Trinuclear nickel(II) amino acid Schiff base complex containing phenolato and acetato bridges: Structural and functional resemblance of urease

Article abstract of DOI:10.1016/j.ica.2020.120109

A novel phenolato and acetato bridged trinuclear Ni(II) complex [Ni₃L₂] was synthesized by the reaction between the amino acid Schiff base, N-o-vanillylidene-L-histidine(L) and Ni(OAc)₂·2H₂O. Single crystal X-ray analysis reveals that the trinuclear structure of Ni₃L₂ is built by linking two terminal NiN₂O₄ cores with one NiO₆ centre core (NiN₂O₄?NiO₆?NiN₂O₄) and each Ni(II) ion exists in an octahedral environment. Variable-temperature magnetic susceptibility data represent the antiferromagnetic interactions of the Ni₃L₂ mediated by a single Ni-O-Ni bridge. The bond length and bond angles around each terminal nickel centre match the nickel centre in urease enzyme of native bacteria Klebsiella aerogenes, Bacillus pasteurii and Helicobacter pylori. The catalytic activity of Ni₃L₂ on the hydrolysis of urea into ammonia and carbon dioxide was established by the absorption spectral method. The urea binding propensity of the nickel complex Ni₃L₂ was compared with jack bean urease by using molecular docking studies.

Full text of DOI:10.1016/j.ica.2020.120109

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Trinuclear nickel(II) amino acid Schiff base complex containing phenolato
and acetato bridges: Structural and functional resemblance of urease
S. Thalamuthu, M.A. Neelakantan
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https://doi.org/10.1016/j.ica.2020.120109
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Please cite this article as: S. Thalamuthu, M.A. Neelakantan, Trinuclear nickel(II) amino acid Schiff base
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Trinuclear nickel(II) amino acid Schiff base complex containing phenolato
and acetato bridges: Structural and functional resemblance of urease
S. Thalamuthu and M.A. Neelakantan*
Chemistry Research Centre, National Engineering College, K.R. Nagar, Kovilpatti - 628 503, Thoothukudi District,
Tamil Nadu, India.
*Corresponding Author
E-mail address (MAN): drmaneelakantan@gmail.com

Abstract
A novel phenolato and acetato bridged trinuclear Ni(II) complex [Ni3L2] was synthesized
by the reaction between the amino acid Schiff base, N-o-vanillylidene-L-histidine(L) and
Ni(OAc)2∙2H2O. Single crystal X-ray analysis reveals that the trinuclear structure of Ni3L2 is
built by linking two terminal NiN2O4 cores with one NiO6 centre core (NiN2O4∙∙∙NiO6∙∙∙NiN2O4)
and each Ni(II) ion exists in an octahedral environment. Variable-temperature magnetic
susceptibility data represent the antiferromagnetic interactions of the Ni3L2 mediated by a single
Ni-O-Ni bridge. The bond length and bond angles around each terminal nickel centre match the
nickel centre in urease enzyme of native bacteria Klebsiella aerogenes, Bacillus pasteurii and
Helicobacter pylori. The catalytic activity of Ni3L2 on the hydrolysis of urea into ammonia and
carbon dioxide was established by the absorption spectral method. The urea binding propensity
of the nickel complex Ni3L2 was compared with jack bean urease by using molecular docking
studies.
Keywords: Trinuclear nickel complex; X-ray structure; Urease activity; Molecular Docking

1. Introduction
The search for new polynuclear metal complexes mimicking enzyme activity has attracted
many researchers during the last several years due to their significance as multielectron transfer
centers in biological systems [1-7]. Polynuclear metal complexes exist as an active site of
metallohydrolases involved in catalyzing the hydrolysis of peptide and phosphate ester bonds in
amidohydrolases, amidinohydrolases, and peptide hydrolases [8,9]. A common structural feature
of the metallohydrolases like leucine aminopeptidase, urease, methionine aminopeptidase and
arginase is the presence of a dinuclear metal active site (Zn(II), Ni(II), Co(II) or Mn(II)) with
carboxylate bridges [10-12].
Urease, a nickel-containing enzyme, was the first enzyme crystallized from Jack bean
(Canavalia ensiformis) is one of the essential metallohydrolases [13]. The primary function of
urease is the hydrolysis of urea into ammonia and carbamate [14]. It also catalyzes small amides
such as formamide and acetamide to their corresponding carboxylic acids. These hydrolytic
products have been shown as a critical virulence determinant in the pathogenesis of many
clinical conditions, which is detrimental for both human and animal health [15] and agriculture
[16]. Urease participates in the environmental cycling of various nitrogen compounds, including
urea-based fertilizers [17]. The enzyme also plays a role in seed germination by degrading the
urea formed through arginase activity [18]. Perhaps the most unfamiliar pathogenic organism
that exploits urease is Helicobacter pylori (H. pylori). The mechanistic interaction of vitamin C
and urease of H. pylori is a key factor in the preventative or the therapeutic agent for treating H.
pylori-related infections [19]. The multi-faceted significance of this enzyme has stimulated
widespread interest in its characterization and mechanism of action.
The crystal structure of the active site of various bacterial ureases from K. aerogenes, B.
pasteurii, and H. pylori revealed common structural features. The active site of K. aerogenes is
shown in Scheme 1 [20,21]. The active site of urease contains two Ni2+ ions separated by 3.5-3.7
Å, bridged by the oxygen molecules of a carbamylated lysine residue, and bound to two
histidines. One Ni2+ is moreover bound to aspartate carboxylate oxygen. The coordination
geometry of the Ni2+ is furnished by a water molecule terminally bound to each metal ion and by
a nickel-crossing over hydroxide bridge. These ligand coordinations yield one penta-coordinated
Ni2+ ion with distorted square-pyramidal geometry and another Ni2+ ion with hexa-coordination
and pseudo-octahedral geometry [22]. The hydrolysis rate of urea by urease is 1014 times faster

