A unique nickel system having versatile catalytic activity of biological significance

Article abstract of DOI:10.1021/ic901546t

A new dinuclear nickel(ll) complex, [Ni₂(LH₂)(H ₂O)₂(OH)(NO₃)](NO₃)₃ (1), of an "end-off" compartmental ligand 2,6-bis(N-ethylpiperazine- iminomethyl)-4-methyl-phenolato, has been synthesized and structurally characterized. The X-ray single crystal structure analysis shows that the piperazine moieties assume the expected chair conformation and are protonated. The complex 1 exhibits versatile catalytic activities of biological significance, viz. catecholase, phosphatase, and DNA cleavage activities, etc. The catecholase activity of the complex observed is very dependent on the nature of the solvent. In acetonitrile medium, the complex is inactive to exhibit catecholase activity. On the other hand, in methanol, it catalyzes not only the oxidation of 3,5-ditert-buty !catechol (3,5-DTBC) but also tetrachlorocatechol (TCC), a catechol which is very difficult to oxidize, under aerobic conditions. UV-vis spectroscopic investigation shows that TCC oxidation proceeds through the formation of an intermediate. The intermediate has been characterized by an electron spray ionizaton-mass spectrometry study, which suggests a bidentate rather than a monodentate mode of TCC coordination in that intermediate, and this proposition have been verified by density functional theory calculation. The complex also exhibits phosphatase (with substrate p-nitrophenylphosphate) and DNA cleavage activities. The DNA cleavage activity exhibited by complex 1 most probably proceeds through a hydroxyl radical pathway. The bioactivity study suggests the possible applications of complex 1 as a site specific recognition of DNA and/or as an anticancer agent.

Full text of DOI:10.1021/ic901546t

Inorg. Chem. 2010, 49, 3121–3129 3121
DOI: 10.1021/ic901546t
A Unique Nickel System having Versatile Catalytic Activity of
Biological Significance
Tanmay Chattopadhyay,†,r Madhuparna Mukherjee,† Arindam Mondal,‡ Pali Maiti,† Arpita Banerjee,†
Kazi Sabnam Banu,† Santanu Bhattacharya,§ Bappaditya Roy,‡ D. J. Chattopadhyay,‡ Tapan Kumar Mondal,
Munirathinam Nethaji,^ Ennio Zangrando,*,# and Debasis Das*,†
†Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata-700 009, India,
‡Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata-700 019,
India, §Department of Chemistry, Maharaja Manindra Chandra College, Kolkata-700 003, India,
Department of Chemistry, Jadavpur University, Jadavpur, Kolkata-700 032, India, ^Department of Inorganic
and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India, and #Dipartimento di Scienze
Chimiche, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy. rPresent address: Nanotube Research
Centre (NTRC), AIST, Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-805, Japan.
Received August 4, 2009
A new dinuclear nickel(II) complex, [Ni2(LH2)(H2O)2(OH)(NO3)](NO3)3 (1), of an “end-off” compartmental ligand
2,6-bis(N-ethylpiperazine-iminomethyl)-4-methyl-phenolato, has been synthesized and structurally characterized.
The X-ray single crystal structure analysis shows that the piperazine moieties assume the expected chair conformation
and are protonated. The complex 1 exhibits versatile catalytic activities of biological significance, viz. catecholase,
phosphatase, and DNA cleavage activities, etc. The catecholase activity of the complex observed is very dependent on
the nature of the solvent. In acetonitrile medium, the complex is inactive to exhibit catecholase activity. On the other
hand, in methanol, it catalyzes not only the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) but also tetrachloro-
catechol (TCC), a catechol which is very difficult to oxidize, under aerobic conditions. UV-vis spectroscopic
investigation shows that TCC oxidation proceeds through the formation of an intermediate. The intermediate has been
characterized by an electron spray ionizaton-mass spectrometry study, which suggests a bidentate rather than a
monodentate mode of TCC coordination in that intermediate, and this proposition have been verified by density
functional theory calculation. The complex also exhibits phosphatase (with substrate p-nitrophenylphosphate) and
DNA cleavage activities. The DNA cleavage activity exhibited by complex 1 most probably proceeds through a hydroxyl
radical pathway. The bioactivity study suggests the possible applications of complex 1 as a site specific recognition of
DNA and/or as an anticancer agent.
Introduction
of nickel enzymes known to date are of plant or bacteria
origins.1-8 Here, it is noteworthy that it had taken about
50 years to identify the first nickel metalloenzyme since the
date of crystallization (1926)9 of jack-bean urease, the first
enzyme ever isolated as a crystalline material.10 The aspects
pointed out above lead to at least two considerations: first, it
is premature to exclude the possible presence of a nickel
enzyme in animals, and second, the apparent lack of interest
to explore the biochemistry of nickel. During the past few
years, we have been interested in searching for small coordi-
nation molecules that exhibit enzymatic activity to mimic
the structure as well as the functional properties of metallo-
biosites. Our recent work on the dinuclear CuII-system
(here system stands for the “end-off” compartmental ligand
In spite of the very rich coordination chemistry of nickel,
its biological significance is not well developed. All types
*To whom correspondence should be addressed. E-mail: dasdebasis2001@
yahoo.com.
(1) Walsh, C. T.; Orme-Johnson, W. H. Biochemistry 1987, 26, 4901.
(2) The Bioinorganic Chemistry of Nickel; Lancaster, J. R., Jr., Ed.; VCH:
New York, 1988.
(3) Nickel and its role in biology. In Metal Ions in Biological systems;
Sigel, H., Ed.; Marcel Dekker: New York, 1988; Vol. 23.
(4) Haussinger, R. P. Biochemistry of nickel. In Biochemistry of the
Elements, Frieden, E., series Ed.; Plenum Press: New York, 1993; Vol. 12.
(5) Cammack, R.; van Vliet, P. Catalysis by nickel in biological system.
In Bioinorganic Catalysis; Redijik, J., Bouernans, L., Eds.; Marcel Dekker:
New York, 1998.
