Hydrolysis of the RNA model substrate catalyzed by a binuclear ZrIV-substituted Keggin polyoxometalate

Article abstract of DOI:10.1039/c5dt02077h

The reactivity and solution behaviour of the binuclear Zr^IV-substituted Keggin polyoxometalate (Et₂NH₂)₈[{α-PW₁₁O₃₉Zr(μ-OH)(H₂O)}₂]·7H₂O (ZrK 2 : 2) towards

Full text of DOI:10.1039/c5dt02077h

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Hydrolysis of the RNA model substrate catalyzed
IV
by a binuclear Zr -substituted Keggin
Cite this: DOI: 10.1039/c5dt02077h
polyoxometalate†
a
a
a,b
Thi Kim Nga Luong, Gregory Absillis, Pavletta Shestakova and
a
Tatjana N. Parac-Vogt*
IV
The reactivity and solution behaviour of the binuclear Zr -substituted Keggin polyoxometalate
2 2 8 2 2 2
(Et NH ) [{α-PW11O39Zr(µ-OH)(H O)} ]·7H O (ZrK 2 : 2) towards phosphoester bond hydrolysis of the
RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP) was investigated at different
reaction conditions (pD, temperature, concentration, and ionic strength). The hydrolysis of the phospho-
1
ester bond of HPNP, followed by means of H NMR spectroscopy, proceeded with an observed rate
constant, kobs = 11.5(±0.42) × 10− s−1 at pD 6.4 and 50 °C, representing a 530-fold rate enhancement in
5
1
31
comparison with the spontaneous hydrolysis of HPNP. H and P NMR spectra indicate that at these reac-
tion conditions the only products of hydrolysis are p-nitrophenol and the corresponding cyclic phosphate
ester. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest rate observed at pD 6.4.
= 455 M− ) and catalytic rate constant (k
1
= 42 × 10−5 s−1) for the HPNP–ZrK
The formation constant (K
f
c
) of 63.35 ± 1.82 kJ mol− , enthalpy of activation (ΔH ) of 60.60 ±
1
‡
2
2
: 2 complex, activation energy (E
a
.09 kJ mol− , entropy of activation (ΔS ) of −133.70 ± 6.13 J mol
(ΔG ) of 102.05 ± 0.13 kJ mol at 37 °C were calculated from kinetic experiments. Binding between ZrK
1
‡
−1 −1
K , and Gibbs activation energy
‡
−1
Received 1st June 2015,
Accepted 30th July 2015
31
2
: 2 and the P–O bond of HPNP was evidenced by the change in the P chemical shift and signal line-
DOI: 10.1039/c5dt02077h
31
31
broadening of the P atom in HPNP upon addition of ZrK 2 : 2. Based on P NMR experiments and
www.rsc.org/dalton
isotope effect studies, a mechanism for HPNP hydrolysis in the presence of ZrK 2 : 2 was proposed.
reactivity of newly developed artificial phosphatases and
Introduction
6
–12
nucleases.
The advantage of these model DNA substrates is
Polyoxometalates (POMs) are generally described as a large the presence of good leaving groups in their structures,
class of inorganic oxoclusters that contain early transition increasing their reactivity in comparison to for example DNA
metals (V, Nb, Ta, Mo and W) in their highest oxidation state. and RNA biomolecules. Phosphoester bond hydrolysis in RNA
POMs have applications in a broad range of research domains is an intramolecular transesterification, supported by the
1
,2
3
4,5
including material science,
medicine and catalysis.
attack of the 2′-OH group to the phosphate backbone. As a
IV
Recently our group discovered that a series of Zr -substituted result, RNA is hydrolyzed faster than DNA, which lacks such
POM complexes could act as artificial phosphosesterases. Zr
IV
internal nucleophile. Because of the presence of an OH-group
is ideally suited as an active center in artificial hydrolytic 2-hydroxypropyl-4-nitrophenylphosphate (HPNP, Scheme 1) is
metalloenzymes because of its high Lewis-acidity and oxo- often used as a RNA model substrate to test the reactivity of
1
3,14
philic properties, allowing both the coordination and acti- new artificial nucleases.
IV
vation of the substrate and nucleophile. Model DNA
compounds such as 4-nitrophenyl phosphate (NPP) and bis-4-
nitrophenyl phosphate (BNPP) are often used when testing the
A mononuclear Zr -substituted Wells–Dawson type POM
15H[Zr(α -P ]·25H O was demonstrated to be able to
K
2
2
W
17
O
61
)
2
2
a
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium.
