Synthesis, physicochemical properties and in vitro catalytic activity of a dinuclear nickel(II) complex with a N5O-hexadentate ligand: A functional model for phosphohydrolases

Article abstract of DOI:10.1016/j.poly.2016.02.013

The development of artificial nucleases has been stimulated by the realization that hydrolytically active metal complexes may potentially find utility as robust versatile replacements for restriction enzymes in molecular biology research and as nucleic ac

Full text of DOI:10.1016/j.poly.2016.02.013

Accepted Manuscript
Synthesis, physicochemical properties and in-vitro catalytic activity of a dinu-
clear nickel(II) complex with a N O-hexadentate ligand: A functional model for
5
phosphohydrolases
Fernando R. Xavier, Ademir Neves
PII:
S0277-5387(16)00098-X
http://dx.doi.org/10.1016/j.poly.2016.02.013
POLY 11828
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Accepted Date:
16 September 2015
8 February 2016
Please cite this article as: F.R. Xavier, A. Neves, Synthesis, physicochemical properties and in-vitro catalytic activity
of a dinuclear nickel(II) complex with a N O-hexadentate ligand: A functional model for phosphohydrolases,
5
Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2016.02.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, physicochemical properties and in-vitro catalytic
activity of a dinuclear nickel(II) complex with a N O-hexadentate
5
ligand: A functional model for phosphohydrolases
a
by Fernando R. Xavier * and Ademir Neves
b
a
Departamento de Química, Universidade do Estado de Santa Catarina, Joinville –
b
SC, Brazil. CEP 89219-710; Departamento de Química, Universidade Federal de
Santa Catarina, Florianópolis – SC, Brazil. CEP 88040-900; *Corresponding
author: fernando.xavier@udesc.br
Abstract
The development of artificial nucleases has been stimulated by the
realization that hydrolytically active metal complexes may potentially find utility
as robust versatile replacements for restriction enzymes in molecular biology
research and as nucleic acid-targeting therapeutics. In this regard, a novel
unsymmetrical dinucleating ligand HL (where L is the 2-[(4,7-diisopropyl-1,4,7-
triazonan-1-yl)methyl]-4-methyl-6-[(pyridin-2-ylmethylamino)methyl]phenolate
1
O-donnor set was synthesized and characterized by IR and H-
moiety) with an N
5
NMR spectroscopy. The reaction of HL with nickel(II) perchlorate led to the
formation of a µ-phenoxo-bridged dinuclear Ni(II) complex, [Ni (L)(µ-
OAc) ]BPh • H O (1). Physicochemical measurements (molar conductometry, IR
2
2
4
2
and UV-visible spectroscopy and electrochemistry) were performed for 1 and
1

these properties are compared with those presented by similar compounds in the
literature. ESI-MS measurements in positive and negative modes confirmed the
complex composition as well as the presence of tetraphenylborate counterions,
based on their mass and isotopic distribution patterns. Potentiometric titration
3 2
studies of 1 were carried out (CH CN/H O 50% v/v) and we probed its acid/base
properties, which are crucial to gaining an understanding of the phosphodiester
catalytic cleavage mechanism. Finally, the ability of 1 to mimic the functional role
of the dinuclear metalloenzymes was evaluated through studies on its reactivity
toward the model-substrate bis(2,4-dinitrophenyl)phosphate (2,4-bdnpp). When
compared with similar previously described compounds, complex 1 helped to
probe the influence of the aromatic/aliphatic N-donor moieties, such as
macrocyclic rings, pyridine and imidazole, through their catalytic efficiency
-1 -1
parameter (E / L mol s ).
1. Introduction
Phosphate esters are omnipresent in the area of biology, where they fulfil a
number of key roles. They are found in the hydrophilic “head” group of
phospholipids (the major component of cell membranes) and in many small
molecules that play a role in metabolism, such as ATP, coenzyme A and
cAMP.[1] However, probably the most notable biological structures that employ
phosphate esters moieties are deoxyribonucleic acid (DNA) and ribonucleic acid
(
RNA). Chemically, these biopolymers are comprised of ribonucleotides or
deoxyribonucleotides in RNA and DNA, respectively, and the monomers are
connected through their 3’,5’-phophodiester bonds [1] .
2

The uncatalyzed hydrolysis of phosphodiesters is favored
thermodynamically but in terms of kinetics it is exceedingly slow, making them
particularly robust in their structural roles in biology. In particularly, in DNA
these bonds have an estimated half-life of 30,000,000 years (pH 6.8, 25 ◦C),
calculated from the rate of hydrolysis of dineopentyl phosphate measured under
-16 -1
these conditions (7×10 s ) [2] . The nucleophilic cleavage of phosphodiester
bonds entails the attack of a nucleophile on the phosphorus atom of a
phosphodiester moiety, via a pentacoordinate, trigonal bipyramidal, transition
state in which the attacking nucleophile and departing alkoxide generally occupy
the apical positions [3] .
In this context, the search for low molecular weight catalysts for
phosphodiester cleavage has attracted much interest among scientists, since these
compounds may help to elucidate several issues such as the cleavage mechanism,
the role of metals in these biological systems, and the design of more effective
synthetic hydrolases [4] [5] . Since many hydrolytic enzymes contain two metal
centers at their active site, which operate in a cooperative fashion to cleave their
substrates, it is not surprising that in recent decades there has been intense interest
in the development of binuclear metal complexes as a route to generating more
active synthetic cleavage agents [6] .
In particularly, dinucleating ligands containing pyridyl pendant arms or N-
donor macrocyclic motifs are among the most studied compounds, where their
metal complexes have shown prominent catalytic activity [4] [6] . However, given
that dinuclear metalloenzymes usually present an unsymmetrical feature around
3

