Comparisons of phosphorothioate and phosphate monoester transfer reactions: Activation parameters, solvent effects, and the effect of metal ions

Article abstract of DOI:10.1021/ja983862x

The thermodynamics, pH dependency, solvent effects, and the effect of divalent metal ions have been examined for the hydrolysis reactions of the phosphorothioate monoester p-nitrophenyl phosphorothioate (pNPPT) and compared with those of the corresponding phosphate monoester, p-nitrophenyl phosphate (pNPP). The pH dependency for pNPPT hydrolysis mirrors that for typical phosphate monoesters, with the monoanion being the most reactive species. The ratio of the rate constants for pNPPT hydrolysis to that for pNPP is 1380 for the monoanion reactions and 12.6 for the dianion reactions at 39 °C. The free energy of activation for hydrolysis of the pNPPT dianion in water is 27.9 kcal/mol versus 29.5 kcal/mol for pNPP; for the monoanion reactions the values are 22.2 kcal/mol for pNPPT and 26.8 kcal/mol for pNPP. The free energies of solvation for pNPPT and pNPP are within 0.1 kcal/mol of each other despite the poorer hydrogen bonding ability of sulfur versus oxygen. This minimal difference in ground-state energies thus does not account for the difference in activation energies, which must therefore arise from transition state effects. The more favorable ΔG? for pNPPT dianion hydrolysis versus pNPP is entropie in origin; the enthalpic barrier is greater in the pNPPT reaction but is more than offset by a more favorable entropy of activation that arises from the fully dissociative mechanism followed by pNPPT. By contrast, the more favorable ΔG? for hydrolysis of the pNPPT monoanion compared to the pNPP monoanion is enthalpic in origin. The hydrolysis rate of the pNPPT dianion increases by nearly 10⁷-fold as DMSO content is changed from O to 95%. The rate acceleration is due to a lower enthalpy of activation in the mixed solvent. In contrast, the rate of hydrolysis of the monoanion of pNPPT in aqueous DMSO remains almost unchanged from the aqueous value; the enthalpic barrier in 95% DMSO is decreased but is offset by an increase in the entropie barrier. The kinetic effects on hydrolysis of the pNPPT dianion caused by complexation with magnesium, which coordinates with oxygen in phosphorothioates, and cadmium, which coordinates with sulfur, were found to be similar with each metal, resulting in a small decrease in the rate constant.

Full text of DOI:10.1021/ja983862x

2156
J. Am. Chem. Soc. 1999, 121, 2156-2163
Comparisons of Phosphorothioate and Phosphate Monoester Transfer
Reactions: Activation Parameters, Solvent Effects, and the Effect of
Metal Ions
Irina E. Catrina and Alvan C. Hengge*
Contribution from the Department of Chemistry and Biochemistry, Utah State UniVersity,
Logan, Utah 84322-0300
ReceiVed NoVember 6, 1998
Abstract: The thermodynamics, pH dependency, solvent effects, and the effect of divalent metal ions have
been examined for the hydrolysis reactions of the phosphorothioate monoester p-nitrophenyl phosphorothioate
(pNPPT) and compared with those of the corresponding phosphate monoester, p-nitrophenyl phosphate (pNPP).
The pH dependency for pNPPT hydrolysis mirrors that for typical phosphate monoesters, with the monoanion
being the most reactive species. The ratio of the rate constants for pNPPT hydrolysis to that for pNPP is 1380
for the monoanion reactions and 12.6 for the dianion reactions at 39 °C. The free energy of activation for
hydrolysis of the pNPPT dianion in water is 27.9 kcal/mol versus 29.5 kcal/mol for pNPP; for the monoanion
reactions the values are 22.2 kcal/mol for pNPPT and 26.8 kcal/mol for pNPP. The free energies of solvation
for pNPPT and pNPP are within 0.1 kcal/mol of each other despite the poorer hydrogen bonding ability of
sulfur versus oxygen. This minimal difference in ground-state energies thus does not account for the difference
q
in activation energies, which must therefore arise from transition state effects. The more favorable ∆G for
pNPPT dianion hydrolysis versus pNPP is entropic in origin; the enthalpic barrier is greater in the pNPPT
reaction but is more than offset by a more favorable entropy of activation that arises from the fully dissociative
q
mechanism followed by pNPPT. By contrast, the more favorable ∆G for hydrolysis of the pNPPT monoanion
compared to the pNPP monoanion is enthalpic in origin. The hydrolysis rate of the pNPPT dianion increases
7
by nearly 10 -fold as DMSO content is changed from 0 to 95%. The rate acceleration is due to a lower enthalpy
of activation in the mixed solvent. In contrast, the rate of hydrolysis of the monoanion of pNPPT in aqueous
DMSO remains almost unchanged from the aqueous value; the enthalpic barrier in 95% DMSO is decreased
but is offset by an increase in the entropic barrier. The kinetic effects on hydrolysis of the pNPPT dianion
caused by complexation with magnesium, which coordinates with oxygen in phosphorothioates, and cadmium,
which coordinates with sulfur, were found to be similar with each metal, resulting in a small decrease in the
rate constant.
Introduction
in interpreting differences in enzymatic reactions in terms of
mechanistic effects.
O-phosphorothioate analogues of phosphate monoesters have
historically been used to probe kinetic and stereochemical
aspects of phosphoryl transfer. Both uncatalyzed reactions in
solution and enzymatic reactions have been carried out, the latter
most notably with alkaline phosphatase. The differences ob-
served in alkaline phosphatase activity have been used in
Phosphate monoesters typically react in solution by mecha-
nism A in Figure 1. This is a concerted ANDN reaction in which
6
the nucleophile enters and the leaving group departs in a single
transition state. This pathway for phosphate monoester dianions
7
is supported by a very small entropy of activation, a large
8
7
(
-1.2) âlg, and a small ânuc, and the occurrence of inversion
1-3
9
attempts to infer mechanistic details of this enzymatic reaction.
Alternative interpretations of such differences, termed thio
of configuration when the phosphoryl group is made chiral.