E-mail: Tatjana.Vogt@chem.kuleuven.be; http://www.chem.kuleuven.be/lbc/
b
NMR Laboratory, Institute of Organic Chemistry with Centre of Phytochemistry,
Bulgarian Academy of Sciences, Acad. G. Bontchev Str., B1.9, 1113 Sofia, Bulgaria
†
Electronic supplementary information (ESI) available: Data on the percentage
31
of HPNP as a function of the reaction time, P NMR spectrum, kinetic data on
the influence of pD, temperature. See DOI: 10.1039/c5dt02077h
Scheme 1 Hydrolysis of HPNP promoted by ZrK 2 : 2.
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catalytically enhance the hydrolysis rate of the DNA model such as pD, temperature, initial concentration, ionic strength
8
12
compound NPP by nearly two orders of magnitude. In a pre- and substrate concentration, the speciation behavior of ZrK
IV
vious study we also found that the dinuclear Zr -substituted 2 : 2 at the conditions used to study HPNP hydrolysis was
Keggin type POM (Et
ZrK 2 : 2, Fig. 1) efficiently promoted hydrolysis of the extre- kinetic experiments and to propose a mechanism for HPNP
mely stable phosphodiester BNPP, with a 320-fold rate hydrolysis in the presence of ZrK 2 : 2 POM.
2
NH
2
)
8
[{α-PW11
O
39Zr(µ-OH)(H
2
O)}
2
]·7H
2
O
studied in detail. These results are needed to understand the
(
1
2
enhancement in comparison with spontaneous hydrolysis.
In addition, it was shown that the monomeric [α-PW O Zr-
1
1 39
4
−
(
2
µ-OH)(H O)] (ZrK 1 : 1) species is present in aqueous solu-
Results and discussion
Aqueous study of ZrK 2 : 2
tion at near-neutral pD and that this species is responsible for
the hydrolysis of the phosphoester bond in BNPP. ZrK 1 : 1 is
6
considered to be more catalytically active when compared to Depending on the pD, temperature, reaction time, starting
3
1
ZrK 2 : 2 as its Zr(IV) ion has more coordinated water molecules concentration, and ionic strength, ZrK 2 : 2 having a single
P
2
0
that can be replaced by the substrate or that can act as a resonance at −13.49 ppm (Fig. 1a) can convert into the cata-
3
1
nucleophile. Theoretical studies have shown that when ZrK lytically less active ZrK 1 : 2 (Fig. 1b), characterized by two
P
1
2,21,22
1
: 1 interacts with NPP or BNPP, it prefers to form monoden- resonances at −14.67 and −14.77 ppm.
However, recent
P DOSY study on a 3.0 mM aqueous solution of ZrK 2 : 2 also
showed the presence of the 1 : 1 Zr -substituted Keggin type
Although natural nuclease enzymes play a critical role in species (Fig. 1c) with a P resonance at −13.7 ppm. More
various biotechnology applications, efficient artificial importantly, the DFT calculations identified this species as the
3
1
tate complexes which are more stable than the corresponding
bidentate complexes.
6
IV
3
1
1
5
nucleases are still needed. For example, artificial restriction active species responsible for phosphoester bond hydrolysis in
enzyme can be made sequence specific by encoding sequence BNPP at pD 6.4 and 60 °C. Therefore, the aim of this aqueous
6
specificity into the cleavage agent or by linking it to an appro- solution study is to further optimize reaction conditions in
1
5
priate DNA binding agent. Such enzymes are not only very such a way that the ZrK 1 : 1 concentration is increased and to
useful for molecular biological field such as DNA cutting at a apply these conditions to determine the kinetics of HPNP
site not recognized by current restriction enzymes, but also for hydrolysis in the presence of ZrK 2 : 2. To the best of our
genomic analysis. In addition, artificial nucleases can be syn- knowledge the synthesis and isolation of pure ZrK 1 : 1 at
1
6
thesized in large amounts and are often used to elucidate neutral pD conditions has not been reported. ZrK 1 : 1 is only
1
5
6
the precise role of active metal ions in natural nucleases. The present as a single species in acidic solutions (pH ≤ 3.4),
metal ion accelerates the hydrolysis process by activation of while the monolacunary Keggin species, a precursor of ZrK
2
3
the phosphodiester bond, activation and/or delivery of the 1 : 1, only exists at near-neutral pH.
nucleophile, and by stabilization of the pentacoordination
transition state and the leaving group.
