Kinetic studies of the dimerization, alkylation and enzyme kinetic isotope effects of adenine

Abstract

Tautomeric and anionic forms of nucleic acids are potentially involved in mutations and replication and translational errors; however, research into the chemistry of deprotonated nucleobases is limited. This research focused on the ion-pairing and self-aggregation ability of the adeninate anion in DMSO. Through 1H NMR and UV spectroscopy and theoretical studies using IQA and REP-FAMSEC, it was shown that ion pairs of sodium and potassium salts of adenine exist in solution with cation coordination sites at the N3 and N9 atoms of the purine ring. Self-aggregation of these ion-pairs is governed by π-π stacking followed by higher order aggregation governed by coordination to metal cations. Our study provides insight into the interactions of the molecule and provides a building block for future studies on the adeninate system. Much research has been dedicated to understanding the site preference for the alkylation of the adeninate ion as it yields mixtures of regio-isomers during the synthesis of biologically active compounds, yet no kinetic data have been obtained to understand the thermodynamics governing the reaction. The thermodynamic control of the regio-selectivity of benzylation of the adeninate ion was investigated. Through dynamic 1H NMR spectroscopy and theoretical modelling, it was shown that benzylation occurs via an SN2 mechanism in which the enthalpy of formation for N9-benzyladenine is higher than for the N3 isomer. However, this increase in the barrier height is compensated for by the increase in the entropy resulting in a looser transition state structure than that of the N3 isomer which experiences an entropic bottleneck. The derived thermodynamic properties of the adeninate anion will enable reaction conditions to be optimized for better control of synthesis at research and industrial level. A primary KIE from ATP deuterated at the C8 position (C8D-ATP) on several kinase enzymes has been reported, potentially providing new insight for drug design towards Mycobacterium tuberculosis. However, the KIE on the V and V/K were not obtained. This research aimed to develop a high resolution mass spectroscopy method to study the KIE of C8D-ATP on Mycobacterium tuberculosis shikimate kinase. To this end, a high resolution MS method was developed which showed high sensitivity, precision and specificity. A KIE on V and V/K of 1.5 and 1.4, respectively, were observed for Mycobacterium tuberculosis shikimate kinase showing a secondary isotope effect. The MS method can be used for quantitative data collection of enzyme kinetics without sample modification. Our study implies that the adenine moiety in ATP facilitates molecular recognition only and that the C8‒H bond of ATP is not involved in bond cleavage during phosphorylation.