Design of a dinuclear nickel(II) bioinspired hydrolase to bind covalently to silica surfaces: Synthesis, magnetism, and reactivity studies

Article abstract of DOI:10.1021/ic300018t

Presented herein is the design of a dinuclear Ni^II synthetic hydrolase [Ni₂(HBPPAMFF)(μ-OAc)₂(H₂O)] BPh₄ (1) (H₂BPPAMFF = 2-[(N-benzyl-N-2-pyridylmethylamine) ]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl)])-4-methyl-6-formylphenol) to be covalently attached to silica surfaces, while maintaining its catalytic activity. An aldehyde-containing ligand (H₂BPPAMFF) provides a reactive functional group that can serve as a cross-linking group to bind the complex to an organoalkoxysilane and later to the silica surfaces or directly to amino-modified surfaces. The dinuclear Ni^II complex covalently attached to the silica surfaces was fully characterized by different techniques. The catalytic turnover number (k_cat) of the immobilized Ni ^IINi^II catalyst in the hydrolysis of 2,4- bis(dinitrophenyl)phosphate is comparable to the homogeneous reaction; however, the catalyst interaction with the support enhanced the substrate to complex association constant, and consequently, the catalytic efficiency (E = k _cat/K_M) and the supported catalyst can be reused for subsequent diester hydrolysis reactions.

Full text of DOI:10.1021/ic300018t

Article
pubs.acs.org/IC
Design of a Dinuclear Nickel(II) Bioinspired Hydrolase to Bind
Covalently to Silica Surfaces: Synthesis, Magnetism, and Reactivity
Studies
Clovis Piovezan,† Jaqueline M. R. Silva,† Ademir Neves,*,† Adailton J. Bortoluzzi,† Wolfgang Haase,‡
Zbigniew Tomkowicz,§ Eduardo E. Castellano,∥ Tessa C. S. Hough,⊥ and Liane M. Rossi*,⊥
†Departamento de Química, Laborator
Catarina, SC, 88040-900 Florianopolis, Brazil
‡Institut fur Physikalische Chemie, Technische Universitat Darmstadt, Petersenstrasse 20, D-64287-Darmstadt, Germany
́ ̂
io de Química Bioinorganica e Cristalografia (LABINC), Universidade Federal de Santa
́
̈
̈
§Institute of Physics, Reymonta 4, Jagiellonian University, PL-30-059 Krakow, Poland
∥Instituto de Física, Universidade Federal de Sao Carlos, Sao Carlos, SP 13560-970, Brazil
̃
̃
⊥Instituto de Química, Universidade de Sao Paulo, Sao Paulo 05508-000, Brazil
̃
̃
S
* Supporting Information
ABSTRACT: Presented herein is the design of a dinuclear
NiII synthetic hydrolase [Ni2(HBPPAMFF)(μ-OAc)2(H2O)]-
BPh4 (1) (H2BPPAMFF = 2-[(N-benzyl-N-2-pyridylmethyl-
amine)]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl)])-4-
methyl-6-formylphenol) to be covalently attached to silica
surfaces, while maintaining its catalytic activity. An aldehyde-
containing ligand (H2BPPAMFF) provides a reactive func-
tional group that can serve as a cross-linking group to bind the
complex to an organoalkoxysilane and later to the silica
surfaces or directly to amino-modified surfaces. The dinuclear
NiII complex covalently attached to the silica surfaces was fully
characterized by different techniques. The catalytic turnover
number (kcat) of the immobilized NiIINiII catalyst in the
hydrolysis of 2,4-bis(dinitrophenyl)phosphate is comparable to the homogeneous reaction; however, the catalyst interaction with
the support enhanced the substrate to complex association constant, and consequently, the catalytic efficiency (E = kcat/KM) and
the supported catalyst can be reused for subsequent diester hydrolysis reactions.
metal-hydroxide, is critical to an effective hydrolysis proc-
ess.5−12
INTRODUCTION
■
The development of unsymmetrical polydentate ligands and
their use for the preparation of mono- and dinuclear metal
complexes with catalytic activity as metallohydrolases and/or
metalloproteases are examples of synthetic bioinspired
systems.1,2 Nucleases are natural catalysts much more efficient
and specific than those synthesized in the laboratory and their
biological role includes the degradation or digestion of
polymers, modification of nucleic acids, DNA repair, and viral
defense (restrictive enzymes).3 For many nucleases, the metal
ions are essential cofactors and are directly involved in the
hydrolysis of phosphodiesters. They increase the catalytic rate
of the hydrolytic cleavage of the phosphodiester bond by a
factor of 1012 with respect to the noncatalyzed reaction.4 The
effectiveness of metal ions in promoting the hydrolysis of
phosphate bonds and DNA cleavage in biological systems is a
function of many factors, including the nature of the metal ion,
the phosphatediester species, and the compositions and
structures of complexes formed between them. The availability
of a nucleophile, such as a properly positioned coordinated
The search for low molecular weight molecules able to
catalytically cleave DNA has attracted much interest among
scientists, although a great number of nucleases are well-known.
This interest embraces the elucidation of the cleavage
mechanism, the role of metals in biological systems, and the
design of more effective synthetic hydrolases, as well as the use
of these new compounds as catalysts, conformational probes,
and synthetic restriction enzymes.13
The study of the catalytic activity of synthetic metal-
locomplexes in solution, inspired by the catalytic properties
of biological systems, has been reported by several groups who
found that these substances provide very efficient systems for
catalytic oxidation reactions,14−16 controlled drugs deliv-
ery,17−19 biodiesel transesterification reaction,20,21 and bleach-
ing process.22−24
Received: January 4, 2012
Published: May 15, 2012
© 2012 American Chemical Society
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A constant theme in these challenging areas is the formation
of structured assemblies containing complexes located in well-
defined chemical environments. The creation of a supported
metal center that is accessible to large organic substrates is an
essential property for the biomimetic catalytic application of
supported metallocomplexes. The immobilization of metal-
locomplexes associated with the production of an easily
recyclable solid such as an inorganic support; e.g., silica
would greatly enhance the usefulness of such complexes for
diverse new uses. The combination of efficient catalysts with
immobilization on solid supports can result in efficient and
selective catalysts because the matrix support can impose shape-
selectivity, promoting a special environment for the approach of
the substrate to the active species. In addition, immobilization
may prevent molecular aggregation or bimolecular self-
destruction reactions, which lead to deactivation of the catalytic
active species.25,26
The immobilization of such biomimetic complexes on the
surface of solid supports by covalent bonds rather than by
electrostatic attraction requires a step of ligand design.
Additionally to the functional groups placed in the ligand to
mimic the groups present in the active site (first coordination
sphere of the metal) of a given metalloenzyme, the ligand itself
should provide a reactive functional group that can serve as a
cross-linking group to bind the complex to the solid surface. It
is worth drawing the attention to the fact that the changes in
the ligand should maintain the corresponding complex
properties as expected to mimic the metalloenzyme, such as
bond distances and angles. Still, the introduction of reactive
functional groups in the ligand must be such that this group will
not make part of the coordination sphere of the metal;
therefore, the approach of this group to the metal center must
be sterically hindered.
bioinspired nuclease, another ligand has been designed to
maintain as much as possible the chemical environment for the
metal ions coordination besides the addition of a reactive
functional group that can serve as a cross-linking group to bind
the corresponding dinuclear NiII complex to the surface of
silica.
