The spontaneous hydrolysis of 2-pyridyl phosphate is a good model for the special mechanism for the hydrolysis of phosphate monoester monoanions

Article abstract of DOI:10.1002/poc.3165

The microscopic pK_a of the pyridine N of the phosphate monoester 2-pyridyl phosphate (MPP) is 2.76, significantly higher than that expected for the leaving group (RO) oxygen of a typical monoester ROPO₃ ⁼. This favours the prototropic equilibrium considered to be the key step in the rapid hydrolysis of a typical monoester, and the observed rate of hydrolysis of the monoanion MPP^- is shown to be close to that expected for the protomer MPP⁺⁼, with neutral 2-pyridone as the leaving group. Copyright 2013 John Wiley & Sons, Ltd. The prototropic equilibrium is a key step in the hydrolysis of monoester MPP^-, allowing neutral 2-pyridone to be the leaving group from MPP⁺⁼. Copyright

Full text of DOI:10.1002/poc.3165

Special Issue Article
Received: 20 April 2013,
Revised: 14 May 2013,
Accepted: 05 June 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/poc.3165
The spontaneous hydrolysis of 2-pyridyl
phosphate is a good model for the special
mechanism for the hydrolysis of phosphate
†
‡
monoester monoanions
a
a
b
Michelle Medeiros , Alex M. Manfredi , Anthony J. Kirby *
a
and Faruk Nome **
a
The microscopic pK of the pyridine N of the phosphate monoester 2-pyridyl phosphate (MPP) is 2.76, significantly
=
higher than that expected for the leaving group (RO) oxygen of a typical monoester ROPO3 . This favours the
prototropic equilibrium considered to be the key step in the rapid hydrolysis of a typical monoester, and the ob-
À
+=
served rate of hydrolysis of the monoanion MPP is shown to be close to that expected for the protomer MPP , with
neutral 2-pyridone as the leaving group. Copyright © 2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this paper.
Keywords: phosphate monoesters; hydrolysis; S 2(P) mechanism
N
pre-equilibrium appears to account quantitatively for the ob-
INTRODUCTION
[
2]
served rates of the reaction.
We have been interested recently in the mechanisms of hy-
Two defining points for the reactivity of phosphate monoesters,
established many years ago, are the rate maximum in the region
of pH = 4 for the hydrolysis of the typical monoanion and the
very high sensitivity of the rates of hydrolysis of the dianion to
[
4,7]
drolysis of esters of 2-hydroxypyridine.
Tri-2-pyridyl phos-
phate is hydrolysed to the di-pyridyl DPP without significant
involvement of the pyridine nitrogens, because these are very
[
1–3]
[4]
the pK
a
of the leaving group.
The high sensitivity observed
weakly basic in the triester (pK
(pK = 2.73) pyridine nitrogens of DPP do appear to play a role,
a
a
= À0.22). But the more basic
for the reactions of phosphate ester dianions, combined with a
sensitivity to the basicity of the nucleophile that is close to zero,
can be understood in terms of a transition state for the S
actions of monoester dianions characterised by weak bonding to
both incoming nucleophile and leaving group (Scheme 1).
Our recent work on the reactivity of phosphate triesters has fo-
cussed attention on the contributions of the non-leaving groups
to the phosphate transfer process. In the case of triesters, this
can be comparable with that of the leaving group itself, while
the single non-leaving group present in the corresponding reac-
as general bases, in the hydrolysis of the diester. Which is
hydrolysed at 25 °C some 10000 times faster than expected for a
N
2(P) re-
[
5]
diaryl phosphate with leaving groups of pK = 9.09. The immedi-
a
[
2,3]
ate product of this reaction (Scheme 4) is the monoester 2-pyridyl
phosphate (MPP), but this is hydrolysed faster than DPP over al-
most the complete pH region (Fig. 1), and so does not accumulate
[
4]
* Correspondence to: A. J. Kirby, University Chemical Laboratory, Department of
Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
E-mail: ajk1@cam.ac.uk
[
5]
tions of diesters has minimal effect on reactivity. This simple
analysis excludes reactions of monoesters because there are no
non-leaving groups present. None, that is, apart from the OH
*
* Correspondence to: F. Nome, Departamento de Química, Universidade Federal
de Santa Catarina, Florianópolis, SC, 88040-900 Brazil.
E-mail: Faruk.Nome@ufsc.br
À
and O groups attached to P, which are always present and can-
not be varied except by isotopic substitution. However, these
groups also have major effects on reactivity and are indeed re-
sponsible for the defining points referred to earlier.
