Charge Dispersion and Its Effects on the Reactivity of Thiamin-Derived Breslow Intermediates Michael Bielecki, Graeme W. Howe and Ronald Kluger* Davenport Chemistry Laboratories, Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6 Canada *E-mail:
[email protected] Table of Contents Image
Supporting Information Placeholder ABSTRACT: The enzymic decarboxylation of 2-ketoacids proceed via their C2-thiazolium adducts of thiamin diphosphate (ThDP). Loss of CO2 from these adducts leads to reactive species that are known as Breslow intermediates. The protein-bound adducts of the 2-ketoacids and ThDP are several orders of magnitude more reactive than the synthetic analogues in solution. Studies of enzymes are consistent with formulation of protein-bound Breslow intermediates with localized carbanionic character at the reactive C2α position, reflecting the charge-stabilized transition state that leads to this form. Our present study reveals that nonenzymic decarboxylation of the related thiamin adducts proceeds to the alternative charge-dispersed enol form of the Breslow intermediate. These differences suggest that the greatly enhanced rate of decarboxylation of the precursors to Breslow intermediates in enzymes arise from maintenance of carbanionic character at the position from which the carboxyl group has been cleaved, avoiding charge dispersion by stabilizing electrostatic interactions with the protein as formulated by Warshel. Applying Guthrie’s no-barrier theory also leads to the conclusion that maintaining the tetrahedral carbanion at C2α of the resulting adduct minimizes associated kinetic barriers by avoiding re-hybridization as part of steps to and from the intermediate. Finally, maintenance of the reactive energetic carbanion agrees with the concepts of Albery and Knowles as the outcome of evolved enzymic processes.
Enzymes that catalyze the decarboxylation of 2-ketoacids utilize the cofactor thiamin diphosphate (ThDP) to form covalent adducts of their substrates.1 This induces the necessary reversal of polarity to allow accumulation of electron density from loss of CO2 at what had been the carbonyl carbon. However, while synthesized adducts of 2-ketoacids and thiamin undergo decarboxylation in neutral solutions, those rates are considerably slower than for similar enzyme-bound adducts of ThDP.1-3 The immediate product from loss of
CO2 is a reactive species known as a Breslow intermediate and can be represented either as an enol or zwitterionic carbanion (Scheme 1). Breslow used chemical models to deduce the nature of the intermediates in the decarboxylation of 2-ketoacids in enzymes that arise upon loss of CO2. The term has been generalized to include related adducts derived from N-heterocyclic carbenes in which the heterocycle is derived from a diazole
or triazole rather than from the thiazole (“enol” shown in Scheme 1.4-6
Scheme 1. General scheme for formation of Breslow intermediates from ThDP and 2-ketoacids.
Structures formulated as enols derived from adducts of thiamin diphosphate (as in Scheme 1) have been assigned from X-ray crystallographic and spectroscopic analyses.7-11 However, other studies of the structure and reactivity of enzymic Breslow intermediates bound to ThDP-dependent enzymes conclude that enzyme-stabilized intermediates are present as charge-separated carbanions (“carbanion “ in Scheme 1).12-16 Formation of charged intermediates implies a polar transition state (TS) for the decarboxylation step. The rate constant for the enzymic decarboxylation step from these intermediates is at least six orders of magnitude larger than that for the corresponding step in the nonenzymic decarboxylation of adducts of 2-ketoacids and thiamin.1 These results led us to consider to what extent does an enzyme maintain the carbanionic form of the Breslow intermediate and what catalytic advantage (if any) does this provide. Recent reports from Berkessel reveal that Breslow intermediates (derived from 1,3-diazoliums) are enols in the crystalline state.17-19 Only limited experimental data exists that can be used to interpret the nature of Breslow intermediates derived from thiazolium compounds in solution.20, 21 None of the examples is from an intermediate derived from thiamin. Furthermore, the degree to which charges remain localized at the transition state for a decarboxylation process that leads directly to a Breslow intermediate in solution is also unknown. In order to understand the basis of the substantial acceleration provided by association of ThDP adducts with proteins, we need to know the charge distribution of a comparable species in solution. The magnitude of the Hammett ρ for the decarboxylation of mandelylthiamin (MTh, Scheme 2: Z = H), a benzoylformate-thiamin adduct, will reflect the degree of charge build-up at the transition state and in the resulting energetic Breslow intermediate. This result forms the basis for comparison with the previously reported ρ value of 4.4 for
2
an analogous enzymic decarboxylation.22 Thus, we prepared a series of phenyl-substituted analogues of MTh, an accurate comparator for the corresponding enzymic pre-decarboxylation intermediate derived from ThDP (Scheme 1, R=Ph). Our improved synthetic route to MTh serves as the basis for producing phenyl-substituted derivatives of MTh (Scheme 2: Z = p-OCH3; pCH3; p-F; p-Cl; m-Br).23, 24 The results of kinetic studies on these compounds allow us to make a confident assessment of the charge distribution in the Breslow intermediate and TS that leads to it. We find that the results are consistent with an induced electrostatic environment on the enzyme that maintains a reactive intermediate that minimizes surrounding intrinsic barriers. The results are consistent with proposals for evolved catalytic efficiency.25, 26
Scheme 2. MTh spontaneously undergoes decarboxylation in neutral solutions to produce the corresponding Breslow intermediate and CO2.