Silica is a very versatile material that can be prepared with
good control of particles size and morphology. Sub-100 nm
silica nanospheres can be prepared by means of micro-
emulsions,28,29 or by a modified Stober sol−gel synthesis,30
̈
with narrow size distribution. Silica spheres in the nano- and
microsize range have been extensively used for the immobiliza-
tion or entrapment of many kinds of organic molecules, such as
dyes,28,29,31−33 biomolecules,34−36 DNA,37 and bioinspired
model complexes for nucleases.38 Silica has been also used
for coating different nanomaterials, such as magnetic nano-
particles,39−42 metal nanoparticles,43−45 and quantum dots,46 in
order to bring different properties such as chemical stability and
easy surface functionalization for targeting purposes. Silica
surfaces contain reactive silanol groups that are easy to
functionalize by reacting the silica matrix with commercially
available organoalkoxysilane reagents33,47 or organic molecules
previously modified with organoalkoxysilane reagents.48−50
This strategy assures chemical attachment of target molecules
to the inorganic matrix by stable covalent bonds.
In this Article, we will describe the design of a dinuclear NiII
synthetic nuclease [Ni2(HBPPAMFF)(μ-OAc)2(H2O)]BPh4
(1) to be covalently attached to silica surfaces while
maintaining its catalytic activity. Interestingly, complex 1
immobilized on functionalized silica has been successfully
applied in the construction of a novel sensor for the
determination of fisetin by square-wave voltammetry.51 Herein,
experimental details for the syntheses of the ligand
H2BPPAMFF and its dinuclear complex 1 are also described.
In Scheme 1, there is an example of a bioinspired ligand and
its dinuclear nickel(II) complex,27 a biomimetic structural
model for nucleases, which displays catalytic activity in
hydrolysis of phosphate esters. The complex hydrolyses the
substrate bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) at a
rate 138.400 faster than the noncatalyzed reaction at 25 °C and
pH 9. Taking into account the structure and activity of this
EXPERIMENTAL SECTION
■
Physical Measurements. Electronic spectra were recorded on a
Perkin-Elmer UV−Vis/NIR Diode Array spectrophotometer in the
280−800 nm range. IR spectra were recorded on a Bomem MB100
FT-IR spectrometer in the 400−4000 cm−1 range. Cyclic voltammetry
was performed using an Epsilon EC 2000 Potentiostat (working
electrode, gold; reference electrode, Ag/Ag+; support electrode,
platinum; support electrolyte, tetrabutylammonium hexafluorophos-
phate; internal standard, ferrocene). Elemental analyses were
determined on the CHN Perkin-Elmer 2400 analyzer. 1HNMR
spectra were recorded on a Varian-FT spectrometer (400 MHz) in
CDCl3 using tetramethylsilane as the internal standard. Kinetic
measurements were recorded on a Varian Cary 50Bio UV−Vis
Diode Array spectrophotometer coupled to a thermostatic bath.
Transmission electron microscopy (TEM) images were performed
using a Philips CM 200 microscope. Samples for TEM observations
were prepared by placing a drop containing the nanoparticles in a
carbon-coated copper grid. ESI-MS of the complexes dissolved in
ultrapure acetonitrile/water (1:1, v/v) solution (500 μL) were
analyzed using an amaZon X Ion Trap MS instrument (Bruker
Daltonics) with an ion spray source using electrospray ionization in
positive ion mode. The ion source conditions were an ion spray
voltage of 4500 V. Nitrogen was used as nebulizing gas (20 psi) and
curtain gas (10 psi). The samples were directly infused into the mass
spectrometer at a flow rate of 300 μL/h. The scan range was m/z
200−3000. Magnetic DC measurements were performed with a
Quantum Design SQUID magnetometer, model MPMS 5XL, in
magnetic field of the induction of 0.1 and 2.0 T. No field effect was
observed. The measured samples were pure complex 1, SiO2-1, and
pure SiO2. Additionally, the capsule container was measured.
Scheme 1. Example of a Bioinspired Synthetic Nuclease27
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X-ray Crystallography of Complex 1b. A small fragment with
dimensions of 0.05 × 0.02 × 0.02 mm3 was selected from a crystalline
sample of complex 1b for crystallographic analysis. X-ray diffraction
data were measured on a Kappa-CCD diffractometer with graphite-
monochromated Mo Kα (λ = 0.71073 Å) radiation, at room
temperature. Diffraction data were collected (φ and ω scans with K-
offsets) with COLLECT.52 Integration and scaling of the reflections
were performed with HKL DENZO-SCALEPACK software package.53
The unit cell parameters were obtained by least-squares refinement
based on the angular settings for all collected reflections using HKL
SCALEPACK.53 Because of the very small size of the crystal,
absorption correction was not applied to the intensities. The structure
was solved by direct methods54 and refined by full-matrix least-squares
on F2.55 All non-hydrogen atoms were refined with anisotropic
displacement parameters, except for carbon atoms of one isopropanol
solvate. Perchlorate anion is partially disordered in which two oxygen
atoms occupy two alternative positions. Water solvate are also
disordered. Hydrogen atoms bonded to C atoms were placed at their
idealized positions using standard geometric criteria. Just one H atom
of the coordinated water molecule was found from the Fourier
difference map. H atoms of the isopropanol and water solvate could
not be located. Selected crystallographic information is presented in
the experimental solutions were adjusted to 0.100 mol·L−1 of ionic
strength by the addition of KCl. Computations of the triplicate results
were carried out with the BEST7 program, and species diagrams were
obtained with SPE and SPEPLO programs.57
Materials. All chemicals were purchased from Aldrich, Sigma, or
Merck and were of analytical grade. 2-Chloromethyl-4-methyl-6-
formylphenol,58 2,4-BDNPP,59 and 2,4-DNPP59,60 were synthesized
by previously described methods.
Syntheses. Synthesis of the Ligand 2-[(N-Benzyl-N-2-pyridylme-
thylamine)]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl)])-4-
methyl-6-formylphenol (H2BPPAMFF). N-Benzyl-N-2-pyridylmethyl-
amine (BPA). This amine was synthesized using a procedure similar to
that previously described.61 2-(Aminomethyl)pyridine (4.9 mL, 47
mmol) was dissolved in 20 mL of methanol and cooled to 0 °C. A
methanolic solution of benzaldehyde (5.30 g, 50 mmol, in 30 mL) was
added dropwise with stirring. After 24 h, was added carefully sodium
tetrahydroborate (1.13 g, 30 mmol) with stirring. The reaction mixture
was further stirred for 4 h at room temperature. The mixture was
concentrated under reduced pressure, and after the addition of HCl 2
mol·L−1 (30 mL), the reaction mixture was washed with 5 × 10 mL of
CHCl3 to remove the excess of benzaldehyde. The water layer was
made alkaline with saturated aqueous Na2CO3 (pH 10), extracted with
5 × 10 mL portions of CHCl3, dried over Na2SO4, and concentrated
under a reduced pressure. A pale yellow oil was obtained. Yield, 75%.