than the spontaneous hydrolysis of urea [23]. The resolving catalytic pathway of this fascinating
metalloenzyme remains a subject of extraordinary research. Therefore, it encourages further
efforts to provide analogs of the urease active site and exploit and mimic its unique reactivity
ultimately.
Several researchers reported many urease dinuclear model complexes for mimicking the
active site of urease [24,25]. However, to our knowledge, the report of trinuclear nickel
complexes having urease activity is little in the literature [26-28]. Chattopadhyay et al. reported
the dinuclear nickel complex with a compartmental ligand, 2,6-bis-(N-ethylpiperazine-
iminomethyl)-4-methyl-phenolato exhibits biological significance such as catecholase,
phosphatase, and DNA cleavage activities [29]. Recently many trinuclear model coordination
complexes are reported for their magnetic and enzymatic behavior [30-33]. We have been
interested in searching for small coordination complexes exhibiting enzymatic and anticancer
activities [34-36]. In the current commitment, we report the synthesis, spectral studies, and
crystallographic characterization of phenolato and acetato bridged trinuclear Ni(II) complex
[Ni3L2]. The catalytic effect of Ni3L2 on the hydrolysis of urea was studied by the absorption
spectral titration method. A molecular docking study was used to identify the binding modes of
urea with Ni3L2.
2.
Experimental part
2.1.
Materials
Water was purified with a MilliQ distillation purification system. All the solvents used
were analytical grade. o-vanillin, L-histidine, Ni2(OAc)2∙2H2O, jack bean urease, HEPES buffer,
urea, thiourea, phenol red, and methanol were purchased from Sigma-Aldrich and were of
analytical reagent grade. All the chemicals were used without further purification.
2.2.
Physical measurements
Elemental analyses were performed in a Vario EL III elemental analyzer. Infrared spectra
were recorded on a Shimadzu FT-IR 8400S spectrophotometer in the 400-4000 cm–1 region
using KBr pellets. UV-Vis spectra were obtained on Shimadzu UV-Vis 2450 spectrophotometer.
The Electron Spray Ionization (ESI) mass spectrum was recorded on a MICROMASS

QUATTRO II triple quadrupole mass spectrometer. Thermal gravimetric analysis of the sample
o
o
was carried out from room temperature to 1000 C at a heating rate of 20 C min-1 using the
Perkin-Elmer Diamond TG/DTA system. The magnetic susceptibility measurements of the
powdered sample were measured using a superconducting quantum interference device vibrating
sample magnetometer (SQUID-VSM, Quantum Design) with an applied field of 1000 Oe over
the temperature range of 5-300 K.
2.3.
Synthesis of the [Ni3L2]
The ligand, N-o-vanillylidiene-L-histidine (L), was synthesized by the condensation of o-
vanillin and L-histidine, as reported in our earlier work [37]. The L (0.3073 g, 1.0 mmol) was
dissolved in 10 mL of methanol: water mixture (1:1). To this solution, the methanolic solution of
nickel acetate Ni(OAc)2·2H2O (0.250 g, 1.0 mmol) was added dropwise at room temperature
with constant stirring and refluxed for 2 h. The resulting solution was allowed to stand at room
temperature for one week in a closed flask. The pale green crystals were separated and washed
with hot ethanol. The compound was further dried under vacuum.
Yield: 72%. M.Pt. >573 K. Anal. calcd. for (Ni3C32H38N6O14): C, 42.39; H, 4.22; N, 9.27.
Found: C, 42.44; H, 4.28; N, 9.31. IR (KBr pellet, cm–1, s (strong), m (medium), b (broad)):
ν(OH+NH), 3400(b), νasym(COO-), 1608, 1580(s), νsym(COO-), 1440, 1380 (m), ν(C=N), 1633 (s), ν(C-O)
1260 (s) , ν(M-O), 408(w), ν(M-N), 570(w). ESI-MS: Found m/z (M+H)+ = 904.12 (calc. = 904.19).
,
2.4.
Single crystal X-ray analysis
X-ray data collection was performed with Bruker AXS Kappa Apex II CCD
diffractometer equipped with graphite monochromated Mo (Kα) (λ = 0.7107 Å) radiation.
Crystal fixed at the tip of the glass fiber using cyanoacrylate adhesive was mounted on the
goniometer head with the aid of a video microscope and optically centered at the sphere of
confusion of the goniometer axes. The automatic cell determination routine with 36 frames at
three different detector orientations was employed to collect unit cell determination reflections.
Further, intensity data for structure determination were collected through an optimized strategy,
which gave an average 4-fold redundancy. The program APEX2-SAINT (Bruker, 2004) was
used for integrating the frames.

