Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imino groups of DNA bases

Abstract

We describe the equilibrium and kinetic aspects of the formaldehyde reaction with the endocyclic imino groups of derivatives of thymine, uracil, and a series of halogenated uracils, as well as poly(uridylic acid) and poly(inosinic acid). The main results are: (i) the equilibrium constants for forming a hydroxymethyl adduct remain quite constant at about 2-2.5 (M-1) for all the compounds studied, independent of their pK; (ii) both forward and reverse rate constants with 5'-TMP are specific base catalyzed in the pH range of about 4-9; (iii) the response of the rate constants to temperature and to several solvent additives are measured; (iv) at neutral pH, for the series of pyrimidine compounds, a linear free energy relation is observed between the logarithm of both the forward and the reverse rate constant and the pK for deprotonation; (v) the unstructured polynucleotides, poly(U) and poly(I) react very similarly to their constituent monomers; (vi) a reaction mechanism is proposed; and (vii) some implications for polynucleotide studies are discussed. In an appendix, a method of spectral analysis is derived to obtain accurate estimates of the quite small equilibrium constants; this should be applicable to all similar two-component systems in which the final product is unobtainable, either by isolation or by saturation. Together with the results of the previous paper on the formaldehyde reaction with exocyclic amino groups (J. D. McGhee and P. H. von Hippel, preceding paper), these results form a reasonably comprehensive account of the basic chemical controls required to use formaldehyde as a quantitative probe of DNA structure.