EXPERIMENTAL SECTION Synthesis
Methyl benzoylformate was purchased from commercial sources. Substituted derivatives of methyl benzoylformate were synthesized from commercially available substituted acetophenones by oxidation with selenium dioxide in pyridine,27 followed by esterification with dimethyl sulfate. Liquid isobutylene was prepared from acidic dehydration of t-butanol. This was used to produce t-butyl esters with sulfuric acid as a catalyst.28 Esters of the phenyl-substituted MTh derivatives were synthesized by condensing the substituted benzoylfor-
mate esters with O-t-butyl-diphenylsilyl-thiamin chloride.24 The generalized synthetic route is summarized in Scheme 2 (see SI for details). Scheme 2. Preparation of phenyl-substituted derivatives of mandelylthiamin.
thiamin and benzaldehyde in the presence of organic bases first reported by Oka and coworkers.30, 31
Figure 1. Products from decarboxylation of MTh and derivatives in neutral buffers include those from fragmentation.
The rates of decarboxylation of MTh derivatives were monitored for reactions in pH 7.0 HEPES buffers (0.10, 0.20, 0.30 and 0.40 M) at 25.0 (±0.1) o C with ionic strength maintained at 1.00 M with potassium chloride. The reaction progress was followed by monitoring changes in UV-VIS spectra. Data were obtained in triplicate. The observed first order rate coefficients were calculated from non-linear regression analysis of the absorbance at 330 nm from PTK. The changes in absorbance with time were fit to the integrated first order rate law to obtain first order rate constants. Repeated wavelength scans gave stable isosbestic points over the course of the reactions. Rate constants at zero buffer concentration were obtained by extrapolation of plots of observed rate coefficients vs. buffer concentration. Products were characterized by assignments of their 1H NMR and MS in larger scale runs as well as UV-Vis spectra.
Results and Discussion MTh and its phenyl-substituted derivatives undergo rate-limiting decarboxylation followed by rapid protonation at C2α to give derivatives of 2-(1-hydroxylbenzyl)thiamin (HBnTh, Figure 1). Unlike the enzymic process, the reactions in solution form additional products upon loss of CO2, principally the result of fragmentation to substituted phenyl thiazole ketones (PTKs) and 2,5dimethyl-4-amino-1,3-pyrimidine (DMAP) (Figure 1).29 These are consistent with products from reactions of
1 0.8 0.6 Log(kx /kH)
Kinetic and product studies
The absorbance near 330 nm arises from phenyl-substituted derivatives of PTK, a product of Oka fragmentation of the Breslow intermediates.29 The rate constant for decarboxylation of MTh (k~10-4 s-1) is considerably smaller than the rate constant for subsequent fragmentation (k~105 art 40C and 104 at 25 ) of the resulting Breslow intermediate.32 Thus, the rate of appearance of the fragmentation products provides data for the rate of the decarboxylation step.32 A Hammett plot of log (kX/kH) as a function of σ gives ρ = 0.6(±0.1) with R2 = 0.89 (Figure 2).
0.4 0.2 0 -0.2 -0.4 -0.4
-0.2
0
0.2
0.4
Figure 2. Hammett plot of first order rate constants for decarboxylation of MTh and derivatives at 25 oC.
