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Decarboxylation, CO2 and the Reversion Problem Ronald Kluger* Davenport Laboratories, Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada CONSPECTUS: Decarboxylation reactions occur rapidly in enzymes but usually are many orders of magnitude slower in solution, if the reaction occurs at all. Where the reaction produces a carbanion and CO2, we would expect that the high energy of the carbanion causes the transition state for C−C bond cleavage also to be high in energy. Since the energy of the carbanion is a thermodynamic property, an enzyme obviously cannot change that property. Yet, enzymes overcome the barrier to forming the carbanion. In thinking about decarboxylation, we had assumed that CO2 is well behaved and forms without its own barriers. However, we analyzed reactions in solution of compounds that resemble intermediates in enzymic reaction and found some of them to be subject to unexpected forms of catalysis. Those results caused us to discard the usual assumptions about CO2 and carbanions. We learned that CO2 can be a very reactive electrophile. In decarboxylation reactions, where CO2 forms in the same step as a carbanion, separation of the products might be the main problem preventing the forward reaction because the carbanion can add readily to CO2 in competition with their separation and solvation. The basicity of the carbanion also might be overestimated because when we see that the decarboxylation is slow, we assume that it is because the carbanion is high in energy. We found reactions where the carbanion is protonated internally; CO2 appears to be able to depart without reversion more rapidly. We tested these ideas using kinetic analysis of catalytic reactions, carbon kinetic isotope effects, and synthesis of predecarboxylation intermediates. In another case, we observed that the decarboxylation is subject to general base catalysis while producing a significant carbon kinetic isotope effect. This requires both a proton transfer from an intermediate and C−C bond-breaking in the rate-determining step. This would occur if the route involves the surprising initial addition of water to the carboxyl, with the cleavage step producing bicarbonate. Interestingly, some enzyme-catalyzed reactions also appear to produce intermediates formed by the initial addition of water or a nucleophile to the carboxyl or to nascent CO2. We conclude that decarboxylation is not necessarily a problem that results from the energy of the carbanionic products alone but from their formation in the presence of CO2. Catalysts that facilitate the separation of the species on either side of the C−C bond that cleaves could solve the problem using catalytic principles that we find in many enzymes that promote hydrolytic processes, suggesting linkages in catalysis through evolution of activity.
1. INTRODUCTION Carboxylic acids are normally highly stable compounds with little tendency to undergo spontaneous decarboxylation. This contrasts with the rapid loss of the carboxyl group from the substrates of decarboxylase enzymes. We expect decarboxylases to provide a reaction pathway that reduces the energies of transition states, utilizing functional groups of the protein and its cofactors to stabilize the residual carbanion accompanying the formation of CO2 (Scheme 1). Guided by the Hammond Postulate, we also expect that whatever lowers the energy of the highly energetic carbanionic product will also reduce the energy of the associated transition state. In considering the mechanisms that can lead to a faster reaction, we ignore the one product that all the reactions have
in common: CO2. Conventional wisdom tells us that such a stable entity should not require any accommodation in order for enzymes that produce it to achieve their catalytic prowess. Yet, as we examined decarboxylation reactions that were accessible to catalysis by enzymes, we found, to our surprise, that some of the same reactions are also subject to catalysis in the absence of enzymes by proton donors and acceptors. Our in-depth examinations by kinetics, product analysis, and isotope effects led us to discover the why, when, and how of dealing with CO2.
2. ENZYMES THAT UTILIZE THIAMIN DIPHOSPHATE DECARBOXYLATE 2-KETOACIDS Our earlier work led us to obtain readily quantitative measures of enzymes that promote decarboxylation of 2-ketoacids.1−3 Those enzymes utilize thiamin diphosphate (ThDP) as a cofactor.3 In the absence of enzymes, decarboxylation of 2ketoacids does not occur spontaneously at any measurable rate,
Scheme 1. Decarboxylation via Formation of a Carbanion and CO2
Received: June 22, 2015
© XXXX American Chemical Society
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DMAP (Scheme 4).14−16 PTK has a strong absorbance at 330 nm, providing a convenient indicator to measure the rate of
even at high temperatures. However, ThDP-dependent decarboxylases produce very rapid reactions of 2-ketoacids. These enzymic reactions avoid formation of high-energy acyl carbanions by conjugation of 2-ketoacids and ThDP. This provides an energetically accessible thiazolium-stabilized carbanion along with CO24 (Scheme 2).