2.5.
Urease activity Measurements
The urease activity of the Ni3L2 was studied using absorption spectroscopy. The stock
solution of Ni3L2 was prepared by dissolving 90 mg of Ni3L2 in 10 ml of 50% DMSO:water
solution. The stock solution of phenol dye (0.01 M) was prepared by dissolving 35 mg of the
phenol red indicator with 3 ml of 0.1 N sodium hydroxide solution and then diluted with
deionized water to 10 ml. The 0.5 M of urea solution was prepared in water. The reaction
mixture was prepared in 10 ml of volumetric flask. The concentration of urea and phenol red
used for urease activity was 500 mM and 0.01 mM, respectively. The 100 L of 0.5 M urea and
10 L of 0.01 mM phenol red dye solutions were taken in 10 ml of volumetric flask. Similarly,
13 series of solutions were prepared using the same method. Then, 0-1300 uL of Ni3L2 was
added to each flask and diluted to 10 ml using 50% DMSO and 0.1mM HEPES buffer solution.
For the urea hydrolysis, each flask was incubated for 1 h at 37°C. The experiments were done by
measuring phenol red absorbance at 560 nm with an increasing concentration of Ni3L2 (0-130
μM), a fixed concentration of phenol red, and urea in buffer solution. While measuring the
absorption spectra, an equal amount of the complex was added to the reference solution to
eliminate the complex's absorbance at 560 nm. Comparing the absorption spectrum of the
complex in DMSO and in buffer medium demonstrates that the complex has high stability in the
buffer medium (Fig. S1).
2.6.
Molecular docking
A molecular docking study was carried out to demonstrate the interface, binding models,
and structural similarities of interaction between the complex Ni3L2 and jack bean urease (PDB
code: 3LA4) with urea (Zinc code: 08214514). The hetero atoms, including water molecules,
were removed, and polar hydrogen atoms and Kollman charges were added to the receptor
molecule. Every single other bond was permitted to be rotatable. In the docking investigation,
the coupling site was assigned over the whole minor and significant scores of the urea molecule,
which was enclosed in a box with the number of grid points in x×y×z directions of 40×40×40
[grid box] and a grid spacing 0.375 Å. Docking studies were conducted using AutoDock Tools
(ADT) version 1.5.6 and AutoDock vina program. The docked structures were exported into the

discovery studio for visual inspection of the possible binding mode interactions of the urease
with urea. A similar procedure was used for the docking of the Ni3L2 complex with urea.
3.
Results and discussion
3.1.
Synthesis and spectral characterization
The ligand, N-o-vanillylidiene-L-histidine (L), was synthesized by the condensation of o-
vanillin and L-histidine, as reported in our earlier work [37]. The reaction of L with nickel
acetate yielded the trinuclear Ni3L2 complex, [Ni3L2(OAc)2(H2O)2] (Scheme 2). The complex
contains di-µ-phenoxo and di-µ-acetato bridged structure and other metal-binding sites in the
ligand (azomethine, imidazole, carboxylate, and methoxy), which create octahedral geometry
around each Ni2+ ion. The Ni3L2 complex is soluble in DMSO and DMF. The low molar
conductance value of Ni3L2 (8 Ω−1 cm2 mol−1) indicates its non-electrolytic nature [38].
IR spectrum of Ni3L2 exhibits broad band in the region of 3400-2700 cm−1 is due to the
presence of coordinated water molecules. The C=N stretching vibration of Ni3L2 observed at
1630 cm–1 is shifted by 10 cm–1 to a lower frequency than that of free ligand(L), indicating imine
nitrogen of L is coordinated to the metal center (Fig. S2). The phenolic C−O stretch of L at 1240
cm−1 is shifted to 1260 cm−1 in the Ni(II) complex. The methoxy group displays an asymmetrical
C−O−C stretching at 1224 cm–1 with symmetrical C−O−C stretching at 1098 cm−1. Both these
stretching vibrations are shifted to higher values (1245 and 1115 cm−1) in Ni3L2, indicating that
the methoxy group of L is involved in the coordination. The two new bands observed in the
complex at 1580 cm−1 and 1440 cm−1 are assigned to asymmetric and symmetric carboxylate
(COO¯) stretching frequencies of bridging acetate groups [39, 40]. The magnitude of separation
between asymmetric and symmetric carboxylate (COO¯) stretching frequency of the acetate
group (<200 cm−1) indicating that the acetate group is involved in coordination with nickel ion
through the bridging mode [41]. The ligand coordination to the nickel ion through the oxygen
and nitrogen atoms is substantiated by the presence of new medium and weak bands in the 420-
390, and 570-560 cm–1 ranges are due to ν(M−O) and ν(M−N) vibrations, respectively.
In the UV region of the complex, the ligand-based transitions undergo hypochromism
and a slight bathochromic shift indicating L forms complex with the Ni(II) ion (Fig. S3 (a)).
3
3
Three broad absorption bands are expected for octahedral d8 ions due to the A2g3Tl (P), T1g