(6) Maroney, M. J.; Davidson, G.; Allan, C. B.; Figlar, J. Struct. Bonding
(Berlin) 1998, 92, 1.
(7) Fontecilla-Camps, J. C. Struct. Bonding (Berlin, Ger.) 1998, 91, 1.
(8) Ragsdale, W. S. Curr. Opin. Chem. Biol. 1998, 2, 208.
(9) Sumner, J. B. J. Biol. Chem. 1926, 69, 435.
(10) Jabri, E.; Carr, M. B.; Hausinger, R. P.; Karplus, P. A. Science 1995,
268, 998.
r
2010 American Chemical Society
Published on Web 03/01/2010
pubs.acs.org/IC

3122 Inorganic Chemistry, Vol. 49, No. 7, 2010
Chattopadhyay et al.
2,6-bis(N-ethylpiperazine-iminomethyl)-4-methyl-phenol, LH)
enabled us to find out the most active model of catechol
oxidase known to date.11 It not only catalyzes the oxidation
of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butyl-
benzoquinone (3,5-DTBQ) (with a kcat value as high as
2.88 ꢀ 104 h-1) but also that of tetrachlorocatechol (TCC)
to tetrachlorobenzoquinone (TCQ), a process never reported
earlier by using dinuclear copper(II)-complexes. In this
paper, we report the synthesis and the comprehensive char-
acterization by routine physicochemical studies and by X-ray
single crystal structure analysis of a unique dinuclear nickel-
(II) complex [Ni2(LH2)(H2O)2(OH)(NO3)](NO3)3, 1, having
the same compartmental ligand. The complex, besides being
a model compound for the met form of the active site of
catechol oxidase, it exhibits a broad spectrum of catalytic
activities of biological significance, in particular, it catalyzes
the hydrolysis reaction of the phosphate monoester p-nitro-
phenylphosphate (PNPP) and causes the cleavage in plasmid
DNA.
Table 1. Crystallographic Data and Details of Refinement for Complex 1
empirical formula
formula weight
crystal system
space group
C21H40N10Ni2O16
806.05
hexagonal
P61
9.9402(13)
57.2290(15)
4897.1(9)
6
1.640
1.240
2520
˚
a (A)
˚
c (A)
3
volume (A )
˚
Z
calc density (g/cm3)
M (Mo-KR) (mm-1
F(000)
)
θ range (ꢀ)
2.14-27.98ꢀ
12 428
4948
0.0604
3112
collected reflections
indep reflections
Rint
observed reflections I>2σ(I)
parameters
R1 [I>2σ(I)]
458
0.0531
wR2 [I>2σ(I)]
0.0737
0.885
0.463, -0.359
goodness of fit on F2
-3
˚
residuals (e A
)
Triton X-100) were included. Cells were grown in the wells for
24 h, up to 70-80% confluence. The wells were then inoculated
aseptically with 50 μL of different dilutions of the samples and
the negative and positive controls, and the plates were incubated
at 37 ꢀC in a humid atmosphere containing 5% CO2 for 30 h.
After the incubation period, 50 μL of an aqueous solution of
MTT (2 mg/mL) was added to each well, and the plates were
incubated further for 4 h at 37 ꢀC in a humid atmosphere
containing 5% CO2. After this time, the liquid medium in the
wells was removed, 200 μL of dimethylsulfoxide (DMSO) was
added to each well, and the absorbance at 450 nm was deter-
mined in a microplate reader. The toxic effect of complex 1 on
the VERO cell line was calculated from the following (eq 1):
Experimental Section
Physical Methods and Materials. All materials were obtained
from commercial sources and used as purchased. Solvents were
dried according to standard procedure and distilled prior to use.
The 2,6-diformyl-4-methylphenol was prepared according to
the literature method.12 Nickel nitrate hexahydrate (Merck) and
N-(2-aminoethyl)piperazine (Aldrich) were purchased from
commercial sources and used as received. Nickel was estimated
gravimetrically with dimethylglyoxime. Elemental analyses (carbon,
hydrogen, and nitrogen) were performed using a Perkin-Elmer
240C analyzer. Infrared spectra (4000-400 cm-1) were recorded
at 28 ꢀC on a Shimadzu FTIR-8400S using KBr as a medium.
Electronic spectra (800-200 nm) were obtained at 27 ꢀC using a
Shimadzu UV-3101PC, where dry acetonitrile/dry methanol
were used as a medium as well as a reference. The electrospray
mass spectra were recorded on a MICROMASS Q-TOF mass
spectrometer. The electron paramagnetic resonance (EPR) ex-
periment was done at both 25 and -135 ꢀC in pure methanol
using a Bruker EMX-X band diffractometer. The measurements
of the variable-temperature magnetic susceptibility and the field
dependence of magnetization were carried out on a Quantum
Design MPMS-XL5 SQUID magnetometer. Susceptibility data
was collected using an external magnetic field of 0.2 T in the
temperature range of 2-300 K. The experimental susceptibi-
lities were corrected for diamagnetism (Pascal’s tables).13
Experimental Procedure of the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazonium bromide (MTT) Assay. Monkey kidney
fibroblast (VERO) cells and A549 cells (a carcinomas human
alveolar basal epithelial cell line) were maintained in Dulbecco’s
modified eagle medium (DMEM) media supplemented with 5%
(v/v) calf serum, 2 mmol/L glutamine, and 1% (v/v) penicillin
(streptomycin).Cells (T-25 flask) were cultured at 37 ꢀC in a
humid atmosphere containing 5% CO2 and subcultured every
2-3 days. Cells were harvested with trypsin, and a suspension
containing 2 ꢀ 105 cells per mL was prepared. This assay was
performed in a 96-well, flat-bottomed, γ-irradiated, microliter
plates with lids. Filter sterilized (0.22 μM), phosphate buffer
saline (PBS) soluble complex 1 (1 mg/mL) (50 μL) was serially
diluted. Both negative (50 μL PBS) and positive controls (50 μL
%viability ¼ ðmean absorbance of the sample
=mean absorbance of the controlÞ ꢀ 100
ð1Þ
X-ray Data Collection and Crystal Structure Determination.
