Secondary Deuterium Isotope Effects Research Articles

Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers

Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.

Comment on “The role of electrostatic induction in secondary isotope effects on acidity” by E. A. Halevi, New J. Chem., 2014, 38, 3840

We reject Halevi's interpretation and reaffirm our conclusion that secondary deuterium isotope effects on acidity are due to n–σ* delocalization.

Half a century of research, teaching, service

This is a summary of the research of Charles L. Perrin and his coworkers in the areas of aromatic mercuration, vibronic borrowing, ipso factors in electrophilic aromatic substitution, malonic anhydrides, mechanisms of proton exchange in amides, stereoelectronic control, the so‐called reverse anomeric effect, an NMR titration method for highly accurate measurement of relative acidities, secondary deuterium isotope effects on acidity, symmetry of hydrogen bonds in solution, and nucleophilic addition to a para‐benzyne diradical, along with Perrin's contributions to teaching and to service. Copyright © 2014 John Wiley & Sons, Ltd.

Cα–H Carries Information of a Hydrogen Bond Involving the Geminal Hydroxyl Group: A Case Study with a Hydrogen-Bonded Complex of 1,1,1,3,3,3-Hexafluoro-2-propanol and Tertiary Amines

Experimental measurement of the contribution of H-bonding to intermolecular and intramolecular interactions that provide specificity to biological complex formation is an important aspect of macromolecular chemistry and structural biology. However, there are very few viable methods available to determine the energetic contribution of an individual hydrogen bond to binding and catalysis in biological systems. Therefore, the methods that use secondary deuterium isotope effects analyzed by NMR or equilibrium or kinetic isotope effect measurements are attractive ways to gain information on the H-bonding properties of an alcohol system, particularly in a biological environment. Here, we explore the anharmonic contribution to the C-H group when the O-H group of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) forms an intermolecular H-bond with the amines by quantum mechanical calculations and by experimentally measuring the H/D effect by NMR. Within the framework of density functional theory, ab initio calculations were carried out for HFP in its two different conformational states and their H-bonded complexes with tertiary amines to determine the (13)C chemical shielding, change in their vibrational equilibrium distances, and the deuterium isotope effect on (13)C2 (secondary carbon) of HFP upon formation of complexes with tertiary amines. When C2-OH was involved in hydrogen bond formation (O-H as hydrogen donor), it weakened the geminal C2-H bond; it was reflected in the NMR chemical shift, coupling constant, and the equilibrium distances of the C-H bond. The first derivative of nuclear shielding at C2 in HFP was -48.94 and -50.73 ppm Å(-1) for anti and gauche conformations, respectively. In the complex, the values were -50.28 and -50.76 ppm Å(-1), respectively. The C-H stretching frequency was lower than the free monomer, indicating enhanced anharmonicity in the C-H bond in the complex form. In chloroform, HFP formed a complex with the amine; δC2 was 69.107 ppm for HFP-triethylamine and 68.766 ppm for HFP-d2-triethylamine and the difference in chemical shift, the ΔδC2 was 341 ppb. The enhanced anharmonicity in the hydrogen-bonded complex resulted in a larger vibrational equilibrium distance in C-H/D bonds. An analysis with the Morse potential function indicated that the enhanced anharmonicity encountered in the bond was the origin of a larger isotope effect and the equilibrium distances. Change in vibrational equilibrium distance and the deuterium isotope effect, as observed in the complex, could be used as parameters in monitoring the strength of the H-bond in small model systems with promising application in biomacromolecules.

Secondary Isotope Effects on 13C and 15N Chemical Shifts of Schiff Bases Revisited

Abstract An analysis is presented of secondary deuterium isotope effects on 15N and 13C chemical shifts of the methylamine Schiff base of 4,6-dimethoxysalicylaldehyde. It is shown that for this compound existing as an equilibrium between an OH and a NH-form, the secondary isotope effects depend on the OH/NH ratio giving a function with a maximum and a minimum. Secondary deuterium isotope effects on 13C and 15N chemical shifts are compared in a broader range of compounds and the above trend is confirmed. Changes in the dielectric constants of the medium with temperature are found to be rather unimportant for the change in equilibrium. Equilibrium isotope effects on chemical shifts are shown to be important. The magnitudes of the isotope effects are correlated both to intrinsic and equilibrium isotope effects. The latter correlate to the shape of the two-potential well, the energy difference and the difference in chemical shifts of the two tautomers.

