Questioning the paradigm of metal complex promoted phosphodiester hydrolysis: [Mo7O24]6- polyoxometalate cluster as an unlikely catalyst for the hydrolysis of a DNA model substrate

Article abstract of DOI:10.1039/b714860g

The first example of a phosphodiester bond cleavage promoted by a highly negatively charged polyoxometalate cluster has been discovered: the hydrolysis of the phosphodiester bond in a DNA model substrate bis(p-nitrophenyl)phosphate (BNPP) is promoted by t

Full text of DOI:10.1039/b714860g

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Questioning the paradigm of metal complex promoted phosphodiester
hydrolysis: [Mo7O24]62 polyoxometalate cluster as an unlikely catalyst
for the hydrolysis of a DNA model substrate{
Els Cartuyvels, Gregory Absillis and Tatjana N. Parac-Vogt*
Received (in Cambridge, UK) 26th September 2007, Accepted 8th November 2007
First published as an Advance Article on the web 16th November 2007
DOI: 10.1039/b714860g
phosphoanhydride bonds in ATP.5 Although the complexation
The first example of a phosphodiester bond cleavage promoted
between [Mo7O24]62 and phosphate-containing compounds has
by a highly negatively charged polyoxometalate cluster has
been well established, the interactions between [Mo7O24]62 and
been discovered: the hydrolysis of the phosphodiester bond in a
bis-substituted phosphates, including phosphodiesters, have been
DNA model substrate bis(p-nitrophenyl)phosphate (BNPP) is
promoted by the heptamolybdate anion [Mo7O24]62 with rates
virtually unexplored, most likely because they have been presumed
to be extremely weak or virtually non-existent.6a
which represent an acceleration of nearly four orders of
magnitude compared to the uncatalyzed cleavage.
The mechanism by which metal complexes accelerate the rate of
phosphate ester hydrolysis is generally described as an interplay of
several factors: (i) Lewis acid activation through the coordination
of phosphoryl oxygen(s) to the metal ion, (ii) nucleophile
activation of coordinated water or hydroxide ligand, (iii) leaving
group activation via coordination of the leaving group oxygen to
the metal, (iv) general base catalysis of solvent water through
metal-coordinated hydroxide ligand and (v) acting of metal
The [Mo7O24]62 is formed by acidification of aqueous
coordinated water molecules as an intramolecular general acid
catalyst.1 As a result, metal complexes that act as artificial
molybdate solutions to pH below 6 (eqn (1)). Its structure has
been confirmed by numerous studies both in solution and in the
solid state.6 The thermodynamic parameters published elsewhere
overall positive charge, free coordination site for the binding of
phosphoesterases have to meet a number of criteria such as an
can be used for estimation of [Mo7O24]62 concentration under
phosphoryl oxygen, the presence of coordinated water or
different reaction conditions.6,7 It has to be noted that the accurate
hydroxide, and the presence of functional groups for acid–base
experimental determination of [Mo7O24]62 concentration is still
catalysis, among others.1,2 In recent years a range of such
complexes, containing most notably Co3+, Fe3+, Zn2+, Cu2+
,
rather difficult, although models for the speciation of molybdate
Zr4+, Ln3+, have been shown to exhibit remarkable activity toward
phosphodiester cleavage.3 With the exception of the work by
Yatsimirsky and his co-workers, in which charge neutral
lanthanide complexes have been implicated as hydrolytically active
species3h it has been generally assumed that only positively charged
metal complexes can act as artificial phosphoesterases. However,
in this study we demonstrate, that despite its high negative charge,
coordination saturation, and lack of coordinated water or
hydroxide, the polyoxomolybdate [Mo7O24]62 cluster efficiently
hydrolyzes the phosphodiester bond in commonly used DNA
model substrate bis(p-nitrophenyl)phosphate (BNPP).
