Kemp Elimination Reaction Catalyzed by Electric Fields

Abstract

The Kemp elimination reaction is the most widely used in the de novo design of new enzymes. The effect of two different kinds of electric fields in the reactions of acetate as a base with benzisoxazole and 5‐nitrobenzisoxazole as substrates have been theoretically studied. The effect of the solvent reaction field has been calculated using the SMD continuum model for several solvents; we have shown that solvents inhibit both reactions, the decrease of the reaction rate being larger as far as the dielectric constant is increased. The diminution of the reaction rate is especially remarkable between aprotic organic solvents and protic solvents as water, the electrostatic term of the hydrogen bonds being the main factor for the large inhibitory effect of water. The presence of an external electric field oriented in the direction of the charge transfer (z axis) increases it and, so, the reaction rate. In the reaction of the nitro compound, if the electric field is oriented in an orthogonal direction (x axis) the charge transfer to the NO2 group is favored and there is a subsequent increase of the reaction rate. However, this increase is smaller than the one produced by the field in the z axis. It is worthwhile mentioning that one of the main effects of external electric fields of intermediate intensity is the reorientation of the reactants. Finally, the implications of our results in the de novo design of enzymes are discussed.

Catalytic Effect of Electric Fields on the Kemp Elimination Reactions with Neutral Bases

“…Table 2 presents, for the reactions of 5-nitrobenzisoxazole with methylamine or imidazole in acetonitrile or water, the partition of ΔE' � into its three components: the potential energy barrier (ΔE � ) and the electrostatic and non-electrostatic terms of the solvation free energy (ΔG solv el� and ΔG solv nonÀ el� , respectively). For comparison, the values for the reaction with acetate [19] have also been included. In the last case, in which an anionic base is used, ΔE' � is 9.2 kcal mol À 1 larger in water than in acetonitrile, the dominant component being essentially the electrostatic solvation free energy (+ 10.0 kcal mol À 1 ).…”

“…[36] According to Shaik, [11] the reaction axis is the one "along which the electron pairing of the reactants evolves to the electron pairing of the products". In our previous study [19] of the Kemp elimination reactions of benzisoxazole or 5-nitrobenzisoxazole with acetate, the reaction axis was taken as the one which linked the O1' atom of acetate with the O1 atom of the substrate. However, this choice may be questioned in the case of the nitro compound, since the acceptor NO 2 group also receives electronic charge from the base.…”

“…When an anionic base is used. [19] the catalysis is due to the relative stabilization of the reactant complex with respect to the TS. As the negative charge is more localized in the RC than in the TS, and the stabilization will be larger in the reactant complex as far as the dielectric constant of the solvent is increased.…”

“…In a previous paper, [19] we studied the effect of solvent reaction fields and of external electric fields on the rate of the Kemp elimination reaction [20] which is usually taken as a benchmark for the design of new enzymes. [21][22][23][24][25][26][27] In particular, we used 5-nitrobenzisoxazole as substrate and acetate as the base that withdraws a proton from the substrate (see Scheme 1).…”

Protein electrostatics: From computational and structural analysis to discovery of functional fingerprints and biotechnological design

“…An accurate analysis of the electrostatic environmental effects may open new routes toward the rational design and optimization of efficient catalysts; however, when using electric field alignments in the reactive centers of complex catalytic systems, much more predictive capacity is needed for computational design providing results transferable to the experimental field [92] . Electrostatic preorganization has been brought into focus by works studying artificial enzymes to be used in biocatalysis and, in particular, diverse variants of Kemp eliminase, an unnatural enzyme used as benchmark for testing protocols for de novo design [92] , [93] , [94] . Kemp elimination, that is the conversion of benzisoxazoles into salicylonitriles, takes place in solution via a delocalized transition state in which a proton transfer and a bond breaking in a 5-membered ring occur simultaneously; no natural enzyme capable of sustaining such conversion as primary and specific reaction has been identified so far.…”