The high sensitivity to leaving group capability for the hydro-
lysis of monoester dianions, which makes esters of phenols with
†
This article is published in the Journal of Physical Organic Chemistry as a special
issue on In Memoriam, edited by Michael Page, University of Huddersfield,
Chemistry, Queensgate, Huddersfield, HD1 3DH, United Kingdom.
‡
[
2]
This paper is part of a special edition dedicated to the memory of Professor
Rory More O'Ferrall.
pK < 5.45 more reactive than the corresponding monoanions,
a
is the result of the multiple n–σ*P—O interactions available to a
dianion, which lengthen and weaken the P—OAr bond in the
ground state (Scheme 2).
The same mechanism is thought to account also for the much
faster hydrolysis of alkyl monoester monoanions, which are
believed to react via an initial proton transfer equilibrium to
a M. Medeiros, A. M. Manfredi, F. Nome
Departamento de Química, Universidade Federal de Santa Catarina,
Florianópolis, SC, 88040-900, Brazil
[
6]
b A. J. Kirby
University Chemical Laboratory, Department of Chemistry, University of
Cambridge, Cambridge CB2 1EW, UK
+
=
+=
form the highly reactive (ROH) PO
3
form (M , Scheme 3). This
J. Phys. Org. Chem. (2013)
Copyright © 2013 John Wiley & Sons, Ltd.

M. MEDEIROS ET AL.
O(–)
O
O
+
O
O
–
O
–
–
(
+)
(–)
OAr
+
–
Nu:
P
OAr
Nu
P
Nu
P
O
+
OAr
–
-2
O
–
O
N
Scheme 1. The transition state for S 2(P) reactions of the dianions of
phosphate monoesters is characterised by weak bonding to both leaving
group and incoming nucleophile
-
-
-
4
6
8
Scheme 2. Multiple n–σ*P—O interactions assist the cleavage of mono-
ester dianions
-10
Scheme 3. The prototropic pre-equilibrium mechanism that accounts for
-2
0
2
4
6
8
10
12
14
the pH-rate maximum for the hydrolysis of a typical phosphate monoester
pH/H0
Figure 1. pH-rate profiles for the spontaneous hydrolysis reactions of
monoester 2-pyridyl phosphate (MPP) and DPP, at 25 °C and ionic
strength 1.0 M (KCl). The points are experimental, and the curves are cal-
culated using the data of Tables 1 and S.1 (Supporting Information)
were followed, at constant temperature and ionic strength 1.0 M (KCl), by
monitoring the appearance of pyridone at 294 nm for 4 days on a Varian
Cary 50 spectrophotometer (Mulgrave, Victoria, Australia) equipped with a
thermostatted cell holder. Rate constants were obtained by fitting to the first
order equation, or, where appropriate, by the initial rate method.
Scheme 4. The hydrolysis of the DPP monoanion to inorganic
phosphate
[5]
at pH's below 10. We needed to understand the kinetic proper-
ties of the monoester to calculate the accurate rate constants for
the hydrolysis of DPP, and here report the details of this work,
which offers a unique insight into the general mechanism of
Scheme 3 for the hydrolysis of phosphate monoesters.
RESULTS AND DISCUSSION
The hydrolysis of monoester 2-pyridyl phosphate
The pH-rate profile (Fig. 1) for the hydrolysis of MPP shows an
acid-catalysed reaction at low pH, minimal reactivity at high pH
and the rate maximum near pH = 4 expected for the hydrolysis
of a phosphate monoester.
EXPERIMENTAL SECTION
The pH-rate profile reflects both the mole fractions of the var-
ious ionic forms present (Scheme 5) and their reactivity as func-
tions of pH. We consider the five distinct ionic species shown in
General
Organic solvents were carefully dried, and synthetic reactions carried out
in strictly anhydrous conditions under argon. Chemicals and inorganic
salts were of the highest purity available and were used as purchased.
Scheme 5, which includes the pK
a
s obtained by analysing the
kinetic results shown in Table 1.
+
The reactions at low pH, involving the cation MPP and the
Monoester 2-pyridyl phosphate
±
zwitterion MPP , proceed at rates similar to the hydrolysis of
The MPP was prepared by hydrolyzing the pure lithium salt of the diester
the diester DPP, as might be expected. The predominant ionic
form between pH = 2.62 and 5.62, the region of the rate maxi-
mum of Fig. 1 near pH = 4, is the monoanion MPP . As discussed
[5]
DPP at 80 °C in 1 M KOH and diluting to give a stock solution. The
monoester dianion does not react at a significant rate with hydroxide an-
ion and is stable under the conditions.