solutions are well-established and based on reliable and repro-
ducible data.{
7[MoO4]22 + 8H+ = [Mo7O24]62 + 4H2O
(1)
The cleavage of phosphodiester model substrate BNPP
proceeded smoothly in the presence of an equimolar amount of
Na6[Mo7O24] (25 mM, pH 5.5, 50 uC). The release of 2 equivalents
of p-nitrophenol (NP) monitored by 1H NMR spectroscopy
afforded calculation of the rate constant for the cleavage of BNPP
(kobs = 2.3 ¡ 0.1 6 1026 s21 at 50 uC). This represents an
acceleration of almost four orders of magnitude compared to the
uncatalysed cleavage of BNPP (kobs = 3 6 10210 s21).8 The
absence of any paramagnetic species during the course of reaction
excludes the possibility of oxidative cleavage and the reduction of
Mo(VI) to Mo(V). The monophasic kinetic profile suggests that the
cleavage of the first phosphoester bond in BNPP is much slower
than the cleavage of the phosphoester bond in the first product of
hydrolysis (p-nitrophenyl)phosphate (NPP), and is the rate
determining step. Indeed, the separate experiments involving
NPP and Na6[Mo7O24] showed that the cleavage of the
phosphoester bond in NPP proceeded nearly 40 times faster than
the cleavage of the phosphodiester bond in BNPP.
Our interest in the hydrolytic properties of polyoxomolybdate
has been provoked by several reports describing the interaction of
[Mo7O24]62 with mono-substituted phosphates, including nucleo-
tides.4 It has been recognized long ago that the presence of
molybdate leads to hydrolysis of the labile ‘‘high energy’’
Katholieke Universiteit Leuven, Department of Chemistry,
Celestijnenlaan 200F, B-3001 Leuven, Belgium.
E-mail: Tatjana.Vogt@chem.kuleuven.be; Fax: +32 16 327992;
Tel: +32 16 32761
{ Electronic supplementary information (ESI) available: 95Mo NMR
spectrum of the sample. See DOI: 10.1039/b714860g
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The effect of the pH on the cleavage reaction was examined in
order to correlate the rate–pH profile with the species distribution
diagram.§ As seen in Fig. 1, the pH dependence of the kobs exhibits
a bell-shaped profile, with the fastest cleavage observed at pH =
5.3. While depending on the conditions, slightly different
speciation models can be found in the literature, however, there
is very little doubt that at concentrations of molybdate greater
than 1 mM [Mo7O24]62 is the only polyoxoanion that exists at
pH = 5.3.6 Indeed, 95Mo NMR spectroscopy, which recently has
been proved as an excellent and accurate tool for the examination
of equilibria involving molybdate,9 showed the presence of a peak
at d = 34 ppm, confirming that at pH 5.3 [Mo7O24]62 is the only
polymolybdate form present in solution. Comparison of the rate
profile with the concentration profile of molybdate species, whose
existence has been previously well established in solution, shows
striking overlap of the kobs profile with the concentration of
hydrolytic activity of [P2Mo5O23]62 toward BNPP was also tested,
and no observable cleavage of the phosphoester bond occurred
even after prolonged reaction times. Variable temperature 31P
NMR spectra of a mixture containing BNPP and [Mo7O24]62
recorded immediately upon mixing, revealed broadening of the
BNPP 31P resonance upon an increase of temperature, suggesting
a dynamic exchange process between free and bound BNPP at
higher temperatures (Fig. 2).
The binding constant between BNPP and the [Mo7O24]62 was
determined by carrying out kinetic experiments using a fixed
amount of BNPP and increasing amounts of [Mo7O24]62 at pH 5.3
and 50 uC (Fig. 3).
By taking into account the fact that the release of the first
equivalent of NP from BNPP is the rate limiting step (eqn (2)), the
values for the rate constant for cleavage of the phosphodiester
bond k2 (3.33 6 1026 s21), and the association constant for the
BNPP–[Mo7O24] complex K1 (28.7 M21), were obtained by a
[Mo7O24]62 6,7,9
Although within the whole range of examined pH
.
other species, such as MoO422 and [Mo8O26]42 as well as various
protonated forms of them, are present in solution, they seem to be
catalytically much less active. Since at pH 8, where MoO422 is the
only Mo(VI) species found in solution, BNPP hydrolysis hardly
occurred it is reasonable to assume that this monomeric complex
does not cause hydrolysis. Similarly, slow hydrolysis at the lower
pH values implies low catalytic activity of [Mo8O26]42 which is the
most abundant polyoxoanion in the pH range from 2 to 4.6,7,9
The significant increase of the rate of BNPP hydrolysis in the
presence of heptamolybdate at pH = 5.3 implies that interaction
between [Mo7O24]62 and BNPP must take place in the solution.
The hydrolysis reaction followed by 31P NMR spectroscopy
revealed that upon addition of Na6[Mo7O24] to BNPP the 31P
NMR signal of BNPP shifted only slightly upfield by 0.15 ppm.