The effect of pD on the solution behaviour of 1.0 mM ZrK
2 : 2 was recently reported. It was concluded that ZrK 2 : 2 and
1
7,18
In this study we explore the phosphoesterase activity of ZrK ZrK 1 : 1 are present in equilibrium in solutions with pD of 5.4
: 2 towards the RNA model system HPNP, a stable phospho- and 6.4. At pD values higher than 6.4, the dominant species is
2
diester characterized by a half-life of hundreds of days at pH ZrK 2 : 2, while at pD values lower than 5.4, the dominant
1
9
6
8
.0 and 30 °C. As a previous study showed that the solution species is ZrK 1 : 1. In this study the effect of temperature on
speciation of this POM is highly dependent on several factors the speciation equilibria was determined at nearly neutral pD
pD 6.4).
A ZrK 2 : 2 solution was kept at temperatures ranging from
(
3
1
3
7 °C to 80 °C, before assessing its species distribution by
NMR. Fig. S1† shows that both ZrK 2 : 2 (−13.49 ppm) and
ZrK 1 : 1 (−13.7 ppm) are present after mixing for 1 h without
P
1
2
6
heating. Unfortunately, partial overlap of the ZrK 1 : 2 and ZrK
1
: 1 resonances makes integration and therefore quantifi-
cation difficult. Temperature has no significant effect on the
speciation of ZrK 2 : 2 after heating for 1 h up to 60 °C. From
70 °C upward only minor amounts (19%) of ZrK 1 : 2 were
detected, ZrK 1 : 1 is present up to 70 °C, and at 80 °C its inten-
sity decreases significantly.
To further observe the influence of concentration and incu-
bation time on the ZrK 1 : 1 species distribution, a 1.0 mM and
3
.0 mM solution of ZrK 2 : 2 at pD 6.4 were kept at 50 °C for a
Fig. 1 Structures of ZrK 2 : 2 (a), ZrK 1 : 2 (b) and ZrK 1 : 1 (c). The WO
groups are represented by green octahedra, while the internal PO
6
4
prolonged period of time. Fig. S2† indicates that for 1.0 mM of
ZrK 2 : 2 both ZrK 2 : 2 and ZrK 1 : 1 were detected after mixing
and ZrK 1 : 1 was still present in the solution after heating for
2
groups are represented by pink tetrahedra. Zr(IV), H O molecules and
OH group are represented by blue, violet and red balls respectively.
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7
h. For a 3.0 mM ZrK 2 : 2 solution it can be seen from
Fig. S3† that no ZrK 1 : 1 was detected after mixing and after
2
h 25 min, a very small amount of ZrK 1 : 1 was observed.
Previous studies show that ionic strength has an effect on
the POM solution behavior and/or the hydrolysis rate of
1
0,12,24,25
different molecules such as NPP, BNPP and dipeptide.
Therefore, the influence of ionic strength on the ZrK 1 : 1
species distribution was also studied for a 1.0 mM sample of
3
1
ZrK 2 : 2. P NMR spectra of a 1.0 mM solution of ZrK 2 : 2 in
the presence of increasing concentrations (0.25 M to 2.0 M) of
NaClO at pD 6.4 were recorded after mixing. Fig. S4† shows
4
4
that an addition of 0.25 M NaClO lead to the disappearance
of ZrK 1 : 1 signal while the signals of ZrK 2 : 2 and ZrK 1 : 2
were clearly detected. This means that NaClO4 can promote
1
2,26,27
the conversion of ZrK 1 : 1 into ZrK 2 : 2 or ZrK 1 : 2
and
higher concentrations of NaClO
ZrK 2 : 2 into ZrK 1 : 2.