Scheme 4. Fragmentation of Decarboxylation Product from Mandelylthiamin
Scheme 2. Thiamin Diphosphate Forms Conjugates with 2Ketoacids on Enzymes
The diphosphate portion of ThDP serves as a site for binding its nonreacting extended side chain to a metal ion that binds within the associated protein.5−8 The ongoing association of protein and cofactor allows them to function as a truly coordinated catalytic system. Thiamin, vitamin B1, is a functional analogue of ThDP with a hydroxyl group in place of the diphosphate of ThDP.
decarboxylation of MTh because the unimolecular fragmentation reaction is much faster than decarboxylation. Ian Moore measured the rate of cleavage of the carbanion from HBnTh.17−19 The rate is about 2 orders of magnitude larger than the enzymic kcat. How can an enzyme accelerate one reaction and slow another? Clearly, the enzyme does not cleave the cofactor but the carbanion that fragments rapidly must occur on the same pathway as that which follows the loss of CO2. We found that the answer is that the reaction that suppresses the fragmentation reaction also increases the rate of C−C bond cleavage in decarboxylation.
3. ENLIGHTENMENT FROM THE PROFICIENCY OF BENZOYLFORMATE DECARBOXYLASE (BFD) Benzoylformate decarboxylase (BFD) catalyzes a similar reaction to the well-known decarboxylation of pyruvate.9−11 Miriam Hasson and her co-workers had determined its highresolution crystal structure,12 while Kenyon, Cleland, Cook and their co-workers carried out studies on its kinetics, sequence, and alternative reactions.9−11,13 The ThDP-derived intermediate from benzoylformate, mandelylThDP (MThDP), loses its substrate-derived carboxyl group to form a thiazolium-stabilized carbanion. Protonation of that carbanion gives the enzymebound intermediate (2−1-hydroxybenzyl-ThDP, HBnThDP), followed by release of benzaldehyde and regeneration of enzyme-bound ThDP (Scheme 3).
5. INACTIVATION OF BFD BY A PHOSPHONATE ANALOGUE OF THE SUBSTRATE Another interesting result came from analysis of BFD’s structure with what we would expect to be a stable analogue of the substrate. Benzoylphosphonate should be a reversible inhibitor of BFD, functioning by addition of ThDP to its carbonyl group.20 Yet, Hasson and co-workers found that benzoylphosphonate irreversibly inactivates benzoylformate decarboxylase, forming the phosphate ester derived from the serine hydroxyl at the active site of BFD (Scheme 5).12 How can a stable C−P bond cleave when it reacts with ThDP on an enzyme?
