COMMUNICATION
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The hydrolysis of phosphate diesters in cyclohexane and acetonew
Randy B. Stockbridge and Richard Wolfenden*
Received 4th March 2010, Accepted 15th April 2010
First published as an Advance Article on the web 6th May 2010
DOI: 10.1039/c0cc00229a
The hydrolysis of phosphate diesters is one of the most difficult
reactions known. Here we show that in acetone or cyclohexane,
(10 mL) of cyclohexane with vigorous stirring overnight, after
which equilibrium was found to have been established. The
5
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concentrations of DNP and TBA in the cyclohexane phase
at 25 1C, phosphodiesters undergo hydrolysis 5 ꢀ 10 and
9
2
ꢀ 10 -fold more rapidly than in water, respectively, and that
were then determined by back-extracting the clear cyclohexane
1
this rate enhancement is achieved by lowering the enthalpy of
activation.
layer into D O (0.6 mL) for H NMR analysis (see ESIw for
2
additional experimental details). A second extraction of the
+
cyclohexane layer yielded no additional TBA or DNP . The
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The hydrolysis of unactivated alkyl phosphodiester monoanions,
such as those present in the backbone of DNA, proceeds with
resulting equilibrium constant for DNP transfer from water
+
ꢁ5
to cyclohexane was 3.9 (ꢂ0.4) ꢀ 10 at 25 1C, and TBA and
7
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a half-life of 3 ꢀ 10 years at 25 1C in the absence of a
DNP were present in the cyclohexane layer at equimolar
1
catalyst. The restriction enzyme EcoRV reduces that half-life
concentrations. The observed distribution coefficient was
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2
to 0.24 ms. Among the catalytic effects that may be responsible
unaffected by varying the concentration of DNP in the
for the rate enhancements produced by enzymes is desolvation
3
of the substrate, and recent experiments have shown that the
aqueous phase, indicating that the DNP was fully dissociated
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in the cyclohexane phase (ESIw). DNP :TBA transfer
became more favorable as temperature was raised from
10 and 50 1C, yielding a linear van’t Hoff plot (ESIw) that
was used to estimate the thermodynamic changes associated
extraction of a phosphate monoester dianion from water into
cyclohexane increases its second order rate constant for
12 4
hydrolysis by a factor of B10 . The extent to which nonpolar
surroundings might also accelerate phosphodiester hydrolysis
is unclear. The hydrolysis of a phosphate diester with a weakly
basic leaving group, bis(4-nitrophenyl) phosphate, has been
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with transfer of DNP :TBA from water to cyclohexane
(Table 1).
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To determine the rate of DNP hydrolysis in wet cyclo-
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shown to be accelerated B15-fold in DMSO, but little
hexane, portions (10 mL) of the cyclohexane layer (prepared
as described above) were incubated in Teflon-lined acid digestion
bombs (Parr Instruments Co. #276AC) for various time
intervals ranging from 16 hours to 21 days at temperatures
ranging from 130–210 1C. After heating, the cyclohexane layer
experimental information appears to be available about solvent
effects on the hydrolysis of unactivated phosphodiesters. We
therefore decided to examine the hydrolysis of dineopentyl
phosphate (DNP) in nonpolar solvents, for comparison with
1
its uncatalyzed hydrolysis in water. In this molecule, steric
was back-extracted with H
evaporated to dryness (the volatile neopentanol was removed
during that process), and the residue was dissolved in D O for
2
O (1 mL), the aqueous layer was
hindrance precludes nucleophilic attack at carbon. Here, we
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show that the tetrabutylammonium (TBA ) salt of DNP
2
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3
1
enters wet cyclohexane (containing 4.2 ꢀ 10 M water at
analysis by H NMR using added dioxan as an internal
integration standard. The extent of hydrolysis was determined
by monitoring the decomposition of the diester using the
integrated intensities of the peaks arising from DNP, and
separate experiments showed that reaction followed first order
kinetics in both cyclohexane and acetone at several temperatures.
The activation energies are such that hydrolysis of the monoester
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saturation) at concentrations sufficient to permit measurement
ꢁ
of its rate of hydrolysis. The results indicate that DNP
hydrolysis proceeds much more rapidly as the solvent becomes
more nonpolar, and that at 25 1C, the second order rate
constant for hydrolysis is enhanced by
a
factor of
ꢀ 10 when the reaction is transferred from water to wet
cyclohexane.
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2
4
(neopentyl phosphate, NP) in cyclohexane occurs almost
To determine the distribution coefficient of DNP from water
to cyclohexane, a small volume (0.1 mL) of an aqueous
instantaneously in the temperature range (130–210 1C) over
which DNP hydrolysis was monitored in the present experiments
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solution of DNP :TBA (0.05 M DNP Na , titrated to
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(Fig. 1B). Identical rate constants were obtained when the
extent of reaction was measured either by the disappearance of
DNP using proton NMR, or by the release of inorganic
phosphate determined spectrophotometrically using an acid
+
pH 9 with TBA OH ) was first extracted with a large volume
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8
Department of Biochemistry and Biophysics, University of North
Carolina, Chapel Hill, North Carolina 27599, USA.
E-mail: water@med.unc.edu; Fax: +1 919-966-2852;
Tel: +1 919-966-1203
molybdate procedure.
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To determine the rate of DNP hydrolysis in acetone,
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DNP :TBA was dissolved (0.01 M) in acetone containing
1% water (v/v), sealed in quartz tubes under vacuum, and
incubated for varying time periods ranging from 11 hours to
3 days at temperatures ranging from 155 to 230 1C. After
w Electronic supplementary information (ESI) available: Detailed
experimental methods; van’t Hoff analysis of equilibria for transfer
of DNP from water to cyclohexane; effect of ester concentration on
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ꢁ
distribution coefficients observed for transfer of DNP from water to
cyclohexane; detail of Arrhenius plots for DNP hydrolysis in cyclo-
hexane and acetone. See DOI: 10.1039/c0cc00229a
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incubation, reaction mixtures were diluted in DMSO-d for
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1
analysis by H NMR. The same results were obtained when
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306 | Chem. Commun., 2010, 46, 4306–4308
This journal is ꢃc The Royal Society of Chemistry 2010