3
(F), and T2g (F) transitions. The complex shows two bands in the visible region, one at 635 nm
( = 30 M–1 cm–1) and another weak absorption at 755 nm ( = 8 M–1 cm–1) are assigned to
3
3A2g→3T1g (F), and spin-forbidden A2g1Elg transitions, respectively (Fig. S3(b)). The low
energy transition 3A2g→3T2g (F) is observed above 800 nm. The third band at 412 nm (3A2g→3T1g
(P)) couples with the UV-region charge transfer band. The active site of Ni2+ ion in urease also
displays three prominent bands in the visible and near-IR region at 407, 745, and 1060 nm [42].
This indicates that the active site of urease having the Ni(II) in the octahedral environment. The
electronic transition values (412 nm, 635, and >800 nm) of Ni3L2 demonstrate that each terminal
nickel ion is similar to the active site of urease with the octahedral environment [Ni(N2O4)]. The
Racah parameter (B), splitting energy (Dq), and nephelauxetic ratio (β) were calculated using the
above three electronic transitions values. The Racah parameter (B) and splitting energy (Dq)
were calculated using equations 1 and 2.
휈2 + 휈3 ― 3휈1
........................................................................................................................(1)
퐵 =
15
................................................................................................................(2)
휈3 = 15퐵 +30퐷푞 ― 휈2
The values D = 951 cm-1, B = 766 and β = 0.709 indicates that the inter electronic
repulsion is decreased during the process of complexation. These values well agree with the
reported acetato bridged Ni(II) complex [43].
The ESI mass spectrum of Ni3L2 in DMSO shows a moderate-intense peak at 904 m/z,
demonstrating the existence of trinuclear [Ni3L2(OAc)2(H2O)2] complex (Fig. S4). The other
fragmentation peaks observed at m/z = 368 and m/z = 715 are due to the presence of mono and
dinuclear complex aggregates, respectively (Fig. S4). The thermal decomposition of Ni3L2 starts
from 30-200°C with 9% weight loss (theoretical loss=9.5%) corresponding to the elimination of
physically adsorbed solvent molecules and coordinated water molecules. The subsequent
degradation with the weight loss of 15 % in the range of 200-300°C is assigned to the removal of
acetate moiety with the liberation of carbon dioxide (Fig. S5). The other degrading steps with
weight loss ~ 26% indicate that one ligand molecule is removed from the temperature range 300-
1000°C. The remaining 50% of the metal complex is not degraded up to 1000o C, demonstrates
that Ni3L2 has high thermal stability.