Scheme 3. Mandelylthiamin (MTh, R2 = CH2CH2OH) and the Corresponding Diphosphates Undergo Decarboxylation Followed by Protonation with the Expected Products As Shown
Scheme 5. Inactivation Reactions of Phosphonate Conjugate of ThDP on Enzyme
4. FRAGMENTATION OF THE DECARBOXYLATION PRODUCT FROM MANDELYLTHIAMIN The carbanion that results directly from the decarboxylation of MTh is accessible by deprotonation of HBnTh at C2α. As a carbon acid, its deprotonation is subject to general base catalysis.14 It appears that as the carbanion delocalizes to form an enamine, it rapidly undergoes irreversible fragmentation into derivatives of thiamin’s two heterocyclic constituents, PTK and
6. REACTIONS OF MANDELYLTHIAMIN Clearly, the reactions of MTh can provide a basis for answering questions about catalysis in this enzyme. Qingyan Hu developed a synthesis for MTh, using that material for kinetic and product studies related to its decarboxylation.21−23 Hu found that adding Brønsted acids reduces fragmentation from the carbanion derived from decarboxylation of MTh (Scheme 3).21,22 Moreover, Hu found that pyridine/pyridinium buffers B
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of a barrier for the combination of the carbanion and CO2 creates a comparable barrier to the forward reaction. Can a catalyst function simply by facilitating product formation through reducing the extent of reversion? The Hammond Postulate had guided us only in thinking about the importance of the energy of the carbanion in the nature of the transition state. The completion of a decarboxylation reaction requires the separated solvation of the products.26 The rate law that considers formation of the immediate products of decarboxylation in terms of the steady state approximation clearly shows that the critical competition in the observed rate of a reaction depends on the size of the commitment factor,27 the extent to which the reaction proceeds toward product formation from a reactive intermediate. We also found another line of evidence from the work of Cox and Jencks. They studied reactions in which a Brønsted acid or base is an effective catalyst only if it associates with the reactant prior to the rate-determining step of the reaction.28,29 Without preassociation of the catalyst and reactant, the catalytic transition state becomes inaccessible due to limitations of diffusion. Applying this idea to Hu’s observations, we considered the importance of the potential for protonated pyridine to π-stack with the aromatic rings in MTh as a means of achieving preassociation with the reactant.30 This would allow the preassociated protonated pyridine to facilitate protonation of the carbanion in competition with the carbanion’s addition to CO2. This drives the reaction forward.23
increase the rate of decarboxylation of MTh while providing uniquely effective suppression of fragmentation.21,22 These observations gave us the critical lead into approaching catalysis in decarboxylation in terms of the properties of CO2. The highly efficient suppression of fragmentation by pyridine’s conjugate acids implied that the pyridinium acid must be available to protonate the critical carbanion. How can this increase the rate of the reaction? Hu investigated the effects of other conventional buffers, and none increased the observed rate of the reaction. The only other effective catalysts were the acid components of C-alkyl substituted pyridine buffers. However, all were equally effective with no correlation to their acidities, suggesting that this catalysis involves a thermodynamically favorable transfer to a localized carbanion.24 To see whether the acceleration is due to electrostatic stabilization of the transition state for decarboxylation without proton transfer, she tested N-ethylpyridinium ion and found that it had no effect on the rate or products of reaction. Therefore, transfer of the proton from a pyridinium acid is likely to be essential for catalysis as well as for trapping the product to prevent its fragmentation (Scheme 6). Scheme 6. Trapping a Carbanion by a Pre-associated Brønsted Acid Blocks Reversion
8. TESTING ACCELERATION BY PROTONATION USING CHANGES IN CARBON KINETIC ISOTOPE EFFECTS If the rate of C−C bond-breaking increases relative to reversion by the preassociated acid trapping of the carbanion, the measured carbon kinetic isotope effect for the reaction will increase in the presence of the associated acid.13,27,31 That is, the observed magnitude of a carbon kinetic isotope effect depends on the intrinsic isotope effect of that step and the commitment factor associated with that step.27 On the other hand, if the catalyst functions by lowering of energy through the carbanion of the transition state,32 based on measurements on analogous reactions, the CKIE should not be affected.33 With these criteria as critical kinetic standards, Scott Mundle determined carbon kinetic isotope effects for the decarboxylation of MTh with and without protonated pyridine in solution.34 He found that the addition of pyridine/pyridinium buffers increases the CKIE for decarboxylation of MTh, consistent with the predicted outcome from protonation of the carbanion by the preassociated Brønsted acid being the source of the rate acceleration.34−37
7. THE CRITICAL ENERGY PROFILE: DECARBOXYLATIONS THAT PRODUCE CARBANIONS AND CO2 Hu’s results led us to reconsider the nature of decarboxylation reactions. We analyzed our results in terms of the theoretical analysis of Major and Gao.25 They profiled the free energy for the decarboxylation of N-methyl picolinate, calculating a barrier to breaking the C−C bond, including a plot of the free energy of the system as a function of the length of that bond (Figure 1).