Diffraction data for the structure reported were collected at
room temperature on a BRUKER SMART APEX diffract-
˚
ometer (Mo-KR radiation, λ = 0.71073 A) equipped with a
charge-coupled device (CCD). Cell refinement and indexing and
scaling of the data sets were carried out using packages Bruker
SMART APEX and Bruker SAINT.14 The structure was solved
by direct methods and subsequent Fourier analyses15 and
refined by the full-matrix least-squares method based on F2
with all observed reflections. The contribution of hydrogen (H)
atoms at calculated positions was introduced in the final cycles
of refinement. Crystallographic data and details of refinement
are reported in Table 1. All the calculations were performed
using the WinGX System, version 1.70.01.16
Synthesis of Complex 1. A methanolic solution (5 mL) of
N-(2-aminoethyl)piperazine (0.258 g, 2 mmol) was added drop-
wise to a heated methanolic solution (10 mL) of 2,6-diformyl-4-
methylphenol (0.164 g, 1 mmol), and the resulting mixture was
boiled for 30 min. Then, a methanolic solution (15 mL) of
Ni(NO3)2 6H2O (0.673 g, 2.5 mmol) was added, and the result-
3
ing mixture was refluxed for two hours. After cooling, the clear
deep-green solution was kept in a CaCl2 desiccator in the dark.
Rectangular-shaped blue crystals of 1, suitable for X-ray data
collection, were obtained after a few days. Yield: 0.48 g (72%).
FT-IR: ν(CdN) 1621 cm-1; ν(skeletal vibration) 1546 cm-1
;
(11) Banu, K. S.; Chattopadhyay, T.; Banerjee, A.; Bhattacharaya, S.;
Suresh, E.; Nethaji, M.; Zangrando, E.; Das, D. Inorg. Chem. 2008, 47, 7083.
(12) Gagne, R. R.; Spiro, C. L.; Smith, T. J.; Hamann, C. A.; Thies,
W. R.; Shiemeke, A. K. J. Am. Chem. Soc. 1981, 103, 4073.
(13) (a) Dutta, R. L.; Syamal, A. Elements of Magnetochemistry, 2nd ed.;
East West Press: Manhattan Beach, CA, 1993. (b) Kahn, O. Molecular Magne-
tism; VCH : New York, 1993.
(14) SMART, SAINT Software Reference Manual Bruker; Bruker AXS
Inc.: Madison, WI, 2000.
(15) Sheldrick, G. M. SHELX97 Programs for Crystal Structure Analysis
€
€
(Release 97-2); University of Gottingen: Gottingen, Germany, 1998.
(16) Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.

Article
Inorganic Chemistry, Vol. 49, No. 7, 2010 3123
ν(H2O) 3399.4 cm-1. UV-vis (MeOH)/nm: 395 (sh, ε =
6728 M-1cm-1), 599 (sh, ε = 1040 M-1cm-1), 968 (sh, ε =
402 M-1cm-1). Anal. calcd for C21H40N10O16Ni2: C, 31.26; H,
4.96; N, 17.37; Ni, 14.56%. Found: C, 31.36; H, 4.73; N, 17.39;
Ni, 14.58%.
Computational Details. Density functional theory (DFT)
calculations were carried out with the Gaussian 03 package.17
The exchange functional of Becke and the correlation functional
of Lee, Yang, and Parr (B3LYP)18,19 were employed in combi-
nation with the Stuttgart-Dresden (SDD) effective core poten-
tials20 for the Ni atoms and the 6-31G(d) basis set21 for the
remaining elements. Frequency calculations were performed
within the harmonic approximation to check whether the opti-
mized geometries correspond to energy minima (NIMAG = 0)
on the potential energy surface. The J value for 1 was calculated
following the previously reported method,22 using the broken-
symmetry approach to estimate the energy of the low-spin
states. We have used H = -2JS1S2 convention for the exchange
Hamiltonian, the equation for calculating the J value is J =
[E(QT) - E(BSS)]/6, where E(QT) is the energy of the quintet
state of the dinuclear Ni complex and E(BSS) is the energy of its
broken-symmetry singlet state. Vertical electronic excitations
based on UB3LYP optimized geometry were computed using
the time-dependent density functional theory (TD-DFT) form-
alism23 in methanol using a conductor-like polarizable conti-
nuum model (CPCM).24
Figure 1. ORTEP view of the cationic dinuclear complex of 1.