3.2.
X-ray description of Ni3L2
Crystallographic data and structural refinement details, hydrogen bond lengths, and bond
angles for Ni3L2 are given in Tables 1 and S1. The molecular structure of the complex consists
of a discrete centrosymmetric trinuclear unit formula [Ni3L2(OAc)2(H2O)2] together with solvent
water molecules. Fig. 1 shows the ORTEP representation of Ni3L2 with atoms drawn as a 50%
probability ellipsoid. Ni3L2 crystallizes in a tetragonal system with space group P41212 and half
of the formula unit in the asymmetric unit. The molecule has two-fold rotation symmetry with
the crystallographic two-fold axis passing through Ni(2) atom. The complex contains three Ni
ions bridged by two μ-phenoxido and syn-syn acetate groups, and the bridging is between the
terminal (Ni2 and Ni2i) and the central (Ni1) nickel ions imply that the two terminal
mononuclear units [Ni(N2O4)] are connected by a central [Ni(O6)] unit. Each terminal
coordination sphere of nickel centres is completed by one imidazole from histidine, azomethine,
carboxylate and a water molecule in a pseudo-octahedral environment. In Ni3L2, two nickel ions
are held at a separation of 3.074 Å by the bridging of acetate residue and phenoxide group, and
this separation is longer than intermetallic interactions.
The two nickel ions separation of 3.074 Å is comparable to the values reported for the
trinuclear octahedral complex [Ni3(H2L)2(NO3)2] [44]. Similarly, the two nickel ions in the active
site of native bacterial urease are separated by 3.60 Å and bridging by carboxylate group of a
carbamylated lysine residue and hydroxide group. The distance between nickel ions in Ni3L2 is
3.074 Å shorter than that found for native urease. This deviation is caused by the phenoxide
bridging angle between the terminal and central nickel atom (Ni(1)–O(2)–Ni(2)) is 115.81°. It is
lower than that of native urease (119°) [8]. The basal plane of terminal Ni(II) atoms (Ni2 and
Ni2i) are furnished by one of the oxygen atoms O5 of bridging acetate group, an oxygen atom of
carboxylate (O3), a bridging phenoxide oxygen atom (O2), and the imine nitrogen atom (N1)
from the L with the bond distance in the range 1.995(4) - 2.069(4) Å. The bond lengths of these
basal plane values around the terminal Ni(1) are comparable to the reported trinuclear nickel
complexes (Table S1) [45,46].
The axial positions of terminal nickel atoms (Ni2 and Ni2i) are saturated by imidazole
nitrogen atom (N2) from L and oxygen atom (O7) from the water molecule. The axial bond
lengths (2.071 and 2.145 Å) are comparatively more prolonged than the equatorial bond lengths.

Also, the histidine part of L is in facial arrangement with terminal Ni(II) ions. The central nickel
atom (Ni1) is bounded by two μ2-phenoxido oxygen atoms O(2) and O(2)i in the basal plane
with a bond distance of 2.026 Å. One of the methoxy and bridging acetate oxygen atoms (O1 and
O6) are bonded with the central Ni1 atom with a distance of 2.123 Å and 2.026 Å in the basal
plane. These values are comparable to those found for the octahedral complex [Ni(μ-L)(μ-
OAc)Ni(H2O)(N3)] [47]. The axial positions are contributed by another methoxy and bridging
acetate oxygen atoms (O1i and O6i) with the same bond distance. The bond angles around the
Ni1 vary from 78.3°-178.3°, and Ni2 varies from 80.7°-178.3°. The phenoxide bridging angle
between terminal and central nickel atoms (Ni(1)–O(2)–Ni(2)) is 115.81°. This larger deviation
of Ni–O–Ni angle from 90° demonstrates the magnetic coupling between two Ni(II) centers as
antiferromagnetic [20]. This antiferromagnetic coupling between two Ni(II) centers has also been
observed in the active site of urease. Due to the bridging mode of phenoxide and acetate oxygen
atoms, the trans bond angles O(2)-Ni(2)-O(3) and N(1)-Ni(2)-O(5) decreases to 166.38° and
170.90° from the ideal octahedral trans bond angle 180°. Similarly, the cis bond angles O(5)-
Ni(2)-O(2) and O(2)-Ni(2)-N(2) increases to 100.90° and 95.04° from the normal cis bond angle
90° demonstrates that the bridging mode of phenoxide and acetate oxygen atoms leads to
distortion from the ideal octahedral. The two nickel atoms Ni1 and Ni2 are separated by 3.074 Å
indicates the absence of any bond between the two nickel centers.
The crystal packing of the metal complex shows that 12 disordered water molecules are
in the unit cell (Fig. 2). The crystal packing is stabilized through water-mediated intermolecular
and intramolecular-H bonds (Table S2). Fig. S5 a & b describes the intermolecular H-bonding
and intramolecular-H bonding in Ni3L2.
3.3. Magnetic properties
The temperature dependence magnetic behavior of Ni3L2 was studied to predict the
antiferromagnetic interaction between the nickel centers (Ni(1) and Ni(2). The χmT value of
Ni3L2 is 3.57 cm3 K mol-1 at 300 K. This value is a little higher than the spin-only (3.00 cm3 K
mol-1) value for the complex having two unpaired electrons (S = 1 spins) with g = 2.2. The
magnetic property of the trinuclear nickel complex was shown under the form of a plot of χmT
versus T in Fig. 3. Upon cooling up to 100 K, χmT value slowly decreases, and further cooling

falls to 0.24 cm3 K mol-1 at 25 K. This low-temperature decrease of χmT values show the weak
antiferromagnetic interaction between the Ni(II) centres. The shape of the plot also indicates an
antiferromagnetic behavior with different strength of magnetic couplings at different temperature
regions. This behavior arises due to the negligible interaction between the terminal and center
Ni(II) ions (Ni1 and Ni2) owing to the significant distance (3.074 Å) between them.
Taking into account the trinuclear centrosymmetric structure of Ni3L2, we tried to fit its
magnetic behavior through the isotropic spin Hamiltonian, eq 1
3
∑
…………………………….(3)
(푔푁푖 푆 퐻 + 푆 퐷 푆 )
퐻 = ― 2퐽(푆1푆2 + 푆2푆3) +

