Thermodynamic origin of the increased rate of hydrolysis of phosphate and phosphorothioate esters in DMSO/water mixtures

Article abstract of DOI:10.1021/jo060896b

The hydrolysis rates of the dianions of phosphate and phosphorothioate esters are substantially accelerated by the addition of polar aprotic solvents such as DMSO and acetonitrile. The activation barrier ΔG? is smaller due to a lower enthalpy of activatio

Full text of DOI:10.1021/jo060896b

Thermodynamic Origin of the Increased Rate of Hydrolysis of
Phosphate and Phosphorothioate Esters in DMSO/Water Mixtures
Kerensa Sorensen-Stowell and Alvan C. Hengge*
Department of Chemistry and Biochemistry, Utah State UniVersity, Logan, Utah 84322-0300
hengge@cc.usu.edu
ReceiVed April 28, 2006
The hydrolysis rates of the dianions of phosphate and phosphorothioate esters are substantially accelerated
by the addition of polar aprotic solvents such as DMSO and acetonitrile. The activation barrier ∆Gq is
smaller due to a lower enthalpy of activation. The enthalpy of transfer of p-nitrophenyl phosphate (pNPP)
and p-nitrophenyl phosphorothioate (pNPPT), from water to 0.6 (mol) aq DMSO (60 mol % water in
DMSO) were measured calorimetrically. The enthalpies of activation for the hydrolysis reactions in the
two solvents permitted the calculation of the enthalpy of transfer of the transition states. This transfer is
thermodynamically favorable for both the reactants and the transition states but is more favorable for the
transition states. In the case of pNPP, the enthalpy of transfer of the reactant is -23.9 kcal/mol, compared
to -28.3 for the transition state. The difference is greater for pNPPT, where the enthalpy of transfer of
the reactant is -23.2 kcal/mol and that for the transition state is -35.3. The results show that the reduced
enthalpies of activation in both hydrolysis reactions arise not from a destabilization of the reactants in
the mixed solvent, but from the fact that the enthalpy of transfer of the transition states to the mixed
solvent is significantly more negative than the enthalpy of transfer of the reactants.
Introduction
p-nitrophenyl phosphate (pNPP) dianion is accelerated by up
to 106 in a solution that is 95% DMSO, 5% water (which will
be designated as 0.95 (v) aq DMSO), and similar rate accelera-
tions occur in other mixed solvents.2 The hydrolysis of the
phosphorothioate analogue p-nitrophenyl phosphorothioate (pN-
PPT) is also accelerated by addition of DMSO, by nearly 7
orders of magnitude in 0.95 (v) aq DMSO.3 In both cases, the
reduction in ∆Gq arises from a sharply reduced enthalpy of
activation in the mixed solvent. In the hydrolysis of the pNPP
Phosphoryl transfer reactions are one of the most common
reactions in biochemistry. The enzymatic hydrolysis of phos-
phate monoesters, most commonly tyrosine, serine, and threo-
nine phosphates, is involved in cell signaling and regulation.
The substrates for most phosphatases are believed to be the
dianion forms, which have very slow rates of uncatalyzed
hydrolysis; the estimated half-life for attack by water on alkyl
phosphate dianions is 1012 years at 25 °C.1
The rate of hydrolysis of phosphomonoester dianions is
strongly affected by the medium. The rate of hydrolysis of the
(2) Abell, K. W. Y.; Kirby, A. J. Tetrahedron Lett. 1986, 27, 1085-
1088.
(1) Lad, C.; Williams, N. H.; Wolfenden, R. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 5607-10.
(3) Catrina, I. E.; Hengge, A. C. J. Am. Chem. Soc. 1999, 121, 2156-
2163.