An essential difference that occurs in the phosphoryl transfer
from the dianion of p-nitrophenyl phosphate (pNPP) to anhy-
drous tert-butyl alcohol, where this acceptor also is the reaction
solvent, is the observation of racemization at phosphorus when
the reactant was made chiral by the use of oxygen isotopes.9
This outcome was interpreted to indicate the formation of a free
metaphosphate intermediate in the DN + AN mechanism B in
4
,5
effects, in enzymatic reactions have been discussed.
While a considerable body of kinetic and thermodynamic data
have been collected for the phosphoryl transfer reactions of
monoesters, less is known about some aspects of thiophosphoryl
transfer. In particular, the activation parameters, the effect of
metal ions, and the solvent effects on thiophosphoryl transfer
reactions have not been reported. Knowledge of how phospho-
rothioates differ from phosphates in these aspects is important
9
Figure 1. This outcome was in contrast to that in more
(6) For a description of the IUPAC nomenclature for reaction mecha-
nisms, see: Guthrie, R. D.; Jencks, W. P. Acc. Chem. Res. 1989, 22, 343-
(
(
1) Breslow, R.; Katz, I. J. Am. Chem. Soc. 1968, 90, 7376-7377.
2) Chlebowski, J. F.; Coleman, J. E. J. Biol. Chem. 1974, 249, 7192-
349.
(7) Kirby, A. J.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 3209-3216.
(8) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415-
423.
(9) Friedman, J. M.; Freeman, S.; Knowles, J. R. J. Am. Chem. Soc.
1988, 110, 1268-1275.
7
202.
(
(
(
3) Han, R.; Coleman, J. E. Biochemistry 1995, 34, 4238-4245.
4) Hollfelder, F.; Herschlag, D. Biochemistry 1995, 34, 12255-12264.
5) Herschlag, D.; Jencks, W. P. Biochemistry 1990, 29, 5172-5179.
1
0.1021/ja983862x CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/24/1999

Phosphorothioate and Phosphate Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 10, 1999 2157
Figure 1. Pathways observed for phosphoryl transfer from monoester dianions (top) and monoanions (bottom). A is the concerted A
with a dissociative transition state with the bond to the leaving group largely broken and a small degree of bond formation with the nucleophile.
B is a stepwise D + A mechanism proceeding by way of a (thio)metaphosphate intermediate. At the bottom is a mechanism for reaction of the
N N
D mechanism
N
N
monoanionic species; proton transfer may occur in a preequilibrium step as shown before nucleophilic attack, or proton transfer can occur simultaneous
with leaving group departure, depending upon the basicity of the leaving group.
nucleophilic solvents such as methanol, where the product had
inverted configuration consistent with nucleophilic participation
In contrast to stereochemical results with chiral phosphate
monoesters, stereochemical studies with the dianion and the
1
0
18 16
in the transition state of the concerted mechanism A. The
observation of racemization in the tert-butyl alcohol reaction
could be due to a bimolecular displacement of the nitrophenolate
by tert-butyl alcohol followed by a succession of displacements
of the bridge-protonated tert-butyl phosphate prior to the
deprotonation step forming the stable product. However, the
finding of a significantly more positive entropy of activation
monoanion of chiral p-nitrophenyl [O, O ]phosphorothioate
1
4,15
16,17
show that ethanolysis
and hydrolysis
both proceed with
a large degree of racemization, indicating the formation of free
thiometaphosphate as an intermediate. The observation of a large
volume of activation for the hydrolysis of the dianion of 2,4-
dinitrophenyl thiophosphate, in contrast to the near zero value
measured with the corresponding phosphate ester, also is
indicative of a difference in mechanism and is consistent with
q
11
for the tert-butyl alcohol reaction (∆S ) +24.5 eu) compared
q
7
17
to the aqueous reaction (∆S ) + 3.5 eu) is more consistent
with a switch to the fully dissociative DN + AN mechanism in
tert-butyl alcohol.
formation of a thiometaphosphate intermediate. These results
indicate that phosphorothioates typically react via mechanism
B in Figure 1. This is an SN1 (DN + AN) type of mechanism in
which a (thio)metaphosphate intermediate is formed in the rate-
determining step; this highly reactive species is then attacked
by a nucleophile in a subsequent rapid step.
Thus in contrast to phosphoryl transfer, where a metaphos-
phate intermediate forms only in the absence of a competent
potential phosphoryl acceptor, thiophosphate transfer in solution
routinely occurs via a thiometaphosphate intermediate. The
notion that thiometaphosphate possesses greater stability than
The reactions of phosphate monoanions are believed to
proceed by a mechanism in which the proton is transferred to
the leaving group either in a preequilibrium step as shown in
Figure 1 or, for less basic leaving groups, simultaneously with
8
P-O bond cleavage. This aqueous reaction also shows a very
8
small entropy of activation, and the reaction of pNPP in
methanol proceeds with inversion of configuration at phospho-
10
rus.
Experimental12 and theoretical13 evidence regarding the
metaphosphate is supported by the fact that while the latter has
bonding in phosphorothioates indicates that structure 1 is the
18,19
been directly detected only in the gas phase
trithiometa-
phosphate has been isolated as the tetraphenylarsonium salt.2
0
With this information as background, the aim of this study
was to discover further information about the chemistry of
phosphorothioate monoesters compared with phosphate coun-
terparts. So-called “thio effects”, the ratio of reaction rates of
corresponding phosphate and phosphorothioates, have been used
to infer mechanistic information for the enzymatic catalysis of
most accurate representation of the charge distribution and
bonding, with greater negative charge and less double bond
character on the sulfur atom than each of the two nonbridge
oxygens. A study of the hydrolysis reactions of O-aryl phos-
phorothioate dianions using linear free-energy relationships
(
14) Cullis, P. M.; Iagrossi, A. J. Am. Chem. Soc. 1986, 108, 7870-
871.
(15) Cullis, P. M.; Misra, R.; Wilkins, D. J. J. Chem. Soc. Chem.
Commun. 1987, 1594-1596.
16) Domanico, P.; Mizrahi, V.; Benkovic, S. J. In Mechanisms of
7
4
found a value for âleaving group of -1.1 which suggests that the
transition states resemble those for reactions of the aryl
(
enzymatic reactions: stereochemistry; Frey, P. A., Ed.; Elsevier: New York,
1986; pp 127-137.
phosphate ester counterparts, which exhibit a value of -1.2 for
8
this parameter.
