Computational studies decarboxylation

These three main studies of the gas phase behavior of orotate derivatives show that the 4-protonation pathway is always favored over the 2-protonation pathway. When the barriers are calculated relative to a common reference point of orotic acid, as was done in the Singleton-Beak-Lee study, the 4-protonation pathway is favored by a considerable amount, due mostly to the higher basicity of the 4-oxygen over the 2-oxygen in orotate. Still, the 4-protonation pathway also seems to be favored intrinsically, as evidenced by the consistently lower barriers computed for decarboxylation of the 4-protonated zwitterion 6, regardless of the Nl-R group. 

So far, three computational studies of isotope effects related to the ODCase mechanism have been published Singleton, Beak and Lee used 13C isotope effects to elucidate the mechanism by which the uncatalyzed decarboxylation of orotic acid takes place.46 Phillips and Lee calculated 15N isotope effects and compared them to known experimental values to show that oxygen-protonation mechanisms are viable for the enzyme-catalyzed process.47 Kollman and coworkers focused on the 15N isotope effect associated with C5-protonation.27 Each study is described further below. 

In addition to these quantum mechanical studies on the energetics of decarboxylation, several computational studies focused on isotope effects associated... 

Calculations have so far answered some questions related to the decarboxylation of orotic acid derivatives in different media, but these same computational studies have also opened up many additional areas of controversy. 

Using the EVB approach, which was calibrated to the aqueous reaction [28], Warshel et al. also reproduced the experimental free energy of activation for the catalyzed (OMP) reaction in ODCase. The EVB energy profile and barrier for the decarboxylation reaction in the gas phase was not reported. Although the computed barrier heights for the enzyme reaction are similar in the two computational studies [16, 28], a major difference is that the... 

The same catalyst 14 was found to promote enantioselective decarboxylative protonation reaction. Recent computational study used DFT/PM3... 

Nonetheless, there is still hope that quantum mechanical studies may play a key role in deducing the ODCase mechanism. What these studies have shown is that several mechanisms are energetically viable. They have also provided structural models of transition states and their complexes with active site groups that can be used to design experiments for distinguishing between the several mechanisms that remain in the running. One particularly promising experiment that has already been proposed is the measurement of the 1SN decarboxylation isotope effects for the N3 site of OMP. Phillips and Lee have made the computational prediction that while decarboxylation via 2-protonation and without pre-protonation should have normal isotope effects (1.0027 and 1.0014, respectively), the 4-protonation pathway should display an inverse IE of 0.9949.47 Thus, the combination of computationally predicted and experimentally measured IE values may ultimately lead to elucidation of the enzyme mechanism. 

Warshel and coworkers used EVB72 and FEP71 calculations to predict the free energy of activation for ODCase-catalyzed decarboxylation and to examine its origins. This study differs from that of Gao and coworkers in two fundamental ways (besides in the details of the computational methods used in each). First, Warshel and coworkers explored the effects of changing the protonation states of several important residues in ODCase. Second, in some of their simulations, the ammonium group of Lys73 (Fig. 2) was treated quantum mechanically. 

So far, the several reported studies in which free energy computations have been applied to the mechanism of OMP decarboxylation have not produced an answer to the question of where the rate acceleration provided by ODCase comes from. 

The enzyme mechanism, however, remains elusive. Quantum mechanical models generally disfavor C6-protonation, but 02, 04, and C5-protonation mechanisms remain possibilities. Free energy computations also appear to indicate that C5-protonation is a feasible mechanism, as is direct decarboxylation without preprotonation O-protonation mechanisms have yet to be explored with these methods. Controversy remains, however, as to the roles of ground state destabilization, transition state stabilization, and dynamic effects. Because free energy models do take into account the entire enzyme active site, a comprehensive study of the relative energetics of pre-protonation and concerted protonation-decarboxylation at 02, 04, and C5 should be undertaken with such methods. In addition, quantum mechanical isotope effects are also likely to figure prominently in the ultimate identification of the operative ODCase mechanism. 

The efficient photodecarboxylation of the keto acids (77) has been studied. The reactions involve the formation of the carbanions (78). Aqueous solutions of fenofibric acid (79) at pH 7.4 show the formation of two intermediates when subjected to laser excitation. The study has indicated that the triplet state of the acid in water is of a jtji type. Photoionization is an important process in the aqueous medium. New photoreactive phenylalanine analogues (80) and (81) have been prepared. These were incorporated into position 5 of the pentapeptide, thymopentin. The resultant derivatives were photolabile and underwent decomposition on irradiation at 365 nm. Computational methods have been used to analyse the photoreactivity of the tryptophan derivative (82). The calculations were directed towards an understanding of the quenching of the fluorescence. The results indicate that hydrogen transfer alone does not quench the fluorescence, but that an aborted decarboxylation path is involved. Proton transfer... 

In analyzing the origin of enzyme catalysis, Warshel and others have advocated the importance of comparing the enzymatic reaction with a reference reaction in water [32]. In addition, it is also necessary to study the reference reaction in the gas phase in order to understand the intrinsic reactivity and the effect of solvation. Thus, to understand enzyme catalysis fully, we must compare results for the same reaction in the gas phase (intrinsic reactivity), in aqueous solution (solvation effects), and in the enzyme (catalysis). This is not possible when there is no model reaction for the uncatalyzed process in the gas phase and in water, or if the uncatalyzed reaction is a bimolecular process as opposed to a unimolecular reaction in the enzyme active site. None of these problems apply to the ODCase reaction. Furthermore, OMP decarboxylation is a unimolecular process, both in water and the enzyme, providing an excellent opportunity to compare directly the computed free energies of activation [1] this is the approach that we have undertaken [16]. Warshel et al. used an ammonium ion-orotate ion pair fixed at distances of 2.8 or 3.5 A as the reference reaction in water to mimic an active site lysine residue [32]. 

Using both computational and experimental techniques, initial studies demonstrated that the position of reaction with a given electrophile was dependent on its size [39]. Related studies, using the prochiral cation derived from decarboxylation of Mosher s acid as a probe, computationally estimated the difference in energy between the adducts formed from Re and Si face attack on this cation as an indicator of stereoinductive potential [42]. 

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