H-Bonding in Alcohols Is Reflected in the Cα−H Bond Strength:  Variation of C−D Vibrational Frequency and Fractionation Factor

Abstract

The variation of the C-D vibrational stretching frequency in primary and secondary alcohols containing the D-C-O-H functionality has been examined for cases in which the alcohol functions as a proton donor in an H-bond. The C-D stretching frequency is a function of the H-bond enthalpy of formation determined by Hartree-Fock calculations, decreasing by approximately 5 cm -1 per kcal/mol. This decrease in frequency is attributed to the increase in the overlap of the O-H bonding electrons with the C-D antibonding orbital as the H-bond is strengthened. The Raman spectra of [1-D]trifluoroethanol and [1-D]trifluoroethoxide in aqueous solution serve as an example; the alcohol has two separate C-D stretches that differ by 45 cm -1 and deprotonation results in an average 78 cm -1 decrease in the C-D stretching frequency. A measured deuterium equilibrium isotope effect on the acid ionization constant of [1-D 2 ]trifluoroethanol of 1.13 is consistent with a decreased fractionation factor of the C-1 protons due to the decrease in the C-D stretching frequency.A model nucleoside complexed with the H-bonding residues at the active site of nucleoside hydrolase indicates that H-bond formation can explain the anomalous secondary isotope effects reported for the hydrolysis of [5′-3 H]inosine (Horenstein et al. Biochemistry 1991, 30, 10788-10795). The correlations of both the C-D stretching frequency and the fractionation factor with the conformation and H-bond strength with primary and secondary alcohols as donors should serve as tools for the characterization of these important interactions in biological systems.

Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

“…kX1000 s À1 , so that the conformational/protonation state isotopic equilibrium is maintained during the time course of the enzymatic reaction. These equilibria can be associated with 2 H equilibrium isotope effects as large as 1.15-1.20 (Williams, 1983;Anet and Kopelevich, 1986;Gawlita et al, 2000;Lewis and Schramm, 2001 are well known especially with regards to sugar metabolism (Schray and Benkovic, 1978). Lewis and Schramm have provided a comprehensive review of such influences on the observed V/K isotope effect (Lewis and Schramm, 2006).…”

“…The initial binding step requires the desolvation of the alcohol. Both theoretical and experimental investigations indicate that the loss of water as an H-bond donor to the oxygen results in a significant decrease in the stretching frequency of the adjacent C-H bond (Gawlita et al, 2000;Maiti et al, 2003). These observations predict a depletion of D in the desolvated initial enzyme-substrate complex (a normal binding effect).…”

“…Unfortunately, the widely adapted formulation for the interpretation of kinetic isotope effects on complex enzymatic reaction mechanisms presupposes that only one step, usually the step involving changes in covalent bonding, be isotopically sensitive. Complications, including two bond cleavage steps have been considered (Blanchard and Cleland, 1980); however, analysis of the potential for isotope effects on conformational changes (Cook, et al, 1981;Schramm, 2001, 2003) and hydrogen-bond formation (Gawlita, et al, 2000;Lewis and Schramm, 2003) lead to the necessary conclusion that isotope effects can exist on numerous steps in a serial enzyme reaction, and in principle will occur on every step. In this paper, we derive a new and more intuitive way of writing the expression for V/K isotope effects for un-branched mechanisms when all steps can be considered isotopically sensitive.…”

β-Secondary deuterium equilibrium and kinetic isotope effects on nucleophilic attack by methanol at a carbonyl: Computational estimation of deviation from the rule of the geometric mean

“…The isotope effects in this figure are based on gas phase calculations and can therefore be regarded as slightly more inverse than might be expected for a solvated hydroxymethylene reactant undergoing nucleophilic attack in an enzyme active site. This is because the solvated alcohol will function as a hydrogen-bond acceptor with water, which will reduce the electron density at the hydroxyl thereby stiffening the C-H bonds of the methyl group [23,54]. Desolvation will therefore lead to a loosening of the bonds and a normal isotope effect.…”

“…The stiffness of these bonds is sensitive to both the orientation and H-bonding status of the adjacent hydroxyl. In aqueous solution complete ionization leads to secondary deuterium isotope effects on the order of 6% per C-H bond [23]. Likewise, desolvation and H-bonding in the active site of thymidine phosphorylase has been shown to produce a tritium binding equilibrium isotope effect on the order of 6% per C-H bond [24,25].…”