The effects of substituents on rates of decarboxylation reflect the extent of carbanion character that develops at C2α in the transition state as the C-C bond cleaves. Since the enzymic results are consistent with the formation of a localized carbanion, the substituent effects that we have determined provide a comparative basis for the corresponding reactions in solution. Applying the Hammond postulate enables us to deduce the extent to which charge remains localized at C2α in the energetic Breslow intermediates, despite their transient existence. Decarboxylation leading to the immediate carbanion in the Breslow intermediate rather than to the subsequent enol would result in a much larger sensitivity to substituents. Values of ρ > 3 are typical for E1cb reactions with
3
rate-determining formation of carbanions.33, 34 Transfers of benzylic protons give ρ = 4.035 and the anionic polymerization of styrene gives ρ = 5.36 In contrast, the deprotonation of 1-phenylnitromethane, in which the developing charge is internally neutralized, has ρ = 1.2.37 In that case, there is a lag in charge delocalization relative to the initial charge development (imperfect synchronization38). Still, the value of ρ is small in comparison to that for production of ultimately localized carbanions. Thus, we conclude that the ρ value of 0.6 for the decarboxylation of MTh is clearly significantly smaller than expected for reactions that produce carbanions. The extent of charge-delocalization at the transition state for the departure of CO2 is very extensive in solution and this leads to the uncharged enollike character of the reactive Breslow intermediate. Studies of the enzymic decarboxylations of analogous adducts of ThDP and phenyl-substituted benzoylformates provide evidence for a dramatically different form of the Breslow intermediate from that in solution. Schutz et al. reported results that they present as a Hammett plot for the decarboxylation of para-substituted phenyl derivatives of benzoylformate by IPDC (indole pyruvate decarboxylase).22 That enzyme efficiently promotes the decarboxylation of benzoylformate and its para-substituted analogues. The Hammett plot of kcat vs. is non-linear with concave downward curvature at its midpoint. The authors reasonably ascribe this curvature to a change in rate-limiting step due to the effects of substituents on a reactive intermediate. The section with a positive slope (ρ = 4.4) occurs with substrates with electron-donating substituents (methyl, ethyl, methoxy). The portion with a negative slope (ρ = -2.5) occurs with substrates with electron-withdrawing substituents (fluoro, bromo, chloro, nitro). The ρ value of 4.4 is substantial and is consistent with a transition state leading to the localized carbanion of the charge-separated intermediate. Of the various steps in the catalytic pathway of ThDP-dependent decarboxylases, only the decarboxylation leading to a localized carbanion could account for such a large value. The loss of CO2 from electron-rich para-substituted benzoylformates on benzoylformate decarboxylase (BFDC) is partially rate-limiting as deduced from carbon-isotope effects.39 Therefore, a rate-limiting decarboxylation step in IPDC with electron-rich substrates is reasonable. In IPDC, kcat for the bulkier and less electron-donating p-CH3CH2benzoylformate is larger than kcat for the smaller but stronger electron-donating p-CH3-benzoylformate. This indicates that the observed substituent effects are primarily the result of electronic interactions. The value of ρ = 4.4 for the enzymic process contrasts with the non-enzymic ρ = 0.6 for decarboxylation of derivative of MTh that we have now determined. Since the carbanion is formed on the enzyme, it is reasonable that
4
electrostatic interactions contribute to stabilizing the enzyme-bound state.40 This is consistent with direct observations of localized carbanion-like intermediates on enzymes.12-16 In solution, localized carbanions would be considerably higher in energy than the readily accessed enol that forms upon relaxation. Furthermore, in the enzymic cases derived from ThDP, the presence of the diphosphate facilitates binding without interfering with catalysis. In solution, the diphosphate provides no role for a catalytic effect.41, 42 As noted above, the protein-bound form favors a path to an otherwise more energetic form of the Breslow intermediate. Warshel has elucidated the importance of electrostatic stabilization of transition states and reactive intermediates that occur through relief of electrostatic stress within the associated protein.40, 43-45 This would allow the localized carbanion to be maintained upon cleavage of the C-C bond that leads to formation of CO2. Warshel’s formulation establishes the overall importance of significant electrostatic effects for catalytic processes within proteins that utilize release of locally induced stress.46, 47 Why would an enzyme evolve to stabilize the intrinsically more energetic form of