4
favoured the conversion of
1
Fig. 2 H NMR of the hydrolysis reaction of 1.0 mM of HPNP in the
presence of 1.0 mM of ZrK 2 : 2 at different time intervals at pD 6.4 and
In this study, the influence of the substrate HPNP and the
inhibitor disodium diphenyl phosphate (DPP) on the equili-
°
5
0 C.
IV
bria between the different Zr -substituted Keggin type POMs
was also examined. The concentration of ZrK 2 : 2 was kept
constant (1.0 mM) and the concentration of HPNP was
3
1
P NMR spectra (Fig. 3) recorded during the course of the
3
1
increased from 1.0 mM to 50.0 mM. As can be seen from reaction show that cyclic phosphate (CycP), with a P reso-
Fig. S5,† an increase in HPNP concentration lead to the nance at 18 ppm, is formed.1
9,28
At the end of hydrolysis, no
1
31
gradual disappearance of ZrK 1 : 1 and promoted the conver- HPNP resonances were detected in both H and P NMR
sion of ZrK 2 : 2 into ZrK 1 : 2. The same trend was seen when spectra, indicating full conversion of HPNP into cyclic phos-
using DPP in the concentration range 1.0 mM to 40.0 mM phate and NP.
(Fig. S6†). These experiments suggest that the interaction of
Several control experiments were performed. At pD 6.4 and
ZrK 2 : 2 with HNPP and DPP ligands also plays an important 50 °C, the reaction between HPNP and the monolacunary
7
−
role in the ZrK 1 : 1 species distribution.
Keggin POM ([α-PW O ] ) did not show any change in
1
1 39
The above results show that most ZrK 1 : 1 was observed observed rate constant when compared to spontaneous HPNP
using a 1.0 mM solution of ZrK 2 : 2 in the absence of salt at cleavage. This experiment indicates that the monolacunary
pD 6.4 and 50 °C. Therefore, these conditions will be used in POM does not promote HPNP hydrolysis and that the
the kinetic studies of HPNP hydrolysis.
Hydrolysis of HPNP in the presence of ZrK 2 : 2
The hydrolysis of HPNP in the presence of ZrK 2 : 2 (Scheme 1)
1
is conveniently followed by means of H NMR spectroscopy.
P–O bond hydrolysis can be observed by the disappearance of
the aromatic resonances of HPNP (8.28 ppm and 7.38 ppm)
and the appearance of the aromatic p-nitrophenyl (NP) reso-
nances at 8.19 ppm and 6.96 ppm. Fig. 2 shows an example of
1
the H NMR spectra for HPNP hydrolysis in the presence of
ZrK 2 : 2 at different time intervals.
1
Based on the H NMR integration values from Fig. 2, the
percentage of HPNP at different time increments was calcu-
lated in order to obtain the observed rate constant, and half-
life. The percentage of HPNP and NP as a function of reaction
time are shown in Fig. S7† and the natural logarithm of the
concentration of NPP as a function of time (Fig. S8†) was fitted
to a first-order linear decay function. At pD 6.4 and 50 °C an
−
5 −1
observed rate constant of 11.5(±0.42) × 10
.7 h were calculated. The presence of equimolar amounts of
ZrK 2 : 2 resulted in a 530-fold rate acceleration in comparison
s
and half-life of
3
1
Fig. 3
the presence of 1.0 mM of ZrK 2 : 2 after (a) mixing, (b) 40 min, (c) 1 h
0 min, (d) 2 h 25 min, (e) 6 h 50 min at pD 6.4 and 50 °C (left) and
respectively zoomed upfield region of the spectra (right). (400 MHz,
O, 293 K, NS = 1024, 25% H PO ).
P NMR of the hydrolysis reaction between 1.0 mM of HPNP in
1
2
−
7
with the spontaneous hydrolysis of HPNP (2.17(±0.11) × 10
−
1
s
) under the same reaction conditions.
D
2
3
4
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6
embedded Zr(IV) ions are responsible for the observed reactiv- come from either the POM or solvent water molecules. The
ity. Under identical conditions HPNP hydrolysis promoted by deprotonation of water molecules is hindered at pD values
ZrK 1 : 2 was about 12 times slower (kobs = 9.20(±0.56) × 10−
6
smaller than 6.4, resulting in a decrease of kobs at low pD
values. The formation of OH nucleophiles is facilitated by an
−
1
−
s
) in comparison to ZrK 2 : 2 promoted hydrolysis.
