The H-phosphonate approach to oligonucleotide synthesis. An investigation on the mechanism of the coupling step

Abstract

A kinetic study on the pivaloyl chloride promoted H-phosphonate condensation step in the presence of differently substituted pyridines has been carried out. The kinetics of the system follow second order kinetics with a 1 ∶ 1 ratio of nucleoside 3′-H-phosphonate and nucleoside component (with free 5-OH). Irrespective of the pyridine derivative used, the reaction rate is dependent on the concentration of this as well as of the hydroxy component and H-phosphonate. A reactive intermediate is indirectly identified from the kinetic evidence. This intermediate is suggested to be a pyridinium adduct formed by attack of the pyridine derivative on the initially formed mixed phosphonic carboxylic anhydride. Considerably lower rates of condensation in the presence of sterically hindered pyridines further support the existence of nucleophilic catalysis in the reactions with non-hindered pyridines. In addition, the rate of reaction in the presence of pyridines with pK_a values above ∼4.8 is enhanced by an increase in pivaloyl chloride concentration. The main reason for this enhancement is most likely the fact that pivaloyl chloride removes pivalate ion, which retards the reaction by influencing the equilibrium between the mixed anhydride and the pyridinium intermediate. Although the observed rate constants are composed of several constants, their temperature dependence gives some indication of the nature of the transition-state of the rate-limiting step. Entropies of activation are estimated to be slightly positive, suggesting a transition state arising from attack of the hydroxy component on the pyridinium intermediate but involving a fair degree of bond breakage to the leaving group, i.e. the pyridine.