Altered transition state for the reaction of an RNA model catalyzed by a dinuclear zinc(II) catalyst.

The cyclization of 2-(hydroxypropyl)-4-nitrophenyl phosphate (HpPNP) catalyzed by the dinuclear zinc complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (1) proceeds by a transition state that is different from that of the uncatalyzed reaction. Kinetic isotope effects (KIEs) measured in the nucleophilic atom and in the leaving group show that the uncatalyzed cyclization has a transition state (TS) with little phosphorus-oxygen bond fission to the leaving group ((18)k(lg) = 1.0064 +/- 0.0009 and (15)k = 1.0002 +/- 0.0002) and that nucleophilic bond formation occurs in the rate-determining step ((18)k(nuc) = 1.0326 +/- 0.0008). In the catalyzed reaction, larger leaving group isotope effects ((18)k(lg) = 1.0113 +/- 0.0005 and (15)k = 1.0015 +/- 0.0005) and a smaller nucleophile isotope effect ((18)k(nuc) = 1.0116 +/- 0.0010) indicate a later TS with greater leaving group bond fission and greater nucleophilic bond formation. These observed nucleophile KIEs are the combined effect of the equilibrium effect on deprotonation of the 2'-hydroxyl nucleophile and the KIE on the nucleophilic step. An EIE of 1.0245 for deprotonation of the hydroxyl group of HPpNP was obtained computationally. The different KIEs for the two reactions indicate that the effective catalysis by 1 is accompanied by selection for an altered transition state, presumably arising from the preferential stabilization by the catalyst of charge away from the nucleophile and toward the leaving group. These results demonstrate the potential for a catalyst using biologically relevant metal ions to select for an altered transition state for phosphoryl transfer.

The effects of sulfur substitution for the nucleophile and bridging oxygen atoms in reactions of hydroxyalkyl phosphate esters.

The effects of sulfur substitution on the reactions of hydroxyalkyl phosphate esters are examined. These compounds are models for the intramolecular phosphoryl transfer reaction involved in the cleavage of the internucleotide bond in RNA. The models studied here lack the ribose ring and their conformational flexibility results in greater stability and the availability of different reaction pathways. Sulfur in the nucleophilic position shows no nucleophilic reaction at phosphorus, instead rapidly attacking at the beta carbon atom, forming thiirane with departure of a phosphomonoester. Sulfur substitution at either of the two bridging positions leads to cleavage of the diester via formation of a cyclic intermediate, but with significant rate acceleration when compared to the oxygen analogues. The bridge-substituted models react substantially slower than the analogous ribose compounds with sulfur substitution at comparable positions. Kinetic isotope effects reveal significant differences in the transition state depending on which bridging position sulfur occupies. When sulfur is in the scissile bridging position, a highly associative transition state is indicated, with a largely formed bond to the nucleophile and the scissile P-S bond is little changed. When sulfur occupies the other bridging position, the isotope effects imply a very early transition state in a concerted reaction.

A nonhydrolyzable analogue of phosphotyrosine, and related aryloxymethano- and aryloxyethano-phosphonic acids as motifs for inhibition of phosphatases.

Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl and aryloxy (or thio) ethyl phosphonic acids of the general formula RX-(CH(2))(n)-PO(3)H(2) (where X=O or S and n=1 or 2), have been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases (two alkaline phosphatases, a protein-tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs. The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties toward the protein-tyrosine phosphatase from Yersinia. The K(i) for the d isomer is 33-fold lower than that of the l isomer, and is more than an order of magnitude lower than the reported K(m) of the substrate l-phosphotyrosine.