10.1021/jo060896b CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/23/2006
7180
J. Org. Chem. 2006, 71, 7180-7184

Hydrolysis of Phosphate and Phosphorothioate Esters in DMSO/Water
dianion, ∆Hq ) 30.6 kcal/mol in water, but is reduced to
20.7 ( 0.6 kcal/mol in 0.95 (v) aq DMSO.4 In the hydrolysis
of pNPPT, ∆Hq is reduced from 37.0 ( 1 kcal/mol in water to
22.9 ( 0.7 kcal/mol in 0.95 (v) aq DMSO.3 Similar consider-
ations apply for the pNPPT reaction, although the increases in
pNPPT hydrolysis rates are not as dramatic at the highest DMSO
percentages as for pNPP.3
In the original report2 of the medium effect on pNPP
hydrolysis, it was speculated that the rate acceleration might
be due to the disruption of hydrogen bonding to the phosphoryl
group, resulting in a weakening of the P-O ester bond. This
can be rationalized by a contribution from an extreme resonance
form consisting of the phenolate ion and metaphosphate. The
loss of stabilizing hydrogen bonds between the phosphoryl group
and the solvent might be expected to enhance contribution from
such a resonance form. The reduced enthalpy of activation is
consistent, though not uniquely so, with a weaker scissile bond
in the mixed medium. Similar kinetic isotope effects and
Brønsted âlg values4 indicate that the hydrolysis of pNPP
proceeds through a similar transition state in both solvent
systems; thus, the rate acceleration does not result from a
mechanistic change.
Subsequent experimental results have not supported the notion
that DMSO or other solvents significantly weaken the scissile
P-O ester bond.5,6 Cheng et al.5 used Raman spectroscopy on
pNPP in water/DMSO mixtures and determined that the bridging
P-OR bond lengthens by only ∼0.013 Å in 0.95 (v) aq DMSO.
Further investigations using isotope shifts on 31P NMR in
various solvents6 found no evidence of significant weakening
of the ester bond upon transfer from water to aprotic solvents.
These results suggest that the rate increase arises from some
other factor. One hypothesis is that the DMSO/water solvent
mixture might better solvate the transition state, with its more
dispersed charge, relative to the reactant.5 To test this hypothesis,
the enthalpies of transfer for the transition states for the
hydrolysis of pNPP and of pNPPT from water to a DMSO/
water mixture were obtained, and compared with those of the
reactants. Because the dominant contribution to the reduced free
energy of activation in the mixed solvent is enthalpic, the
enthalpies of transfer should be directly relevant to the source
of the faster rate of reaction in water/DMSO mixtures.
FIGURE 1. Diagram of a hypothetical reaction coordinate showing
the relationship between the thermodynamic parameters in eq 1.
notation in eq 1 follows these precedents.7-12 The relationship
in eq 1 is shown pictorially in Figure 1.
When the reactants are ionic, such as the phosphate ester
dianions examined in this study, it is necessary to obtain the
enthalpy of transfer of the reactant ion free of its counterions.
This is accomplished using an extrathermodynamic assumption
involving ion pairs consisting of two symmetric species, in
which the central charge is shielded from solvent by a sizable
organic fragment.13 The enthalpy of solution (∆Hsol) of such
salts is assumed to consist of equal contributions from each ion.
Tetrabutylammonium tetrabutylborate is one such commonly
used pair, others being tetraphenylarsonium tetraphenylborate,
and similar species. To obtain the enthalpy of solution for an
anion using this method, the ∆Hsol of tetrabutylammonium
tetrabutylborate is measured in the solvent of interest. Half of
this quantity is assigned to the tetrabutylammonium ion. The
∆Hsol of the tetrabutylammonium salt of the anion of interest
is then measured, and the contribution from the cation(s) is
subtracted. Since its introduction by Grunwald,13 values of ∆Hsol
for a number of single ions have been obtained using this
method.7-12,14 The enthalpy of transfer from one solvent to
another is the difference between the enthalpies of solution in
the two solvents of interest.