As ZrK 1 : 2 does not show any conversion into other POM increase of pD, leading to a gradual increase of kobs. After
1
2
species up to concentrations of 6.0 mM (pD 6.4 and 60 °C),
reaching a maximum at near neutral pD a decrease of kobs is
the observed hydrolysis is exclusively the result of the presence usually observed due to the formation of a hydrolytically in-
3
1
of ZrK 1 : 2. This difference can be explained by the fact that in active HPNP coordination complex. P NMR shows that
the case of ZrK 2 : 2, ZrK 2 : 2 partially converted into a more during the reaction performed at pD 4.5 (Fig. S9†) only ZrK
active ZrK 1 : 1 species and this species promoted the reaction 1 : 1 was observed, suggesting that indeed lower reaction rates
resulting in the higher rate constant, while in the case of ZrK are not due to the conversion of catalytically more active ZrK
1
: 2, no ZrK 1 : 1 species was detected. Under the same con- 1 : 1 into ZrK 2 : 2 or ZrK 1 : 2, but rather due to the low concen-
−
ditions, the reaction between the ZrCl
2 2
O·8H O and HPNP was tration of OH nucleophiles present at low pD. The deprotona-
also examined. Under these conditions, the formation of in- tion of water nucleophiles as well as the promotion of the
2
9,30
soluble Zr(IV) hydroxyl polymeric gels
ing in a rate constant of 2.83(±0.30) × 10
constant represents a 40-fold decrease in comparison to HPNP lysis starts to decrease.
was observed, result- conversion of ZrK 2 : 2 into less active ZrK 1 : 2 become more
−
6
−1
s
. This rate evident at pD values higher than 6.4 when kobs of HPNP hydro-
hydrolysis in the presence of ZrK 2 : 2.
The conversion of ZrK 2 : 2 into ZrK 1 : 2 during the course
of reaction was evidenced in the reaction mixtures studied at
different pD values and 50 °C. Fig. 3 shows that only 11% of
ZrK 1 : 2 was observed after 2 h 25 min at pD 6.4, while in
Effect of pD on HPNP hydrolysis
Since the reactivity and speciation of ZrK 2 : 2 is strongly highly alkaline solutions (pD 9.5) further decomposition of
affected by pD, the effect of pD on the rate constant of HPNP ZrK 2 : 2 into the hydrolytically inactive monolacunary species
hydrolysis in the absence and in the presence of ZrK 2 : 2 was was observed immediately after mixing (Fig. S10†), resulting in
determined in the pD range 4.5 to 9.5. In the absence of ZrK a complete loss of catalytic activity.
2
: 2 at pD 4.5 and 5.5, no hydrolysis of HPNP was observed
Effect of temperature on HPNP hydrolysis
after 3 months, while in the pD range 6.4 to 9.5, the rate con-
stants are at least two orders of magnitude slower than that in As the temperature also influences the ZrK 2 : 2 solution equili-
the presence of ZrK 2 : 2 (Table S1†). The pD-rate constant bria, the effect of temperature on the hydrolysis reaction rate
profile for HPNP hydrolysis in the presence of ZrK 2 : 2 is bell- was examined on a solution containing equimolar amounts of
shaped, with fastest hydrolysis observed at pD 6.4 (Fig. 4).
HPNP and ZrK 2 : 2 (1.0 mM) at pD 6.4 in the temperature
This bell-shaped profile further supports a Lewis-acid cata- range from 37 °C to 80 °C. From the data shown in Fig. S11a,†
lyzed hydrolysis mechanism because if hydrolysis would be the activation energy (63.35 ± 1.82 kJ mol− ) was calculated
Brønsted acid mediated, the rate constants would increase at from the Arrhenius equation, which is significantly lower than
low pH values. The bell-shaped profile can be explained in the the one in the absence of ZrK 2 : 2 under identical reaction
following way. HPNP hydrolysis in the presence of this POM conditions (131.64 ± 2.73 kJ mol− ).
1
1
requires OH− as an active nucleophile and this OH group can
Based on the Eyring equation (Fig. S11b†), the enthalpy of
‡
−1
activation (ΔH = 60.60 ± 2.09 kJ mol ) and entropy of acti-
‡
−1
vation (ΔS = −133.70 ± 6.13 J mol K) were calculated. The
negative entropy of activation results from the coordination of
HPNP to the ZrK POM catalyst. Out of these data, the Gibbs
‡
activation energy (ΔG ) was calculated to be 102.05 ± 0.13 kJ
−
1
‡
mol at 37 °C. This ΔG value is similar to that of BNPP
−
1
12
hydrolysis (111.12 kJ mol at 37 °C) by ZrK 2 : 2 and of NPP
hydrolysis (96.94 kJ mol− at 37 °C) by the Wells–Dawson type
1
8
K
2 2 17 61 2 2
15H[Zr(α -P W O ) ]·25H O POM.