À
earlier, the rate maximum near pH = 4 is typical of a phosphate–
monoester, but the rate observed for the hydrolysis of MPP
(Table 1) is a great deal faster than that expected for an ester
with a comparable leaving group. (300 000 times faster, at 25°C,
than that observed for the hydrolysis of the monoanion of
Kinetic methods
Reactions were initiated by adding 10 or 30 μL of the stock solution of
À3
the substrate MPP (5 × 10 M) to 1 or 3 mL of buffered aqueous solution
[
2]
–
monophenyl phosphate at 39 °C. ) What is special about MPP is
the basic nitrogen of the 2-pyridyl leaving group, which makes
at the appropriate pH. Buffers used were HCl (pH < 2), chloroacetate
(pH = 2–3.5), acetate (pH = 4–5.5), BIS-TRIS (pH = 6–7), TRIS (pH = 8–9),
+=
À
CHES (pH = 8.7–10.4), carbonate (pH ≈ 10) and KOH (pH > 10). Reactions
the equilibrium K = [MPP ]/[MPP ] much more favourable.
e
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. (2013)

2
-PYRIDYL PHOSPHATE HYDROLYSIS
a
Scheme 5. The ionic forms of monoester 2-pyridyl phosphate (MPP) significant in the pH region. The apparent pK s shown are derived from the
kinetics (Fig. 1)
-
-
-
-
-
-
1
2
3
4
5
6
Table 1. Kinetic data (rate constants and pK s) derived from
the pH-rate profile of Fig. 1 for the hydrolysis of monoester
a
2
-pyridyl phosphate (MPP), at 25 °C and ionic strength 1.0 M
(KCl)
À1
Ionic form
pK
a
(observed)
khyd/s
+
MPP
À1.02 ± 0.3
2.62 ± 0.3
5.62 ± 0.2
2.87 ± 1.04e-2
1.02 ± 0.70e-3
1.32 ± 0.38e-2
±
MPP–
+=
MPP ⇌ MPP
Full kinetic data appear in Table S.1 of the Supporting
2
À
Information. The dianion MPP
is stable to hydrolysis
+
±
under the conditions used, while MPP and MPP carry the
reaction in strong acid and near pH = 0, respectively.
2
3
4
5
6
7
8
9
10
pH (pD)
Figure 2. The hydrolysis of monoester 2-pyridyl phosphate (MPP) in the
region of the pH-rate maximum shows an inverse solvent deuterium iso-
tope effect
Scheme 6. Microscopic dissociation constants for the ionic forms of
monoester 2-pyridyl phosphate (MPP) significant in the pH region
a
Scheme 7. Kinetic (blue) and microscopic (red) pK s derived for the various ionic forms of monoester 2-pyridyl phosphate (MPP) present in the pH
region. See the text
J. Phys. Org. Chem. (2013)
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M. MEDEIROS ET AL.
+
=
+=
Scheme 8. MPP , with a partial positive charge on the leaving group oxygen, is kinetically equivalent to the O-protonated form M of a phosphate
monoester (Scheme 3)
+
=
The curve fit for the pH-rate profile for the hydrolysis of the
monoester MPP (Fig. 1) gives a reasonable set of rate constants
system with a much less basic leaving group than MPP , and
an unfavourable equilibrium constant for the prototropic
À11
and three apparent pK
gion, pK and pK , are composed of pairs of microscopic con-
stants. Of the four ionic forms present in water near pH = 7
a
s (Table 1), of which the two in the pH re-
pre-equilibrium of the order of 10 . The estimated rate
constant was one order of magnitude larger than necessary
to account for the observed rate. The corresponding pre-
equilibrium for the MPP system is unfavourable by only some
2
3
[
2]
+
=
(
Scheme 6), MPP is of particular interest as it can be cleaved
by a uniquely readily accessible version of the mechanism
a
3 pK units (Scheme 5), so that the equivalent calculation can
(
Scheme 3) generally accepted for the hydrolysis of the
be made with greater confidence. The convincing conclusion
is the same in both cases that M is the species that carries
[
2]
+=
monoanions of phosphate monoesters.
The mechanism of Scheme 3 involves a pre-equilibrium
protonation of the weakly basic oxygen of the leaving group of
the monoester dianion, to generate very small amounts of the
the reaction.