1H NMR δH (400 MHz; CDCl3): 8.5 [1H, Py]; 7.6−7.1 [8H, Ar];
Table 1. Crystal Data and Structure Refinement for Complex
1b
3.9−3.8 [4H, CH2] in ppm.
2-(N-Benzyl-N-2-pyridylmethylamine)-4-methyl-6-formylphenol
(BPAMFF). To a solution of BPA (1.5 g, 7.5 mmol in 20 mL of
CH2Cl2) was slowly added a solution of 2-chloromethyl-4-methyl-6-
formylphenol (1.38 g, 7.5 mmol in 30 mL of CH2Cl2) under stirring,
and the reaction mixture was stirred for 24 h. The desired compound
was washed with 5 × 30 mL of saturated aqueous Na2CO3 (pH 10),
and the organic layer was dried over Na2SO4 and concentrated under a
reduced pressure. A yellow oil was obtained. Yield: 92%. 1H NMR δH
(400 MHz; CDCl3): 10.3 [1H, aldehyde]; 8.5 [1H, Py]; 7.6−7.1
[10H, Ar]; 3.8−3.7 [6H, CH2]; 2.2 [3H, CH3] in ppm.
empirical formula
formula weight
temperature
C59H70ClN5Ni2O15.50
1250.07
293(2) K
wavelength
0.71073 Å
crystal system
space group
monoclinic
P21/c
unit cell dimensions
a = 14.7830(3) Å
b = 18.4790(6) Å
c = 22.9010(8) Å
β = 97.235(2)°
6206.2(3) Å3
4
2-[(N-Benzyl-N-2-pyridylmethylamine)]-4-methyl-6-[N-(2-pyridyl
methyl)aminomethyl)]phenol (BPAPyFF). A solution of 2-(amino
methyl)pyridine (0.7 mL, 6.7 mmol in 30 mL of CH3OH) was added
dropwise to a solution of the BPAMFF (2.4 g, 6.9 mmol, in 20 mL of
CH3OH) and stirred for 4 h. After 4 h, was added carefully sodium
tetrahydroborate (0.25 g, 6.7 mmol) with stirring. The reaction
mixture was stirred for another 4 h at room temperature. The mixture
was concentrated under a reduced pressure and after added 30 mL of
HCl 2 mol·L−1. The aqueous phase was washed with 5 × 10 mL of
CHCl3. The water layer was made alkaline by saturated aqueous
Na2CO3 (pH 10), extracted with five 10 mL portions of CHCl3, dried
over Na2SO4, and concentrated under a reduced pressure. A yellow oil
was obtained. Yield: 72%. 1H NMR δH (400 MHz; CDCl3): 8.5 [2H,
Py]; 7.6−6.8 [13H, Ar]; 3.9−3.6 [10H, CH2]; 2.2 [3H, CH3] in ppm.
2-[(N-Benzyl-N-2-pyridylmethylamine)]-4-methyl-6-[N-(2-pyridyl
methyl)aminomethyl)])-4-methyl-6-formylphenol (H2BPPAMFF). To
a solution of BPAPyFF (2.11 g, 4.8 mmol in 20 mL of CH2Cl2) was
slowly added a solution of 2-chloromethyl-4-methyl-6-formylphenol
(0.88 g, 4.8 mmol in 30 mL of CH2Cl2), under stirring. After the
addition, the reaction was stirred for 24 h. The compound was washed
with 5 × 30 mL of saturated aqueous Na2CO3 (pH 10), and the
organic layer was dried over Na2SO4 and concentrated under a
volume
Z
density (calculated)
absorption coefficient
F(000)
1.338 Mg/m3
0.718 mm−1
2624
crystal size
0.05 × 0.02 × 0.02 mm3
theta range for data collection 2.61 to 25.00°
index ranges
−17 ≤ h ≤ 17; −21 ≤ k ≤ 18; −24 ≤ l ≤ 27
30871
reflections collected
independent reflections
absorption correction
refinement method
10685 (Rint = 0.0725)
none
full-matrix least-squares on F2
data/restraints/parameters
goodness-of-fit on F2
final R indices [I > 2σ(I)]
R indices (all data)
10685/52/764
1.080
R1 = 0.0915; wR2 = 0.2436
R1 = 0.1150; wR2 = 0.2653
1.012 and −0.727 e·Å−3
largest diff. peak and hole
Table 1, and full tables of crystallographic data (except structure
factors) were deposited at Cambridge Structural Database (CCDC
843653), and these data are available free of charge at www.ccdc.cam.
ac.uk.
Potentiometric Titrations. The potentiometric studies were
carried out with a Corning-350 research pH meter fitted with
blueglass and Ag/AgCl reference electrodes, in acetonitrile/water
(50:50, v/v) solutions. The pKw of the acetonitrile/water (50:50% v/
v) containing 0.2 mol·L−1 of KCl used was 15.40.56 Equilibrium
measurements were performed in a thermostatted cell, purged with
argon, containing 50.00 mL of the acetonitrile/water (50:50) solution
and 0.05 mmol of complex. The temperature was 25.00 0.05 °C, and
1
reduced pressure. A yellow solid was obtained. Yield: 70%. H NMR
δH (400 MHz; CDCl3): 10.2 [1H, aldehyde]; 8.5 [2H, Py]; 7.6−6.8
[15H, Ar]; 3.8−3.6 [12H, CH2]; 2.2 [6H, CH3] in ppm. CHN%
(C37H38N4O3) found (calcd.) C 75.2 (75.7), H 6.1 (6.5), N 9.5 (9.2);
fp (57−62 °C).
Synthesis of the Complex [Ni2(HBPPAMFF)(μ-OAc)2(H2O)]BPh4
(1). Complex 1 was synthesized in methanolic solution by mixing
Ni(ClO4)2·6H2O (0.73 g, 2 mmol, in 20 mL) and the H2BPPAMFF
ligand (0.58 g, 1 mmol, in 20 mL) with stirring and mild heating (60
°C). After 15 min, NaCH3COO·3H2O (0.16 g, 2 mmol) was added,
and a green solution was obtained. NaBPh4 (0.34 g, 1 mmol) was then
added; immediately, a light green solid was formed, filtered off, and
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Scheme 2. Synthesis of the Ligand H2BPPAMFF
dried under vacuum for 24 h. Yield: 58%. IR (KBr), in cm−1: ν(C−HAr
and C−HAliph) 3431−2922; ν(BPh4) 1606; νass(OAc) 1554;
νsym(OAc) 1427; ν(H−Ophen) 1317; ν (C−O) 1263; δ(C−HAr) 734
and 705. CHN% Calcd. for Ni2C65H67N4O8B (found): C 67.2 (67.6);
H 5.8 (5.7) N 4.8 (4.9). ΛM =120 Ω−1·cm2·mol−1 in CH3CN
electrolyte 1:1.
stored quantitatively to determine the amount of complex 1
immobilized on the support by comparing the UV−vis absorption
maximum near 400 nm for the alkoxysilane-complex solutions
obtained before and after immobilization. Typically, the immobiliza-
tion is quantitative, which corresponds to a complex 1 loading of 6 ×
10−6 mol·g−1
Caution! Perchlorate salts with organic ligands are potentially explosive
and must be handled in small amounts with care.
Reactivity Studies. Phosphatase-like activity of complex 1 was
determined through the hydrolysis reaction of the model substrate
bis(2,4-dinitrophenyl)phosphate [2,4-BDNPP] under substrate excess.
