (TD) mechanism of dimerization where poly( C) acts as the template to bind 8-MeImpdG in a cooperative manner and lead, for the first time, to the formulation of principles that govern template- directed chemistry. Analysis of the kinetic data via a proposed TD cooperat ive model provides association constants for the affinity between polymer a nd monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate s ynthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first syst em that could serve as a textbook example of a TD reaction under conditions such that the template is fully saturated by monomers and under conditions that it is not.