9. GENERAL BASE CATALYSIS IMPLICATES CARBOXYL HYDRATION AND THE DEPARTURE OF BICARBONATE Graeme Howe found that the decarboxylation of MTh is unexpectedly subject to general base catalysis with β = 0.26.38 Also, the reaction shows a significant solvent kinetic isotope effect. The results are consistent with a mechanism in which there is partial transfer of a proton in the rate-determining step, occurring along with C−C cleavage (the reaction has a significant carbon kinetic isotope effect34). If addition of water to the carboxyl group precedes C−C cleavage, this would
Figure 1. Calculated energy profile for the formation of CO2 and Nmethylpyridine zwitterion from N-methyl picolinate in water.25
The energy of the reactant increases and then flattens at the highest energy point as the bond lengthens to the distance expected for separation of CO2 and the carbanion. However, there is no change in energy as the C−C bond extends beyond where they expect the formation of CO2 and the carbanion to be complete. This implies that there is little or no barrier to the recombination of the carbanion and CO2. In that case, the lack C
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Accounts of Chemical Research lead to formation of a tetrahedral addition intermediate,39,40 giving the carbanion and bicarbonate (Scheme 7).38
measuring the basicity of carbanions.49 Instead, we use the more accessible but less consistent “kinetic pKA”. If this is a single step, the relative rates of proton removal can yield the relative acidity of the compounds. However, the rate of the reverse reactions and specific kinetic issues may distort the comparison.24 Whenever a base accepts a proton from carbon, it competes for that proton with the nascent carbanion.50,51 To the extent that the return of the proton to the carbanion slows the overall process, the rate of exchange with the isotopic solvent as a measure of rate is slower than it would be in a truly direct process.50,51 This leads to an overestimation of the actual basicity of the carbanion when using only kinetics of proton exchange as a basis for estimating the pKA of a carbon acid.24
Scheme 7. Decarboxylation via Brønsted Base Catalysis in the C−C Cleavage Step
Bicarbonate is less electrophilic and more soluble than CO2. Because bicarbonate is anionic, electrostatic repulsion can promote its separation from the product carbanion. We know that catalytic arrays in enzymes facilitate formation and decomposition of tetrahedral intermediates in proteases and esterases.41 Evolved differences could promote decarboxylation via “tetrahedral intermediates” to promote decarboxylation (Scheme 8).42
12. DECARBOXYLASES AND ALTERNATIVES TO CO2 Replacing alanine with serine at the active site of benzaldehyde lyase converts that ThDP dependent enzyme, which is not a decarboxylase, to an effective benzoylformate decarboxylase.52,53 The change to serine could lead to addition of its hydroxyl to the carboxyl of benzoylformate. In that case, cleavage of the C−C bond would occur without initial formation of CO2 as shown in Scheme 6. Instead, the reaction would produce a monocarbonate ester of serine that can lose CO2 in a location that is more remote from the carbanion. The mutant enzyme, BAL A28S, also reacts at serine with benzoyl phosphonate, forming the phosphate ester, leading to its inactivation. This parallels the inactivation of BFD by the same substrate analogue.52 Ding and co-workers reported the unexpected structural similarity of the active sites of isoorotate decarboxylase (IODC) and carboxypeptidase.54 They propose that IODC functions by addition of water and release of bicarbonate, paralleling the mechanism of carboxypeptidase A (Scheme 9). Smith and colleagues investigated the mechanism and products of fatty acid synthase, including a decarboxylation step.55,56 Using a coupled assay with PEP carboxylase, which requires bicarbonate as a substrate, Smith demonstrated that the decarboxylase produces bicarbonate. The mechanism he proposed is similar to that of ribonuclease, utilizing histidine bases in alternating states of protonation to promote the addition of water to the carboxyl that is removed.56 Oikawa and co-workers reported the structure of 2,6dihydoxybenzoate decarboxylase.57 A parallel with the mechanism proposed for IODC via hydration and formation of bicarbonate would be reasonable.54 An interesting aspect is that enzyme can also catalyze the carboxylation reaction.57 A logical bicarbonate-based mechanism for the carboxylation is presented in a very broad study of biocatalysis by Faber and coworkers.58