Results and Discussion
The enthalpy of transfer of the transition state, δ∆Hqtrans, can
be calculated from the enthalpy of transfer of the reactant and
the enthalpy of activation:
Figure 1 shows one example of how a transfer from one
solvent to another (from solvent 1 to solvent 2 in the example
shown) would result in a reduced enthalpy of activation, if the
enthalpy of transfer of the reactants is more thermodynamically
unfavorable than the transfer of the transition state. The goal
of this work was to determine if this, or some other scenario,
occurs in the phosphate ester hydrolysis in the two solvents.
δ∆Hqtrans ) δ∆Hsol + δ∆Hq
(1)
In this equation, δ∆Hsol is the difference between the enthalpy
of solution of the reactant in the two solvents, which is the
enthalpy of transfer. δ∆Hq is the difference between the enthalpy
of activation in the two solvents. A number of workers,
particularly Haberfield, have obtained the enthalpy of solvent
transfer of transition states for a number of reactions, and the
(7) Haberfield, P. J. Phys. Chem. 1983, 87, 1423-1426.
(8) Haberfield, P.; Fortier, D. J. Org. Chem. 1983, 48, 4554-4557.
(9) Haberfield, P.; Friedman, J.; Pinkston, M. F. J. Am. Chem. Soc. 1972,
94, 71-75.
(10) Haberfield, P.; Kivuls, J.; Haddad, M.; Rizzo, T. J. Phys. Chem.
1984, 88, 1913-1916.
(4) Grzyska, P. K.; Czyryca, P. G.; Golightly, J.; Small, K.; Larsen, P.;
Hoff, R. H.; Hengge, A. C. J. Org. Chem. 2002, 67, 1214-20.
(5) Cheng, H.; Nikolic-Hughes, I.; Wang, J. H.; Deng, H.; O’Brien, P.
J.; Wu, L.; Zhang, Z. Y.; Herschlag, D.; Callender, R. J. Am. Chem. Soc.
2002, 124, 11295-306.
(11) Haberfield, P.; Nudelman, A.; Bloom, A.; Romm, R.; Ginsberg, H.
J. Org. Chem. 1971, 36, 1792-1795.
(12) Haberfield, P.; Pessin, J. J. Am. Chem. Soc. 1982, 104, 6191-6194.
(13) Grunwald, E.; Baughman, G.; Kohnstam, G. J. Am. Chem. Soc. 1960,
82, 5801-5811.
(6) Sorensen-Stowell, K.; Hengge, A. C. J. Org. Chem. 2005, 70, 8303-
8.
(14) Fuchs, R.; Bear, J. L.; Rodewald, R. F. J. Am. Chem. Soc. 1969,
91, 5797-5800.
J. Org. Chem, Vol. 71, No. 19, 2006 7181

Sorensen-Stowell and Hengge
TABLE 1. Enthalpies of Solution
TABLE 3. Activation Parameters for Hydrolysis of p-NPP and
p-NPPT in Water and in Mixed Solvent in Which the Water
Component Is pH 10.0, 256 mM CAPS Buffer
H2O ∆Hsol
kcal/mol
,
0.6 mol aq DMSO
∆Hsol, kcal/mol
compd
0.6 (mol) aq DMSO
H2O
[(Bu)4N][(Bu)4B]a
[(Bu)4N]3PO4
[(Bu)4N]2pNPP
[(Bu)4N]2PP
Not soluble
-44.7 ( 0.5
-39.1 ( 0.2
-36.5 ( 0.2
-36.5 ( 0.3
14.0 ( 0.3
-48.3 ( 0.3
-41.4 ( 0.3
-38.1 ( 0.3
-38.1 ( 0.5
esters
∆Hq (kcal/mol)
∆Sq( eu)
∆Hq (kcal/mol) ∆Sq (eu)
p-NPP
p-NPPT
26.2 ( 0.5
29.0 ( 0.1
-1.5 ( 0.5
25.7 ( 0.1
30.6a
3.5a
37 ( 1b
29 ( 3b
[(Bu)4N]2PPT
a
Literature values.17 b Literature value.3
a Literature value.9
TABLE 2. Enthalpies of Solvent Transfer, δ∆Hsol, of Compounds
unexpected. It has long been known that the enthalpies of
transfer from water to neat DMSO are exothermic for large,
polarizable or delocalized anions such as iodide and perchlor-
ate.14,15 The only anion for which the enthalpy of transfer has
been obtained for both neat DMSO and the mixed solvent used
in the present study is iodide, for which the enthalpy of transfer
to the mixed solvent (-5.5 kcal/mol)7 is more exothermic than
for transfer to neat DMSO (reported values range from -2.6 to
-4.9).15 The observation that inorganic phosphate follows the
same trend demonstrates that the aromatic moiety is not
responsible for the observed exothermicities in Table 2.