Table 2. Selected Coordination Bond Lengths (Angstroms) and Angles (Degrees)
for 1 with Esds in Parentheses
Ni(1)-N(1)
2.218(5)
2.011(5)
2.039(4)
1.995(5)
2.104(5)
2.185(5)
2.970(1)
83.2(2)
169.30(19)
104.1(2)
93.0(2)
87.12(19)
88.36(19)
171.9(2)
88.1(2)
88.3(2)
84.0(2)
93.3(2)
86.05(19)
94.95(19)
88.53(19)
176.4(2)
Ni(2)-N(4)
Ni(2)-N(5)
Ni(2)-O(1)
Ni(2)-O(2)
Ni(2)-O(4)
Ni(2)-O(5)
2.231(6)
2.004(6)
2.047(4)
1.996(5)
2.109(6)
2.138(5)
Results and Discussion
Ni(1)-N(2)
Ni(1)-O(1)
Description of Crystal Structure. The X-ray structural
determination of compound 1 reveals a dinuclear nickel
cationic complex and a nitrate anion. An ORTEP view of
μ-phenoxo-dinickel(II) complex of 1 with an atom label-
ing scheme is shown in Figure 1. The nickel ions exhibit a
distorted octahedral coordination sphere comprised in
the equatorial plane of the phenoxido-bridged oxygen,
the hydroxyl group, and the imine and amine nitrogen
donors. The axial positions are occupied by an aqua
and a nitrate oxygen. The Ni-N(amine) bond distances
Ni(1)-O(2)
Ni(1)-O(3)
Ni(1)-O(6)
Ni(1)-Ni(2)
N(2)-Ni(1)-N(1)
O(1)-Ni(1)-N(1)
O(2)-Ni(1)-N(1)
O(3)-Ni(1)-N(1)
O(6)-Ni(1)-N(1)
N(2)-Ni(1)-O(1)
O(2)-Ni(1)-N(2)
N(2)-Ni(1)-O(3)
N(2)-Ni(1)-O(6)
O(2)-Ni(1)-O(1)
O(1)-Ni(1)-O(3)
O(1)-Ni(1)-O(6)
O(2)-Ni(1)-O(3)
O(2)-Ni(1)-O(6)
O(3)-Ni(1)-O(6)
N(5)-Ni(2)-N(4)
O(1)-Ni(2)-N(4)
O(2)-Ni(2)-N(4)
O(4)-Ni(2)-N(4)
O(5)-Ni(2)-N(4)
N(5)-Ni(2)-O(1)
O(2)-Ni(2)-N(5)
N(5)-Ni(2)-O(4)
N(5)-Ni(2)-O(5)
O(2)-Ni(2)-O(1)
O(1)-Ni(2)-O(4)
O(1)-Ni(2)-O(5)
O(2)-Ni(2)-O(4)
O(2)-Ni(2)-O(5)
O(4)-Ni(2)-O(5)
83.4(2)
169.1(2)
104.7(2)
92.9(2)
86.2(2)
88.31(19)
171.9(2)
86.8(2)
88.5(2)
83.79(18)
93.6(2)
86.56(17)
92.2(2)
92.62(19)
175.2(2)
˚
(2.218(5), 2.231(6) A) are significantly longer than the
(17) Frisch, M. J.;Trucks, G. W.; Schlegel H. B.; Scuseria G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li,
X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J .B.; Ortiz, J. V.; Cui, Q.; Baboul, A.G.;
Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; M. A. Al- LahamPeng,
C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen,
W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision D.01;
Gaussian, Inc., Wallingford CT, 2004.
˚
Ni-N(imine) ones (2.011(5), 2.004(6) A), and the Ni-O
bond lengths appear slightly shorter for the phenoxido
oxygen when compared with the hydroxyl group (2.043(4) vs
1.996(5) A, mean values). On the other hand, the axial
Ni-ONO2 and Ni-OH2 distances average to 2.161(5)
and 2.106(6) A, respectively. The bond angles Ni(1)-
O(1)-Ni(2) and Ni(1)-O(2)-Ni(2) of 93.26(17) and
96.2(2)ꢀ, respectively, lead to an intermetallic separation
of 2.970(1) A (see Table 2). The piperazine moieties
˚
˚
˚
assume the expected chair conformation and are proto-
nated, as deduced for the charge balance and the occur-
rence of H-bond distances. The crystal packing shows an
extended H-bonding scheme (see Supporting Infor-
mation). In fact, protonated piperazine nitrogens and
coordinated water molecules, acting as H-donors toward
nitrate anions, lead to a three-dimensional (3D) supra-
molecular arrangement.
(18) Becke, A. D. Phys. Rev. A: At., Mol., Opt. Phys. 1988, 38, 3098.
(19) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter 1988,
37, 785.
(20) Dolg, M.; Wedig, U.; Stoll, H.; Preuss, H. J. Chem. Phys. 1987, 86,
866.
(21) Petersson, G. A.; Al-Laham, M. A. J. Chem. Phys. 1991, 94, 6081.
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J. Comput. Chem. 2003, 24, 982.
(23) (a) Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. J. Chem. Phys.
1998, 109, 8218. (b) Casida, M. E.; Jamorski, C.; Casida, K. C.; Salahub, D. R.
J. Chem. Phys. 1998, 108, 4439.
Magnetic Study. The variable temperature magnetic
data of the complex were collected on a polycrystalline
sample in the temperature range of 300-2 K, using an
(24) (a) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995. (b) Cossi,
M.; Barone, V. J. Chem. Phys. 2001, 115, 4708. (c) Cossi, M.; Rega, N.;
Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669.

3124 Inorganic Chemistry, Vol. 49, No. 7, 2010
Chattopadhyay et al.
Figure 2. χM versus T plot of complex 1 and the χMT versus T plot is
shown in inset. The solid line represents the theoretical curve using the
equation for Ni2 dimer.
Figure 3. Spin density plot (isosurface cutoff value = 0.003).
applied magnetic field of 0.2 T. The plots of χM and χMT
versus T data are shown in Figure 2. The χMT value at
300 K for the dimeric complex is 3.4 cm3 mol-1 K, which
is slightly higher than the theoretical value (3.0 cm3 mol-1 K)
for two noninteracting ions of a S=1 spin state. The χMT
value gradually increases upon decreasing temperature
and reaches a maximum (4.65 cm3 mol-1 K) at 10 K.
Further cooling resulted in a sudden drop in χMT values.
The room temperature χMT value as well as the increasing
nature of the χMT values with decreasing temperature is a
clear signature of the presence of a global ferromagnetic
interaction in the molecule. The decrease of the χMT value at
a temperature below 10 K is probably due to zero-field
splitting and to intermolecular antiferromagnetic interaction.
To fit the magnetic susceptibility versus T data, we first
attempted using a simple model of the dimer with a S=1
spin state without considering zero-field splitting. The
quality of fitting was not satisfactory, particularly in the
low-temperature region. However, reasonable fitting was
obtained when we considered the zero-field splitting, and
the best-fitting parameters obtained were g = 2.2, J =
44.26 cm-1, and D = -4.6 cm-1. A similar magneto-
structural correlation for the dinuclear nickel(II) com-
plexes have been reported by some other groups.25
The singly occupied molecular orbitals (SOMOs) ob-
tained by the ROB3LYP level calculation (see Supporting
Information) reveal that they are essentially dz2 and
dx2-y2 characters with reduced contribution from the
bridging μ-oxo, μ-phenoxido, and μ-NO3 groups. The
Mulliken spin density plot obtained by the UB3LYP level
calculation (Figure 3) also clearly indicates the ferromag-
netic interaction between the two Ni(II) centers is a super-
exchange phenomenon through the μ-oxo and μ-pheno-
xido groups, and the Jvalue obtained from the calculation is
47.2 cm-1 with a slight overestimation from the experi-
mental value (Jexp = 44.26 cm-1).