For an independent test of this mechanistic conclusion, we
measured the solvent deuterium isotope effect for the hydrolysis
of MPP in the region of the pH-rate maximum. The pH-rate profile
(Fig. 2) indicates clearly that the kinetic isotope effect is inverse:
curve fitting gives a ratio kH2O/kD2O = 0.73 ± 0.22, consistent with
the value close to unity expected for the proposed prototropic
pre-equilibrium of Scheme 3 but inconsistent with a mechanism
+=
kinetically equivalent, highly reactive species M , with the very
2
À
good leaving group ROH attached to the PO3 phosphorus-
centre. In the MPP system, the corresponding equilibrium
–
+=
MPP ⇌ MPP (K , Scheme 6) is substantially more favourable,
e
because the pK s of the phosphate OH and the pyridinium N of
a
±
À
the leaving group of MPP (K22 and K , Scheme 6) are much
involving the spontaneous attack of solvent water on MPP .
21
closer. We can derive reliable values for these microscopic pKas
from estimates of the pK s of the phosphoric acid groups of the
a
À
±
–
ArOPO OH systems of MPP and MPP , as follows. Measured
pK values for a series of monoaryl phosphates ArOPO OH
2
CONCLUSIONS
À
a
2
show a good linear free energy relationship with the pK s of
the parent phenols ArOH. This correlation provides estimates
for pK22 and pK31 (Scheme 6) of 3.17 and 5.46, respectively,
a
The positive charge produced by protonation of the pyridine
[
8]
2
À
N of MPP is partially delocalised onto the leaving group
oxygen (Scheme 8), making the system a simple model for
the kinetically equivalent but far less accessible O-protonated
based on the literature values of 9.09 and 0.75 for the pK s of
a
[
9]
+=
2
-hydroxypyridine and its conjugate acid. Full details are
intermediate M of Scheme 3, considered to account for the rate
maximum in the region of pH= 4 observed for the hydrolysis of
phosphate monoesters.
presented in the Supporting Information.
The complementary values of pK21 and pK32 are readily obtained
2 3
from the expressions K = K21 + K22 and 1/K = (1/K31 + 1/K32) as 2.76
and 5.11, respectively. This allows the calculation of the
–
+=
equilibrium constant K
e
for MPP ⇌ MPP
as 0.39 ± 0.08
Acknowledgements
(
Scheme 7), indicating that 28% of the monoanion is present in
+=
what we can assume to be the reactive form MPP
.
We are grateful to INCT-Catálise, PRONEX, FAPESC, CNPq, and CAPES
in Brazil for support of this work.
The rate constant obtained for the hydrolysis of the monoanion
À
À1
of the monoester MPP (Table 1) is 1.32 ± 0.38 e-2 s : so if the
reaction is carried out – as we would expect – exclusively by the
+
=
2
8% of MPP present, this must be hydrolysed with a rate con-
REFERENCES
À1
stant of 4.7± 0.6e-2 s . The linear free energy relationship for
the dependence of the rate constant for hydrolysis of the dianions
[
[
1] W. W. Cleland, A. C. Hengge, Chem. Rev. 2006, 106, 3252.
2] A. J. Kirby, A. G. Varvoglis, J. Am. Chem. Soc. 1967, 89, 415.
of phosphate monoesters on the pK
the leaving group (log khyd = 0.70 ±0.44–1.19 ± 0.087pKLG
a
of the conjugate acid of
[3] J. K. Lassila, J. G. Zalatan, D. Herschlag, Annu. Rev. Biochem. 2011,
[2]
À1
8
0, 669.
s
at
[
4] A. J. Kirby, M. Medeiros, P. S. M. Oliveira, E. S. Orth, T. A. S. Brandão,
3
9 °C; see Supporting Information) predicts a rate constant
E. H. Wanderlind, A. Amer, N. H. Williams, F. Nome, Chem-Eur. J.
+=
À1
for the hydrolysis of MPP of 0.64 s at 39 °C, corresponding at
2
011, 17, 14996.
À1
25 °C to a figure of 0.13 (between 0.04 and 0.41) s . This is in rea-
[5] A. J. Kirby, M. Medeiros, J. R. Mora, P. S. M. Oliveira, T. A. S. Brandão,
A. Amer, N. H. Williams, F. Nome, J. Org. Chem. 2013, 78, 1343.
[6] A. J. Kirby, Adv. Phys. Org. Chem., 1994, 29, 87
sonable agreement with the observed value of approximately
À1
0
.05 s . (For a detailed discussion of the short extrapolation lead-
[
7] A. J. Kirby, M. Medeiros, P. S. M. Oliveira, T. A. S. Brand¼o, F. Nome,
Chem-Eur. J. 2009, 15, 8475
ing to this conclusion, see the Supporting Information).
The original calculation supporting the mechanism of Scheme 3
was for the hydrolysis of the methyl phosphate monoanion, a
[
8] N. Bourne, A. Williams, J. Org. Chem. 1984, 49, 1200.
[9] A. Albert, J. N. Phillips, J. Chem. Soc. 1956, 1294.
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