During the course of hydrolysis the concentration of the initial
complex decreased, and appearance of NPP, which is the first
product of hydrolytic reaction was observed. Interestingly, 31P
NMR spectra did not show evidence for the presence of inorganic
phosphate, which is expected as the final product of the hydrolytic
reaction. Instead, the final species present in solution had the same
spectroscopic features as a separately prepared pentamolybdodi-
phosphate ion [P2Mo5O23]62, which typically forms in mildly
acidic solutions containing molybdate and phosphate ions.10 The
computer generated least-squares fit of eqn (3) to kobs
.
ð2Þ
6{
k2½Mo7O24 ꢀ0
kobs
~
(3)
6{
k{1=k1z½Mo7O24 ꢀ0
At first glance the interaction between [Mo7O24]62 and BNPP
seems counterintuitive, since highly negatively charged metal
clusters are not expected to bind or interact with the negatively
charged anions. However, the anion–anion interactions in
polyoxometalate chemistry are not without precedent, given that
interaction between negatively charged polyoxometalate anions
have been previously detected both in solid state and in solution.11
The intriguing question is then by which mechanism does the
cleavage of the phosphoester bond occur?
Although the bell-shaped pattern of the pH profile is usually
indicative of either a deprotonation step of two acidic functions in
the metal complex which have opposite effect, or an equilibrium
involving metal bound water and metal bound hydroxide that acts
as an active nucleophile,1,2 both mechanisms are highly improb-
able since [Mo7O24]62 does not contain any acidic functions or
coordinated water ligands.6 In addition, the polyoxometalate
anions are known to be very weak conjugate bases with low
negative charge densities.6
Fig. 1 pH dependence of kobs (&, solid line) for the cleavage of BNPP
(25 mM) in the presence of Na2MoO4 (175 mM starting conc.). Fractions
of Mo species have been added for comparison (points taken from ref. 9).
Fig. 2 Half-width (n1/2) of the BNPP 31P NMR resonance in the
presence of Na6[Mo7O24] as a function of temperature.
86 | Chem. Commun., 2008, 85–87
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experiments are not available, an approximation had to be made by
applying the thermodynamic parameters that were obtained in 0.6 M
NaClO4 solution.
§ The pH experiments were performed in the absence of any buffer. The
pH of the solutions was measured before and after the hydrolysis reaction
and the difference in pH was always less than 0.1 pH unit. The hydrolysis
of BNPP in the absence of metal complex was measured at each pH and
the final values of the kobs have been adjusted for the background cleavage.
" Addition of equimolar amounts of phenyl phosphonate or methyl
phosphonic acid inhibited BNPP hydrolysis (kobs = 1.1 6 1026 and 9.1 6
1027 s21, respectively) suggesting competition for binding to [Mo7O24]62
.
I At 37 uC only 30% of BNPP was cleaved after 70 h, and at room
temperature the BNPP hydrolysis was extremely slow implying very weak
interactions with [Mo7O24]62
.
1 N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res.,
1999, 32, 485.
2 (a) E. L. Hegg and J. N. Burstyn, Coord. Chem. Rev., 1998, 173,
133; (b) J. R. Morrow and O. Iranzo, Curr. Opin. Chem. Biol., 2004, 8,
192.
Fig. 3 Influence of [Mo7O24]62 concentration on kobs for cleavage of
BNPP (20 mM, pH = 5.3, T = 50 uC).
3 (a) F. Mancin, P. Scrimin, P. Tecilla and U. Tonellato, Chem. Commun.,
2005, 2540 and references therein; (b) R. Ott and R. Kra¨mer, Angew.
Chem., Int. Ed., 2000, 39, 3255; (c) A. M. Fanning, S. E. Plush and
T. Gunnlaugsson, Chem. Commun., 2006, 3791; (d) G. Feng, D. Natale,
R. Prabaharan, J. C. Mareque-Rivas and N. H. Williams, Angew.
Chem., Int. Ed., 2006, 45, 7056; (e) H.-J. Schenider, J. Rammo and
R. Hettich, Angew. Chem., Int. Ed. Engl., 1993, 32, 1716; (f)
A. O’Donoghue, S. Y. Pyun, M. Y. Yang, J. R. Morrow and
J. P. Richard, J. Am. Chem. Soc., 2006, 128, 1615; (g) M. Komiyama,
S. Kina, K. Matsumura, J. Sumaoka, S. Tobey, V. M. Lynch and
E. Anslyn, J. Am. Chem. Soc., 2002, 24, 13731; (h) F. Aguilar-Perez,
P. Gomez-Tagle, E. Collado-Fregoso and A. K. Yatsimirsky, Inorg.
Chem., 2006, 45, 9502.