(17) Burgess, J.; Blundell, N.; Cullis, P. M.; Hubbard, C. D.; Misra, R.
J. Am. Chem. Soc. 1988, 110, 7900-7901.
(10) Buchwald, S. L.; Friedman, J. M.; Knowles, J. R. J. Am. Chem.
(18) Meyerson, S.; Harvan, D. J.; Hass, J. R.; Ramirez, F.; Maracek, J.
J. Am. Chem. Soc. 1984, 106, 6877-6883.
Soc. 1984, 106, 4911-4916.
(
11) Hoff, R. H.; Hengge, A. C. J. Org. Chem. 1998, 63, 6680-6688.
(19) Henchman, M.; Viggiano, A. A.; Paulson, J. F.; Freedman, A.;
Wormhoudt, J. J. Am. Chem. Soc. 1985, 107, 1453-1455.
(20) Roesky, H. W.; Ahlrichs, R.; Brode, S. Angew. Chem., Int. Ed. Engl.
1986, 25, 82-83.
(12) Iyengar, R.; Eckstein, F.; Frey, P. A. J. Am. Chem. Soc. 1984, 106,
8
309-8310.
13) Liang, C.; Allen, L. C. J. Am. Chem. Soc. 1987, 109, 6449-6453.
(

2158 J. Am. Chem. Soc., Vol. 121, No. 10, 1999
Catrina and Hengge
phosphoryl transfer. Thus we were particularly interested in
examining properties of thiophosphate chemistry with relevance
to enzymatic reactions. In this study we report the pH
dependency of the aqueous hydrolysis and the activation
parameters for the reactions of the dianion and monoanion of
p-nitrophenyl phosphorothioate (pNPPT), and the comparative
free energies of solvation of pNPPT and the pNPP dianions.
We have also examined the effect of added DMSO on the rates
and activation parameters of the reactions of pNPPT and
compared the results with a prior analogous study of the
phosphate ester. This added cosolvent accelerates the hydrolysis
of phosphate monoesters by an effect that has been implicated
to be associated with enzymatic catalysis. We also report the
mechanistic and kinetic effects of divalent metal ions on the
aqueous hydrolysis reaction of the pNPPT dianion, using both
an oxygen- and a sulfur-preferring metal ion.
solutions were filtered and assayed at time intervals to determine when
saturation had been reached, which was 15 min or longer. For pNPPT
the concentrations were measured after total hydrolysis of an aliquot
in 0.2 M glycine at pH 2, from total p-nitrophenol (pNP) concentration.
The pNP released was determined by taking an aliquot in 0.1 N NaOH
and measuring the absorbance at 400 nm. The concentrations at
saturation for pNPP were similarly measured, with the total hydrolysis
carried out with alkaline phosphatase in 0.1 M TRIS buffer at pH 9, 1
2+
2+
mM Zn and Mg
.
Mixed Solvent Rate Studies. The DMSO content of the aqueous
solutions was varied from 0 to 95% in kinetic studies of the hydrolysis
of the pNPPT, under conditions similar to those previously used to
study reactions with pNPP.22 Both the pNPPT dianion and monoanion
rate constants were determined by following the release of p-
nitrophenolate anion at 400 nm or of protonated p-nitrophenol at 335
nm, respectively. The dianion reactions were run at concentrations of
0
0
.02 M NaOH, and the monoanion reactions in solutions containing
.02 M formate. All reactions were run at 39 °C with the exception of
the dianion reaction in 95% DMSO. This reaction at this temperature
was too fast for accurate measurement of the rate constant, so its value
was extrapolated from a linear Eyring plot constructed from reactions
at lower temperatures.
Experimental Section
Materials. Reagents and solvents were commercial products and
were used as received unless otherwise noted. Thiophosphoryl chloride
was distilled under nitrogen before use. Pyridine was distilled from
calcium hydride.
Synthesis. The bis(cyclohexylammonium) salt of p-nitrophenyl
phosphate was prepared and purified by recrystallization from 95%
The rate constants for hydrolysis of the dianion and the monoanion
in 95% DMSO/water were measured at a range of temperatures to
construct Eyring plots, from which the activation parameters were
determined as described for the aqueous reactions.
2
1
ethanol by using the method of Bourne and Williams. pNPPT was
prepared as the bis(cyclohexylammonium) salt by the same method
and was purified by washing with cold distilled water in lieu of
recrystallization, which resulted in substantial hydrolysis of the
phosphorothioate ester. Both products were characterized by proton
and by phosphorus NMR spectroscopy. The pNPPT showed no
detectable levels of pNPP by NMR.
2 3 2
The Effect of Metal Ions. Stock solutions of MgCl and Cd(NO )
at 1 M were used in these experiments. The formation of metal-pNPPT
complexes was determined from changes in the UV-vis spectrum at
constant substrate concentration and the metal concentration was varied
(
metal was present both in the reference and experiment cells). The
2
+
stability constant for the complex between pNPPT and Cd was
determined from the change in λmax as a function of Cd2 concentration
by using eqs 1 and 2
+
Kinetics. (a) pH-Rate Profile. First-order rate constants for the
aqueous hydrolysis of pNPPT were measured over the pH range from
-
1 to 15 at 39 °C and µ ) 1 M (KCl). The reactant concentration
KML ) [ML]/([M ] - [ML])([L ] - [ML])
(1)
(2)
varied from 0.1 to 4 mM. Buffers used were as follows: pH 2, 0.2 M
t
t
glycine or oxalate; pH 3, 0.2 M formate; pH 4-5, 0.2 M acetate; pH
[
ML] ) [L ]{(W - W )/(W - W )}
6
-7, 0.2 M MES; pH 7-9, 0.2 M TRIS; pH 10-11, 0.2 M carbonate.
t 0 1 2 1
Appropriate concentrations of HCl and NaOH were used respectively
for pH values below 1 and above 13.
where [M
t
t
] and [L ] are the total concentrations of metal and ligand (in
At pH 5 and above the initial rate method was used to determine
the rate constants. The release of p-nitrophenol was followed by adding
aliquots of the reaction mixtures to 0.1 N NaOH and measuring the
absorbance of the nitrophenolate anion at 400 nm with an extinction
this case pNPPT) in all forms, [ML] is the concentration of metal-
ligand complex, W is the observed λmax, and W and W are the λmax
values in the absence and at saturating metal concentrations, respec-
0
1
2
tively.