The experiments were carried out in triplicate and spectrophoto-
metrically monitored at 400 nm (ε = 12 100 L·mol−1·cm−1) using a
Varian Cary 50 BIO UV−Vis spectrophotometer coupled to a
thermostat bath. Reactions were monitored to less than 5% conversion
of 2,4-BDNPP to 2,4-DNP, and the data was treated using the initial
rate method. Initial rates were obtained directly from the plot of the
2,4-dinitrophenolate concentration (2,4-DNP) versus time under the
same experimental conditions. In these experiments, all of the
solutions were prepared in aqueous/buffer media, except the complex
stock solution (3.0 × 10−4 mol·L−1 in acetonitrile). Studies regarding
the effects of pH on the hydrolysis reaction were performed in the pH
range 5.00−10.5 (MES pH 4.00−6.50; HEPES pH 7.00−8.50; CHES
pH 9.00−10.5; I = 0.1 mol·L−1 with LiClO4), under a 100-fold excess
of substrate, at 25 °C. Experiments to determine the dependence of
the reaction rate on the substrate concentration were carried out at 25
°C and pH 9.00 for both substrates (2,4-BDNPP and 2,4-DNPP).
The heterogeneous reaction was performed under the same
conditions as described above. The reactions were carried out in a 4
mL thermostatic glass reactor equipped with a magnetic stirrer. In a
typical reaction employing the heterogeneous catalyst, SiO2-1 (2.2 ×
10−8 mol) was weighed in a vial flask. The solid was suspended (0.15
mL of buffer and 0.14 mL of acetonitrile), and 0.1 mL of the substrate,
2,4-BDNPP, was added, resulting in a catalyst/substrate molar ratio of
1:100. The hydrolysis reaction was carried out during a controlled time
interval (30 min) under magnetic stirring. The solution containing the
reaction products was separated from the solid catalyst by
centrifugation and analyzed by UV−vis. The same procedure was
followed for the control reactions using the corresponding silica
supports without the complex.
Synthesis of the Complex [Ni2(HBPPAMFF)(μ-OBz)2(H2O)]ClO4
(1b). Complex 1b was synthesized as described for complex 1 but
with the use of sodium benzoate instead of sodium acetate. Suitable
crystals for X-ray analysis were obtained after two weeks by slow
diffusion of isopropanol in an acetonitrile solution of 1b. It is
important to mention here that the yield of this reaction is very low
when compared to that of 1, and thus, it was used only to fully
characterize the structure of 1b in which both NiII centers have similar
coordination environments as those proposed for the μ-acetate
complex 1.
Synthesis of Silica Nanospheres (SiO2). This preparation is a
modification of the reverse microemulsion reported elsewhere.39 In a
500 mL round-bottomed flask, 15.5 g of polyoxyethylene (5)
nonylphenylether (IGEPAL CO-520, Aldrich Co.) was dissolved in
250 mL of cyclohexane. The mixture was sonicated for 3 min, and
then, 2.75 mL of TEOS (tetraethylorthosilicate) and 5.4 mL of
ammonium hydroxide solution (29%) were added. The reaction
mixture was kept under magnetic stirring for 24 h. The material was
then precipitated with methanol, collected by centrifugation (7000
rpm, 30 min), and washed 3 times with ethanol. The material was
oven-dried at 100 °C.
Preparation of Organoalkoxysilane and Immobilization of
Complex 1 on SiO2 Nanospheres (SiO2-1). (3-Aminopropyl)-
triethoxysilane (APTES) (0.3 μL) was added to 3 mL of complex 1
solution (2.1 × 10−4 mol·L−1 in acetonitrile), corresponding to 1:1
amine/aldehyde molar ratios. The mixture was stirred at room
temperature for 15 min in order to prepare the alkoxysilane of
complex 1. To the solution obtained above was added 100 mg of SiO2
nanospheres. The reaction mixture was kept under magnetic stirring at
room temperature for 24 h and then separated by centrifugation and
washed three times with acetonitrile. The supernatant solution was
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Scheme 3. Proposed Equilibria Observed for Complex 1 in Acetonitrile/Water (50:50) Solution
mol·L−1·cm−1) d−d transition, respectively (Figure S2,
Supporting Information).
RESULTS AND DISCUSSION
■
Synthesis of the Ligand H2BPPAMFF. Silica or other
Cyclic voltammograms were recorded in acetonitrile in the
potential range −2.0 to 2.0 V versus Ag/AgCl, using
[TBA][(PF)6] as supporting electrolyte. In the cyclic
voltammograms for complex 1, two nonreversible cathodic
waves were observed at −1.27 and −1.64 V versus NHE, at 100
mV·s−1 (Figure S3, Supporting Information). These redox
potentials can be attributed to the electron transfer processes
NiIINiII → NiIINiI/NiIINiI → NiINiI. Interestingly, the redox
process observed at −1.27 V in 1 is in close agreement with the
value observed for the FeIINiII/FeIINiII redox couple (−1.41 V)
in a dinuclear model complex in which the NiII center has a
similar coordination environment as in 1.12 The nonreversi-
bility of these processes is indicative of the instability of the
totally reduced form in solution. Repetitive CV scans of 1
showed the maintenance of the CV curve profile with no
significant decrease in either cathodic or anodic waves.
The measured molar conductivity of 1 in acetonitrile solution
at 25 °C was 120 S·cm2·mol−1, which is characteristic of a 1:1
electrolyte.63
Potentiometric titration studies of 1 (water/acetonitrile,
50:50) showed the neutralization of 3 mol of KOH per mol of
complex in the pH range 3−12. After fitting the data with the
BEST7 program (Figure S4, Supporting Information), three
protonation constants were obtained at 3.91, 6.19, and 9.75.
The dissociation of the bridging carboxylate groups of 1 in
aqueous medium is quite in line with its well-documented
lability, which is facilitated by increasing the pH of the
solution.64,65
nanomaterials coated by silica can be easily reacted with 3-
aminopropyltriethoxysilane (APTES) to generate amino-
functionalized surfaces, and in this case, the design of a ligand
with a free aldehyde group could be the best choice for binding
the desired ligand or complex to the silica matrix via an imine
bond. Alternatively, the ligand or complex can be first reacted
with APTES (equimolar) to prepare an organoalkoxysilane that
can easily react with silanol groups of silica surfaces. On the
basis of the structures of the ligand HL1 and the biomimetic
NiII complex with very interesting catalytic activities previously
reported (Scheme 1),27 a new ligand was designed containing
similar functional groups and an additional free aldehyde group
to allow the covalent linkage to amino-functionalized surfaces
or amino-alkoxysilanes. This new ligand contains an aldehyde
group sterically hindered to be part of the coordination sphere
of the metal center, and therefore, after complexation with
metal ions, it will remain free for anchoring the silica matrix.
The step by step approach for the synthesis of the ligand is
depicted in Scheme 2. The last step involves an alkylation
reaction of a secondary amine with 2-chloromethyl-4-methyl-6-
formylphenol which gives rise to the desired aldehyde group
opposite to the coordinating groups in the hexadentate
dinucleating ligand. The purity and structure of H2BPPAMFF
1
was confirmed by H NMR and elemental analysis.
