Mechanistic studies for transesterification of RNA analogues catalyzed by a dinuclear zinc(II) complex

RNA cleavage, as catalyzed by self-cleaving ribozymes, is dependent on metal ions involved in the active sites. To better understand the roles of the metal ions and to develop efficient artificial RNA-cleaving agents, small-molecule-metal-complex mimics are being studied. Among the metal complexes studied in Morrow group, a dinuclear Zn(II) complex, Zn 2(L), was shown to exhibit extraordinary catalytic activity toward the minimal RNA analog, 2-hydroxylpropyl-4-nitrophenyl phosphate (HpPNP ). To further characterize this catalyst, the cleavage of uridine 3'- p-nitrophenyl phosphate (UpPNP) and uridine 3'-alkylphosphate (UpR) were also examined. Surprisingly, a lower rate enhancement was observed for the Zn2(L)-catalyzed UpPNP transesterification compared with that of HpPNP in our kinetic studies. This difference reveals that using HpPNP as a model substrate for RNA cleavage study might overestimate the catalytic power of a catalyst. The pH profile of second-order rate constants obtained for the Zn2(L)-catalyzed UpPNP transesterification is consistent with two catalytic pathways. The study of deuterium solvent isotope effect for UpPNP cleavage was carried out to elucidate the catalytic mechanism. The lack of significant kinetic isotope effect ( kH/kD ∼ 0.8) rules out the presence of concerted Bronsted acid/base catalysis and indicates the relevance of direct cation-anion interactions between the zinc cations in Zn 2(L) and the oxyanion in the transition state of the reaction. Moreover, the binding of Zn2(L) catalyst to monomethylphosphate dianion as a transition state analog was measured to probe the stabilization contributed from the interactions between phosphorane oxyanions and Zn2(L). The pH dependence of the binding supports the zinc aquo complex, Zn2(L)(H2O), as the catalytically active species. Furthermore, to study the involvement of Zn2(L) in the leaving group departure of the phosphodiester cleavage reaction, a structure-activity correlation study was performed by using substrate UpR with various substituents on the leaving alcohol group. A Bronsted coefficient (betalg) of -0.72 was derived for the Zn 2(L)-catalyzed UpR transesterification. Compared with the betalg value of -1.28 for the uncatalyzed reaction reported in literature, the less negative betalg value obtained here suggests that Zn2(L) catalyst effectively neutralizes the negative charges on the leaving alcohol group at the transition state. A hypothetical catalytic mechanism is presented.