-1
coefficient of 18 300 M . Below pH 5 reactions were run to completion
greater than 10 half-lives) and rate constants determined by a fit of
The rate constants for pNPPT hydrolysis reactions in the presence
(
2+
2+
of Mg and Cd were determined by using the initial rate method,
assaying for release of p-nitrophenol. The reaction mixtures were filtered
before absorbance measurement to remove precipitated metal-inorganic
phosphorothioate complex, and the reaction solution absorbance was
measured at 400 nm in quartz cells. The buffer used was 0.18 M
HEPES, pH 8.0 for the reactions with both metal ions (µ ) 1.1 with
KCl for the reactions with magnesium ion and µ ) 1 M with KCl for
those with cadmium ion). The concentration of pNPPT was 1 mM,
and metal ion concentrations were 0.3 M for magnesium (which was
near saturation under these conditions) and 0.05 M for cadmium. The
dependence on the hydroxide ion concentration was studied for the
the full time course data. The rapid reactions around pH 2 were followed
in situ at 330 nm with use of a Cary 1Bio spectrophotometer equipped
with a temperature controller.
(b) Thermodynamic Studies. The rate constants for the aqueous
hydrolysis of the pNPPT dianion were determined at four different
temperatures (30, 50, 60, and 70 °C) at a substrate concentration of 4
mM in 0.2 M CHES buffer at pH 10 and µ ) 1 M (KCl). The initial
rate method was used. The reactions were followed at 400 nm with a
Cary 1Bio spectrophotometer.
The rate constants for the aqueous hydrolysis of pNPPT monoanion
reactions were determined at four different temperatures (20, 30, 39,
and 50 °C). The reactions were run in 0.2 M glycine buffer at pH ) 2,
µ ) 1 M (KCl) and at 0.1 mM pNPPT concentration. At 20 °C the
initial rate method was used; for the other three temperatures the
reactions were run until completion (>10 half-lives). All reactions were
followed at 330 nm with use of a Cary 1Bio spectrophotometer.
Solubility Experiment. The bis(cyclohexylammonium) salts of
pNPPT and of pNPP were used for these experiments. The concentra-
tions at saturation of the two salts in water were measured at four
different temperatures. These experiments were performed at temper-
atures lower than 25 °C to avoid measurable hydrolysis of pNPPT
during the time necessary for solubility measurements. The stirred
2+
hydrolysis reaction in the presence of Mg . For this study the rate
constants at three pH values were determined; the buffer used was 0.18
M HEPES at pH ) 8.0, 8.3, and 8.6, with ionic strength maintained at
1
.1 with KCl.
Results
Values of the first-order rate constants for hydrolysis of
p-nitrophenyl phosphorothioate (pNPPT) were plotted as a
function of pH in Figure 2. The data show the bell-shaped pH-
(
22) Abell, K. W. Y.; Kirby, A. J. Tetrahedron Lett. 1986, 27, 1085-
(21) Bourne, N.; Williams, A. J. Org. Chem. 1984, 49, 1200-1204.
1088.

Phosphorothioate and Phosphate Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 10, 1999 2159
Table 2. Activation Parameters for Hydrolysis of
p-Nitrophenylphosphorothioate (pNPPT) and p-Nitrophenyl
Phosphate (pNPP)
pNPP
aq)
pNPPT
(aq)
pNPPT
(95% DMSO)
(
dianion
q
30.67
∆
∆
∆
H , kcal/mol
37.0 ( 1.0
+29 ( 3
22.9 ( 0.7
+12 ( 3
q
7
S , eu
+3.5
q
29.5a
27.9 ( 1.0a
a
G , kcal/mol
19.1 ( 0.7
monoanion
q
25.4 8
∆
∆
∆
H , kcal/mol
22.0 ( 0.6
-1 ( 2
18.8 ( 0.3
-12 ( 1
22.7 ( 0.3
q
8
S , eu
-4.5
q
26.8a
22.2 ( 0.6a
a
G , kcal/mol
a
q
q
q
Calculated from ∆G ) ∆H - T∆S at 39 °C.
Figure 2. The pH dependence of the first-order rate constants (s-1
)
for hydrolysis of pNPPT at 39 °C. The line represents the fit to the
data by using the equation log Y ) log[C/(1 + H/K + K /H)] with
a
b
2
3
use of the program BELL of Cleland.
Table 1. Rate Constants for Reactions of
p-Nitrophenylphosphorothioate (pNPPT) and p-Nitrophenyl
Phosphate (pNPP) at 39 °C
species
pNPPT
pNPP
pNPPT/ pNPP
k k
monoanion
-
1
6
1470a
1.078
k (s ) × 10
1380
12.6
dianion
-
1)
6
0.195a
7538
0.01557
69
k (s × 10
k
monoanion/kdianion
Figure 4. The temperature dependence of the solubility in water of
the dicyclohexylammonium salts of p-nitrophenyl phosphate (∆) and
p-nitrophenyl phosphorothioate (O).
a
Experimental data from this work; see Experimental Section for
details. Other data noted with superscripts denote data from the
referrences indicated.
q
q
free energies of activation were calculated from ∆G ) ∆H -
q
q
T∆S at 39 °C. The free energy of activation ∆G was found
q
to be 27.9 (1 kcal/mol for the dianion reaction, with ∆H )
q
3
7 ( 1 kcal/mol and ∆S ) +29 ( 3 eu. For the monoanion
q
q
reaction, ∆G ) 22.2 ( 0.6 kcal/mol with ∆H ) 22.0 ( 0.6
q
and ∆S ) -1 ( 2 eu. These values together with the literature
values for the activation parameters for the hydrolysis of
p-nitrophenyl phosphate for comparison are given in Table 2.