Synthesis and Characterization of [Ni2(HBPPAMFF)(μ-
OAc)2(H2O)]BPh4 (1) and [Ni2(HBPPAMFF)(μ-OBz)2(H2O)]-
ClO4 (1b). The reaction of Ni(ClO4)2·6H2O with the ligand
H2 BPPAMFF in methanol in the presence of
NaCH3COO·3H2O and NaBPh4 affords complex 1 as a light
green solid. The IR spectrum of 1 shows bands at νass(COO−)
at 1554 cm−1 and νs(COO−) at 1427 cm−1 indicating the
coordination of a carboxylate group in a bridging mode (Δ =
127 cm−1).62The terminal phenolic vibration (δO−H)
observed in the free ligand in 1375 cm−1 was observed in the
complex (1317 cm−1) indicating that this group was not
deprotonated (Figure S1, Supporting Information) in agree-
ment with the X-ray structure of 1b. Magnetic susceptibility
data for the free complex 1, collected as a function of
temperature (4.5−300 K) at a constant magnetic field, reveal a
weak antiferromagnetic coupling (J = −3.1 K), which is in
agreement with a structure containing the {NiII(μ-phenoxo)(μ-
OAc)2} moiety (vide infra).27
These pKa values are consistent with the following equilibria
obtained for a similar dinuclear NiIINiII complex without a
coordinated terminal phenol/phenolate group:27 complex 1
[ L H ( O H 2 ) N i I I ( μ - A c O ) 2 N i I I ( O H 2 ) ]
↔
[ L -
(OH2)3NiIINiII(OH2)] ↔ [L(OH2)2NiII(μ−OH)NiII(OH2)]
↔ [L(OH2)(OH)NiII(μ−OH)NiII(H2O)], as shown in
Scheme 3 (H2BPPAMFF = H2L). Thus, pKa1 is assigned to
deprotonation of the coordinated phenol ligand (also observed
in the crystal structure of complex 1b), while pKa2 and pKa3 are,
respectively, attributed to the formation of a NiII(μ−OH)NiII
species and deprotonation of a NiII-bound water molecule,
resulting in the catalytically active species (vide infra). In fact,
this proposal is strongly supported by the ESI-MS data of 1
under kinetic experimental conditions (vide infra).
In order to obtain a crystal for structural characterization of
complex 1, we tried many different crystallization techniques
such as changing counterions and exogenous bridging groups of
complex 1. Finally, we obtained a crystal suitable for X-ray
The electronic spectrum of 1 measured in acetonitrile
solution exhibits features at 308 nm (ε = 4300 mol·L−1·cm−1)
and 390 nm (ε = 4700 mol·L−1·cm−1), which can be attributed
to pπ → dπ* intraligand charge transfer, and 604 nm (ε = 20
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diffraction analysis of the di-μ-benzoate complex [NiNi-
(HBPPAMFF)(μ-OBz)2(H2O)]+ (1b). Complex 1b crystallizes
as green single crystals in acetonitrile/isopropanol diffusion that
belong to the monoclinic crystal system and space group P21/c.
An ORTEP view of the cation complex is presented in Figure 1.
and by the phenoxo endogenous bridge. Both NiII centers are
in an N2O3 coordination environment arranged in distorted
octahedral geometries, as evidenced by all of the angles around
them that deviate from 180° and 90° (Table 2). The Ni1 center
is bound to two nitrogen atoms [N1 (tertiary amine) and N32
(pyridine)] and by the terminal protonated O20 phenol
oxygen, which are arranged in a facial mode and belong to the
pendant arm of the binucleating ligand H2BPPAMFF. It is
important to note that the Ni1−O20 bond length (2.175(5) Å)
is significantly longer compared to the Ni−Ophenolate distances
in other dinuclear NiIINiII and mononuclear NiII complexes
where the phenol group is deprotonated (Ni−Ophenolate bond
distances are in the range 1.97 to 2.00 Å).66,67 In fact, in
complex 1b, the presence of the H atom on the terminal phenol
leads to the weaker interaction between Ni1 and O20 (Ni1−
O20 = 2.175 Å) than that between Ni1 and the bridging
phenolate O10 (Ni1−O10 = 1.997 Å). Besides the nitrogen
atoms, the coordination sphere of the Ni1 center is completed
by three oxygen atoms, O61 and O71, provided by the
exogenous benzoate bridges, and O10 from the endogenous
phenoxo group. The coordination sphere of the Ni2 center is
composed of the amine nitrogen N4, trans positioned to the
oxygen atom O62, the nitrogen pyridine N42 trans to the
oxygen O72, and the oxygen OW1 (from an water molecule)
trans to the oxygen O10 (from an phenoxo bridge).
Figure 1. ORTEP view of [NiNi(HBPPAMFF)(μ-OBz)2(H2O)]+
(1b). Ellipsoids are drawn at 40% probability level. For clarity, only
a partial labeling scheme is shown (Figure S5, Supporting Information,
shows all atoms labeled).
On the basis of the characterization of complex 1 by different
techniques, the X-ray structure of complex 1b, the similarity of
H2BPPAMFF to ligands reported previously in the literature,27
and the ligand geometry that favors the coordination of two
metal centers, complex 1 can be formulated as
[Ni2(HBPPAMFF)(μ-AcO)2(H2O)]BPh4 as shown in Figure
2.
The crystallographic data and the main bond distances/angles
are given in Tables 1 and 2. The resolution of the crystal
Table 2. Selected Bond Lengths (Å) and Angles (deg) for
Complex 1b
Ni1−Ni2
Ni1−O10
Ni1−O71
Ni1−O61
Ni1−N32
Ni1−N1
Ni1−O20
O10−Ni1−O71
O10−Ni1−O61
O71−Ni1−O61
O10−Ni1−N32
O71−Ni1−N32
O61−Ni1−N32
O10−Ni1−N1
O71−Ni1−N1
O61−Ni1−N1
N32−Ni1−N1
O10−Ni1−O20
O71−Ni1−O20
O61−Ni1−O20
N32−Ni1−O20
N1−Ni1−O20
O72−Ni2−O10
3.3968(12)
1.997(4)
2.022(5)
2.028(5)
2.068(6)
2.119(5)
2.175(5)
96.55(19)
93.02(18)
100.2(2)
171.5(2)
91.9(2)
Ni2−O72
Ni2−O10
Ni2−O62
Ni2−N42
Ni2−O1W
Ni2−N4
2.029(5)
2.039(4)
2.044(5)
2.087(6)
2.089(5)
2.184(5)
O72−Ni2−O62
O10−Ni2−O62
O72−Ni2−N42
O10−Ni2−N42
O62−Ni2−N42
O72−Ni2−O1W
O10−Ni2−O1W
O62−Ni2−O1W
N42−Ni2−O1W
O72−Ni2−N4
O10−Ni2−N4
O62−Ni2−N4
N42−Ni2−N4
O1W−Ni2−N4
Ni1−O10−Ni2
94.8(2)
94.18(18)
173.2(2)
92.02(19)
91.5(2)
Figure 2. Proposed structure for complex 1.