It is noted by comparing the data for phenyl and p-nitrophenyl
phosphates that there is a negligible difference in the heat of
transfer imparted by the nitro group. Thus, it seems a safe
assumption that the enthalpy of transfer for pNPPT should
closely approximate the value for PPT.
Because the activation parameters for the hydrolysis of pNPP
and pNPPT have been reported in a 95% (v) aq DMSO,
somewhat different from the 0.6 (mol) aq DMSO used here,
the rates of hydrolysis of these esters were measured as a
function of temperature to construct Eyring plots (Figure 2),
which were used to determine the activation parameters. The
lines represent the best linear least squares fit to the data. The
slopes and intercepts were used to calculate the enthalpy and
entropy of activation for each ester and their standard errors.
These values along with literature values for the activation
parameters for the hydrolysis reactions in water are reported in
Table 3. The activation parameters revealed a lower enthalpy
of activation in 0.6 (mol) aq DMSO, as previously found for
0.95 (v) aq DMSO. However, in the case of pNPP, ∆Hq is less
dramatically reduced in 0.6 (mol) aq DMSO than when the
reaction medium is 0.95 (v) aq DMSO. This observation is
consistent with the previous finding that the hydrolysis rate of
pNPP increases most dramatically at very high DMSO percent-
age.2 The rate enhancement is ∼24-fold upon increasing DMSO
from 80% to 90 vol %, compared to an ∼1800-fold enhance-
ment caused by the change from 80% to 95% DMSO.16
The transition states for the hydrolysis reactions of both pNPP
and pNPPT are loose, with little nucleophilic participation by
water in the pNPP reaction, and none in the case of pNPPT,
which reacts via a thiometaphosphate intermediate.18,19 The
large, positive entropy of activation in the DMSO/water solvent
indicates that the dissociative mechanism for pNPPT hydrolysis
is maintained in this solvent. Thus, the transition state resembles
metaphosphate and the p-nitrophenoxide leaving group. KIEs
and Ions (kcal/mol)
solvent transfer
H2O f 0.6 (mol) aq DMSO
species of interest
(Bu)4N+ a
δ∆Hsol (kcal/mol)
10.8 ( 0.1
-3.6 ( 0.9
-36.0 ( 0.9
-2.3 ( 0.4
-23.9 ( 0.3
-1.6 ( 0.4
-23.2 ( 0.4
-1.6 ( 0.5
-23.2 ( 0.6
3(Bu)4N+ PO4
3-
3-
PO4
2(Bu)4N+ pNPP2-
pNPP2-
2(Bu)4N+ PP2-
PP2-
2(Bu)4N+ PPT2-
PPT2-
a Literature value.7
The first goal was to obtain enthalpies of solution of pNPP
and pNPPT in water and in aqueous DMSO. The solubility
requirements to permit accurate calorimetric measurement of
the enthalpy of solution of the bis(tetrabutylammonium) salts
limited the DMSO composition of the mixture to 60% DMSO/
40% H2O by mole fraction (0.6 (mol) aq DMSO). This
corresponds to 0.85 (v) aq DMSO. The mole fraction notation
will be used to describe results in this solvent mixture, in
keeping with the notation used in previous work in the same
medium, some of which is referenced below.
