Magnesium ion catalyzed phosphorus-nitrogen bond hydrolysis in imidazolide-activated nucleotides. Relevance to template-directed synthesis of polynucleotides

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Abstract
Magnesium, an ion necessary in enzymatic as well as in nonenzymatic template-directed polynucleotide-synthesizing reactions, has been found to catalyze the hydroxide ion attack on the P-N bond of selected 5''-monophosphate imidazolide derivatives of nucleotides, such as guanosine 5''-monophosphate 2-methylimidazolide (2-MeImpG), guanosine 5''-monophosphate imidazolide (ImpG), and adenosine 5''-monophosphate 2-methylimidazolide (2-MeImpA). Calcium ion behaves similarly, but quantitatively the effects are smaller. Pseudo-first-order rate constants of 2-MeImpG and ImpG hydrolysis as a function of Mg2+ concentration have been obtained in the range 6 .ltoreq. pH .ltoreq. 10 at 37.degree.C. Mg2+ catalysis is particularly effective around pH 10 where a 0.02 M concentration leads to 15-fold acceleration and a 0.2 M concentration to a 115-fold acceleration of the rate. At other pH values Mg2+ catalysis is less dramatic, mainly because the noncatalyzed reactions is faster. Mg2+ catalysis is attributed to the reaction of the zwitterionic form of the substrate (SH.+-., imidazolide moiety protonated) with OH- rather than reaction of the anionic form (S-, imidazolide moiety deprotonated) with water. This conclusion is based on a study of the N-methylated substrates N-MeImpG and 1,2-diMeImpG, respectively, which were generated in situ by the equilibrium reaction of ImpG with N-methylimidazole and 2-MeImpG with 1,2-dimethylimidazole, respectively. In contrast, in the absence of Mg2+ the reaction of S- with water completes with the reaction of SH.+-. with OH-. The present study bears on the mechanism of the Mg2+-catalyzed template-directed synthesis of oligo- and polynucleotides derived from 2-MeImpG and on the competition between oligonucleotide synthesis and hydrolysis of 2-MeImpG.