Serine Protease Mechanism-Based Mimics. Direct Evidence for a Transition State Bridge Proton in Stable Potentials

Abstract

We have synthesized 1-(2-hydroxyacetyl)piperidine-2-one (2) and 1-(2-hydroxyacetyl)azepan-2-one (3). Equilibrium (K(f)) between the free alcohol (open form) and the tetrahedral intermediate (cyclol) is readily established, and both forms are observed in the D(2)O (1)H NMR spectra of 2 and 3. Therefore, their interconversion can be considered as an almost thermoneutral non-identical one. Pseudo-first-order rate constants (k(obs)) were obtained by simulating the AB (1)H NMR system observed for the cyclol. By best fitting the experimental points of a k(obs) versus pD profile to the equation k(obs) = 0.5k(0r) + 0.5k(r) K(ac)/(K(ac) + [D(+)]) + 0.5k(f)K(ao)/(K(ao)+ [D(+)]), the parameters involved were obtained: rate constants of rupture and formation (k(0r) and k(0f) = K(f)k(0r)) catalyzed by water, rate constants of rupture (k(r)) and formation (k(f)) from the conjugated bases of the cyclol form and the open form, and their acidity equilibrium constants K(ac) and K(ao). The system studied mimics the serine alcohol attack on the peptide bond and its reverse reaction in serine protease enzymes. In fact, the reaction rates are similar or perhaps even faster than the ones obtained for enzymatic reactions. The results also show the participation of water molecules forming catalytic proton bridges in stable potentials with the two interconverted forms. The position change of the bridged proton is sensitive to lactam ring size, and it is manifested by considerable change in the pKa values of both cyclol and open forms. Other evidence such as kinetics, DeltaS degrees , DeltaS, and proton inventory experiments and semiempirical molecular calculations support this proposal.

Cleavages in stable tetrahedral intermediates' bonds and the role of water

“…In this work we report the measurement of Δ S o for n = 6. Previously,12 we had reported this value for n = 7. For n = 5, we may estimate this value assuming a weak water hydrogen bond interaction.…”

“…Therefore, an evolved enzyme will fulfil this condition in order to guarantee the minimum energy barrier in both directions of the catalytic processes. We have shown12 that the rate constants measured ( k 2 ) using 1 HNMR line shape analysis following the broadening of the STI 1 HNMR AB signals (CH 2 , attached to the endocyclic oxygen) are of the same order of magnitude (up to 60 s −1 ) as values reported21for enzyme reactions (up to 280 s −1 ). These results are consistent with the proposal that the STI indeed mimics the serine protease mechanism.…”

“…From the above plots, a pKa of 11.9 has been kinetically measured for the STI where X = O and n = 5. However, for X = O, n = 5 and 6, we have recently found12 that, indeed, an almost thermoneutral equilibrium between the open form and the STI is established. This equilibrium allowed us to propose that this system mimics the serine protease enzymic mechanism.…”

“…However, the measured (from k obs versus pK a profile) pKa values for n = 6 and 7 are, respectively, 7.3 and 5.1. This unusual decrease in pK a has been attributed12 to a OHOH 2 O cyclic interaction that makes the ‐OH proton more acidic. This interaction is favoured in the order n = 7 > n = 6 ≫ n = 5.…”

“…In the second part, previous results10, 11 were used to illustrate our suggestions. In part three, the lactam 1‐(2‐hydroxyacetyl)‐piperidine‐2‐one ( n = 6) was synthesized as previously described 12. A 400 MHz JEOL eclipsed plus NMR instrument was used.…”