10. GENERALIZING: CO2 VS BICARBONATE IN DECARBOXYLATION REACTIONS We have noted that decarboxylation can become problematic where CO2 and a carbanion form simultaneously. The basis of the problem is the intrinsic nature of the components: the carbanion is likely to be an energetic nucleophile26 and CO2 is a very reactive electrophile.43 As a nonpolar compound, CO2 has low solubility in water.44 Decarboxylations that can generate CO2 directly should occur where the carbanion delocalizes into other portions of the residual species or where an acid can quench the carbanion. Reactions where the carbanion has a resonance contributor from an iminium ion or where an irreversible fragmentation is accessible as the carbanion forms are examples of such intrinsic avoidance of reversion.45−47 Enzymes that catalyze decarboxylation reactions that allow simultaneous relocation of the excess electronic density from the carbanion will not be subject to the barrier to producing CO2 directly. Examples include the nonenzymic decarboxylation of 3-ketoacids48 and the enzymic decarboxylation of enzyme-generated 3-iminoacids,45,46 where the electron pair delocalizes as the C−C bond is broken. 11. PREDICTING DECARBOXYLATION RATES FROM BASICITY The relative energies of carbanions relate directly to the magnitude of the pKA of their conjugate acids. Where carbanions are the product of decarboxylation, we expect the rate of the reaction to reflect the basicity of the carbanion. Where we can obtain the value of a pKA from data acquired by titration at equilibrium, we can reliably compare the energies of carbanions. However, titration is usually not possible for
13. DECARBOXYLATION BY TRANSCARBOXYLASES Biotin-dependent transcarboxylases promote a decarboxylation reaction in a substrate where the carboxyl group is transferred
Scheme 8. Serine Protease-Like Mechanism for BFD
D
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Accounts of Chemical Research Scheme 9. Decarboxylation and Peptide Hydrolysis by a Common Catalytic Apparatus
to the N1′ position of biotin.59,60 That carboxyl group formally transfers to the carbanion derived from the second substrate. In this enzyme, the decarboxylation produces a carbamate that permits CO2 to be available for transfer to the second substrate and for the initial decarboxylation to occur in the presence of a reactive carbanion. Sauers, Groh, and Jencks introduced the concept of “low entropy” CO2 as a carboxylation agent for biotin to explain the role of ATP in biotin-dependent carboxylases that utilize bicarbonate as a cosubstrate.61 CO2 that forms by C−C bond cleavage of transcarboxylase in the donor substrate will necessarily have “low-entropy” character, because it forms locally without ATP. Alternatively, the conjugate base of biotin at N1′ could add to the carboxyl of the substrate, followed by C−C cleavage, to produce the same result. In either case, the carboxylation of biotin not only provides an intermediate for subsequent transfer of the carboxyl, it would avoid nonproductive internal return of CO2. The addition product of biotin and CO2 is simply a tamed form of CO2 that allows controlled transfer from donor to acceptor.
Scheme 10. Hydroxide-Catalyzed Decarboxylation Generates Carbonate
15. CHALLENGES A reviewer disagreed with some aspects of the mechanisms we propose that would reduce the extent of reversion. In particular, that reviewer expects that the free energy of a hydrated carboxyl must be higher than the energy of the transition state for C−C bond cleavage. That would make the proposed mechanism via hydration to produce bicarbonate and a carbanion unattainable. However, reports of related exchange rates66 indicate that the energies of substituted carboxyl hydrates are likely to be below that of transition states for C−C bond cleavage. Thus, since no alternative mechanism that is consistent with the reported results has been proposed, the current schemes remain valid. We look forward to ongoing work in the area that will address these fundamental challenges.