No Contribution of an Inductive Effect to Secondary Deuterium Isotope Effects on Acidity

Effect and cause: Secondary deuterium isotope effects on the acidity of the deuterated compounds 1–4 were measured by using an NMR titration method applicable to a mixture and capable of very high accuracy. Variable-temperature experiments show that these isotope effects are due only to changes in vibrational frequencies. These findings refute an inductive origin for these isotope effects.

No Contribution of an Inductive Effect to Secondary Deuterium Isotope Effects on Acidity

Wirkung und Ursache: Sekundäre Deuteriumisotopeneffekte auf die Acidität der deuterierten Verbindungen 1–4 wurden mithilfe einer NMR-Titrationsmethode gemessen, die auf Mischungen anwendbar und durch sehr hohe Genauigkeit gekennzeichnet ist. Temperaturabhängige Messungen ergaben als alleinige Ursache der Isotopeneffekte Änderungen der Schwingungsfrequenzen, was eine induktive Ursache ausschließt.

Investigating Inner-Sphere Reorganization via Secondary Kinetic Isotope Effects in the C−H Cleavage Reaction Catalyzed by Soybean Lipoxygenase: Tunneling in the Substrate Backbone as Well as the Transferred Hydrogen

This work describes the application of NMR to the measurement of secondary deuterium (2° (2)H) and carbon-13 ((13)C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° (2)H and (13)C KIEs on k(cat)/K(m) directly for 11,11-d2-LA, whereas the values for the 2° (2)H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° (2)H and (13)C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° (2)H KIEs.

Secondary deuterium isotope effects on the acidity of glycine

Secondary deuterium isotope effects (IE) on the acidity (p K a ) of glycine were measured by 13C NMR titration. It was found that deuteration decreases the p K a by 0.034 ± 0.002. The experimental data are supported by theoretical calculations, which, in turn, allowed to relate the acidity decrease to the lowering of glycine vibrational frequencies upon deuteration.

Position-Specific Secondary Deuterium Isotope Effects on Basicity of Pyridine

Secondary isotope effects (IEs) on basicities of various deuterated pyridine isotopologues and of 2,6-lutidine-2,6-(CD(3))(2) have been accurately measured in aqueous solution by an NMR titration method applicable to a mixture. Deuteration at any position of pyridine increases the basicity, but the IE per deuterium is largest for substitution at the 3-position and smallest for the 2-position, which is closest to the site of N-protonation, smaller even than that for 2-CD(3) substitution. Computations at the B3LYP/cc-pVTZ level overestimate the magnitude of the measured IEs but largely reproduce the variability with isotopic position. Because the calculated IEs are based on changes in vibrational frequencies on N-protonation, the correspondence between calculated and experimental IEs implies that they arise from zero-point energies, rather than from inductive effects.

Deuterium isotope effects on 13C and 15N chemical shifts of intramolecularly hydrogen-bonded enaminocarbonyl derivatives of Meldrum’s and Tetronic acid

Secondary deuterium isotope effects on 13C and 15N nuclear shieldings in a series of cyclic enamino-diesters and enamino-esters and acyclic enaminones and enamino-esters have been examined and analysed using NMR and DFT (B3LYP/6-31G(d,p)) methods. One-dimensional and two-dimensional NMR spectra of enaminocarbonyl and their deuterated analogues were recorded in CDCl 3 and CD 2Cl 2 at variable temperatures and assigned. 1 J NH coupling constants for the derivatives of Meldrum’s and tetronic acids reveal that they exist at the NH-form. It was demonstrated that deuterium isotope effects, for the hydrogen bonded compounds, due to the deuterium substitution at the nitrogen nucleus lead to large one-bond isotope effects at nitrogen, 1Δ 15N(D), and two-bond isotope effects on carbon nuclei, 2ΔC(ND), respectively. A linear correlations exist between 2ΔC(ND) and 1Δ 15N(D) whereas the correlation with δNH is divided into two. A good agreement between the experimentally observed 2ΔC(ND) and calculated dσ 13C/dR NH was obtained. A very good correlation between calculated NH bond lengths and observed NH chemical shifts is found. The observed isotope effects are shown to depend strongly on Resonance Assisted Hydrogen bonding.

