Phosphate monoester hydrolysis in cyclohexane

Article abstract of DOI:10.1021/ja907967y

(Chemical Equation Presented) The hydrolysis of simple phosphate monoesters is among the most difficult reactions that are subject to catalysis by enzymes, and it has been suggested that extraction of the substrates from solvent water may contribute to th

Full text of DOI:10.1021/ja907967y

Published on Web 12/03/2009
Phosphate Monoester Hydrolysis in Cyclohexane
Randy B. Stockbridge and Richard Wolfenden*
Department of Biochemistry and Biophysics, UniVersity of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27514
Received September 23, 2009; E-mail: water@med.unc.edu
With an estimated half-life of 1011 years at 25 °C, the spontane-
ous hydrolysis of a monoalkyl phosphate dianion may be the most
difficult reaction that is catalyzed by an enzyme.1 At the active
sites of hydrolytic enzymes, this reaction proceeds with a half-life
of 17 ms.1 It has been suggested,2 although not universally agreed
upon,3 that the extraction of substrates from solvent water may be
among the factors that contribute to the rate enhancements produced
by enzymes in general. Abell and Kirby’s demonstration4 that the
hydrolysis of 4-nitrophenyl phosphate proceeds >106-fold more
rapidly in wet DMSO than in water and subsequent observations
by Hengge and his associates5 furnish experimental support for the
possibility that desolvation effects figure prominently in the action
of phosphate monoesterases. Here, we show that the dianion of
neopentyl phosphate (NP2-) enters wet cyclohexane as its tetrabu-
tylammonium (TBA+) salt. In cyclohexane, the second-order rate
constant for phosphomonoester hydrolysis is enhanced by a factor
of 2.5 × 1012 compared with the second-order rate constant for
hydrolysis of NP2- in water.7
Figure 1. Arrhenius plots showing the hydrolysis of NP2- in cyclohexane.
Observed first-order rate constants (k) are not corrected for water concentra-
tion. For comparison, the rates of reaction in water are shown as a broken
line with the temperature range over which data were collected indicated
by the gray box (A). Data obtained by NMR are shown as solid points, and
data collected by the molybdate assay for inorganic phosphate are shown
as open points (B). In A, the vertical axis on the right corresponds to 25
°C.
Distribution coefficients were determined by stirring a small
volume (1 mL) of aqueous NP2- (0.1 M, titrated to pH 11 with
TBA hydroxide) for 6 h with a large volume (100 mL) of
cyclohexane and then back-extracting the organic layer into a small
volume (0.65 mL) of D2O (Supporting Information (SI)) for analysis
by proton NMR. The distribution coefficient observed for NP2-
transfer from water to cyclohexane in the presence of excess TBA+
was 7.0 ((0.4) × 10-6 at 25 °C, and TBA+ was found to be present
in cyclohexane in a molar ratio of 2 parts TBA to 1 part of NP.
This value was unaffected by varying the concentrations of solutes
in the aqueous phase, implying that TBA salts are fully dissociated
in cyclohexane.6 Thermodynamic changes associated with water-
to-cyclohexane transfer were determined from a van’t Hoff plot of
these distribution coefficients as a function of changing temperature
in the range from 10 to 50 °C, which was linear over the range
examined. The distribution coefficient increased with increasing
temperature (SI).
1) from which rate constants at 25 °C and the corresponding
thermodynamics of activation were estimated (Table 1).
In wet cyclohexane, NP2- hydrolysis proceeded with a rate
constant of 3.8 × 10-12 s-1 at 25 °C, exceeding the estimated rate
constant for hydrolysis of the dianion in water (2 × 10-20 s-1)1 by
a factor of 1.9 × 108. Although it is possible that NP2- retains
waters of hydration in cyclohexane,8 experiments involving a
mixture of NP2- in wet cyclohexane with varying amounts of dry
cyclohexane showed that the rate of hydrolysis of NP2- in
cyclohexane varied in proportion to the concentration of water that
was present (Figure 2). This implies that hydrolysis is bimolecular,
as it is for the aqueous reaction (in water, phosphate monoester
hydrolysis proceeds through a concerted, dissociative transition
state).9 With the concentrations of water present in water-saturated
cyclohexane (4.2 × 10-3 M)10 and pure water (55.5 M) corrected
for, the second-order rate constant for water attack on NP2- in
cyclohexane (8.8 × 10-10 s-1 M-1) exceeds the rate constant for
attack in water (3.6 × 10-22 s-1 M-1) by a factor of 2.5 × 1012.
Remarkably, the source of this rate enhancement is entirely entropic
in origin (Table 1).
To determine the rate of monoester hydrolysis in wet cyclohex-
ane, portions (10 mL) of the clear cyclohexane layer, prepared at
25 °C as described above, were incubated in Teflon-lined acid
digestion bombs for various time periods. After heating, samples
in cyclohexane (10 mL) were back-extracted into water (1 mL),
the aqueous phase was evaporated to dryness (any neopentanol was
removed by that process), and the residue was dissolved in D2O
containing dioxan as an integration standard, for analysis by NMR.
The course of hydrolysis was monitored by comparing the integrated
intensity of the peak arising from NP2- in the starting material and
reacted samples. Hydrolysis was also monitored by the release of
inorganic phosphate, measured spectrophotometrically using acid-
molybdate,7 with identical results. Hydrolysis was found to proceed