푖
푖
푖 푖 푖
푖 = 1
where S1 and S3 identify the two terminal Ni(II) ions, while the central Ni atom is named
S2. We assume that S1 = S2 = S3 =1 and that gx = gy = gz = g. J represents the magnetic
interaction between adjacent metal ions. According to anisosingle-ion anisotropy, D is assumed
to be the same for the three Ni(II) ions (D1 = D2 = D3 = D). The reason for using single-ion
anisotropy in the Hamiltonian formalism is the drastic decrease in the susceptibility data at low
temperatures [48]. The best fit values (g = 2.2, J= -3.8 cm-1 and D = 10.2 cm-1) obtained from the
experimental data of the trinuclear complex are comparable with the reported similar system
[49]. The presence of a syn−syn acetate bridging pattern in the complex triggers
antiferromagnetic interactions between the metal ions [50]. The complex shows a similar Ni-O-
Ni magnetic exchange pathway reported for phenoxide bridged trinuclear Ni(II) complexes [43,
48-51].
3.4. Electrochemical studies
The redox behavior of nickel centre in biological enzymes, nickel-containing enzymes
are divided into redox and non-redox categories. The nickel enzymes containing anionic S-
donors such as sulfide (as in carbon monoxide dehydrogenase) or thiolate sulfur in the form of
cysteine ligands stabilize the Ni(II/III) redox couple. Thus, S-donor ligands are strongly
associated with Ni redox enzymes to catalyze biological C1 chemistry [52]. The nickel is
employed in enzymes as a Lewis acid catalyst in the other category of enzymes like urease,
acireductone dehydrogenase, and glyoxalase. In contrast to the redox enzymes, these enzymes'
coordination environments are composed exclusively of O/N-donor ligands. In the synthesized
Ni3L2 complex also, the ligation is through N2O4. The coordination chemistry typically favors
six-coordinate Ni(II) complexes that invariably have high-spin electronic structures [53].

The electrochemical properties of Ni3L2 have been studied by cyclic voltammetry. The
Ni3L2 complex shows a quasi reversible Ni(II)/Ni(III) redox couple in the negative potential
range of 0 to -0.8 V. The anodic peak at -0.245 V vs. Ag/AgCl corresponding to the oxidation of
Ni(II) to Ni(III). The formation of Ni(III) species facilitates the formation of intermediate 5 by
the bridging of C=O of urea unit with Ni(II). During the reverse scan, the complex gives the
corresponding reduction peak (Ni(II) to Ni(III)) at -0.578 V. The peak separation between
cathodic and anodic peak (ΔEp > 59 mV), and the ratio of cathodic and anodic current
demonstrating a single quasi reversible one-electron redox wave nature.
3.5. Urease activity
The structural resemblance of Ni3L2 with the active site of urease is predicted by
comparing the crystal data of the complex with the data of bacterial ureases K. aerogenes and
Bacillus pasteurii. Similar to urease, Ni3L2 contains biologically essential moiety such as
histidine, phenolate, and acetate. The two nickel ions in bacterial urease are bridged by a
carbamylated lysine and a hydroxide ion. Each nickel coordinates with two histidine residues
and a water molecule. The coordination sphere of one nickel center, Ni(2), is completed by an
additional terminally bound aspartate resulting in a pseudo-octahedral ligand environment,
whereas another nickel ion, Ni(1), having a vacant site possesses a distorted square pyramidal
geometry. In Ni3L2 the coordination sphere of each terminal nickel ion is similar to the active
site of urease with pseudo octahedral environment [Ni(N2O4)]. Similarly, the bond distance and
bond angle around each terminal nickel ion is comparable to the active site of native Bacillus
pasteurii urease (BPU) and K. aerogenes urease (KAU) (Table 2) [54].
In the present investigation, the urease activity of the synthesized nickel complex was
studied by the absorption spectral titration method. The experiments were done by measuring the
absorbance of phenol red at 560 nm with an increasing concentration of Ni3L2 (0-130 μM), a
fixed concentration of phenol red and urea. Initially, the reaction mixture exists in yellow color.
During the reaction, the reaction mixture turns into red color, and the intensity of color increases
with increasing concentration of the complex. In the absence of Ni3L2, the reaction mixture's pH
is maintained at 6.8, and at this pH, the phenol red exhibits yellow color shows absorption at 440
nm. Upon addition of Ni3L2, the absorbance at 440 nm decreases, and the peak occurs at 560 nm
grows in intensity with complete disappearance of the original peak at 440 nm (Fig. 4), indicates