Symmetry of hydrogen bonds in solution

Abstract A classic question regarding hydrogen bonds (H-bonds) concerns their symmetry. Is the hydrogen centered or is it closer to one donor and jumping between them? These possibilities correspond to single- and double-well potentials, respectively. The NMR method of isotopic perturbation can answer this question. It is illustrated with 3-hydroxy-2-phenylpropenal and then applied to dicarboxylate monoanions. The 18O-induced 13C NMR splittings signify that their intramolecular H-bonds are asymmetric and that each species is a pair of tautomers, not a single symmetric structure, even though maleate and phthalate are symmetric in crystals. The asymmetry is seen across a wide range of solvents and a wide variety of monoanions, including 2,3-di-tert-butylsuccinate and zwitterionic phthalates. Asymmetry is also seen in monoprotonated 1,8-bis(dimethylamino)naphthalenediamines, N,N'-diaryl-6-aminofulvene-2-aldimines, and 6-hydroxy-2-formylfulvene. The asymmetry is attributed to the disorder of the local environment, establishing an equilibrium between solvatomers. The broader implications of these results regarding the role of solvation in breaking symmetry are discussed. It was prudent to confirm a secondary deuterium isotope effect (IE) on amine basicity by NMR titration of a mixture of PhCH2NH2 and PhCHDNH2. The IE is of stereoelectronic origin. It is proposed that symmetric H-bonds can be observed in crystals but not in solution because a disordered environment induces asymmetry, whereas a crystal can guarantee a symmetric environment. The implications for the controversial role of low-barrier H-bonds in enzyme-catalyzed reactions are discussed.

Hydrogen bonding and solution state structure of salicylaldehyde-4-phenylthiosemicarbazone: A combined experimental and theoretical study

Hydrogen bonding in salicylaldehyde-4-phenylthiosemicarbazone ( 1) has been studied by using experimental (NMR, Raman and UV spectroscopies) and quantum chemical (DFT) methods. It has been demonstrated that 1 adopted the hydroxy-thione tautomeric form in solution as found also in the solid state and previously indicated by secondary deuterium isotope effects. Apart from the intra-molecular hydrogen bonds new interactions between 1 and solvent molecules were formed as well. Changes in NMR chemical shifts and calculations have pointed towards a formation of inter-molecular three-centered hydrogen bonds in each of the studied complexes involving OH and NH groups of 1 and associated solvent molecules. Stabilization energies of intra-molecular hydrogen bonds were found to decrease with the increase of the solvent polarity. Two-dimensional NOESY spectra indicated conformational changes in solution with respect to the structure observed in the solid state. These were accounted for by a relatively low barrier of the rotation of the N N single bond thus enabling a molecule to posses a higher conformational flexibility in solution with portions of skewed conformations. The results presented here can help in a better understanding of the role hydrogen bonds can play in bioactivity of related thiosemicarbazone derivatives and their metal complexes.

Nonadditivity of Secondary Deuterium Isotope Effects on Basicity of Trimethylamine

Secondary deuterium isotope effects (IEs) on basicities of isotopologues of trimethylamine have been accurately measured by an NMR titration method applicable to a mixture. Deuteration definitely increases the basicity, by approximately 0.021 in the Delta pK per D. The IE is attributed to the lowering of the CH stretching frequency and zero-point energy by delocalization of the nitrogen lone pair into the C-H antibonding orbital. Because this depends on the dihedral angle between the lone pair and the C-H, a further consequence is a preference for conformations with H antiperiplanar to the lone pair and D gauche. This leads to a predicted nonadditivity of IEs, which is confirmed experimentally. It is found that the decrease in basicity, per deuterium, increases with the number of deuteriums. The nonadditivity of IEs is a violation of the widely assumed Rule of the Geometric Mean.