14. APPLICATION OF THEORY The decarboxylation of trichloroacetate produces chloroform and bicarbonate in basic solutions, an observation reported by Dumas in 1836 and repeated in various forms since that time.62 The review by Urbansky summarizes evidence related to base catalysis of the reaction.63 The presence of base catalysis is indicative of an associative mechanism. Major and Gao’s calculation on the decarboxylation of N-methylpicolinate assumed a dissociative mechanism, resulting in a conundrum due to the lack of a barrier to the reversion process.25 Our own experience with the decarboxylation reaction is from an efficient synthesis of chloroform-d that includes the potential for base catalysis in the decarboxylation of trichloroacetate (in alkaline deuterium oxide).64 Howe’s calculations show that the dissociative decarboxylation mechanism for trichloroacetate is subject to the problem from reversion that we have seen in more complex molecules.65 However, addition of hydroxide and loss of bicarbonate should avoid reversion (Scheme 10). The reaction profile shows a distinct lowering of energy as the C−C bond cleaves, consistent with the observations of Dumas and his successors.65
16. CONCLUDING REMARKS Determining rates and conditions that optimize the rates of decarboxylation reactions informs us of the alternatives to forming CO2 directly in the presence of a carbanion. Reversibility can prevent proceeding via the expected uncatalyzed dissociative mechanism. Where enzymic reactions are subject to in-depth analysis, the chances of finding an alternative mechanism increases. The potential parallel of hydrolytic enzyme reactions and decarboxylation provides for origins in common ancestral enzymes.42 The routes that avoid direct formation of CO2 or that deactivate carbanions on an enzyme overcome reversion and can lead to substantial proficiency.
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Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. E
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(11) Polovnikova, E. S.; McLeish, M. J.; Sergienko, E. A.; Burgner, J. T.; Anderson, N. L.; Bera, A. K.; Jordan, F.; Kenyon, G. L.; Hasson, M. S. Structural and kinetic analysis of catalysis by a thiamin diphosphatedependent enzyme, benzoylformate decarboxylase. Biochemistry 2003, 42, 1820−1830. (12) Hasson, M. S.; Muscate, A.; McLeish, M. J.; Polovnikova, L. S.; Gerlt, J. A.; Kenyon, G. L.; Petsko, G. A.; Ringe, D. The crystal structure of benzoylformate decarboxylase at 1.6 angstrom resolution: Diversity of catalytic residues in thiamin diphosphate-dependent enzymes. Biochemistry 1998, 37, 9918−9930. (13) Weiss, P. M.; Garcia, G. A.; Kenyon, G. L.; Cleland, W. W.; Cook, P. F. Kinetics and mechanism of benzoylformate decarboxylase using carbon-13 and solvent deuterium isotope effects on benzoylformate and benzoylformate analogs. Biochemistry 1988, 27, 2197−2205. (14) Kluger, R.; Lam, J. F.; Kim, C.-S. Decomposition of 2-(1Hydroxybenzyl)thiamin in Neutral Aqueous Solutions: Benzaldehyde and Thiamin Are Not the Products. Bioorg. Chem. 1993, 21, 275−283. (15) Kluger, R.; Lam, J. F.; Pezacki, J. P.; Yang, C.-M. Diverting Thiamin from Catalysis to Destruction. Mechanism of Fragmentation of N(1′)-Methyl-2-(1-hydroxybenzyl)thiamin. J. Am. Chem. Soc. 1995, 117, 11383−11389. (16) Oka, Y.; Kishimoto, S.; Hirano, H. Vitamin B1 and related compounds. CIX. A novel cleavage of thiamin and its homologs by the reaction with aromatic aldehydes. Chem. Pharm. Bull. 1970, 18, 527− 533. (17) Kluger, R.; Moore, I. F. Destruction of Vitamin B1 by Benzaldehyde. Reactivity of Intermediates in the Fragmentation of N1′-Benzyl-2-(1-hydroxybenzyl)thiamin. J. Am. Chem. Soc. 2000, 122, 