Proton inventories

It must be appreciated that the selection of the best model—that is, the best equation having the form of Eq. (6-97)—may be a difficult problem, because the number of parameters is a priori unknown, and different models may yield comparable curve fits. A combination of statistical testing and chemical knowledge must be used, and it may be that the proton inventory technique is most valuable as an independent source capable of strengthening a mechanistic argument built on other grounds. 

At its best, the study of solvent kies by the formalism given can be used to learn about proton content and activation in the transition state. For this reason it is known as the proton inventory technique. The kinetics of decay of the lowest-energy electronic excited state of 7-azaindole illustrates the technique.25 Laser flash photolysis techniques (Section 11.6) were used to evaluate the rate constant for this very fast reaction. From the results it was suggested that, in alcohol, a double-proton tautomerism was mediated by a single molecule of solvent such that only two protons are involved in the transition state. In water, on the other hand, the excited state tautomerism is frustrated such that two water molecules may play separate roles. Diagrams for possible transition states that can be suggested from the data are shown, where of course any of the H s might be D s. 

The proton inventory technique was applied to this system in an attempt to verify further these suggestions. The rate constants for the reactions were evaluated in CH3OH/CH3OD, C2H5OH/C2H5OD, and H2O/D2O mixtures over the full range of deuterium mole fractions. The data for methanol and water are given in Table 9-8. 

Note that prior applications of the proton inventory technique were to reactants in their ground states. The particular example cited, however, refers to an excited state molecule (and indeed was the first of its kind). An implicit assumption made in the... 

Proton inventory technique. 21.9-220 Pseudo-first-order kinetics, 16 Pulse-accelerated-flow method. 255 Pulse radiolysis, 266-268 Pump-probe technique. 266... 

As would be expected, the slopes are almost identical the intercept difference shows that methyl benzoate reacts about 1.5 times as fast as does ethyl benzoate in the standard state, a result easily attributable to the slight increase in steric crowding to the equation (42) hydrolysis in the latter case. The order of the A2 ester hydrolysis reaction in water is thus two, a result quite difficult to obtain in other ways, even in dilute solution, perhaps requiring a proton inventory study of a reaction that is very slow in water. 

As a practical matter it is sometimes impossible to make studies across the entire range of composition (0 < x < 1) because the criterion of 100% enrichment is either too difficult or too expensive to meet. Sometimes, particularly for solvent isotope effects, the linear dependence employed in the derivation of Equation 7.9 is not obeyed, and the deviation from linearity can be employed to elucidate some details of the reaction mechanism given sufficient information on the x dependence of kx, and the absence of trace catalytic impurities. For studies of H20/D20 solvent isotope effects the approach, called proton inventory , has been widely employed. It is discussed in more detail in Section 11.4.3. 

Many rate constants in aqueous solutions are pH or pD sensitive. In particular, enzyme catalyzed reactions often show maxima in plots of pH(pD) vs. rate. The example in Fig. 11.5 is constructed for a reaction with a true isotope effect, kH/kD = 2, and with maxima in the pH(pD)/rate dependences as shown by the bell shaped curves. These behaviors are typical for enzyme catalyzed reactions. When the isotope effect is obtained (incorrectly) by comparing rates at equal pH and pD, the values plotted along the steep dashed curve result. If, however, the rate constants at corresponding pH and pD (pD = pH + 0.5) are employed, a constant and correct value is obtained, kH/kD = 2. Thus for accurate measurements of the isotope effects one must control pH and pD at appropriate values (pD = pH + 0.5 in our example) using a series of buffers. In proton inventory experiments (see below) buffers should be employed to insure equivalent acidities across the entire range of solvent isotope concentration (0 < xD < 1), xD is the atom fraction of deuterium [D]/([H] + [D]). 

Fig. 11.7 Proton inventory curves (plots of k(n)/k(H) vs. n (or x) = atom fraction D) for overall isotope effects of 2 (upper plot) and 10 (lower plot). In each, the three curves (reading from top to bottom) are for single site, two site, and multi-site isotope effects. Error bars of 3% middle curves) are shown in each case. For ko/ki = 2 the technique is unable to distinguish between the curves at this level of precision (3%), but is more than adequate for ko/ki = 10 (Schowen, R. L., J. Label Compd Radiopharm. 50, 1052 (2007), with permission Wiley Interscience)...

This technique is called the proton inventory method and has been employed with success in studies of enzyme reactions. Numerous examples are cited by Quinn (reading list). 

Fig. 11.9 Proton inventory on SrtA catalyzed reaction. The ratio of kcAT = KM (Section 11.2, Equations 11.9 through 11.13) measured in a D2O/H2O solvent mixture of mole fraction n D20 to that measured in H20(n = 0), is plotted vs. n (Frankel, B. A., et al. Biochemistry 44,11188 (2005))...