with first-order kinetics under all conditions examined. Rate
constants obtained for the hydrolysis of NP2- over the temperature
range between 74 and 112 °C yielded linear Arrhenius plots (Figure
In water, the monoanions of alkyl phosphate monoesters are
hydrolyzed ∼1010-fold more rapidly than their dianions.1 That
special reactivity has been attributed to intramolecular hydroxyl
group catalysis of alkoxide elimination through the agency of
intervening water molecules.11 It was therefore of interest to
compare the rates of hydrolysis of NP in its uncharged, monoan-
ionic, and dianionic forms, in wet cyclohexane. An aqueous solution
of the di-TBA salt of NP2- was adjusted with HCl to various pH
values corresponding to different states of ionization of NP (pKa
values 1.8 and 6.8) and extracted with cyclohexane. Proton NMR
showed that the number of equivalents of TBA+ in cyclohexane
corresponded to the number of negative charges on the major
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18248 J. AM. CHEM. SOC. 2009, 131, 18248–18249
10.1021/ja907967y  2009 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Phase Transfer and NP Hydrolysis in the Cyclohexane/
Water System
°C, and that value increases with increasing temperature (see SI
for thermodynamic details).
The present findings imply that the distribution coefficient of
phosphate esters from water to wet cyclohexane increases greatly
as they proceed from the ground state to the transition state in their
hydrolysis. As has been suggested for nucleophilic displacement
reactions in the vapor phase,12 it seems likely that the present
solvent effects arise in part from greater delocalization of charge
in the transition state than in the ground state. Possible changes in
transition state structure, and the extent to which water molecules
remain associated with phosphate esters when they are transferred
from water to cyclohexane, remain to be determined.
Figure 2. Effect of water concentration on rate of NP2- hydrolysis in
cyclohexane at 90 or 100 °C. Water concentration was adjusted by mixing
wet cyclohexane (4.3 × 10-3 M H2O) with dry cyclohexane. Values are
normalized relative to the rate of hydrolysis in water-saturated cyclohexane
at the appropriate temperature.
Table 1. Rate Constants and Thermodynamics of Activation for
Hydrolysis of NP2- in Water1 and Cyclohexane (Thermodynamic
Parameters in kcal/mol)
∆G‡
∆H‡
T∆S‡
k25 (s-1
)
k25 (s-1 M-1
)
H2O
CHX
44.3
32.9 ( 0.53
47.0
47.6
2.7
14.7
2.0 × 10-20
3.6 × 10-22
3.8 × 10-12
8.8 × 10-10
These rate enhancements approach or surpass the rate enhance-
ments produced by many hydrolytic enzymes and accord with the
possibility that desolvation plays a substantial role in the action of
phosphate monoesterases.
ionized form of NP that had been present in the aqueous phase
(Table 2). Distribution coefficients observed for NPH2, NPH-/
TBA+, and NP2-/2TBA+ were found to be similar, and their rate
constants for hydrolysis in wet cyclohexane were distributed over
a surprisingly narrow range (Table 2).
Acknowledgment. We thank N. H. Williams, A. J. Kirby, and
A. C. Hengge for helpful discussions and N. H. Williams for a gift
of neopentyl phosphate used during the initial stages of this work.
We are grateful to the National Institute of General Medical
Sciences (Grant No. GM-18325) for financial support.
Table 2. Rate Constants for Hydrolysis and Coefficients for
Transfer of NP2-, NPH-, and NPH2 from Water to Cyclohexane
Supporting Information Available: Detailed kinetic methods; van’t
Hoff analysis of equilibria for transfer of NP2- and NPH- from water
to cyclohexane; effect of ester concentration on distribution coefficients
observed for transfer of NP2-, NPH-, and NPH2 from water to
cyclohexane; error analysis for distribution coefficients and rate
constants; estimation of ∆H‡ values for phase transfer catalysis. This
material is available free of charge via the Internet at http://pubs.acs.org.
pH (aq.)
TBAc /NPc
species (CHX)
K1
k90 (s-1
)
0
0
NPH2
9.9 × 10-5
5.0 × 10-5
4.7 × 10-6
3.6 × 10-6
7.0 × 10-6
3.6 × 10-6
6.8 × 10-6
1.1 × 10-5
1.8 × 10-5
1.0 × 10-5
1.8
5.5
6.8
12.0
0.09
1.12
1.49
2.0
NPH2 + NPH-
NPH-
NPH- + NP2-
NP2-
In wet cyclohexane, the concentration of water (4.2 × 10-3 M)
is considerably higher than the concentrations of the neopentyl esters
that were present in these experiments (∼10-6 M). Thus, one might
question whether the unexpectedly high apparent reactivity of
monoester dianions in wet cyclohexane arises from a small
population of monoanions that reacts relatively rapidly and that
can be replenished by protons donated by H2O. The similarity of
the observed rate constants for hydrolysis of NPH2, NPH-, and
NP2- in wet cyclohexane seems to render that explanation unlikely.
Transfer to cyclohexane may level the observed rate constants by
removing the intervening water molecules that have been postulated
to participate in intramolecular general acid catalysis,11 tending to
eliminate the special reactivity of monoester monoanions.
For an enzyme acting by desolvation effects, substrate binding
might be compared to solvent transfer in the presence of a phase
transfer catalyst. Scheme 1 shows that for such a catalyst to be
effective, K1 ·K2 ·kCHX must exceed the value of kH2O. In the reactions
described here, the resulting “phase transfer rate enhancement,” as
defined in Scheme 1, is 1.3 × 103-fold for NP2- hydrolysis at 25
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