6145−6150. (18) Moore, I. F.; Kluger, R. Decomposition of 2-(1Hydroxybenzyl)thiamin. Ruling Out Stepwise Cationic Fragmentation. Org. Lett. 2000, 2, 2035−2036. (19) Moore, I. F.; Kluger, R. Substituent effects in carbon-nitrogen cleavage of thiamin derivatives. Fragmentation pathways and enzymic avoidance of cofactor destruction. J. Am. Chem. Soc. 2002, 124, 1669− 1673. (20) O'Brien, T. A.; Kluger, R.; Pike, D. C.; Gennis, R. B. Phosphonate Analogs of Pyruvate - Probes of Substrate Binding to Pyruvate Oxidase and Other Thiamin Pyrophosphate-Dependent Decarboxylases. Biochim. Biophys. Acta 1980, 613, 10−17. (21) Hu, Q.; Kluger, R. Reactivity of intermediates in benzoylformate decarboxylase: avoiding the path to destruction. J. Am. Chem. Soc. 2002, 124, 14858−14859. (22) Hu, Q.; Kluger, R. Fragmentation of the conjugate base of 2-(1hydroxybenzyl)thiamin: does benzoylformate decarboxylase prevent orbital overlap to avoid it? J. Am. Chem. Soc. 2004, 126, 68−69. (23) Hu, Q.; Kluger, R. Making thiamin work faster: acid-promoted separation of carbon dioxide. J. Am. Chem. Soc. 2005, 127, 12242− 12243. (24) Kresge, A. J. What makes proton transfer fast. Acc. Chem. Res. 1975, 8, 354−360. (25) Major, D. T.; Gao, J. L. An integrated path integral and freeenergy perturbation-umbrella sampling method for computing kinetic isotope effects of chemical reactions in solution and in enzymes. J. Chem. Theory Comput. 2007, 3, 949−960. (26) Cram, D. J. Fundamentals of Carbanion Chemistry; Academic Press: New York−London, 1965. (27) Northrop, D. B. Steady-State Analysis of Kinetic Isotope-Effects in Enzymic Reactions. Biochemistry 1975, 14, 2644−2651. (28) Cox, M. M.; Jencks, W. P. General Acid Catalysis of Aminolysis of Phenyl Acetate by a Preassociation Mechanism. J. Am. Chem. Soc. 1978, 100, 5956−5957. (29) Cox, M. M.; Jencks, W. P. Catalysis of the Methoxyaminolysis of Phenyl Acetate by a Preassociation Mechanism with a Solvent Isotope Effect Maximum. J. Am. Chem. Soc. 1981, 103, 572−580. (30) Acharya, P.; Plashkevych, O.; Morita, C.; Yamada, S.; Chattopadhyaya, J. A repertoire of pyridinium-phenyl-methyl cross-
Ronald Kluger is a chemistry professor at the University of Toronto. He was an undergraduate student at Columbia University where he worked with Gilbert Stork (organic synthesis) and Nick Turro (mass spectrometry). His graduate work in chemistry at Harvard University was with Elkan Blout (peptide structure) and Frank Westheimer (phosphate reactivity). He was a postdoctoral researcher with Bob Abeles at Brandeis University, who taught him to think about mechanistically interesting enzymes. His first faculty position was in the chemistry department at the University of Chicago where he was a Sloan Fellow, leaving after only a few years. His research came to fruition in Toronto where he enjoys interactions with excellent coworkers and stimulating colleagues. He is a Fellow of the Royal Society of Canada and a Fellow of the AAAS. He has received awards for his work in both organic chemistry and biological chemistry, as well as the Chemical Institute of Canada Medal.
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ACKNOWLEDGMENTS I am grateful for insights from discussions with Graeme Howe, Jik Chin, Peter Guthrie, Jiali Gao, Richard Schowen, and Kai Tittmann, as well as for the extraordinary efforts of the members of my research group over the years. I thank NSERC Canada for continuing support in the area of this Account through the Discovery Grant program.
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REFERENCES
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DOI: 10.1021/acs.accounts.5b00306 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.accounts.5b00306 Acc. Chem. Res. XXXX, XXX, XXX−XXX
