Hydrogen bonding isotope fractionation factors

It has become clear in the past decade that strong hydrogen bonding has associated with it several characteristic properties. In particular, as hydrogen-bond strength changes, maxima or minima are observed in nmr chemical shifts, the isotope effect on the chemical shift A[5( H) — 8( H)] defined on p. 271, ir Vah/vad band ratios, and in the isotope-fractionation factor, p. 

A large number of different techniques have been used to measure isotopic fractionation factors, and any method that can be used for measuring the equilibrium position of reaction (3) is appropriate. The discussion here will be limited to the more recent methods and those that have been used for studying hydrogen-bonded species. The discussion is further limited to fractionation factors for species in solution. Recent measurements (Larson and McMahon, 1986, 1987, 1988) of

hydrogen-bonded species in the... 

Fig. 10 Possible variation of isotopic fractionation factors with bond strength for hydrogen-bonded species. A,AH".

In low dielectric organic solvents and enzyme active sites a number of hydrogen bonds between groups with similar pKa exhibit highly deshielded 1H NMR peaks (>16 ppm), low isotopic fraction factors and relatively short H-bonds (data on neutron and x-ray diffraction analysis (Gerlt and Gassman, 1992 Zundel, 2000 Cleland and Northrop, 1999). 

Isotopic fractionation factors of hydrogen-bonded protons... 

One way that is used to characterize these hydrogen bonds is to measure a fractionation factor. Given the above analysis, a low isotope fractionation factor is predicted, because the force constant is smaller for the LBHB (look back at the potential wells for LBHBs in Chapters). For example, the observation that the fractionation factors for various substituted phthalate monoanions were between... 

Figures 7-9 show the fractional conversion of methanol in the pulse as a function of temperature for the three catalysts and the three methanol feeds. Evidently the kinetic isotope effect is present on all three catalysts and over the complete temperature range, indicating that the rate limiting step is the breaking of a carbon-hydrogen bond under all conditions. From these experiments, the effect cannot be determined quantitatively as in the case of the continuous flow experiments, but to obtain the same conversion of CD,0D, the temperature needs to be 50-60° higher. This corresponds to a factor of about three in reaction rate. The difference in activity between PfoCL and Fe.(MoO.), is larger in the pulse experiments compared to tHe steady stateJ results.

The value of the fractionation factor for any site will be determined by the shape of the potential well. If it is assumed that the potential well for the hydrogen-bonded proton in (2) is broader, with a lower force constant, than that for the proton in the monocarboxylic acid (Fig. 8), the value of the fractionation factor will be lower for the hydrogen-bonded proton than for the proton in the monocarboxylic acid. It follows that the equilibrium isotope effect on (2) will be less than unity. As a consequence, the isotope-exchange equilibrium will lie towards the left, and the heavier isotope (deuterium in this case) will fractionate into the monocarboxylic acid, where the bond has the larger force constant. 

The equilibrium constant for the isotope-exchange equilibrium can be expressed (6) in terms of the solvent isotope effects on the acid-dissociation constants and of the monocarboxylic acid and dicarboxylic acid monoanion, respectively. It follows that a lower value for the fractionation factor of the hydrogen-bonded proton means that the solvent isotope effect on the acid-dissociation constant will be lower for the dicarboxylic acid monoanion than for the monocarboxylic acid. 

More O Ferrall (personal communication) has recently studied solutions of sodium methoxide in MeOH and MeOD (with a little MeOH) by the NMR method and has derived a fractionation factor for the methoxide ion which must, of course, be due to methanol molecules associated with the solute. This factor is quite far from unity (0-72) and thus emphasizes the importance of lyate-solvent hydrogen bonds, and adds plausibility to the hypothesis of the importance of hydrogen bonding to the aqueous hydroxide ion in connection with hydrogen isotope fractionation in the hydroxide ion. However, it has not yet been shown that the methoxide fractionation is independent of the nature of the cation, and detailed analyses based on this number may be premature. It will be evident that secure knowledge of this parameter and of the fractionation factor of the methanolic hydrogen ion will in due course allow prediction of the ionic product of MeOD relative to that for MeOH and also of ratios for some acids in methanol... 

Acyl-transfer of j8-lactam and acyclic substrates to active site Ser of the f-lactamase of Enterobacter cloacae P99 occurs with a unique mechanism for nucleophilic activation in which the phenolate of Tyr , stabilized in free enzyme by interaction with both the hydroxyl of Ser and by ionic interaction with protonated Ly , acts as general-base [25]. The unusual hydrogen bonding system which exists in free enzyme is thought to manifest as a ground state fractionation factor that is less than unity and account for the inverse solvent isotope effects around 0.7 that have been observed on kc/Km [25-27]. 

Inasmuch as some of the hydrogen atoms are not exchanged readily due to the inertness of their chemical bonds, the isotopic fractionation which involves the easily exchangeable hydrogen atoms in these biological processes must have even larger irichment factors for deuterium and tritium than their measured values would indicate. 

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