Combined Experimental and Theoretical Mechanistic Investigation of the Barbier Allylation in Aqueous Media

The Barbier allylation of a series of para-substituted benzaldehydes with allylbromide in the presence of Zn, In, Sn, Sb, Bi, and Mg was investigated using competition experiments. In all cases, the slope of the Hammett plots indicated a build-up of negative charge in the selectivity-determining step. For Zn, In, Sn, Sb, and Bi, an inverse secondary kinetic isotope effect was found (kH/kD = 0.75-0.95), which was compatible with the formation of a discrete organometallic species prior to allylation via a closed six-membered transition state. With Mg, a significantly larger build-up of negative charge along with a small positive secondary kinetic isotope effect (kH/kD = 1.06) indicated that the selectivity-determining step was the generation of the radical anion of benzaldehyde. The reaction through a six-membered transition state was modeled using density functional theory with the effect of solvent described by a polarized continuum model. The calculated secondary deuterium isotope effects based on this mechanism were found to be in good agreement with experimental values, thus adding further support to this mechanistic scenario.

Experimental Investigation into the Mechanism of the Epoxidation of Aldehydes with Sulfur Ylides

A mechanistic study of the epoxidation of aldehydes with sulfur ylides has been carried out. The DeltaG++ of the reaction was determined to be 22.2 kcal/mol at 298 K. A 13C kinetic isotope effect was determined to be 1.026 for the carbonyl carbon of benzaldehyde. A secondary deuterium isotope effect was determined to be 0.93 for the aldehydic hydrogen atom of benzaldehyde. Substituent effects on reaction rate were studied, and a Hammett rho of +2.50 was found.

Direct Measurements of Deuterium Kinetic Isotope Effects in Anionic, Gas-Phase Substitution and Elimination Reactions

Reactions with dianion nucleophiles were used to determine deuterium kinetic isotope effects (KIE's) in gas-phase E2 and SN2 reactions. This approach allows for the direct identification of elimination and substitution products in the reactions of simple alkyl halides. In general, the E2 reactions give large KIE's (∼7) for perdeutero substrates; however, elimination isotope effects are attenuated in reactions that occur near the collision-controlled limit. In a very slow elimination, a large KIE (>20) is observed, presumably due to lifetime effects in the collision complex. The SN2 reactions all give secondary deuterium isotope effects near unity, with the exception of one system, where suppression of the E2 channel in the perdeutero substrate leads to an enhancement of the SN2 rate and causes a modest inverse isotope effect.

Secondary Deuterium Isotope Effects on the Acidity of Carboxylic Acids and Phenols

Secondary deuterium isotope effects (IEs) on acidities have been accurately measured by an NMR titration method applicable to a mixture of isotopologues. Deuteration definitely decreases the acidity of carboxylic acids and phenols, by up to 0.031 in the DeltapK per D. For aliphatic acids, the IEs decrease as the site of deuteration becomes more distant from the OH, as expected, but a surprising result is that IEs in both phenol and benzoic acid do not decrease as the site of deuteration moves from ortho to meta to para. The experimental data are supported by ab initio computations, which, however, substantially overestimate the IEs. The discrepancy does not seem to be due to solvation. The IEs originate in isotope-sensitive vibrations whose frequencies and zero-point energies are lowered upon deprotonation. In the simplest case, formate, the key vibration can be recognized as the C-H stretch, which is weakened by delocalization of the oxygen lone pairs. For the aromatic acids, delocalization cannot account for the near constancy of IEs from ortho, meta, and para deuteriums, but the observed IEs are consistent with calculated vibrational frequencies and electron densities. Moreover, the ability of the frequency analysis to account for the IEs is evidence against an inductive origin.

A Quantum Mechanics/Molecular Mechanics Study of the Catalytic Mechanism of the Thymidylate Synthase

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.

Kinetic and Chemical Mechanisms of the fabG-Encoded Streptococcus pneumoniae β-Ketoacyl-ACP Reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.