Kinetic studies of the reaction of Z-phenyl cyclopropanecarboxylates (1) with X-benzylamines (2) in acetonitrile at 55 °C have been carried out. The reaction proceeds by a stepwise mechanism in which the rate-determining step is the breakdown of the zwitterionic tetrahedral intermediate, T, with a hydrogen-bonded four-centre type transition state (3). The results of studies of the aminolysis reactions of ethyl Z-phenyl carbonates (4) with benzylamines (2) in acetonitrile at 25 °C were consistent with a four- (5) and a six-centred transition state (6) for the uncatalysed and catalysed path, respectively. The neutral hydrolysis of p-nitrophenyl trifluoroacetate in acetonitrile solvent has been studied by varying the molarities of water from 1.0 to 5.0 at 25 °C. The reaction was found to be third order in water. The kinetic solvent isotope effect was (A h2o/ D2o) = 2.90 0.12. Proton inventories at each molarity of water studied were consistent with an eight-membered cyclic transition state (7) model. 

A change in chemical reactivity occurring as a consequence of changes in the isotopic composition of the solvent. See Proton Inventory Kinetic Isotope Effects... 

PROTON INVENTORY TECHNIQUE Dolichyl-phosphate mannosyltransferase,... 

Proton flow to ATP synthesis/hydrolysis, BINDING CHANGE MECHANISM PROTON INVENTORY TECHNIQUE... 

PROTON INVENTORY TECHNIQUE Transition-state mimic,... 

A computationally derived model for catalysis of the aqueous aldol by nomicotine (41) has been tested (by the same authors) via kinetic isotope effects (KIEs) and thermodynamic measurements.112 A proton inventory indicates that the computational results are not conclusive, and a water molecule is involved in or before the ratedetermining step. 

By proton inventory, a technique that determines whether acid and base groups act simultaneously, we found that hydrolysis of 36 by artificial enzyme 44 involves two protons moving in the transition state [130]. Thus, ImH+ of 46 is hydrogen bonded to a phosphate oxyanion of bound substrate 36 water hydrogen bonded to the Im then attacks the phosphorus, and as the O-P bond forms the ImH+ proton transfers (along with the water proton) to produce the phosphorane monoanion 47. This then goes on to the cleaved product in later catalyzed steps before there is time for pseudo-rotation. These general conclusions have been described and summarized in several publications [131-137]. 

In proton inventory technique, solvent isotope effects are plotted against atomic fractions of deuterium in mixed isotopes of water. A linear plot represents a contribution from a single origin, whereas nonlinear plots may be generated from multiple origins. 

Although at pH 8 the electron distribution favours the formation of flavin semiquinone and reduced iron-sulfur center, the magnetic moments of the two redox centers do not interact. At pH 10, however, 2-electron-reduced TMADH exhibits the EPR spectrum diagnostic of the spin-mteracting state. In a more detailed analysis using the pH-jump technique, the interconversion of three states of TMADH [state 1, dihy-droflavin-oxidised 4Fe-4S center (formed at pH 6) state 2, flavin semi-quinone-reduced 4Fe-4S center (formed at pH 8) state 3, spin interacting state (formed at pH 10)] were studied in both H2O and D2O (Rohlfs et al., 1995). The kinetics were found to be consistent with a reaction mechanism that involves sequential protonation/deprotonation and electron transfer events (Figure 6). Normal solvent kinetic isotope effects were observed and proton inventory analysis revealed that at least one proton is involved in the reaction between pH 6 and 8 and at least two protons are involved between pH 8 and 10. At least three protonation/... 

Q and that the proton inventory for each reaction is linear. Linear plots generally indicate that a single proton is delivered in a single step during the reaction. Thus, the most direct interpretation of these observations is that the two protons required for the overall stoichiometry are delivered during the P to P and P to Q steps of the reaction cycle. The decay of P shows the same pH dependence as the formation of Q as expected if these are successive steps. In contrast, the decay of Q and the steps leading to P do not exhibit a pH dependence, suggesting that proton is only transferred in the two steps that have been identified. 

Anslyn E, Breslow R. Proton inventory of a bifunctional ribonuclease model. J. Am. Chem. Soc. 1989 111 8931-8932. 

Propagation of errors, 40, 48, 248 Propinquity effect, 263, 365 Protol5Tsis, 147, 148 Proton inventory technique, 302 Proton transfer, 166 direct, 148 extent of, 346 fast, 97, 146, 173 isotope effect in, 296 partial, 395 Proximity effect, 365 Pseudo-first-order rate constant, 23 Pseudo-first-order reaction, 61 Pseudo-order rate constant, 23 Pseudo-order reaction, 23 Pseudo-order technique, 26, 78 Pulse NMR, 170... 

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