86.5(2)
84.2(2)
90.63(18)
165.8(2)
91.5(2)
174.52(19)
86.70(19)
93.4(2)
In order to establish unequivocally the catalytically relevant
species for the hydrolysis of diester bonds, ESI-MS studies were
carried out in CH3CN/water solutions (50:50), the same
solvent conditions as those employed for kinetic assays for
complex 1. Figure 3 shows the spectrum in CH3CN/water,
while Figure S6, Supporting Information, shows the observed
and expected isotopic distribution for the assigned species
under these experimental conditions. As can be observed in
Figure 3, four main groups of peaks are observed at mass to
charge (m/z) ratios of 865.20, 759.11, 643.20, and 359.00. The
peak at m/z = 865.20 can be attributed to [Ni2LH(μ-
OAc)2(C2H5OH)]+ (L= HBPPAMFF), which correspond to
a cation similar to the complex 1b shown in Figure 1 with
acetate ions instead of benzoate ions and an ethanol molecule
coordinated to the nickel center instead of water which may
arise from reduction of acetate in the ion spray. The next group
of peaks at m/z = 759.11 is best interpreted in terms of a
dinuclear nickel complex with one μ−OH group and one
80.9(2)
93.15(19)
91.07(17)
170.5(2)
80.42(19)
88.83(19)
114.62(19)
91.72(17)
79.54(19)
175.25(19)
88.8(2)
88.1(2)
90.32(18)
structure of 1b shows an asymmetric unit composed of a cation
complex [Ni2(HBPPAMFF)(μ-OBz)2(H2O)]+ and a perchlo-
rate anion as the counterion. Figure 1 shows the structure of
the cation complex, which is composed of one unsymmetrical
ligand H2BPPAMFF coordinated to two NiII atoms (Ni1 and
Ni2). Each NiII center is six coordinated and bridged by two
exogenous benzoate groups coordinated in the bidentate mode
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spherical particles of ca. 32 nm (Figure S7, Supporting
Information).
The strategy for the immobilization of complex 1 in the silica
nanospheres consists in the modification of complex 1 with (3-
aminopropyl)triethoxysilane (APTES) followed by reaction of
the alkoxysilane-complex formed in situ with the silica
nanospheres. The color of the solution containing the complex
changed from light green to yellow, and after centrifugation and
washing, the silica matrix ended up becoming yellow. The IR
spectrum of SiO2-1 contains broad and strong vibrational bands
of Si−OH, Si−O, and Si−O−Si typical of amorphous silica that
overlay the features corresponding to complex 1 (Figure S8,
Supporting Information). Therefore, we discuss the IR
spectrum in terms of the free ligand compared to the
alkoxysilane-modified ligand formed by reacting the ligand
H2BPPAMFF and APTES. After reaction with APTES, the IR
spectrum is characterized by the disappearance of the carbonyl
band at 1679 cm−1 (νCO) of the aldehyde group and the
appearance of a new band at 1630 cm−1 (νCN), which is a
strong indication of the formation of the imine bond (Figure
4). The electronic spectrum of the alkoxysilane-modified ligand
Figure 3. ESI-MS analysis of complex 1 CH3CN/water solutions
(50:50).
CH3CN ligand ([NiIINiIHL(μ-OH)(CH3CN)]+), indicating
reduction of the NiII center coordinated by the soft side of
the ligand ion under the conditions of the eletrospray ionization
(see electrochemistry), while the group of peaks at 359.08 is
likely due to the dication [Ni2HL(μ-OH)]2+ without exogenous
ligands (i.e., carboxylate, H2O, CH3CN, or C2H5OH). Finally,
the major peak at 643.22 can be assigned to the mononuclear
[NiHL]+ species in which the ligand does not undergo
fragmentation but one nickel atom, most probably from the
bidentate side of the ligand, becomes dissociated from the
dinuclear complex under the conditions of the eletrospray
ionization. It is important to emphasize that the dinuclear
species [Ni2HL(μ-OH)(CH3CN)]+ and [Ni2HL(μ-OH)]2+
identified from the ESI-MS experiments corroborate very well
with the proposed protonation/deprotonation equilibrium
from potentiometric titration (vide supra).
Immobilization of Complex 1 on Silica Nanospheres.
The silica nanospheres used in this study were prepared by
means of a reverse microemulsion formulation with cyclo-
hexane and a nonionic surfactant. The reverse microemulsion is
an isotropic and thermodynamically stable single-phase that
consists of water nanodroplets stabilized by the surfactant
molecules and dispersed in the oil phase. The nanodroplets
serve as nanoreactors for the synthesis of the silica nanospheres
of controlled size. The silica matrix is formed by hydrolysis and
condensation of TEOS by adding ammonium hydroxide to the
reverse microemulsion phase. The transmission electron
microscopy (TEM) analysis of the samples reveals nearly
Figure 4. IR spectra of the ligand H2BPPAMFF and the alkoxysilane-
modified ligand in solid state (KBr pallet) in the range from 1250 to
2000 cm−1. IR spectra in the full range from 400 to 4000 cm−1 are
shown in Figure S9, Supporting Information.
exhibits a strong absorption at 420 nm, absent in the
H2BPPAMFF ligand, which can also be attributed to the
formation of the imine bond (Figure 5c). These results
confirmed the silylation of the ligand, and since the alkoxysilane
groups are likely to hydrolyze and condensate with the silanol
groups on silica surfaces, we can conclude that the ligand was
properly modified to be covalently attached to silica spheres.
After immobilization of complex 1, the color of silica
changed to that characteristic of complex 1. The electronic
spectrum of SiO2-1, measured in solid state (KBr pellets),
exhibits features at 380, 415, and 560 nm (Figure 5d). The
absorption band at 415 nm can be attributed to the
immobilization of complex 1 with the formation of imine
bond, also observed in the alkoxysilane-ligand (Figure 5c),
while the broad band centered at 560 nm can be assigned to d−
d transitions. The spectrum of the pure complex 1 (Figure 5b)
in CH3CN solution shows the corresponding transition at 604
nm, which clearly indicates that the coordination environment
around the NiII centers is affected when complex 1 is anchored
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Figure 6. Temperature dependence of the effective magnetic moment
for complex 1 (open circle) and its contribution extracted from SiO2-1
(solid circle). Solid lines are fits (see text).
Figure 5. UV−vis spectra of (a) H2BPPAMFF ligand, (b) complex 1,
(c) alkoxysilane-ligand in acetonitrile solution, and (d) SiO2-1 in solid
state (KBr pellet).
ferromagnetism to ferromagnetism on introducing slight
changes in the dinuclear core.27
to SiO2. From this information, it seems reasonable to assume
that in SiO2-1, the acetate bridging ligands are replaced by
solvent molecules, most probably with the formation of a
[Ni(μ−OH)Ni] structural unit. In fact, this interpretation is
quite in line with the magnetic results of 1 and SiO2-1 (vide
infra).
In order to follow possible structural modifications when
complex 1 is anchored to silica nanospheres (SiO2-1), magnetic
measurements were carried out. The raw magnetic data showed
paramagnetism of the pure dinuclear NiII complex 1 in the
whole temperature range and diamagnetism of SiO2 and SiO2-1
in the temperature range above 6 and 15 K, respectively. While
the crossover to paramagnetism for SiO2-1 composite is
expected, for SiO2 it probably results from the presence of a
small amount of impurity of unknown origin. In order to obtain
the magnetic contribution of the dinuclear NiII complex 1 in
the SiO2-1 composite, magnetic contribution of SiO2 was
subtracted. This was taken as the susceptibility of the separately
measured SiO2 compound.