the Breslow intermediate? An important insight comes from reports by Kresge and coworkers who observed that transfer of a proton to a localized carbanion has no intrinsic kinetic barrier (following Marcus theory), while transfer to sites with dispersed electron density overcome substantial intrinsic barriers.48 The effects of localization of charge on reactivity are not limited to proton transfers and can be understood from the basis of Guthrie’s No Barrier theory: processes that alter bond lengths and bond angles while other bonds are being formed or broken are the source of intrinsic barriers.49, 50 Interestingly, Guthrie specifically applied no-barrier theory to decarboxylation reactions in terms of the effects of the conversion of the carboxyl group to carbon dioxide.51 The change in hybridization and the requisite concerted atomic movements that avoid impossible intermediates in single-movement steps produce a substantial portion of the intrinsic barrier for formation of CO2 in a decarboxylation process. We extend the application No Barrier theory to the residual structure in decarboxylation. This leads to a conclusion that parallels Kresge’s analysis of the source of barriers to proton transfers from carbon. That is, loss of a carboxyl that leads to a localized carbanion will have a lower intrinsic barrier than those where electron density is delocalized to an enolic species. According to Albery and Knowles, catalysis of an elementary step, as
we see here, is the most sophisticated adaptation in an enzyme's evolution.52 Warshel’s formulation of stressed charges in proteins provides efficient electrostatic stabilization of carbanions (and associated transition states)53 that would be dispersed in solution through rehybridization and general solvation (Figure 3).
does not occur on enzymes. If the fragmentation occurs via the conjugate base of the enolic C2α-OH group as we have proposed,24 the required ionization of the C2αOH for fragmentation would not occur from the localized carbanion due to internal electrostatic repulsion.58, 59 The clear contrast in the nature and resulting reactivity of enzymic and nonenzymic Breslow intermediates that arise from differences in charge dispersion would have provided an evolutionary advantage for intermediates with protein-stabilized localized electrostatic character.
■ Conclusions
Figure 3. Pathways for decarboxylation. According to Guthrie’s No-Barrier theory,” formation of the carbanion involves a minimal intrinsic barrier without rehybridization while formation of the neutral product (enol) requires multiple alterations and a higher intrinsic barrier. With the reactive carbanion bound to the enzyme in a region with positive electrostatic character, the thermodynamic barrier in the transition state that produces such an intermediate can be overcome by attractive interactions that overcome electrostatic stress within the protein following the general principles of enzymic catalysis that have been developed by Warshel (Figure 4).54-57
The small value of ρ for the spontaneous decarboxylation of phenyl-substituted derivatives of MTh in solution indicates that the resulting Breslow intermediates form via transition states that resemble charge-dispersed enols. These structures contrast with the corresponding charge-localized carbanionic Breslow intermediates that bind to enzymes. Proteins can provide environments that favor the zwitterionic carbanion over the enol according to the Warshel formulation.40 Importantly, the pathway via a zwitterionic carbanion would lead to acceleration of the decarboxylation process by minimizing the associated intrinsic Marcus barrier associated with re-hybridization at C2α as proposed in general by Guthrie.60 Maintaining the zwitterionic form also retains the aromatic state of its thiazolium component and prevents the otherwise unavoidable fragmentation that occurs in solution. Finally, the retention of separated charges exemplifies how enzymes evolve to maximize efficiency by maintaining energetic intermediates through structural control in their bound state that would be lost in the unbound state in solution as summarized in Figure 4.25
■ AUTHOR INFORMATION Corresponding Author *Davenport Chemistry Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6 Canada. E-mail:
[email protected]. The authors are grateful for funding from NSERC Canada (Discovery Grant to R.K. and Postgraduate Scholarship to G.W.H.) Notes The authors declare no competing financial interest Figure 4. A hypothetical energy profile for the enzymic decarboxylation of 2-ketoacids via formation of the enol (dashed line) and via the localized carbanion (solid line).
In addition, examination of the charged form of the Breslow intermediate might illustrate why the Oka fragmentation of the ThDP derivatives of benzoylformates
5
■ DEDICATION Our work is based on the scientific heritage given to us by Ronald Breslow and Peter Guthrie.
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