that Ni3L2 hydrolyzes urea with the release of ammonia and carbamate. The release of ammonia
into the solution leads to the rise of pH to around 8. At this stage, phenol red loses its hydrogen
ion from the hydroxide group and results in the negatively charged red ion. The enhancement of
phenol red absorbance intensity at 560 nm is directly related to the compound's urease activity.
The urease activity of Ni3L2 is further confirmed by the inhibition of urease reaction by adding
thiourea to the reaction mixture. Upon adding thiourea to the reaction mixture, the absorbance
intensity of phenol red at 560 nm experiences a sharp decrease (Fig. 5), indicating that thiourea
serves as a competitor to urea for coordination with the complex, and hence inhibits the urease
activity of the complex.
3.5
Molecular docking
The binding model of Ni3L2 with urea obtained from the molecular docking studies using
the AutoDock Vina program is given in Fig. 6. In the model, the coordinated water molecules in
the complex bind with urea through two hydrogen-bonding. The oxygen of urea binds with the
hydrogen of water molecules with a bond distance of 2.76 Å, and the hydrogen of urea binds
with the oxygen of water molecule with 3.07 Å. These hydrogen bonding interactions weaken
the nickel and water coordination and lead to removing a water molecule from the complex and
favors the coordination of urea to the vacant site of nickel atom (Scheme 3). The two hydrogen
atoms of the –NH2 groups in urea interact with the carboxylate –O of the amino acid residue,
arginine-646 (ARG-646) in urease with a bond distance of 3.29 Å and 3.12 Å. The carbonyl
oxygen of urea binds with the amino hydrogen atoms in THR 578 and THR 581with a bond
distance of 3.19 and 3.07 Å. The docking energy of urea with the amino acid residue of urease is
-3.4 kcal/mol. This binding energy value resembles the binding energy obtained for the complex,
Ni3L2, with urea (-1.6 kcal/mol). The molecular docking results reveal that the objective
particles, urea fitted well in the jack bean urease's active site and the terminal center of the Ni3L2.
3.6
Catalytic mechanism
The proposed mechanism for the catalytic activity of Ni3L2 is shown in Scheme 4. The
structural resemblance of Ni3L2 with the native bacterial urease implies that the proposed
mechanism for the catalytic activity of the complex is analogous to the mechanism provided for
the bacterial urease [54, 55]. In the first step, two urea molecules coordinate to the terminal

nickel ions through the O atom of the CO unit with the loss of water molecules (1). Urea
coordinated complex is stabilized by forming a hydrogen bond through the NH2 group to the
bridging phenolic –O atom (2). This hydrogen bond formation facilitates the coordination of urea
nitrogen atoms to the central nickel atom (Ni1 ion). The distance between the closest urea
nitrogen atom and Ni1 is fixed and shortened. During the approach, the Ni1-O-methoxy distance
grew longer until the methoxy groups are finally detached from the Ni1 and form intermediate 3.
A nucleophilic attack on the bridging substrate of the intermediate, 3 shortens the distance
between the urea carbon atom and the bridging oxygen and gives structure 4. The protonation of
uncoordinated urea nitrogen atom facilitates the release of ammonia forms the transition state 5.
The sixth step involves the addition of water molecules on the electron-deficient C=O unit of
urea, leads to bridging coordination of C=O into a unidentate mode of coordination. The H+ ion
transfer from water to –NH2 leads to ammonium carbamate formation and is completely
removed from 5 and creates two vacant sites (Scheme S1). The structure of the complex is
retained by filling the vacant sites with methoxy group and water molecules imply that the
complex has catalytic activity.
4.
Conclusion
In conclusion, we presented a novel trinuclear complex as a model of urease active
enzyme. The amino acid containing Schiff base (Ligand) forms an octahedral trinuclear complex
with Ni(II) ion through phenolato and acetato bridges. The structure of Ni3L2 has been evaluated
by different spectral techniques and single-crystal X-ray analysis. The electronic absorption
spectral studies concluded that the inter electronic repulsion in the nickel centre is decreased
during complexation. The bond length and bond angles around the terminal Ni(II) centre in
Ni3L2 obtained from the single-crystal X-ray analysis matches the active site of native bacterial
urease. The temperature dependence magnetic behavior of Ni3L2 proved the antiferromagnetic
interaction between the nickel centers (Ni(1) and Ni(2). The synthesized complex exhibits urease
activity by converting urea into ammonia and carbamate and efficiently mimicking the urease
enzyme's functional model. The molecular docking studies reveal that the binding energy of urea
with Ni3L2 is comparable to urea with jack bean urease.

