Activating Water: Efficient Intramolecular General Base Catalysis of the Hydrolysis of a Phosphate Triester

Abstract

We have identified the first highly efficient intramolecular general base catalysis (IGBC) of a hydrolysis reaction, in a system where two general bases are available to assist the attack of the same nucleophilic water molecule. The suggested mechanism, available uniquely to a phosphate triester model, is readily available in enzyme active sites, and the results suggest a possible solution to the long-unsolved question of how enzymes are able to activate a water molecule to be a highly effective nucleophile.

Activating Water: Important Effects of Non‐leaving Groups on the Hydrolysis of Phosphate Triesters

“…This shift results from the increased rate constants for the apparent acid‐catalysed reaction that result from protonation of the 2‐pyridyl nitrogen in systems 2 and 3 . Although this centre is not much more basic than the triester PO group, protonation converts the leaving group directly to 2‐pyridone, over 10 8 times less basic than the anion derived from 2‐hydroxypyridine 1. Secondly, with increasing reactivity, typical in hydrolysis reactions, a broad pH‐independent region develops (Figure 1 a).…”

“…The spontaneous hydrolysis of TPP involves GB catalysis of the attack of the effective water nucleophile, as indicated by the substantial solvent deuterium isotope effect, k / k =3.15 1. The GB(s) involved must be hydrogen‐bonded water molecules, because the observed rate is not significantly greater than expected for a triester derived from a phenol of the same p K a , so that any contribution from the pyridine nitrogens, as suggested previously,1 must be minimal. This is abundantly clear from the independent observation that the hydrolysis of the bis‐2‐pyridyl‐4‐nitrophenyl ester (Table 3) gives similar amounts of pyridone and 4‐nitrophenolate (the better leaving group).…”

“…Our original estimate of k 0 =1.5×10 −10 min −1 at 39 °C for the hydrolysis of a triester 1 with a leaving group ArOH of p K a =9.09 was based on the linear free energy relationship of Khan and Kirby 4. The spontaneous hydrolysis of TPP is faster than this estimate, for a triester with two alkyl groups, by a factor of the order of 10 7 –10 8 at 25 °C 1. We expected the activating effect of the two “spectator” aryl groups of TPP to be relatively small: the only direct dialkyl versus diaryl comparison of similar “spectator” groups on a triester, for the endocyclic displacement of salicylate from salicyl phosphate triester, gave a rate difference of 50–60 5.…”

“…We recently reported first results for the unexpectedly reactive triester tris-2-pyridyl phosphate (TPP), which we interpreted as evidence that the three 2-pyridyl groups supported "remarkably efficient hydrolysis to the corresponding diester DPP". [1] This was important, because the likely mechanism, namely, intramolecular general-base (GB) catalysis, had never previously shown the high efficiency in model systems that it evidently deploys in enzyme active sites. [2] We report further work on this system, which makes clear that our estimate, that the neutral hydrolysis of TPP is faster than expected "by a factor of the order of 10 8 at 25 8C", is far too high.…”

Cu(II)‐catalyzed hydrolysis of tris‐2‐pyridyl phosphate assisted by sodium dodecyl sulfate micelles

“…The substrate TPP was synthesized as described previously . Sodium dodecyl sulfate (SDS) was purchased from Sigma‐Aldrich (≥99%) and purified as described elsewhere, while copper(II) sulfate pentahydrate (Sigma Aldrich, ≥98%), sodium acetate trihydrate (Sigma Aldrich, ≥99%) and 2‐( N ‐morpholino)ethanesulfonic acid (MES, Sigma Aldrich, ≥99%) were used as received.…”