TD-DFT calculation has been performed on the optimized
structure of 1 in MeOH, and the calculated transitions are
observed to match well with the experimental bands (Table 3).
The calculation predicts low-energy transitions at 937.6 nm
(f, 0.0058), 921.8 nm (f, 0.0037), 644.2 (f, 0.0045), and 618.4
(f, 0.0034). All these transitions correspond to a mixed d-d
(Ni(dπ) f Ni(dπ)) and to a ligand to metal charge tran-
sition, LMCT, (L f Ni(dπ)) with a partially mixed metal
to ligand charge transfer, MLCT (see Supporting Infor-
mation). The transitions with a high oscillatory factor (f) at
426.3 nm (f, 0.0151) and 412.9 nm (f, 0.0185) correspond
to a LMCT and an intraligand charge transfer transition
(ILCT).
Catechol Oxidase Activity. The catecholase activity of
complex 1 was evaluated by using 3,5-DTBC and TCC
molecules as substrates. The reactions were carried out in
two different solvents, methanol and acetonitrile, at 25 ꢀC
in aerobic conditions and were monitored by means of
UV-vis spectroscopy, following the same technique as
we reported earlier.11 To our surprise, in the acetonitrile
medium, complex 1 is observed to be completely inactive
toward the oxidation of both of the substrates. On the
other hand, in methanol, the oxidation of 3,5-DTBC
proceeds very smoothly and exhibits a saturation kinetics
(kcat = 14 400 h-1) (see Supporting Information). More
interestingly, complex 1 catalyzes the oxidation of TCC to
TCQ, a process never reported for any nickel system, and
the process is observed via the formation of an inter-
mediate (Figure 4).
The crystal structure analyses of 1 and of the analogous
copper derivative11 show that, due to protonation, posi-
tive charge centers are created on the piperazine nitrogen
atoms at the two sides of the compartmental ligand. The
extraordinary activity of the two above-mentioned com-
plexes generates an inquiry as to whether these positive-
charged centers may act as a channel to drive the catechol
moieties, thereby inducing an unprecedented catecholase
activity. A similar scheme was proposed in literature to
explain the activity of CuZn-superoxide dismutase (SOD),
where the CuII lies at the bottom of a narrow channel, and
Electronic Spectra and TD-DFT Calculation. In MeOH,
complex 1exhibits a moderately intense transition at 395 nm
(ε, 6728 M-1cm-1) and a very weak shoulder at 599 nm
(ε, 1040 M-1cm-1). In addition, one moderately intense
shoulder at 968 nm (ε, 402 M-1cm-1) has been resolved.
To predict the nature of electronic transitions, a UB3LYP/
(26) Tainer, J. A.; Getzoff, E. D.; Richardson, J. S.; Richardson, D. C.
Nature 1983, 306, 284.
(25) (a) Nanda, K. K.; Das, R.; Thompson, L. K.; Venkastsubramanian,
K.; Paul, P.; Nag, K. Inorg. Chem. 1994, 33, 1188. (b) Koga, T.; Furutachi, H.;
Nakamura, T.; Fukita, N.; Ohba, M.; Takahashi, K.; Okawa, H. Inorg. Chem.
1998, 37, 989.
(27) Valentine, J. S.; Pantoliano, M. W. Protein-Metal ion interaction in
Cuprozinc Protein (Superoxide dismutase) (R). In Copper Proteins; Spiro,
T. G., Ed.; Krieger Publishing Co.: Malabar, FL, 1981, p. 291.
(28) Valentine, J. S.; Mota de Freitas, D. J. Chem. Educ. 1985, 62, 990.

Article
Inorganic Chemistry, Vol. 49, No. 7, 2010 3125
Table 3. Selected Visible Energy Transitions at the TD-DFT/UB3LYP Level for 1
energy (ev)
1.3224
wavelength (nm)
937.59
osc. factor (f)
principal transitions
character
exp. value (ε, M-1cm-1
)
0.0058
(31%) 144(β) f 151(β)
(29%) 142(β) f 152(β)
(33%) 144(β) f 152(β)
(28%) 142(β) f 151(β)
(35%) 138(β) f 152(β)
(28%) 142(β) f 152(β)
(39%) 145(β) f 152(β)
(31%) 145(β) f 155(β)
Ni(dπ) f Ni(dπ)
L f Ni(dπ)
Ni(dπ) f Ni(dπ)
L f Ni(dπ)
Ni(dπ) f Ni(dπ)
L f Ni(dπ)
Ni(dπ) f Ni(dπ)
Ni(dπ) f L(π*)
L f Ni(dπ)
968(402)
1.3450
1.9246
2.0050
921.80
644.23
618.39
0.0037
0.0045
0.0034
599(1040)
395(6728)
L f L(π*)
2.9084
3.0025
426.30
412.94
0.0151
0.0185
(34%) 150(β) f 153(β)
(28%) 150(β) f 156(β)
(39%) 150(β) f 152(β)
(33%) 150(β) f 156(β)
L(π) f L(π*)
L(π) f Ni(dπ)
L(π) f L(π*)
L(π) f Ni(dπ)
Table 4. Comparison of Catecholase Activity of Complex 1 with an Analogous
Copper Complex
substrate
complex
solvent
3, 5-DTBC
TCC
k )
cat (h-1
√
__
1
1
2
2
MeOH
MeOH
MeOH
MeOH
1.44 ꢀ 104
0.339 ꢀ 104
3.24 ꢀ 104
1.04 ꢀ 104
__
√
__
√
√
__
the dicopper(II) system exhibits much better catecholase
activity than that of the dinickel(II) system.