The enthalpy of solution in 0.6 (mol) aq DMSO, and in water,
for bis(tetrabutylammonium) pNPP was measured and reported
in Table 1. The bis(tetrabutylammonium) salt of pNPPT could
not be obtained. Phosphorothioates are isolated after synthesis
as their cyclohexylammonium salts, but this salt form lacked
sufficient solubility in the mixed medium to carry out calorim-
etry experiments. Attempts to convert pNPPT to the bis-
(tetrabutylammonium) salt were accompanied by extensive
hydrolysis, and pure samples could not be obtained (see the
Supporting Information). The less reactive phenyl phospho-
rothioate (PPT) was successfully converted to the bis(tetrabu-
tylammonium) salt, and calorimetry in water and the mixed
solvent was carried out using this compound. Its oxygen
analogue phenyl phosphate (PP) was also prepared and calo-
rimetry carried out. The enthalpies of solution of these esters,
and of the tetrabutylammonium salt of inorganic phosphate, are
reported in Table 1. The magnitudes are in the range expected
for polyvalent electrolytes.15
To obtain the enthalpies of transfer (δ∆Hsol) of the anionic
species from water to 0.6 (mol) aq DMSO, the contribution of
the tetrabutylammonium ions were subtracted using the literature
value for its enthalpy of transfer7 to give the values in Table 2.
The exothermicity of the transfer of the phosphate esters and
of inorganic phosphate, from water to the mixed solvent, was
(16) Abell, K. W. Y. Ph.D. Thesis, Cambridge University, 1984.
(17) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415-
423.
(18) Hengge, A. C. In ComprehensiVe Biological Catalysis: A Mecha-
nistic Reference; Sinnott, M., Ed.; Academic Press: San Diego, CA, 1998;
Vol. 1, pp 517-542.
(19) Thatcher, G. R. J.; Kluger, R. AdV. Phys. Org. Chem. 1989, 25,
99-265.
(15) Criss, C. M. In Physical Organic Chemistry of Organic SolVent
Systems; Covington, A. K., Dickinson, T., Eds.; Plenum: New York, 1973;
p 23-136.
7182 J. Org. Chem., Vol. 71, No. 19, 2006

Hydrolysis of Phosphate and Phosphorothioate Esters in DMSO/Water
FIGURE 2. Eyring plots for the hydrolysis reactions of p-NPPT (O) and p-NPP (0) in 0.6 (mol) aq DMSO.
FIGURE 3. (Top) transition state for pNPP hydrolysis. The charge of
-2 of the reactant is dispersed nearly equally between the leaving group
and the phosphoryl group. The transition state for pNPPT hydrolysis
is similar but lacks nucleophilic participation.
and LFER data for the hydrolysis of both pNPP and pNPPT
indicate that bond scission is far advanced and that the
phosphoryl group and the leaving group each have about a unit
negative charge in the transition state. These transition states,
with the respective charge distribution, are shown in Figure 3.
From the enthalpies of transfer and the corresponding
enthalpies of activation, the enthalpies of transfer of the
transition states of these two reactions can be calculated using
eq 1. The transfer of the pNPP hydrolysis transition state from
FIGURE 4. Schematic representation of the enthalpies of solution of
water to 0.6 (mol) aq DMSO is exothermic, -28.3 ( 0.5 kcal/
mol. Assuming the enthalpy of solution for pNPPT is similar
to that of PPT, a similar value for the pNPPT transition state of
-31.2 ( 1.2 kcal/mol is obtained. Figure 4 shows these
relationships in pictorial fashion.
pNPP2- (top) and pNPPT2- (bottom), in water and in 0.6 (mol) aq
DMSO, and the enthalpies of transfer for the transition states of their
hydrolysis reactions in the two solvents. Red in the upper diagram
indicates values if the nucleophilic water molecule is included in the
treatment of the pNPP reaction.
