On the question of stepwise vs. concerted cleavage of RNA models promoted by a synthetic dinuclear Zn(ii) complex in methanol: implementation of a noncleavable phosphonate probe

Abstract

To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(II) complex (4) at (s)spH 9.8 and 25 degrees C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k(obs)(6a) = (1.47 +/- 0.09) x 10(-4) s(-1) and k(obs)(6b) = (2.08 +/- 0.09) x 10(-6) s(-1), respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, Kb, and the catalytic rate constant, kcat(max), for the unimolecular decomposition of the 8:4 Michaelis complex. A Brønsted plot of the log (k(cat)(max)) vs. sspKa(phenol) (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log kcat(max) = (-0.80 +/- 0.07)(s)spKa + (10.2 +/- 1.0) which includes the datum for 6a. The datum for 6b, which reacts approximately 70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4.

Overcoming challenges in WAVE bioreactors without feedback controls for pH and dissolved oxygen

“…Finally, we note that our data and interpretation contrast with recent analyses of phosphate diester reactions promoted by related Zn 2+ complexes 21. 47, 48 One of these studies involves the reactions of HPNPP, which is cleaved through the intramolecular attack of an alkoxide to form a similar cyclic diester product. Although kinetic isotope studies suggested that this reaction proceeds by way of an intermediate phosphorane in solution, with the rate‐limiting step the formation of the intermediate, it is suggested that the reaction promoted by dinuclear Zn 2+ complex 1 involves a single step in which nucleophile addition and leaving group departure are concerted 48.…”

“…This study was followed by the examination of a phosphonate substrate (similar to our use of 4 ) that could only be cleaved by intermolecular reaction, and thus could only isomerise through an intermediate phosphorane. Only slow cleavage was observed, demonstrating that the formation of a phosphorane that could pseudorotate is less efficient in this system 47. It is not clear why catalysis by these complexes should be accompanied by a change in mechanism, but it should be noted that in contrast to substrates 5 , HPNPP has a very good leaving group and a less basic, more flexible nucleophile, which might affect how the reaction responds to catalysis.…”

“…Despite their efficacy at promoting cleavage, metal ion based catalysts have not been reported to promote the isomerisation of the phosphate group position; this can be explained if cleavage proceeds via a dianionic phosphorane, which is not stabilised by the metal ions sufficiently to allow pseudorotation to take place 46. In fact, recent results have been presented to suggest that the metal ion promoted cleavage is best described as a concerted process, with a dianionic pentacoordinate transition state rather than an intermediate,21, 41, 47, 48 in which case competing isomerisation is clearly not possible. In one case, based on the analysis of kinetic isotope measurements, it has been proposed that coordination to a dinuclear Zn complex alters the reaction pathway for transesterification of HPNPP from a two step (A N +D N ) addition‐elimination process observed in solution to a concerted displacement (A N D N ) 48.…”

βLG values in mechanistic studies on the transesterification of RNA models and their application in a metal ion complex promoted transesterification

“…The data in Table suggest that the behaviour is, however, a peculiar feature of the catalysis by Zn 2+ 2 − 1 only; Zn 2+ aquo ion and mononuclear complexes appear to behave differently. Hence, it can be suggested that although the reaction catalysed by Zn 2+ 2 − 1 is a step‐wise process, a concerted reaction may still take place in the presence of other metal ion catalysts, as has been proposed by several other studies …”

Mechanistic Studies of Homo- and Heterodinuclear Zinc Phosphoesterase Mimics: What Has Been Learned?

“…Under alkaline conditions the dianionic phosphorane is too short-lived and pseudorotation to an intermediate with the 3′-oxygen and a negatively charged oxygen in the axial positions is too energetically unfavorable for 3′ → 2′ isomerization to occur. Experimental and computational data suggest that the reaction switches to a concerted pathway involving a dianionic pentacoordinate transition state, when the transesterification to the cyclophosphate is catalyzed by metal ions (Bunn et al, 2007; Humphry et al, 2008; Tsang et al, 2009; Edwards et al, 2010). Isomerization is not possible in this case.…”