In order to understand the structure of the intermedi-
ate, we have performed an electron spray ionizaton-mass
spectrometry (ESI-MS) study. The ESI-MS spectrum of 1
in methanol shows the base peak at 581.88 amu, which
corroborates well with the mono positive species 1a (m/z:
calcd 581.95 amu), shown in scheme 1. Immediately after
the addition of TCC to the methanolic solution of 1
(complex:TCC = 1:100), the ESI-MS spectrum shows
dramatic changes, and in this respect, two intermedi-
ates are feasible with the formation of 1b (m/z: calcd
766.85 amu) and/or 1c (m/z: calcd 748.84 amu), where
TCC acts as a monodentate and a bidentate ligand,
respectively, (Scheme 1).
Figure 4. UV-vis spectra (300-500 nm): (i) 1 (1ꢀ 10-4M) in methanol;
(ii) TCC (1ꢀ 10-2M) in methanol; (iii) changes in UV-vis spectra of 1
upon addition of 100-fold TCC observed after each 10 min interval.
the positively charged arginine and lysine residues are
believed to play a role in attracting the anions and guiding
them into the channel.26-29 Further work is required to clarify
these aspects. Another important issue we should address
here is the role of the solvent on the catecholase activity
of our complexes. Our present study reveals that methanol is
a better choice to study the catecholase activity of the
dinickel(II) complex rather than acetonitrile. At present,
we believe that the higher the coordination ability of the
solvent, the lower the catecholase activity of a catalyst in
that solvent and that is most likely due to the competitive
reaction between the solvent and the substrate to make an
association with the catalyst. Since acetonitrile has a
higher coordination ability than methanol, the coordi-
nated water molecules of complex 1 may be substituted by
acetonitrile in the solution, and the resultant species has
little affinity to interact with the substrate, to exhibit any
catalytic activity. However, on comparing the catalytic
efficiency (on the basis of kcat value) of complex 1 with its
analogous copper(II) complex (Complex 2 in Table 4) for
both of the substrates 3,5-DTBC and TCC, as depicted in
Table 4, it may be stated that under identical conditions
Our ESI-MS spectrum clearly indicates 1c (m/z: exptl
748.67 amu) as the better choice. To evaluate whether 1b
and 1c might be real intermediates, we have performed a
series of DFT calculations on the molecular models
built by modifying the molecular crystal structure of 1
(Figure 1). The DFT-optimized geometries of 1c and two
rotamers, 1b and 1b/, are shown in Figure 5. All three
geometries are optimized using the UB3LYP/SDD level,
and the vibrational frequency calculations are performed
to ensure that the optimized geometries represent the
local minima and that there are only positive eigen values.
The optimized structure of 1c is characterized by a Ni-Ni
˚
˚
distance of 3.372 A, while 3.162 and 3.177 A in the two
rotamers 1b and 1b/, respectively. This, however, occurs
at the expense of an increase in the structural distortion of
the rotamer geometries, where the Ni-Ni bond vectors
form an angle of 26.1 and 29.0ꢀ, respectively, for 1b and
1b/ with the plane of the six-member arene rings, whereas
this value reduces to 15.1ꢀ in 1c. The computed zero-point
energies of the two rotamers indicate that 1b is about
8.89 kJ/mol lower in energy with respect to 1b/. This extra-
stabilization might be partly assigned to the presence of two
(29) Bannister, J. V.; Bannister, W. H.; Rotilio, G. Aspects of the
Structure, Function and Applications of Superoxide dismutase (R). CRC
Crit. Rev. Biochem, 1987, 22, 111.

3126 Inorganic Chemistry, Vol. 49, No. 7, 2010
Chattopadhyay et al.
Figure 5. DFT-optimized structures of 1c and two rotamers of 1b and 1b/ (H-atoms are omitted for clarity).
Scheme 1. Possible Compositions of the Complex in Methanol (1a) and the Two Possible Adducts (1b and 1c) upon Addition of TCC to a Methanolic
Solution of Complex 1
Scheme 2. An Alternative Possibility of the Generation of 1c via 1b on the Basis of a DFT Study
˚
strong intramolecular H-bonds at 2.017 and 2.628 A in 1b,
metal center redox participation is responsible for the
catechol to quinone conversion,30,31 though a recent
point of view, based on theoretical calculations, advo-
cates a radical pathway as the most reasonable alterna-
tive.32 However, to date, no experimental evidence gives
support to justify this proposition.
The electrochemical analysis of 1 in both the solvents,
acetonitrile and methanol, is featureless, suggesting no
preference for the nickel(II) ions is present in the complex
to undergo reduction to nickel(I) or oxidation to nickel-
(III). Thus, from the above data, it is not possible to
predict that the metal center redox participation is res-
ponsible for the catecholase activity of complex 1. In
/
which elongates to 2.146 A in 1b . The different stoichiometry
˚
of 1b and 1c, however, prevents us from assessing their
relative stability. An examination of their optimized geome-
tries, however, suggests that despite the difference in their Ni-
Ni distances, the lower strain of the ligand associated to 1c
should favor the formation of this intermediate. Thus, our
DFT calculations suggest that the TCC oxidation, catalyzed
by complex 1, most probably proceeds via the formation of
an intermediate having bidentate coordination of TCC rather
than monodentate coordination or that the formation
of intermediate 1c can take place via the formation of a
secondary intermediate 1b (Scheme 2). This conclusion
agrees with that drawn from the ESI-MS measurements
(vide supra).
(30) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev.
1996, 96, 2563.
(31) Eicken, C.; Krebs, B.; Sacchettini, J. C. Curr. Opin. Struct. Biol. 1999,
9, 677.
(32) Siegbahn, P. E. M. J. Biol. Inorg. Chem. 2004, 9, 577.