To evaluate the comparative free energies of solvation of
p-nitrophenyl phosphate and p-nitrophenyl phosphorothioate in
water, the solubilities of the bis(cyclohexylammonium) salts of
both compounds were measured over a range of temperatures
as described in the Experimental Section. In dilute solution the
activity of a solute is close to unity and the free energy of
solvation is directly proportional to solubility. Thus the relative
free energies of solvation for these two compounds can be
approximated by the ratio of their solubilities. The difference
in the free energies of the solvated reactants ∆∆Gsolvation is then
equal to RT ln(cp/ct), where cp and ct are the concentrations at
Figure 3. Eyring plots for the aqueous hydrolysis reactions of pNPPT
monoanion (∆) and dianion (O).
rate behavior that is typical of phosphate monoesters. The data
were fitted to the equation log Y ) log[C/(1 + H/Ka + Kb/H)]
with use of the program BELL of Cleland23 to give the solid
curve shown. The first and second pKa values were determined
to be 3.68 ( 0.08 and -0.10 ( 0.11.
The rate constants for the hydrolysis of the p-nitrophenyl
phosphorothioate monoanion at pH 2.0 and of the dianion at
pH 10.0 were measured at 39 °C and are compared with the
literature values for the hydrolysis of p-nitrophenyl phosphate
in Table 1. The rate constants for p-nitrophenyl phosphorothioate
hydrolysis were measured as a function of temperature to
construct the Eyring plots (Figure 3) which were used to
determine the activation parameters. The lines represent the best
least-squares linear fit to the data (Sigmaplot, Jandel). The slopes
and intercepts were used to calculate the enthalpy and entropy
of activation in the two reactions and their standard errors. The
24
saturation of the phosphate and phosphorothioate, respectively.
This experiment is a variation of the common method for
determining the difference in free energy of solvation of a
compound in two different solvents from the relationship
∆∆Gsolvation ) RT ln P, where P is the ratio of the solubility of
2
4
the compound in the two respective solvents. The solubility
data as a function of temperature are presented in Figure 4.
Temperatures at or below room temperature were used in this
experiment because of the lability of pNPPT and the need to
keep hydrolysis below detectable levels during the solubility
determinations. The ratio c /c was close to unity at all
p
t
temperatures; the value for ∆∆Gsolvation was found to be
approximately 0.1 kcal/mol at 4 °C.
The effect of added DMSO on the aqueous hydrolysis of the
monoanion and dianion of pNPPT was examined at 39 °C. The
(23) Cleland, W. W. Methods Enzymol. 1979, 63, 103-138.
(24) Marcus, Y. Pure Appl. Chem. 1983, 55, 977-1021.

2160 J. Am. Chem. Soc., Vol. 121, No. 10, 1999
Catrina and Hengge
Figure 5. The dependence on the percentage of DMSO of the rate
constants for hydrolysis of the pNPPT dianion (∆) and monoanion (O)
at 39 °C.
Figure 6. The change in λmax of pNPPT at pH 8.0 in 0.18 M HEPES
buffer, µ ) 1 (KCl), as a function of cadmium ion concentration. The
line represents the best fit of the data with use of eqs 1 and 2.
rate constants were measured for mixtures consisting of from
zero to 95% DMSO, and the results plotted in Figure 5. The
substrate was kept in the dianion form by maintaining a 20 mM
concentration of NaOH in the solvent mixtures. The broad pH-
independent range of the dianion hydrolysis gives strong
assurance that the substrate is fully deprotonated under these
conditions. The monoanion reaction is potentially more prob-
lematic in this regard, as the plateau around pH 2 representing
the monoanion reaction is small and the organic cosolvent could
alter the pKa values and thus the protonation state of the
substrate. This possibility was checked by varying the pH of
the formic acid/formate buffer from 2.6 to 2.8. The rate constants
were the same within experimental error for hydrolysis in the
aqueous DMSO reaction just as was the case in the aqueous
reaction, indicating that the kinetics reflect those of the
monoanion at the peak of the pH-rate profile.
(log K ) 2.50 ( 0.1). This may be compared with the value
a
2
+
reported for the Cd -adenosine monophosphorothioate
2
6
(AMPRS) complex (log K ) 4.62) in which additional
a
interactions between the metal ion and the nucleotide base result
in tighter binding, and with the stability constant with ADPRS
2
5
(log K ) 4.95) where the cadmium ion chelates between
a
the R and â phosphates. The lower pK of the phosphoryl group
a
in pNPPT relative to the nucleotides also reduces its stability
constant relative to these other compounds. The stability constant
2
+
for the Cd -p-nitrophenyl phosphate complex has also been
2
7
reported (log K ) 2.05).
a
2+
The complex between Mg and pNPPT was too weak to
achieve complete complexation even at saturating concentrations
The dianion reaction was substantially accelerated by added
DMSO by up to more than 6 orders of magnitude. The activation
parameters were determined for the reactions in 95% DMSO
from an Eyring plot (Supporting Information, Figure s-1) as
described for the aqueous reactions. For the reaction of the
2+
of Mg , and the stability constant for the complex could not
be determined. It can be assumed that its value will be lower
2+
28
than the reported values for Mg with pNPP (log Ka ) 1.17
27
and 1.29 ) because sulfur substitution for oxygen is known to
lower stability constants for metal ion-phosphate complexes
q
dianion, ∆H ) 22.9 ( 0.7 kcal/mol in 95% DMSO, as
25,26
with oxygen-preferring metal ions.
compared to 37 kcal/mol in the aqueous reaction. The entropy
The effects of each of these metal ions on the rate of
hydrolysis of the dianion of pNPPT were measured at 39 °C.
The experiments with cadmium were performed at concentra-
tions of the metal ion sufficient to result in full complexation
of the pNPPT. Because the complexation of pNPPT with
magnesium was too inefficient to achieve saturation, the kinetic
experiments with this metal ion were conducted at as high a
magnesium concentration as possible under the experimental
conditions. Full complexation of pNPPT with Cd ion results in
q
of activation, ∆S ) +12 ( 3 eu, compared to the value of
+
29 ( 3 eu measured for the aqueous reaction.