Although ZrK 2 : 2 speciation studies have shown that high
temperatures favor conversion of ZrK 2 : 2 into ZrK 1 : 1 and/or
ZrK 1 : 2, the rate of HPNP hydrolysis did increase when the
temperature was increased. This result might be explained by
the fact that conversion of ZrK 2 : 2 into ZrK 1 : 2 is rather slow
and ZrK 1 : 1 is still present during the course of hydrolysis.
Effect of ZrK 2 : 2 concentration on HPNP hydrolysis
In order to observe the influence of catalyst concentration on
the reaction rate, reaction mixtures containing 1.0 mM HPNP
Fig. 4 pD dependence profile of kobs for the cleavage of 1.0 mM of
HPNP in the presence of 1.0 mM of ZrK 2 : 2 at 50 °C.
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of ZrK 2 : 2 were used the conversion to ZrK 1 : 2 was larger and
7
1% of ZrK 2 : 2 was present after 2 h 25 min (Fig. S13†), leading
−6
to 1.06 × 10 mole of ZrK 2 : 2 present in solution.
Scheme 2 General catalytic scheme for HPNP hydrolysis by ZrK 2 : 2.
Effect of ionic strength on HPNP hydrolysis
Since both ZrK 2 : 2 and HPNP are negatively charged at pD
6
.4, the influence of ionic strength on HPNP hydrolysis was
and increasing amounts of ZrK 2 : 2, ranging from 0.25 mM to
examined by adding stepwise NaClO to a mixture containing
4
3
.0 mM were studied at pD 6.4 and 50 °C. The general catalytic
1.0 mM of HPNP and 1.0 mM of ZrK 2 : 2. From Fig. S14,† a
scheme for the hydrolysis of HPNP in the presence of ZrK 2 : 2
is shown in Scheme 2.
significant decrease of the rate constant was observed upon
adding increasing amounts of NaClO . As above control experi-
ments with the monolacunary Keggin POM ([α-PW11
showed that Zr is required factor for the catalytic activity, it is
very likely that the binding between HPNP and POM occurs via
coordination between the negatively charged oxygen of phos-
4
Assuming that the formation of the products (k
slower than reaching the equilibrium between HPNP and ZrK
: 2, we have k + k ≫ k . Because the formation of nitro-
c
) is much
7
−
39
O ] )
IV
2
1
−1
c
phenol (NP) is a first-order reaction, kobs can be written as in
8
,10,11,31
eqn (1).
By fitting the data to eqn (1), the binding constant (K =
IV
phate group of HPNP and the positively charged Zr of POM.
f
/k−1 = 455 M− ) and the catalytic rate constant (k
1
−5 Addition of salt may hinder this interaction, leading lower
k
s
1
c
= 42 × 10
) were obtained (Fig. 5). From this figure it is evident that
−
1
4
reaction rates. However, NaClO also promotes conversion of
ZrK 2 : 2 into ZrK 1 : 2 as can be seen from Fig. S15, S16 and
S17.† Fig. S15† shows that at pD 6.4, 26% of ZrK 1 : 2 is
the POM can hydrolyse an excess of HPNP. Complete hydro-
lysis of HPNP was still observed when 1.0 mM of HPNP and
detected in a reaction mixture containing 0.25 M NaClO
while in the presence of 1.0 M NaClO (Fig. S16†) 70% of ZrK
: 2 was observed. When the concentration of NaClO4 was
4
,
0
.25 mM of ZrK 2 : 2 were used. This suggests that one equi-
4
valent of ZrK 2 : 2 hydrolyses at least 4 equivalents of HPNP,
demonstrating its catalytic activity.
1
increased up to 2.0 M (Fig. S17†), full conversion of ZrK 2 : 2
into ZrK 1 : 2 was observed immediately after mixing. From
kc½ZrK 2:2ꢀ
0
kobs ¼
ð1Þ
these experiments, it is concluded that NaClO not only plays a
kꢁ1=k1 þ ½ZrK 2:2ꢀ
4
0
role in the binding between HPNP and ZrK 2 : 2, but also in
the speciation of this POM in the reaction mixture.