The conversion of catechol to quinone is a two-electron
oxidation process. Most of the investigators working with
copper catechol oxidase model systems believe that the

Article
Inorganic Chemistry, Vol. 49, No. 7, 2010 3127
initial rates, as a function of the concentration of PNPP,
reveals saturation kinetics with Michaelis-Menten-like
behavior. By applying the GraFit32 program for enzy-
matic kinetics, Km and Vmax are calculated from the
graphs (see Supporting Information), and catalyst’s
turn-over number, kcat was determined by dividing Vmax
by the concentration of the complex (kcat=7200 h-1). To
the best of our knowledge, the phosphatase activity of any
nickel complex with our similar ligand system is not yet
reported in the literature, and thus, in order to assess the
catalytic efficiency of the present system, we compare its
kcat value with that of the analogous dicopper(II) com-
plex. To our surprise, we noticed that the dicopper(II)
complex is totally inactive in catalyzing the cleavage of
the phosphate bond (see Supporting Information). The
result indicates that the present dinickel(II) system is a
more efficient catalyst than the dicopper(II) system under
an identical environment, as far as phosphatase activity is
concerned with PNPP as a substrate. Here, it is to note
that the mechanism through which our dinickel(II) sys-
tem catalyzes the phosphate-bond cleavage is not very
clear to us, but we may assume that the Lewis acid (metal
center) base (phosphate moiety) type interaction, as is
proposed in the literature for zinc or copper complexes,33
that catalyzed the phosphate-bond cleavage is operative
also in our system.
Figure 6. UV-vis spectrum (350-500 nm) of: (i) complex 1 (concn 1 ꢀ
10-4 M; in CH3OH); (ii) changes in UV-vis spectra of complex upon
addition of 100-fold PNPP in water-methanol (1:1 v/v) observed after a
5 min interval.
order to verify the other alternative, i.e., radical partici-
pation, we have performed an ambient as well as a low-
temperature (-135 ꢀC) EPR study, although EPR is not a
good method to detect unpaired electrons in a transi-
tion-metal complex with a high D-value. However,
we failed to detect any radical formation during our
EPR experiment. Thus, at this moment, the actual
mechanism through which complex 1 is exhibiting its
extraordinary catecholase activity is not clear to us. This
needs further extensive work, which is underway in our
laboratory.
An interesting remark is that recently polynuclear
coordination complexes of copper, manganese, or nickel
are in use as catalysts, especially for the oxidation of
catechols, to know about the efficiency of a polynuclear
system as catalyst in comparsion to an analogous di/
mononuclear system.34 The results obtained so far are
interesting, but real working mechanisms are not yet
identified.
Phosphatase Activity. One of the criteria for a mole-
cular species to function as chemical nucleases is to provide
the H2O/OH- species as nucleophile. Since our complex
possesses potential a nucleophile constituted by the metal-
bridging hydroxyl group, it would be reasonable to
explore the phosphatase activity of the complex. For this
study, we have chosen PNPP as the test substrate. The
reaction was followed UV-vis spectrophotometrically by
monitoring the increase of the absorption band at 400 nm,
due to the formation of PNP (Figure 6). The kinetics of
the phosphatase activity was determined via the initial
rates method by monitoring the increase of the product,
p-nitriphenol, at 400 nm. The concentration of the sub-
strate PNPP was always kept at least 10 times larger than
that of the Ni(II) complex to maintain the pseudo-first
order condition. All the kinetic experiments were con-
ducted at constant temperature of 20 ꢀC and monitored
with a thermostat. Initially, a series of solutions of the
substrate, PNNP, having different concentrations (1 ꢀ
10-3-5 ꢀ 10-2 mol dm-3), was prepared from concen-
trated stock solution of the substrate using methanol-
water (1:1 v/v) as the solvent. Then 2 mL of such a
substrate solution was poured into a 1 cm quartz cell
kept in the spectrophotometer to equilibrate the tempera-
ture at 20 ꢀC. A 0.04 mL (2 drops) of 5ꢀ10-3 mol dm-3 of
Ni(II) complex in methanol was quickly added and mixed
thoroughly to get an ultimate Ni(II) complex concentra-
tion of 1 ꢀ 10-4 mol dm-3. The absorbance was con-
tinually monitored at 400 nm. The determination of the
DNA Cleavage Activity. On the basis of the encoura-
ging result obtained in the hydrolysis of PNPP, we have
decided to evaluate the influence of complex 1 toward
nucleic acid degradation. Complex 1 is fluorescent, and
its fluorescence intensity was observed to enhance in
presence of DNA. We have exploited this phenomenon
to determine the intrinsic DNA binding constant by
applying the Benesi-Hildebrand model35 (see Support-
ing Information), which evaluates Kintr as 2.69ꢀ103 M-1
.
To assess the DNA cleavage ability of complex 1, super-
coiled (SC) pGEM3Z plasmid DNA (200 ng for each set)
was incubated with different concentrations of the com-
plex in the presence of 10 mM tris buffer (pH 7) at 37 ꢀC
for 1 hour. The reaction was stopped with 1X DNA
loading dye and resolved in 1% agarose gel. Upon gel
electrophoresis of the reaction mixture, a concentration-
dependent DNA cleavage was observed (Figure 7). The
band intensity of the SC, the nicked circular (NC), and the
linear (L) forms was measured through densitometric
analysis (image quant software, Amersham Biosciences)
(33) Molenveld, P.; Engbersen, J. F. J.; Reinhoudt, D. N. Chem. Soc.
Rev., 2000, 29, 75 and references therein.
(34) (a) Alves, W. A; Cerchiaro, G.; Paduan-Filho, A.; Tomazela, D. M.;
Eberlin, M. N.; Da Costa Ferreira, A. M. Inorg. Chim. Acta 2005, 358, 3581.
(b) Majumder, A.; Goswami, S.; Batten, S. R.; Salah El Fallah, M.; Ribas, J.;
Mitra, S. Inorg. Chim. Acta 2006, 359, 2375. (c) Ambrosi, G.; Formica, M.; Fusi,
V.; Giorgi, L.; Micheloni, M. Coord. Chem. Rev. 2008, 252, 1121 and references
therein.
(35) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153.

3128 Inorganic Chemistry, Vol. 49, No. 7, 2010
Chattopadhyay et al.
Figure 7. Gel electrophoresis showing the cleavage of SC pGEM3Z
plasmid DNA (200 ng in base pair) with different concentrations of com-
plex 1, in presence of 10 mM tris buffer (pH 7) at 37 ꢀC for 1 hour.
