title: Kinetics of template-directed pyrophosphate-linked dideoxyguanylate synthesis as a function of 2-MeImpdG and poly(C) concentration: insights into the mechanism.
abstract: Aqueous solutions of deoxyguanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpdG, yield primarily deoxyguanosine 5'-monophosphate, 5'dGMP, and pyrophosphate-linked dideoxyguanylate, dG5'ppdG, abbreviated G2p (see Chart 1). The initial rate of G2p formation, d[G2p]/dt in M h-1, determined at 23 degrees C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid chromatography (HPLC) analysis, exhibits a second-order dependence on 2-MeImpdG concentration, [G]o, indicating a bimolecular mechanism of dimerization in the range 0.02 M < or = [G]o < or =  0.09 M. In the presence of polycytidylate, poly(C), G2p synthesis is accelerated and oligodeoxyguanylate products are formed by incorporation of 2-MeImpdG molecules. The kinetics of G2p formation as a function of both monomer and polymer concentration, expressed in C equivalents, were also determined under the above conditions and exhibited a complex behavior. Specifically, at a constant [poly(C)], values of d[G2p]/dt typically increased with [G]o with a parabolic upward curvature. At a constant [G]o, values of d[G2p]/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 consistent with a template-directed (TD) mechanism of dimerization where poly(C) acts as the template to bind 2-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 cooperative model provides association constants for the affinity between polymer and monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate synthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first system 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
authors.forename: A
authors.initials: A
authors.lastname: Kanavarioti
authors.forename: S
authors.initials: S
authors.lastname: Gangopadhyay
mesh_terms: D003198:Computer Simulation; D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis; D015224:Dideoxynucleosides / Q000138:chemical synthesis; D019281:Dimerization; D011756:Diphosphates / Q000138:chemical synthesis; D019020:Directed Molecular Evolution; D018559:Exobiology; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D011066:Poly C / Q000737:chemistry*; D013698:Templates, Genetic
chemical_list: D003848:Deoxyguanine Nucleotides; D015224:Dideoxynucleosides; D011756:Diphosphates; D011066:Poly C; C036896:guanosine 5'-phospho-2-methylimidazolide; D006157:Guanosine Monophosphate