4 (a) D. E. Katsoulis, A. N. Lambriandou and M. T. Pope, Inorg. Chim.
Acta, 1980, 46, 55; (b) C. F. G. C. Geraldes and M. M. C. A. Castro,
J. Inorg. Biochem., 1986, 28, 319; (c) P. Piperaki, N. Katsaros and
D. Katakis, Inorg. Chim. Acta, 1982, 67, 37; (d) L. M. R. Hill,
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Soc., 1975, 97, 5735.
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286; (b) E. Ishikawa and T. Yamase, J. Inorg. Biochem., 2006, 100,
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49, 121 and references therein.
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Inorg. Chim. Acta, 2002, 331, 322.
8 J. Chin, M. Banzszczyk, V. Jubian and X. Zou, J. Am. Chem. Soc.,
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We propose that the origin of hydrolytic activity of [Mo7O24]62
lies in its high internal lability and a known intramolecular
exchange which results in partial detachment of one MoO4
tetrahedron.9,12 This detachment may allow attachment of the
structurally related phosphodiester tetrahedron into the polyox-
ometalate structure, in a similar manner as in the well-known
process that occurs at higher temperatures in which tetrahedral
molybdate adds to heptamolybdate to yield an octamolybdate
structure.",12a Broadening of 31P NMR resonances of BNPP in the
presence of [Mo7O24]62 upon increase of temperature is consistent
with the interaction between BNPP and [Mo7O24]62 taking place
at elevated temperatures.I The incorporation of the phosphodie-
ster group into the polyxomolybdate skeleton, and sharing of
oxygen atoms with the Mo(VI) centre, may lead to bond strain and
cause polarization of the P–O ester bond and its activation toward
external attack by water. Our hypothesis that the hydrolytic
activity of [Mo7O24]62 may be due to its internal flexibility is
strengthened by the fact that the analogous and isostructural
[W7O24]62 polyoxometalate cluster, which is much more inert and
does not undergo fast intramolecular exchange,9 is virtually
hydrolytically inactive towards hydrolysis of BNPP.
In conclusion, we report the first example of a DNA model
phosphodiester bond cleavage promoted by a highly negatively
charged polyoxometalate cluster. The kinetic studies strongly
suggest that [Mo7O24]62 is the hydrolytically active complex and
that cleavage occurs by a mechanism which is different from that
of all currently known hydrolytically active metal complexes.
Studies involving other types of polyoxometalate complexes such
as artificial phosphoesterases are currently under way. Such studies
are of special interest considering numerous reports that express a
rapidly growing interest in the biological and medicinal application
of polyoxometalates.13
9 R. I. Maksimovskaya and G. M. Maksimov, Inorg. Chem., 2007, 46,
3688.
10 (a) R. Strandberg, Acta Chem. Scand., 1973, 27, 1004; (b)
¨
L. Pettersson, I. Andersson and L.-O. Ohman, Inorg. Chem., 1986,
25, 4726.
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1983, 22, 207; (b) M. Fournier, R. Thouvenot and C. Rocchiccioli-
Deltcheff, J. Chem. Soc., Faraday Trans., 1991, 87, 349.
12 (a) O. W. Horwath and P. Kelly, J. Chem. Soc., Chem. Commun., 1988,
1236; (b) O. W. Horwath and P. Kelly, J. Chem. Soc., Dalton Trans.,
1990, 81.
13 For an overview of the biological activity of polyoxomolybdates
see: (a) J. T. Rhule, C. L. Hill and D. A. Judd, Chem. Rev., 1998, 98,
327; (b) T. Yamase, J. Mater. Chem., 2005, 15, 4733; (c) B. Hasenknopf,
Front. Biosci., 2005, 10, 275; (d) H. U. V. Gerth, A. Rompel,
B. Krebs, J. Boos and C. Lanvers-Kaminsky, Anti-Cancer Drugs,
2005, 16, 101.
Notes and references
{ The ionic strength of the solution was not further adjusted, because it was
noticed that high salt concentration impedes hydrolysis. Since thermo-
dynamic parameters for the exact reaction conditions used in hydrolysis
This journal is ß The Royal Society of Chemistry 2008
Chem. Commun., 2008, 85–87 | 87