Figure 9. Lane 1: pGEM3Z plasmid DNA (200 ng in base pair). Lane 2:
pGEM3Z plasmid DNA (200 ngin base pair) with 150 μM of the complex
1 alone. Lanes 4-6: pGEM3Z plasmid DNA (200 ng in base pair) with
150 μM of the complex 1 in the presence of radical scavenger DMSO.
Lanes8-10: pGEM3Z plasmid DNA (200 ng in base pair) with 150 μMof
the complex 1 in the presence of NaN3. The pGEM3Z plasmid DNA was
also incubated with DMSO (lane3) and NaN3 (lane7) alone to show that
these scavengers do not have any effect on the DNA.
Figure 8. Relative amounts of the different DNA forms in the presence
of increasing concentration of complex 1; SC is violet; NC is brown; and
L is yellow.
and plotted against an increasing concentration of the
complex (Figure 8).
The densitometric analysis data showed that the com-
plex can cause the cleavage of the SC DNA, even at a
20 μM concentration. The Figure shows a decrease of the
SC percentage and an increase of both the NC and L forms,
with respect to the control. Interestingly at a 50 μM
concentration, the SC form is diminished, as expected,
but both the NC and L forms are also decreased. This
particular trend is observed also at higher concentrations.
These experimental facts may be explained in the follow-
ing way: Complex 1 causes the conversion of SC to NC
and L, which were finally cleaved to small oligonucleo-
tides by nonspecific cleavage at various sites. At very low
concentrations of the complex, the rate of the reaction is
slow, and the intermediate formation of the nicked and
linear forms may be observed but at a higher concentra-
tion of the complex; the rate of conversion of the DNA to
small oligonucleotides is too high, and we can only ob-
serve the decrease in the SC form without increasing NC
and/or L. Reports on DNA cleavage by nickel(II) com-
plexes are very scanty.36 The available data reveal that
two mechanisms likely to be involved in DNA cleavage
are the hydrolytic cleavage and free radical mechanisms.
In our case, the DNA cleavage was observed to inhibit, to
a significant extent, when the reaction was carried out
with 150 μM of the complex in the presence of DMSO
(hydroxyl radical scavenger). The use of NaN3, as a
singlet oxygen scavenger, did not inhibit the DNA clea-
vage (Figure 9). The above facts reveal that the ability of
complex 1 to generate hydroxyl radicals is most likely
responsible for the cleavage to DNA.
Figure 10. The effects of complex 1 on VERO cells’ viability. Cells were
treated for 30 h with the compound at the indicated concentrations. Cell
viability was estimated by a MTT assay, as reported in the Experimental
Section. Data are expressedasthe percentage ofviable cells with respectto
untreated controls.
show activity only in the presence of excess of an external
reluctant and dioxygen. Since our complex can bind and
cleave DNA by a self-activating mechanism, its applica-
tion in site-specific recognition of DNA, even in in vivo
conditions, should be very promising.
Bioactivity. To investigate this potentiality, the com-
plex was tested for its effect on the viability of living cells
using a MTT assay.37 VERO (a monkey kidney cell line)
and A549 (a carcinomas human alveolar basal epithelial
cell line) cells were used for the detection of cytotoxicity,
and the results indicate that, at moderate concentrations,
(200-400 μM) the complex shows around a 40% decrease
in the viability of VERO cells (Figure 10), which may be a
consequence of the DNA cleavage activity of the same.
Also the effect of this complex upon the carcinoma cell
line, A549, was similar.
Conclusions
In conclusion, complex 1, the first ever example of this
kind, possesses an inherent property exhibiting versatile
catalytic activities of biological significance. The catecholase
activity of complex 1 is observed to be largely influenced by
The use of metallonucleases in the living cells as DNA
foot-printing agents suffers the deadlock, as most of them
(36) Benelli, C.; Dei, A.; Gatteschi, D.; Pardi, L. Inorg. Chem. 1989, 28,
1476.
(37) Mosmann, T. J. Immuno. Methods 1983, 65, 55.

Article
Inorganic Chemistry, Vol. 49, No. 7, 2010 3129
the nature of the solvent. In a methanol medium, the complex
shows excellent catecholase activity not only with easily
oxidiazable 3,5-DTBC but also with TCC, a substrate whose
oxidation catalyzed by any nickel(II) complex is not known
to date, whereas in the acetonitrile medium, it becomes
inactive, even in oxidizing 3,5-DTBC. An UV-vis spectral
study clearly shows TCC oxidation catalyzed by complex 1
proceeds through the formation of an intermediate, whereas
for 3,5-DTBC, the intermediate, if at all formed, is too
unstable to detect. ESI-MS measurements and DFT calcula-
tions suggest that the coordination mode of TCC in that
intermediate is a bidentate bridging type rather than a
monodentate. The complex 1 also exhibits interesting DNA
cleavage activity, most probably through a hydroxyl radical
pathway. This very property may be crucial for its possible
applications in site-specific recognition of DNA, even in in
vivo conditions, as well as for a promising anticancer drug,
as is evident from the bioactivity study of the complex.
Generally, nickel compounds are considered as moderately
cancerous. However, the report presented in this commu-
nication suggests further extensive investigation with a simi-
lar nickel(II) system to get a breakthrough.
Acknowledgment. We are thankful to Dr. P. S. Mukherjee,
IISc, Bangalore, India, for his help on variable-temperature
magnetic susceptibility measurement and for many fruitful
discussions. The generous help of Mr. Bijan Pal, Research
Scholar, Department of Chemistry, University of Calcutta,
India, to determine the DNA binding constant is also duly
acknowledged.
Supporting Information Available: X-ray crystallographic
data in CIF format of 1, IR spectrum, UV-vis spectroscopic
and kinetic data, and ESI-MS spectra for 1 and its TCC adduct
(intermediate) in methanol, CV for 1 in methanol, DNA clea-
vage and MTT assay, emission spectra, and determination of
DNA binding constant. This material is available free of charge
via the Internet at http://pubs.acs.org.