The essential difference between complex 1 and SiO2-1 lies
in the J and TIP parameters. As can be observed, both are
greater for the dinuclear NiII-complex anchored to SiO2-1. The
stronger antiferromagnetic coupling in the anchored complex
SiO2-1 probably originates due to the formation of a NiII(μ−
OH)NiII moiety, which in principle should approximate the NiII
centers, given rise to a more effective overlapping of the d-metal
and bridging oxygen orbitals, thus increasing the interchange
coupling.68 However, it should be noted that such an
interpretation is merely speculative, given that no structural
information are available for the SiO2-1 anchored system. The
TIP parameters are too large and may result from some
parasitic effects, which have not been taken into account, such
as impurity contained in SiO2 but not present in the SiO2-1
composite. In summary, the magnetic results clearly confirm
that the dinuclear NiII complex was anchored to the silica
nanospheres and that the coordination geometry around the
NiII centers is somewhat modified with respect to the pure
complex 1. Indeed, the kinetic data obtained from the
hydrolysis reaction of the diester phosphate 2,4-BDNPP
catalyzed by 1 and SiO2-1 are in full agreement with this
hypothesis (vide infra).
Reactivity Studies. The catalytic activity of complex 1
immobilized in silica nanospheres (SiO2-1) in the cleavage of
2,4-BDNPP is strongly influenced by the pH of the reaction
mixture. The pH/rate profile curve shown in Figure 7a was
fitted using a Boltzman model to give a pKa value of 8.0, which
can be attributed to deprotonation of a NiII-bound water
molecule to generate the catalytically active species [(OH)Ni-
(μ−OH)Ni(OH2)] when 1 is linked to silica.69
The catalytic activity of complex 1 in the cleavage of 2,4-
BDNPP is also strongly influenced by the pH of the reaction
mixture with an exponential growth pH/rate profile (Figure
7b). However, as expected, for the reaction catalyzed by the
free complex 1, no fully sigmoidal curve could be obtained
since the pKa = 9.75 (see potentiometric titration) to generate
the catalytically active species [(OH)Ni(μ−OH)Ni(OH2)] lies
in the higher end of the investigated pH range. Thus, for both
reactions, these results strongly suggest that the rate of the
catalytic reaction depends on the deprotonation of the NiII
In Figure 6, the temperature dependence of the effective
magnetic moment (μeff = 2.828 (χmolT)1/2) is shown for the
pure dinuclear complex 1, and for the corresponding magnetic
contribution extracted from the SiO2-1 composite.
The data in Figure 6 were fitted to the Hamiltonian given in
eq 1
2
̑
H = −JS1S2 + ΣiDSi + Σigμ HSi i = 1, 2
(1)
i
B
where J is the magnetic exchange constant, S1 = S2 is the spin of
the NiII ion, equal to 1, D is the zero field splitting parameter, g
the spectroscopic factor, H is the magnetic field, and μB is the
Bohr magneton. The parameters obtained from the fits are J =
−3.1 2 K, g = 1.97 0.03, D = 0, and TIP = (2485 800) ×
10−6 emu·mol−1 for the pure complex 1; J = −14.8 3 K, g =
1.97 0.03, D = 0, and TIP = (6101 900) × 10−6 emu·mol−1
for extracted complex 1, where TIP means temperature
independent paramagnetism. D was put equal to zero in
order to avoid overparametrization.
In fact, the weak antiferromagnetic coupling (J = −3.1 K)
obtained for the pure complex 1 is in agreement with a
structure containing the {NiII(μ-phenoxo)(μ-OAc)2} moiety in
which the magnetic interaction generally varies from anti-
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Figure 8. Dependence of the initial reaction rate (V0) on the 2,4-
BDNPP concentration for the hydrolysis reaction promoted by SiO2-1
(a) and complex 1 (b). Conditions: [complex] = 1.0 × 10−5 mol·L−1;
[buffer] = 50 × 10−3 mol·L−1 (CHES, pH) 9.00; I = 50 × 10−3
mol·L−1 (LiClO4) in H2O/CH3CN (50% v/v) at 25 °C.
Figure 7. Dependence of the initial reaction rate (V0) on pH for the
hydrolysis of 2,4-BDNPP promoted by SiO2-1 (a) and complex 1 (b).
Conditions: [complex] = 1.0 × 10−5 mol·L−1; [2,4-BDNPP] = 1.0 ×
10−3 mol·L−1; [buffers] = 50 × 10−3 mol·L−1; I = 50 × 10−3 mol·L−1
(LiClO4) in H2O/CH3CN (50% v/v) at 25 °C.
k
cathomo is about 0.85 for SiO2-1). Nevertheless, considering the
coordinated water molecule of the catalytically active species of
the type [(OH)Ni(μ−OH)Ni(OH2)], as previously suggested
for the NiIINiII analogues.27,70,71 In fact, the distinct pKa values
observed for the pure complex 1 (potentiometic titration) and
the anchored SiO2-1 system are in full agreement with the
magnetochemical results, which clearly reveal that the
coordination geometry around the NiII centers is somewhat
modified when 1 is anchored to the silica nanospheres.
second order rate constant (E = kcat/KM), a distinct catalytic
efficiency with Ehetero/Ehomo of 2 can be observed for SiO2-1,
thus indicating that the heterogeneous system is more efficient
than the soluble catalyst. The higher affinity of 2,4-BDNPP for
the anchored system SiO2-1 presumably originates from some
influence of the solid support. Particularly, for SiO2-1, the
stabilization may occur via interaction with the surface silanol
groups.
The dependence of the initial rates at pH 9.0 on the
concentration of the substrate (0.2−1.8 mmol·L−1) reveals
saturation kinetics with Michaelis−Menten-like behavior
(Figure 8). The kinetic parameters were obtained from
nonlinear square fits using the program Origin 8.0 and the
parameters V0, KM, and kcat are shown in Table 3. BDNPP
bound to free complex 1 hydrolyzes 138,400 times faster than
free BDNPP (kuncat = 3.88 × 10−7 s−1) under identical
experimental conditions,59 while complex 1 immobilized in
silica nanospheres hydrolyzes 2,4-BDNPP 118,500 times faster
than the noncatalyzed reaction. The kinetic parameters Kass
(Kass = 1/KM in Table 3) reveal a higher stabilization of the
substrate−catalyst intermediate for the heterogeneous reaction
than for the reaction in homogeneous media (Kass is about 2
times higher for SiO2-1), while the catalytic turnover constant
However, kcat ≅ 3.0 × 10−3 s−1, obtained for the hydrolysis of
2,4-BDNPP by SiO2-1 at pH = 7.0, lies in the range of values
(4.0 × 10−4 to 3.0 × 10−3 s−1) found for a series of mixed-
valence [FeIIIMII] complexes (MII = Fe, Mn, Co, Ni, Cd) with
the same substrate.10,38,58 In addition, it is important to
emphasize here that, besides its catalytic activity, the anchored
SiO2-1 system has the advantage that it can be reused with
similar efficiency for subsequent diester hydrolysis reactions.