The rate of the monoanion reaction was affected by added
DMSO much less, showing only a slightly decreasing rate with
increasing fractions of DMSO. The activation parameters for
q
this reaction in 95% DMSO were also determined: ∆H ) 18.8
(
0.3 kcal/mol in 95% DMSO, versus 22.0 ( 0.6 kcal/mol in
q
the aqueous reaction. The entropy of activation ∆S ) -12 (
eu, versus the value of -1 ( 2 eu measured for the aqueous
reaction.
Magnesium and cadmium ions each form complexes with
phosphorothioates, as they do with phosphates. In their com-
1
2+
a shift in λmax by about 10 nm; at maximal Mg concentrations
the λmax of pNPPT solution had shifted by about 2 nm.
Assuming that a similar shift results from complexation by the
two metals, then only about one-fourth of the pNPPT was
complexed by Mg ion in the kinetic experiments. Thus the
kinetic consequences of magnesium complexation will be
underestimated in these experiments. The rate constants for the
2+
plexes with phosphorothioates Mg preferentially complexes
with oxygen and Cd2 preferentially binds to sulfur. The
formation of the complex between these metal ions and pNPPT
results in a shift in λmax to lower wavelength. This shift allows
one to follow formation of the metal-pNPPT complex by
measuring the change in λmax as a constant concentration of
pNPPT is titrated with the metal ion. Figure 6 shows the change
in λmax of a solution of pNPPT as it was titrated with cadmium
at constant ionic strength. For Cd2 the stability constant for
+
25
2+
two reactions at pH 8.0 were very similar; for the Cd reaction
-7 -1
k ) 3.02 × 10
s
and for the reaction in the presence of
2+
-7 -1
Mg k ) 2.90 × 10 s . Thus both metal ions were found
to result in a small decrease in the rate constant for hydrolysis.
+
(26) Sigel, R. K. O.; Song, B.; Sigel, H. J. Am. Chem. Soc. 1997, 119,
744-755.
-
1
the complex was determined from these data to be 293 M
(
27) Massoud, S. S.; Sigel, H. Inorg. Chem. 1988, 27, 1447-1453.
(
25) Pecoraro, V. L.; Hermes, J. D.; Cleland, W. W. Biochemistry 1984,
(28) Herschlag, D.; Jencks, W. P. J. Am. Chem. Soc. 1987, 109, 4665-
4674.
2
3, 5262-5271.

Phosphorothioate and Phosphate Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 10, 1999 2161
mirrors the behavior of the phosphate, as does an increase in
the hydrolysis rate at very high pH. In the pNPP reaction this
upward curvature has been attributed to a reaction of the dianion
with hydroxide ion in a nucleophilic aromatic substitution
7
reaction.
Activation Parameters. Since sulfur is much less able to
participate in hydrogen bonding than oxygen, one possible
q
contributor to the greater reactivity (reduced ∆G ) of pNPPT
relative to pNPP might be inferior solvation of the phospho-
rothioate ester in water. To test this hypothesis, the comparative
free energies of solvation of the bis(cyclohexylammonium) salts
of pNPPT and pNPP were estimated from a comparison of the
solubilities of these two compounds, as described in the
Experimental Section. Saturated aqueous solutions of these
compounds are dilute (30 to 40 mM); in dilute solution activities
are close to unity, and free energies of solvation are proportional
to concentration. The difference in solvation between the two
compounds was measured at several temperatures and the largest
difference, at 4 °C, is only about 100 cal/mol. This is far smaller
than the difference in the free energies of activation and indicates
that solvation differences in the ground state are at most a minor
factor in the rate difference between the dianions of pNPPT
and of pNPP.
Figure 7. The dependence of the rate constants for hydrolysis of
pNPPT on the hydroxide ion concentration in the presence (O) and
absence of Mg (∆).
2
+
Figure 7 shows there is no measurable effect of hydroxide
concentration on the rate hydrolysis of the dianion of pNPPT
2+
in the presence of Mg . Because of the limitation on the
solubility of the metal ion at higher pH the kinetics experiments
could not be conducted in a completely pH-independent region
of the profile. Significant precipitation at pH values above 8.0
The breakdown of the activation parameters for the hydrolysis
reactions of the monoanion and dianion of pNPPT into enthalpic
and entropic contributions (Table 2) and a comparison of these
values with those for pNPP hydrolysis reveals some interesting
differences. For the dianion reactions, a less favorable enthalpy
of activation for the phosphorothioate ester is more than offset
by a more favorable entropy of activation. The precise reasons
for the larger enthalpic barrier in the reaction of the phospho-
rothioate ester are not known, but several contributions are
possible. The similarity of the values of âlg for the two reactions
indicates that the degree of P-O bond fission in the transition
states is similar in the two reactions, thus ruling out this as a
source of the difference unless the sulfur substitution results in
an increase in the bond energy of the P-O bond to the leaving
(
the lowest point in Figure 7) precluded the analogous experi-
2
+
ment with Cd .
Discussion
pH-Rate Profile. The aqueous hydrolysis of pNPPT shows
a bell-shaped pH-rate profile (Figure 2) like those of phosphate
monoesters, which indicates that the monoanion is more reactive
than the dianion. However, the difference in rates between the
monoanion and dianion is considerably larger for pNPPT than
for pNPP. At 39 °C, the ratio of the rate constant for hydrolysis
8
7
of the monoanion to that of the dianion of p-nitrophenyl
phosphate is 69, while at the same temperature this ratio for
p-nitrophenyl phosphorothioate is 7500.
q
group in the phosphorothioate ester. The higher ∆H for the
The kinetic effect of sulfur substitution in one of the
nonbridging oxygen atoms (termed a “thio effect”) for the
p-nitrophenyl monoester will be pH sensitive due to the
differences in the pH-rate profiles of the two species. The ratio
phosphorothioate reaction may also partly be due to the absence
of favorable bonding-formation interactions with the nucleophile
that are present in the transition state of the ANDN aqueous
reaction of pNPP but which are absent in the transition state of
the rate-limiting step of the DN + AN reaction of pNPPT. The
internal energy of bond fission in the concerted reaction of pNPP
is the sum of the enthalpic cost of extensive P-O bond cleavage
to the leaving group, partially compensated for by the favorable
bond formation interaction with the nucleophile. The other
interactions which contribute to the enthalpy of the reaction are
hydrogen bonding interactions with the solvent. The very similar
free energies of solvation by water for pNPP and pNPPT
indicate that these compounds are very similarly solvated,
making it seem unlikely that differences in solvation contribute
of rate constants for pNPPT/pNPP has been reported to equal
1
4
8 at 70 °C and pH 8.0, and is 63 at 70 °C and pH 7.0.