Acknowledgment
Authors gratefully acknowledge the financial support received from the Science and Engineering
Research Board (SERB), Department of Science and Technology (DST), Government of India,
New Delhi (EMR/2017/005195). We are grateful to Dr. Babu Varghese, SAIF, IIT Madras, for
the sample's single-crystal X-ray analysis.
Supplementary data
Supplementary data information associated with the manuscript can be found in the online
version. The spectral characterization like IR, UV, ESI-MS, and inter and intramolecular
hydrogen bonding, thermal behavior of complex is given in the supplementary. CCDC:1409315
contains the supplementary crystallographic data for Ni3L2. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-
033; or e-mail: deposit@ccdc.cam.ac.uk.

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Highlights
 Trinuclear nickel(II) complex (Ni3L2) with phenolate and syn-syn acetate bridges

 Characterization of Ni3L2 by various spectral and single crystal X-ray methods
 Structural resemblance of Ni3L2 compared with urease enzyme of native bacteria
 Urease activity of Ni3L2
 Molecular docking of Ni3L2 and urease with urea
Table 1. Crystallographic data and structure refinement of the complex, Ni3L2.
Identification code
Empirical formula
Formula weight
shelxl
Ni3 C32 H42 N6 O17
958.85
Temperature
293(2) K
Wavelength
0.71073 Å
Crystal system, space group
Unit cell dimensions
Tetragonal, P41212
a = 17.0606(5) Å, alpha = 90 deg.
b = 17.0606(5) Å, beta = 90 deg.
c = 14.1454(6) Å, gamma = 90 deg.
4117.2(2) A3
Volume
Z, Calculated density
Absorption coefficient
F(000)
4, 1.547 mg/m3
1.435 mm-1
1984
Crystal size
0.20 x 0.10 x 0.10 mm
2.22 to 24.99 deg.
-20<=h<=13, -19<=k
<=20, -16<=l<=14
20372 / 3605, [R(int) = 0.0458]
98.80%
Theta range for data collection
Limiting indices
Reflections collected / unique
Completeness to theta = 24.99
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
Refinement method
0.871 and 0.755
Full-matrix least- squares on F2
3605 / 18 / 311
Data / restraints / parameters
Goodness-of-fit on F2
1.109
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0379, wR2 =0.1081
R1 = 0.0459, wR2 =0.113
0.01(2)
Absolute structure parameter

Extinction coefficient
0.0011(3)
Largest diff. peak and hole
0.503 and -0.285 e. Å-3

Table 2. Comparison of bond distances and bond angles around the Ni centers in native
ureases with the synthesized complex, Ni3L2.
Ni-Ligand interaction in Native
Native
KAU
2.1
Ni3L2 Ni-Ligand interaction
in complex
Native ureases (Å)
BPU
Ni1-LysO1
2.1
2.1
2.1
2.2
2.2
2.2
2.1
2.2
3.7
2.0
2.0
2.0
2.1
2.0
2.1
2.2
2.1
3.1
Ni1-AcO6
Ni2-AcO5
Ni2-LysO2
2.1
Ni1-OB
2.2
Ni1-phenoO2
Ni2-phenoO2
Ni2-azoN1
Ni2-imdN2
Ni2-waterO
Ni2-carboxlO3
Ni1-Ni2
Ni2-OB
2.0
Ni2-HisN1
2.3
Ni2-HisN2
2.2
Ni2-OW
2.1
Ni2-AspO
2.2
Ni1-Ni2
3.6
Ligand-Ni-Ligand angles (°)
LysO2-Ni2-N1His
LysO2-Ni2-N2His
LysO2-Ni2-Ow
LysO2-Ni2-OAsp
HisN1-Ni2-N2His
OW-Ni2-OB
Ligand-Ni-Ligand angles (°)
94
90
90
93
AcO5-Ni2-N1azo
AcO5-Ni2-N2imd
88
92
91
103
171
110
59
87
AcO5-Ni2-O7water
AcO5-Ni2-O3carboxyl
azoN1-Ni2-N2imd
Water O7-Ni2-O2pheno
Ni1-O2pheno-Ni2
175
113
63
171
85
83
Ni1-OB-Ni2
119
121
115

Lys
His
NH
N
N
His
O
O
NH
N
NH
Ni1
Ni2
N
N
O
H
HN
OH2
OH2
O
O
His
His
Asp
Scheme 1. Schematic representation of the active site of K. aerogenes urease.
[20,21]

Scheme 2. Synthetic scheme of trinuclear Ni(II) complex, Ni3L2.

Fig. 1. ORTEP view of Ni3L2 with ellipsoids at the 50% probability level.

Fig. 2. Crystal packing of Ni3L2.

Fig. 3. Experimental (full circles) and best fit ( continuous line) mT vs T for
Ni3L2.