The above treatment omits consideration of the water
nucleophile in the transition states of the rate-limiting steps of
these reactions. While this is accurate in the dissociative
mechanism for pNPPT hydrolysis, a small amount of nucleo-
philic participation occurs in the hydrolysis of phosphomono-
esters such as pNPP. The literature value for the enthalpy of
transfer of water to 0.6 (mol) aq DMSO is -1.15 kcal/mol.7
Inclusion of this quantity is shown in red in Figure 4. The
enthalpy of transfer for water plus pNPP2- is more negative by
1.15 kcal/mol, and as a result, the enthalpy of transfer of the
transition state is more negative by the same amount.
is -23.9 kcal/mol, compared to -28.3 for the transition state.
The difference is greater for pNPPT, where the enthalpy of
transfer of the reactant is -23.2 kcal/mol, and -31.2 for the
transition state. In each of these reactions the di-negative charge
of the reactant becomes significantly more dispersed in the
transition state, which probably accounts for the greater exo-
thermicity of the transfer to 0.6 (mol) aq DMSO.
Conclusions
These results show that the transfers of the transition states
for the pNPP and the pNPPT hydrolysis reactions are more
exothermic than for transfers of the respective reactants. Thus,
this is the origin of the reduced enthalpy of activation in both
reactions, rather than a scenario such as that outlined in Figure
1. In the case of pNPP, the enthalpy of transfer of the reactant
The results presented here, in conjunction with spectroscopic
investigations on the effects of the solvent medium on P-O
bonds in phosphate esters, indicate that the reduction in the
enthalpy of activation for hydrolysis imparted by 0.6 (mol) aq
DMSO arises from a more favorable enthalpy of transfer of the
transition state relative to the enthalpy of transfer of the pNPP
J. Org. Chem, Vol. 71, No. 19, 2006 7183

Sorensen-Stowell and Hengge
rothioate were accomplished using literature methods as described
in Supporting Information.
or pNPPT reactant. Due to experimental constraints that limited
the percentage of DMSO in the solvent systems that could be
examined, this work cannot exclude the possibility that ad-
ditional effects might operate at higher DMSO fractions, where
rate accelerations are more dramatic.
Thermodynamic Methods. A commercial solution calorimeter
was used to obtain the heats of solution of the phosphate and
phosphorothioate esters in water and in an aqueous DMSO mixture.
The instrument was first standardized using tris(hydroxymethyl)
aminomethane (TRIS), furnished with the calorimeter as a standard-
izing reagent, as described in the Supporting Information. In a
typical experiment, the heat evolved from solution of 250 mg of
solute in 100 g of solvent was measured and used to obtain the
enthalpy of solution. The difference between the enthalpy of solution
in the two solvents is the enthalpy of transfer.
These results demonstrate the potential for medium effects
to significantly reduce the enthalpy of activation for phosphoryl
transfer reactions. This provides a potential means for substantial
rate accelerations to arise from electrostatic interactions in an
enzymatic active site that stabilize the transition state for
phosphoryl transfer. Due to the loose transition state for
phosphomonoester reactions, the entropic barrier is small,
leaving enthalpic considerations as the major avenue to achieve
significant rate accelerations. The results here show that
substantial reductions in the enthalpy of activation result upon
the transfer from a medium of pure water to a DMSO/water
mixture that remains 22 M in water. This is probably modest
in comparison to the environmental effects that can be achieved
in the evolved active site of an enzyme.
Acknowledgment. The authors thank the NIH for financial
support (GM47297) and Tony Kirby for helpful discussions.
Supporting Information Available: Procedures for the syn-
thesis of the phosphate and phosphorothioate esters, calorimetric
procedures and equations used to calculate enthalpies of solution,
and methods used to obtain kinetic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Experimental Section
The synthesis and purification of p-nitrophenyl phosphate, phenyl
phosphate, phenyl phosphorothioate, and p-nitrophenyl phospho-
JO060896B
7184 J. Org. Chem., Vol. 71, No. 19, 2006