However, these ratios are misleading because of differences in
the populations of the more reactive monoanionic and of the
less reactive dianionic forms of the phosphorothioate ester versus
the phosphate ester at these pH values. A better way to evaluate
the thio effect is to compare the rate constants for the dianions
of the two compounds in their respective pH-independent range,
and for the monoanionic species by comparing the rate constants
at the respective pH optima. Doing this with the values in Table
1
1
gives ratios for pNPPT/pNPP for the dianion hydrolysis of
2.6 (in agreement with the previously reported value of 12 )
q
4
significantly to the difference in ∆H .
The DN + AN mechanism for pNPPT hydrolysis avoids the
entropic cost of recruiting a solvent molecule into the transition
state of the rate-limiting step, and as a result the reaction of the
and for the monoanion hydrolysis of 1380. Thus while both
the monoanion and the dianion of pNPPT undergo hydrolysis
faster than the corresponding forms of pNPP, the difference is
much larger between the monoanion reactions than for the
dianion ones.
q
pNPPT dianion exhibits a much more favorable ∆S of +29 eu
compared to +3.5 eu for the reaction of the pNPP dianion. The
more favorable entropy term more than outweighs the enthalpic
The fit of the pH-rate data yields values of 3.68 ( 0.08 and
0.10 ( 0.11 for the pKa values of pNPPT. These results are
q
factor, and the net result is a reduction in ∆G by about 1.6
-
kcal/mol for pNPPT dianion hydrolysis.
in reasonable agreement with previously reported pKa values
of 3.6 and -0.24.16 By comparison, the pKa values for pNPP
are 4.96 and +0.30.21 The pH-independent hydrolysis of the
dianion of the phosphorothioate ester between pH 9.5 and 13
In comparing the monoanion reactions, the dominant energetic
reason for the faster rate of pNPPT is a lower enthalpy of
activation, 22.0 kcal/mol compared with 25.4 kcal/mol for pNPP.

2162 J. Am. Chem. Soc., Vol. 121, No. 10, 1999
Catrina and Hengge
In the monoanion reactions of both compounds the proton is
transferred from a nonbridge oxygen atom to the leaving group.
is considerable speculation that the complexation of metal ions
with the nonbridge oxygen atoms of the phosphate substrate
induces a mechanistic shift away from the dissociative reaction
seen in solution to an associative transition state with more
nucleophilic participation. For example, alkaline phosphatase
8
For leaving groups less basic than phenol, proton transfer is
8
probably concerted with leaving group departure. Since in the
dissociative transition states bond cleavage to the leaving group
2+
is considerable, the leaving group pKa’s in the two transition
requires the presence of two Zn ions. The fact that this enzyme
turns over phosphorothioates much more slowly than phosphate
monoesters has been cited as evidence that this enzyme follows
a different mechanism and transition state than that operative
q
states will be similar. The lower ∆H for the hydrolysis of the
monoanion of pNPPT probably reflects the lower pKa of the
thiophosphoryl group compared with that of the corresponding
phosphoryl group in the pNPP monoanion, resulting in a lower
enthalpic cost for deprotonation. The entropies of activation for
the reactions of the pNPPT and pNPP monoanions are fairly
similar, but that for the phosphorothioate ester is slightly more
favorable, which presumably reflects the fact that the monoanion
reaction proceeds largely via a thiometaphosphate mechanism
as revealed by stereochemical studies.29 The entropic advantage
of this pathway is considerably less in the monoanion reaction
than in the dianion reaction, which suggests that in the
monoanion reactions the major entropic problem is assuming
the proper geometry for the proton transfer. For geometric
reasons this probably occurs via an intervening water molecule
that would allow a six-membered transition state, rather than a
purely intramolecular process via a four-membered transition
state. The combination of enthalpic and entropic factors results
1-3
in the uncatalyzed hydrolysis, where this ratio of reactivities
is reversed. Alternative interpretations of these results have also
4
,5
been discussed.
We compared the effect of divalent metal ions on the aqueous
hydrolysis of pNPPT with prior studies of the effects of calcium
28
and magnesium ions on reactions of phosphate monoesters.
These metal ions each reduce the reactivity of the reactions of
phosphorylated pyridines with pyridine nucleophiles by about
2
-fold. The inhibition by the metal ions was attributed to
complexation with the nonbridge oxygen atoms, reducing their
28
ability to assist in expelling the leaving group. In contrast to
the reaction with pyridine nucleophiles the hydrolysis of the
2+
Mg complexes of phosphorylated pyridines, where the nu-
+
cleophile is a chelated Mg(OH) , are 4 to 6 orders of magnitude
faster than the second-order rate constants for the reaction of
q
in a reduction in ∆G by about 4.6 kcal/mol for the hydrolysis
5
uncomplexed pNPP with water. In that work, the hydrolysis
of the monoanion of pNPPT compared with pNPP.
2+
rate of the Mg complexes demonstrated a strong dependency
Solvent Effects. The addition of aprotic organic solvents
results in large rate accelerations for the aqueous hydrolysis of
the dianions of phosphate monoesters. The rate of the aqueous
hydrolysis of the dianion of pNPP is increased by 6 to 7 orders
of magnitude by added DMSO or HMPA.22 This acceleration
has been attributed to decreased ability of the mixed solvent to
participate in stabilizing hydrogen bonding interactions with the
anionic nonbridge oxygen atoms of the reactant, and the possible
contribution of similar effects to enzymatic catalysis in phos-
phoryl transfer has been pointed out.22
on the concentration of hydroxide ion in contrast to the pH-
independent hydrolysis of free pNPP. In aqueous hydrolysis
2+
2+
reactions of the pNPP dianion, Ca increased the rate but Mg
28
slowed the rate by about 20%.
