Branda˜o et al.
IMPP+, IMPP(, and IMPP-, as well as the relative energies,
∆E (Eb - Ea), between them.
Experimental Section
Materials. Inorganic salts were of analytical grade and were used
without further purification. Liquid reagents were purified by
distillation. 2-(2′-Hydroxyphenyl)imidazole was prepared by the
method of Rogers and Bruice.33
Hydrogen-bonding between the NH and O4 appears to
generate conformers that are more stable than those generated
by hydrogen-bonding between NH and O1. The P1-O1 bond
length, in conformers b, decreases, following the order IMPP-
> IMPP( > IMPP+, and is likely the result of the favored
hydrogen-bond between NH and O1. The degree of ionization
of the phosphate group also determines the degree of the proton-
transfer and lengthening of the P1-O1 bond. An increase of
0.05 Å, from conformer a to b in IMPP-, results from a
hydrogen-bond that is 0.456 Å shorter in conformer b. The
influence of electronic effects of substituents on the imidazolium
group is very high. The imidazolium group exhibits a Hammett
F of ca. 11,31 and with this magnitude of the Hammett similarity
constant, simple protonation of the phosphate group favors
proton-transfer.
Because kinetic effects are strongly dependent on the con-
formational equilibrium between a and b, it is important to note
that in IMPP- the less reactive conformer a is 9.3 kcal‚mol-1
more stable, which slows the reaction. Conversely, in IMPP+
the difference is so small (1.2 kcal‚mol-1) that it is in the
range of energy contribution from thermal energy. Thus, the
freedom of both imidazole and phosphate groups and the
magnitude of ∆E between conformers a (less reactive) and b
(most reactive conformer) are determinant factors for proton-
transfer from the general acid. The rate enhancement and
differences in energy between the conformers follow the order
IMPP+ > IMPP( > IMPP-. As generally observed for organic
compounds where rotational freedom is present, the small energy
differences between a variety of conformers result in a flat
potential energy surface (PES).32 As a consequence of the very
nature of the energy surface, some of the structures obtained in
Figure 5 are not true energy minima on the PES, which shows
a small negative eigenvalue on the Hessian matrices. However,
because of the large energy differences, the qualitative trends
obtained by these results are not affected, and the reported
calculations for isolated molecules are consistent with the
involvement of an intramolecular proton-transfer in the course
of the reaction. Work is in progress to quantitatively describe,
by using a combined quantum mechanics/molecular mechanics
(QM/MM) technique, proton-transfer reactions in enzyme
models.
Synthesis of IMPP. A solution of PCl5 (650 mg, 3.12 mmol) in
CHCl3 (15 mL) was added dropwise to a CHCl3 solution of 2-(2′-
hydroxyphenyl)imidazole (500 mg, 3.12 mmol in 15 mL) in an
ice-water bath. The mixture was stirred at room temperature for
60 min. Water was then added (0.25 mL), and the mixture was left
to react overnight. The solvent was removed under reduced pressure,
and acetone (20 mL) and water (5 mL) were added to the resulting
crude oil. White, fine crystals were immediately obtained and were
collected by filtration. Purification was carried out by dissolving
the crude product in water (30 mL), at pH 7.8 (NaOH), and
unreacted phenol was extracted with CHCl3 (3 × 10 mL). The
resulting water layer was concentrated in vacuo (<40 °C) to 5 mL.
The pH was adjusted to 4.5 with HCl, and methanol (3 mL) was
added. White crystals of NaCl were filtered off. Colorless lapidated,
diamond-like crystals of the product were obtained by slow
evaporation of the solvent at room temperature. Alternatively,
methanol was added directly to the concentrated solution at pH
7.8 to give the sodium salt of IMPP. mp. 222-224 °C (dec)
zwitterionic form. NMR measurements were carried out at pD )
6.25 (monoanionic form): 31P NMR (81 MHz, D2O, external
reference H3PO4 85%) δ -1.10 ppm, s; 13C NMR (100 MHz, D2O,
internal reference TSP) δ 117.9, 122.0, 125.8, 126.5, 131.4, 136.2,
144.5, 154.0; 1H NMR (400 MHz, D2O, internal reference TSP) δ
7.26 (ddd, 1H, J ) 7.5, 7.5, and 3.4 Hz), 7.46 (d, 2H), 7.53 (dd,
1H, J ) 8.0 and 3.5 Hz), 7.57 (m, 1H), 7.70 (dd, 1H, J ) 7.8 and
2.8 Hz).
pKa Determination. Potentiometry. The pKa of IMPP was
determined with a digital pH meter and a combined glass electrode.
