KINETIC DISSECTION OF INDIVIDUAL STEPS IN THE POLY(C)-DIRECTED OLIGOGUANYLATE SYNTHESIS FROM GUANOSINE 5'-MONOPHOSPHATE 2-METHYLIMIDAZOLIDE

A kinetic study of oligoguanylate synthesis on a polycytidylate templa te, poly(C), as a function of the concentration of the activated monom er, guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, is repor ted. Reactions were run with 0.005-0.045 M 2-MeImpG in the presence of 0.05 M poly(C) at 23-degrees-C. The kinetic results are consistent wi th a reaction scheme (eq 1) that consists of a series of consecutive s teps, each step representing the addition of one molecule of 2-MeImpG to the growing oligomer. This scheme allows the calculation of second- order rate constants for every step by analyzing the time-dependent gr owth of each oligomer. Computer simulations of the course of reaction based on the determined rate constants and eq 1 are in excellent agree ment with the product distributions seen in the HPLC profiles. In acco rd with an earlier study (Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahe dron 1984, 40, 39), rate constants, k(i), for the formation of the tet ramer and longer oligomers up to the 16-mer were found to be independe nt of length and somewhat higher than k3 (formation of trimer), which in turn is much higher than k2 (formation of dimer). The k(i) (i great er-than-or-equal-to 4), k3, and k2 values are not true second-order ra te constants but vary with monomer concentration. Mechanistic models f or the dimerization (Scheme I) and elongation reactions (Scheme II) ar e proposed that are consistent with our results. These models take int o account that the monomer associates with the template in a cooperati ve manner. Our kinetic analysis allowed the determination of rate cons tants for the elementary processes of covalent bond formation between two monomers (dimerization) and between an oligomer and a monomer (elo ngation) on the template. A major conclusion from our study is that bo nd formation between two monomer units or between a primer and a monom er is assisted by the presence of additional next-neighbor monomer uni ts. This is consistent with recent findings with hairpin oligonucleoti des (Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317). Our study is the first of its kind that shows the feasibility of a thorough kin etic analysis of a template-directed oligomerization and provides a de tailed mechanistic model of these reactions.