Fig. 5 shows that an increase in ZrK 2 : 2 concentration
leads to an increase in reaction rate. Despite the fact that
higher initial concentrations of ZrK 2 : 2 result in a more favor- Inhibition study
1
2
able conversion to ZrK 1 : 2, the total molar amount of ZrK
: 2 in solution is still much higher, resulting in faster hydro-
lysis. For example, for a 0.5 mL mixture containing 1.0 mM of
HPNP and 0.25 mM of ZrK 2 : 2 (0.13 × 10 mole), no signal of
ZrK 1 : 2 was detected during the course of reaction (Fig. S12†).
Fig. 3 shows that a 0.5 mL mixture of 1.0 mM of HPNP and
As DPP is not hydrolyzed after 4 months both in the absence
2
and in the presence of 1.0 mM of ZrK 2 : 2 at pD 6.4 and
12
6
0 °C, it is a good candidate for inhibitor studies. The hydro-
−6
lysis of 1.0 mM of HPNP was followed in the presence of
1
4
.0 mM of ZrK 2 : 2 and increasing amounts from 1.0 mM to
0 mM of DPP. As it can be seen from Fig. S18,† the rate con-
1
.0 mM of ZrK 2 : 2 contains 81% of ZrK 2 : 2 after 2 h 25 min,
stant of HPNP hydrolysis significantly decreased upon adding
−6
resulting in 0.43 × 10 mole of ZrK 2 : 2. Similarly, when 3.0 mM
increasing amounts of DPP. DPP has a negative effect, as on
IV
the one hand it competes with HPNP for the binding to Zr
and on the other hand it largely shifts equilibrium toward the
formation of ZrK 1 : 2, resulting in reaction rate decrease.
3
1
Binding between HPNP and ZrK 2 : 2 by P NMR
3
1
The interaction between HPNP and ZrK 2 : 2 was studied by
P
NMR. At pD 6.4 (Fig. S19†) ZrK 2 : 2 is characterized by a peak
at −13.49 ppm, while the HPNP signal can be found at
−4.61 ppm (half-width 2.30 Hz). In the presence of 1.0 mM of
ZrK 2 : 2 the peak of HPNP is shifted by 0.14 ppm and the half-
width increased to 5.14 Hz (Fig. 6). Both the change in chemi-
cal shift and the line-broadening support that interaction
between HPNP and ZrK 2 : 2 takes place.
Kinetic isotope effect on HPNP hydrolysis and proposed
mechanism
The kinetic solvent deuterium isotope effect is a powerful tool
Fig. 5 Dependence of the observed rate constant on the concentration
of ZrK 2 : 2 for the hydrolysis of 1.0 mM of HPNP at pD 6.4 and 50 °C.
for mechanistic studies and gives information on the proton
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the solvent, thus leading to cleavage of the P–O bond and the
formation of nitrophenol and cyclic phosphate.
Conclusions
In summary, we report the first example of the hydrolysis of
HPNP promoted by a binuclear Zr(IV)-substituted Keggin type
3
1
polyoxometalate. P NMR measurements offer the evidence
3
1
that the ZrK 1 : 1 species, which is proposed as the active
Fig. 6
P NMR spectra of 1.0 mM of HPNP and the mixture of 1.0 mM
of HPNP and 1.0 mM of ZrK 2 : 2 after adjusting the pD to 6.4 (400 MHz, species, is the major and stable species under the reaction con-
D
2
O, 293 K, NS = 1024, 25% H
3
PO
4
).
ditions. A 530-fold rate increase in comparison to the blank
reaction was observed for HPNP hydrolysis. The present study
demonstrates the potential of Zr(IV)-substituted POMs as artifi-
cial phosphatase and contributes to the further development
of POMs as Lewis acid catalysts for the hydrolysis of bio-
transfer processes in or before the rate-limiting step of the
reaction. RNA model phosphate diesters can be hydrolysed
by two possible mechanisms that can be distinguished by
3
8–40
3
2
molecules.
The results from this study encourage us to
further exploit the hydrolytic activity of this POM towards
other more complex substrates such as adenosine triphosphate
2 2
examining the influence of H O or D O as a solvent on the
(
ATP), DNA/RNA fragments, pesticides such as paraoxon and
kinetics of hydrolysis. When the solvent deuterium kinetic
parathion, and nerve agents such as soman and sarin.
isotope effect (skie), characterized by the kH O/kD2O ratio, is
2
lower than 1.5 a nucleophilic mechanism is expected. At skie
ratios higher than 2 the reaction preceeds via a general base
2
8,33
mechanism.