Titrations were performed in a 150 mL thermostated cell, under
N2 at 25.0 °C, an ionic strength of 0.1 M KCl, and the initial [IMPP]
was 1.0 mM. The solution was titrated with small increments of
0.1008 M KOH, which was CO2 free. All precautions were taken
to eliminate carbonate and CO2 during the titration. The value of
pKw was taken as -13.78. The program BEST734 was used to
calculate the dissociation constants.
Spectrophotometric pH Titration. Absorbance measurements
were made over a pH range with 66.7 µM of IMPP on a diode-
array spectrophotometer with a thermostated cell holder at 25.0
°C. Solutions were buffered with (0.01 M of each) HCOOH (pH
3-4.5), CH3COOH (pH 4-5.5), NaH2PO4 (pH 5.5-7.8), and
H3BO3 (pH 7.8-9.0).
31P NMR Titration was performed at 81 MHz and 25.0 °C.
Chemical shifts were measured in D2O with H3PO4 85% as an
external reference. The IMPP solution (10 mg/mL) was first
adjusted to pH 9.3 with NaOD and then titrated by return with
DCl until pH 4.6 was reached, where IMPP starts to precipitate.
The value of pD was corrected with pD ) pHread + 0.4.35
Kinetics. Hydrolyses of IMPP were followed spectrophotometri-
cally by monitoring the appearance of 2-(2′-hydroxyphenyl)-
imidazole at 310 nm. The temperatures of reaction solutions in
quartz cuvettes were controlled with a thermostated water-jacketed
cell holder. Ionic strengths were 1.0 M with KCl. The reaction was
initiated by the injection of 20 µL of 10 mM stock solutions of
IMPP in water (at pH ∼ 10 and stored in a refrigerator to minimize
hydrolysis) into 3 mL of aqueous solutions to give 66.7 µM IMPP.
Absorbance versus time data were stored directly on a microcom-
Conclusions
The data on IMPP hydrolysis indicate that in water there is
no significant acceleration by the imidazolium moiety whose
effect disappears in going from IMPP+ to IMPP- and IMPP2-
,
where hydrolyses are very slow. This conclusion indicates that
the rate enhancement depends on intramolecular hydrogen-
bonding with the O1 rather than with the phosphoryl oxygen
O4. The near-planar conformation of the imidazolium and
phenyl groups in the activated complex, as in the naphthyl
derivative, 1, is particularly important in considering models
for enzymatic reactions and should be relevant in the design of
new models. The presence of a relatively free NH in the
imidazolium (N2 atom), which can hydrogen-bond to water,
probably decreases the intramolecular catalytic efficiency rela-
tive to that in 1.
(33) Rogers, G. A.; Bruice, T. C. J. Am. Chem. Soc. 1974, 96, 2463-
2472.
(34) Martell, A. E.; Smith, Z. M.; Motekaitis, R. J. NIST Critical Stability
Constants of Metal Complexes Database: NIST Standard Reference
Database 46; NIST: Gaithersburg, MD, 1993.
(31) Charton, M. J. Org. Chem. 1965, 30, 3346-3350.
(32) Floria´n, J.; Warshel, A. J. Phys. Chem. B 1998, 102, 719.
(35) Schowen, K. B. J. In Transition States of Biochemical Processes;
Gandour, R. D., Ed.; Plenum: New York, 1978; pp 225-284.
3806 J. Org. Chem., Vol. 72, No. 10, 2007