In order to assess the possible hydrolysis of the monoester
2,4-DNPP, one of the products formed from the hydrolysis
reaction of the diester 2,4-BDNPP, a stoichiometric reaction
between complex 1 and the 2,4-DNPP substrate, was
monitored. It was observed that 2 equiv of 2,4-DNPP is
released in 12 h for complex 1 at 25 °C, which indicates the
hydrolysis of both the mono- and diester. On the basis of this
information and assuming that the hydrolysis reaction of the
kcat is slightly lower for the heterogeneous reactions (kcathetero
/
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Table 3. Kinetic Parameters for the 2,4-BDNPP Hydrolysis Promoted by Free and Immobilized Complex 1 at 25 °C and pH 9.0
a
b
V0 (mol·L·s−1
)
KM (mol·L−1
)
kcat (s−1
)
Kass (L·mol−1
)
E (L·s−1·mol−1
)
fc
turnover (h−1
)
1
5.37 × 10−7
3.40 × 10−7
2.04 × 10−9
1.57 × 10−3
6.90 × 10−4
5.37 × 10−2
4.6 × 10−2
2.04 × 10−3
637
34.2
66
138,400
118,500
105
33.4
32.7
SiO2-1
1450
1-2,4-DNPP
3.0 × 10−5
33,333
68
a
b
c
Kass = 1/KM. E = kcat/KM (catalytic efficiency). f = (kcat/kuncat); kuncat = 3.88 × 10−7 s−1.
2,4-BDNPP diester leads to the formation of an intermediate
(presumably the monoester coordinated to the catalyst), we
decided to investigate the catalytic activity of complex 1 in the
hydrolysis of the monoester 2,4-DNPP. As can be observed in
Figure 9, the initial rates as a function of substrate
Scheme 4. Proposed Mechanism for the Hydrolysis of 2,4-
BDNPP Promoted by Complex 1
Figure 9. Dependence of the initial reaction rate (V0) on the 2,4-
DNPP concentration for the hydrolysis reaction promoted by complex
1. Conditions: [complex] = 1.0 × 10−6 mol·L−1; [buffer] = 50 × 10−3
mol·L−1 (CHES, pH) 9.00; I = 50 × 10−3 mol·L−1 (LiClO4) in H2O/
CH3CN (50% v/v) at 25 °C.
proceeding through an intramolecular mechanism, in which
the phosphorus atom undergoes a nucleophilic attack
promoted by the coordinated hydroxide ion.
The efficiency of immobilization of complex 1 without using
APTES, which means by noncovalent bonding, is very low.
Moreover, no significant rates of hydrolysis of 2,4-BDNPP were
observed, when compared with uncatalyzed reaction. This
suggests that the immobilization process by adsorption and
most probably electrostatic bonds contributes to blocking the
catalytic metal centers, which can be partially occupied by
interaction with silica and less accessible to the substrate. The
immobilization process by covalent bonding through imine
bond formation reported here is more specific and allowed the
interaction of the complex with the substrate to generate the
active catalytic species to perform phosphatase-like activity with
high turnover rates in the hydrolysis of 2,4-BDNPP. Finally, the
SiO2-1 catalyst was reused in subsequent hydrolysis of 2,4-
BDNPP, after washing the material in a Soxhlet extractor with
1:1 HEPES buffer pH 7/acetonitrile, and the rate of reaction
was similar to that obtained in the first cycle. Therefore, it can
be concluded that the SiO2-1 catalyst is stable for prolonged
periods at least under the experimental conditions employed in
the hydrolysis reactions.
concentration reveal saturation kinetics with Michaelis−
Menten-like behavior. The kinetic parameters, kcat = 2.04 ×
10−3 s−1 and Kass = 33,333 mol−1·L reveal that the association
constant of the monoester with catalyst 1 is about 52 times
greater, while kcat is 26 times lower when compared to those
parameters obtained with the corresponding 2,4-BDNPP
diester. These data allowed us to conclude that distinct
nucleophile groups within complex 1 are acting as the catalysts
in the hydrolysis of the mono- and diester-phosphate
substrates. It is important to note that the spontaneous
hydrolysis of the monoester 2,4-DNPP is about 50 times
greater than the hydrolysis of the diester 2,4-BDNPP under
similar experimental conditions.59 Here, as observed for a
similar dinuclear NiIINiII complex,27 the opposite is observed,
clearly indicating that the hydrolysis reaction of the monoester
is occurring through the attack of a poorer nucleophile than
that responsible for the catalysis of the diester (Scheme 4).72
In order to establish whether the nucleophilic attack on the
phosphorus atom was via the terminal hydroxide ion or a
general base catalysis, isotopic effect was evaluated in the
hydrolysis of 2,4-BDNPP in D2O catalyzed by complex 1 and
SiO2-1. According to Burstyn and co-workers,73 if 0.80 < (kH/
kD) < 1.50, this is indicative that there is no proton transference
involved in the reaction limiting step, and suggests an
intramolecular nucleophilic attack mechanism.74 The kH/kD
ratios obtained for complex 1 and SiO2-1 were all close to 0.95,
which corroborate the presence of a hydrolysis reaction
CONCLUSIONS
■
In summary, a synthetic dinuclear NiII hydrolase, which
contains a carbonyl group located in the ortho position of
the terminal NiII-bound phenolate ligand, was covalently linked
to silica using APTES. The complex attached to the solid
surface of the nanomaterials maintained its phosphatase-like
activity with high turnover rates in the hydrolysis of 2,4-
BDNPP and catalytic rates as high as 118,500 times that of the
uncatalyzed reaction. The heterogeneous system presented
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higher catalytic efficiency (2 times that of the soluble complex),
besides the higher complex to substrate association constant.
Further studies of the complexes 1 and SiO2-1 on the activity of
DNA hydrolysis to generate new possible drugs for
technological applications, e.g, as anticancer agents, are
underway and will be presented in future reports.
ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray crystallographic file in CIF format; Figures S1−S9 and
Table S1 in PDF format. This material is available free of
charge via the Internet at http://pubs.acs.org. Full tables of
crystallographic data of complex 1b (except structure factors)
were deposited at Cambridge Structural Database (CCDC
843653), and these data are available free of charge from
CCDC, 12 Union Road, Cambridge, CB2 1EZ, U.K. (fax, +44-
1223-336-003; e-mail, deposit@ccdc.cam.ac.uk or http://www.
ccdc.cam.ac.uk).
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ademir@qmc.ufsc.br (A.N.); lrossi@iq.usp.br
(L.M.R.).
Notes
The authors declare no competing financial interest.
(28) Zhao, X.; Bagwe, R. P.; Tan, W. Adv. Mater. 2004, 16, 173.
(29) Bagwe, R. P.; Yang, C.; Hilliard, L. R.; Tan, W. Langmuir 2004,
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(30) Silva, P. R.; Vono, L. L. R.; Esposito, B. P.; Baptista, M. S.; Rossi,
́
L. M. Phys. Chem. Chem. Phys. 2011, 13, 14946.
(31) Roy, I.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Bergey, E. J.;
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ACKNOWLEDGMENTS
■
We are grateful for grants that helped to support this research:
CNPq, FAPESC, FAPESP, and INCT-Catalise (Brazil),
BMBF/IB-BRA 07/2007 (Germany). We also thank Professor
Pedro K. Kiyohara (IF-USP) for TEM analysis.
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