Experimental
Materials
In order to propose a mechanism for HPNP hydrolysis, the
skie was calculated. The reaction rates for 1.0 mM HPNP
hydrolysis in the presence of 1.0 mM ZrK 2 : 2 in H O or D O
2 2
The Zr-substituted Keggin POMs (Et NH ) [{α-PW O Zr-
2
2
2 8
11 39
0,34
(
µ-OH)(H O)} ]·7H O (δ = −13.49 ppm)
(ZrK 2 : 2), and
2
2
2
were calculated at identical conditions (pD 6.4 and 50 °C). The
2
1
(
2 2 39 2 2
Et NH )10[Zr(PW11O ) ]·7H O (δ = −14.67 and −14.77 ppm)
hydrolysis in H O proceeded with the rate constant 13.80
2
±0.83) × 10−
of 11.50(±0.42) × 10
5
−1
(ZrK 1 : 2) as well as 2-hydroxypropyl-4-nitrophenyl phosphate
(
s
, while for the reaction in D
2
O a rate constant
was observed. From these results, a
skie effect of 1.2 was calculated and this value of skie effect
3
5,36
−5
−1
(δ = −4.64 ppm)
(HPNP) were synthesized and character-
s
ized according to literature. Disodium diphenyl phosphate
2
8,33 1
(DPP, C H PO Na ·2H O), DCl, and NaOD were purchased
6
5
4
2
2
supports the nucleophilic mechanism.
H spectra (Fig. 2)
from Acros and used without any further purification.
show that one of the products of HPNP hydrolysis is nitro-
3
1
phenol and P spectra (Fig. 3) show that another product of
Kinetic measurements
3
1
HPNP hydrolysis is cyclic phosphate with a P chemical shift
1
9,28
31
Solutions containing 1.0 mM of HPNP and 1.0 mM of ZrK 2 : 2
at 18 ppm.
From P NMR measurements, kinetic experi-
were prepared in D
amounts of 10% DCl and 15% NaOD solutions in D
pH-meter value was corrected by using the equation: pD = pH
2
O. The final pD was adjusted with minor
ments and isotope effect study, the mechanism of HPNP
hydrolysis in the presence of ZrK 2 : 2 was proposed as follow-
ing. As already shown in our previous study, under the reaction
2
O. The
3
7
meter reading + 0.41. The pD of the samples was measured
at the beginning and at the end of hydrolysis, and the differ-
ence was typically less than 0.5 units. The reaction mixture was
conditions there is an interconversion between ZrK 2 : 2, ZrK
6
1
: 1 and/or ZrK 1 : 2. The Zr atom in the ZrK 1 : 2 species is
coordinatively saturated and hardly accessible for the bulky
HPNP ligand, as it is sandwiched by the two Keggin moieties.
1
kept at constant temperature and H NMR spectra were
6
measured at certain time intervals during the hydrolytic reac-
tion to calculate the observed rate constant (kobs) by the inte-
gral method.
Therefore, HPNP coordination to ZrK 1 : 2 is not expected.
ZrK 1 : 1 was found to be more catalytically active when com-
pared to ZrK 2 : 2 as its Zr(IV) ion has more coordinated water
molecules that can be replaced by the substrate or that can act
6
NMR spectroscopy
as a nucleophile. We suggest that ZrK 1 : 1 can act as an active
1
species in this HPNP hydrolytic reaction by coordination to the
H NMR spectra were recorded on a Bruker Avance 400 and
oxygen atom of the phosphate group of HPNP. This suggestion 0.5 mM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TMSP)
3
1
is experimentally supported by the observed shift and line- was used as an internal standard. P NMR spectra were
3
1
broadening of the HPNP signal in the P spectrum (Fig. 6). recorded on a Bruker Avance 400 and Bruker Avance II+ 600
1
This binding would result in a more positive charge on the NMR spectrometer using power-gated H decoupling with 30
phosphorous atom of HPNP, which facilitates the nucleophilic degree flip angle (zgpg30 pulse programme) and 25% phos-
3
1
attack of the OH group of ZrK 1 : 1 or from water molecule of phoric acid was used as a P external reference.
Dalton Trans.
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Paper
1
9 H. Arora, S. K. Barman, F. Lloret and R. Mukherjee, Inorg.
Chem., 2012, 51, 5539–5553.
Acknowledgements
T.N.P-V. and P.S. (BOF + fellowship) thank KU Leuven for the 20 K. Nomiya, Y. Saku, S. Yamada, W. Takahashi, H. Sekiya,
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