In the uncatalyzed aqueous reaction of the pNPPT dianion
there is no nucleophilic participation in the first step; however,
one might expect the Lewis acidity of the metal ion may
influence a mechanistic shift to more resemble the pNPP
reaction. Such coordination might disfavor electron donation
from the nonbridge electrons in the dissociative reaction, while
enhancing the electrophilicity of the phosphorus atom. To
examine the effects of divalent metal ions on the aqueous
hydrolysis of the dianion of pNPPT we chose to study the effects
The rate enhancement on the hydrolysis of the dianion of
pNPPT found to result from added DMSO closely mirrors the
previously reported behavior with pNPP.22 Figure 5 shows the
variation in the rate constants for the reactions of both the
monoanion and the dianion of pNPPT with the fraction of
DMSO up to 95%. Both the degree of rate enhancement and
the overall response to added DMSO found for the dianion of
pNPPT are very similar to those for pNPP. The activation
parameters for the reaction of the dianion of pNPPT in 95%
2+
2+
of added Mg and Cd . Magnesium ion coordinates to oxygen
in complexes with phosphorothioates, while cadmium binds to
25,26
sulfur.
The choice of metal ions for this study was limited
by the need for significant solubility of the metal in the pH
range necessary to study the dianion reaction. Some metal ions
which were soluble under initial conditions formed precipitates
during the course of the reaction.
q
DMSO reveal the acceleration to be enthalpic in origin: ∆H
)
22.9 ( 0.7 kcal/mol, ∼14 kcal/mol less than that for the
Solubility limitations required that the effect of the divalent
metal ions on the hydrolysis be examined at pH 8.5 and lower.
Both metal ions exhibited modest inhibitory effects. More
importantly, no increase in the hydrolysis rate occurs in the
aqueous reaction.
By contrast, the reaction of the monoanion of pNPPT is
22
slowed by added DMSO, as was found to be the case with
pNPP. The entropy of activation is less favorable in 95%
DMSO, which may reflect a less favorable environment for
setting up the hydrogen bonding network for transfer of the
proton from the thiophosphoryl group to the leaving group.
Unlike in the dianion reaction, this is not offset by a large
reduction in the enthalpy of activation. The modest reduction
2+
presence of Mg as the concentration of hydroxide ion increases
Figure 7). The data indicate that these metal ions do not catalyze
the reaction of pNPPT with metal-bound hydroxide as occurs
(
2
+
with Mg complexes of phosphorylated pyridines. The slight
decrease in hydrolysis rate of the metal-pNPPT complexes is
most likely due to the inhibitory effect on electron donation
from the nonbridge oxygen atoms as inferred in the reactions
of phosphorylated pyridines. Thus the hydrolysis reactions of
the metal ion-pNPPT complexes likely remain fully dissociative
DN + AN mechanisms as is the case with free pNPPT. The
reduction in aqueous hydrolysis rate induced by these metal
ions in these solution experiments is much smaller than the 100-
fold slower rate of reaction of phosphorothioate monoesters with
alkaline phosphatase than with phosphate monoesters, so the
q
in ∆H from 22.0 to 18.8 kcal/mol is insufficient to offset the
q
less favorable ∆S , and thus the net result is a slightly larger
q
∆
G in 95% DMSO versus the aqueous reaction.
Effect of Divalent Metal Ions. The effect of metal ions on
the hydrolytic reactions of phosphate monoester dianions is of
interest because many phosphatases are metalloenzymes. There
(
29) Harnett, S. P.; Lowe, G. J. Chem. Soc. Chem. Commun. 1987, 1416-
1
418.

Phosphorothioate and Phosphate Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 10, 1999 2163
Mg2 and Cd , which coordinate preferentially with oxygen
and with sulfur, respectively, each have small inhibitory effects
on the hydrolysis of the dianion. Thus the much slower
enzymatic rate of catalysis by alkaline phosphatase of phos-
phorothioates compared with phosphates is not due simply to
the innate effects of metal ion complexation with the thiophos-
phoryl substrate. One could postulate that in the enzymatic
environment this complexation could be considerably stronger,
although the significantly greater KM values for phosphorothioate
versus phosphate monoesters do not support stronger coordina-
tion of phosphorothioates.
+
2+
slower enzymatic rate is not due simply to the innate effect of
metal ion complexation with the thiophosphoryl substrate.
Conclusions
The substitution of sulfur for a nonbridging oxygen atom in
the p-nitrophenyl phosphate monoester increases the rate of the
monoanion hydrolysis reaction (1380-fold) much more than that
of the dianion (12.6-fold). Although sulfur forms hydrogen
bonds much more weakly than oxygen, the free energies of
solvation of the phosphate and phosphorothioate are nearly the
same and ground state destabilization is not a significant
contributor to the difference in hydrolysis rates of the dianions.
The lower activation energy for hydrolysis of the phospho-
rothioate dianion arises from a more favorable entropy of
activation due to the switch to the fully dissociative DN + AN
mechanism; in contrast, the reaction of the monoanion of the
phosphorothioate is faster than that of the phosphate ester due
to a lower enthalpic barrier. The dianion reaction is significantly
accelerated by increasing fractions of DMSO which causes a
lowering of the enthalpic barrier, which is most likely due to
disruption of stabilizing hydrogen bonding interactions between
the dianionic reactant and the solvent. Similar effects could be
one contribution to the catalysis of the reactions of phosphate
dianions by phosphatases. In contrast, the monoanion reaction
is virtually unaffected by added DMSO. The divalent metal ions
Acknowledgment. Financial support of this work came from
NIH grant GM47297 to A.C.H. The authors thank Dr. V. D.
Parker for helpful discussions regarding the experiments on free
energies of solvation and Dr. W. W. Cleland for assistance in
fitting equilibrium and pH-rate data.
Supporting Information Available: Eyring plot for the
reactions of the monoanion and the dianion of p-nitrophenyl
phosphorothioate in 95% DMSO-water, and the table of rate
constants for aqueous pNPPT hydrolysis with pH (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA983862X