the metal centers in their active sites, there is a significantly smaller number of
catalysts with unsymmetrical bioinspired ligands.
In this regard, for several years our group has been engaged in obtaining
II
symmetrical and unsymmetrical homodinuclear Zn Zn , [7] Cu Cu , [8] -[12]
II
II
II
II
Ni Ni , [13] -[15] Fe Fe , [16] -[18] Mn Mn , [19] -[21] and heterodinuclear
II
III
III/II
III
II
III II
Fe M (M = Mn, Co, Ni, Cu, Zn) complexes which are able to hydrolyze the
II
model substrate 2,4-bdnpp and hydrolytically cleave DNA [5] [22] .
Particularly in the bioinorganic field, the macrocycle 1,4,7-
triazacyclononane (tacn) and a diversity of multidentate functionalized derivatives
have proven to be an outstanding approach to ligand design, mostly due to their
facial coordination mode [4] [6] [8] [23] . Although some unsymmetrical ligands
capable of providing distinct chemical environments to yield mixed-valence
compounds [5] [6] [22] are described in the literature, to date there are only a few
examples of unsymmetrical homodinuclear complexes with a tacn-containing
N3N4
dinucleating archetype. One good example is the ligand L
described by Costas
and coworkers where tacn and pyridyl coordinating units are present [24] .
In this paper, we describe the synthesis and characterization of a new
unsymmetrical tacn-containing dinucleating ligand 2-[(4,7-diisopropyl-1,4,7-
triazonan-1-yl)methyl]-4-methyl-6-{[(pyridin-2-ylmethyl)amino]methyl}phenol
(
HL) and its dinickel(II) complex (1) (Chart 1). It is noteworthy that the
possibility of providing a free coordination site on one of the nickel centers allows
the coordination of an extra labile ligand (e.g., OH groups) which can be easily
substituted by the substrate during the catalytic process.
2
4

<
< Please add Chart 1 here, as a single-column picture >>
Chart 1. Chemical structures of the ligand HL (left) and its nickel(II) cation
complex 1 (right).
2. Experimental Section
2.1 Chemicals and instrumentation
All starting materials were purchased from Aldrich, Acros or Merck and
used as received unless otherwise stated. 2-(aminomethyl)pyridine was distilled
under reduced pressure, tetrabutylammonium hexafluorophosphate was double
recrystallized from hot ethanol.
Elemental analysis (C, H and N) was performed on a Perkin-Elmer 2400
analyzer equipped with an AD-4 autobalance setup.
Infrared spectra were collected on
a
Perkin-Elmer FTIR-2000
-1
spectrophotometer using KBr pellets in the range of 4000-500 cm . The data were
-1
plotted as transmittance (%) as a function of the wavenumber (cm ).
1
H-NMR spectroscopy was carried out with a Varian FT-NMR 400 MHz
3
system, using CDCl as the solvent, at 25 °C. Chemical shifts were referenced to
tetramethylsilane (TMS, δ = 0.000 ppm).
5

The molar conductivity for complex 1 was evaluated in a Schott-Geräte
-2
CG 853 conductivimeter (previously calibrated with an aqueous 1.00 × 10 M
-
3
M
KCl solution; Λ = 1408 µS [25] ) at room temperature using a 1.00 × 10 M
spectroscopic grade acetonitrile solution.
The electrospray ionization-mass spectrometry (ESI-MS) of 1, dissolved in
an ultrapure acetonitrile solution (500 nM), was performed on an amaZon X Ion
Trap MS instrument (Bruker Daltonics) with an ion spray source using
electrospray ionization in positive and negative-ion modes. The ion source
condition was an ion spray voltage of 4.5 kV. Nitrogen was used as the nebulizing
gas (20 psi) and curtain gas (10 psi), and samples were directly infused into the
-1
mass spectrometer at a flow rate of 180 µL h . The m/z scan range was 150 to
000 Da. The simulated spectrum was calculated using the mMass software [26]
27] .
2
[
UV–visible spectra were obtained at room temperature using a Perkin
Elmer Lambda 19 spectrophotometer operating in the range of 300–1000 nm with
-1
-1
quartz cells. Values of ε are given in M cm . The UV-Vis spectrum of 1 was
recorded in acetonitrile (spectroscopic grade) solution.
The electrochemical behavior of 1 was investigated with a Bioanalytical
Systems Inc (BASi) potentiostat/gavanostat. Square-wave voltammograms were
obtained at room temperature in acetonitrile solutions containing 0.1 M of n-
Bu
4
NPF
6
as the supporting electrolyte under argon atmosphere. The
electrochemical cell employed was comprised of three electrodes: gold (working),
+
platinum wire (auxiliary) and Ag/Ag (reference). The ferrocene/ferrocinium
6

+
redox couple Fc/Fc (E = 400 mV vs NHE) [28] was used as the internal
0
standard.
The protonation constants for complex 1 were investigated with a Corning
350 pH meter fitted with a glass-combined electrode (Ag/AgCl) calibrated to read
+
log [H ] directly, designated as pH. The experiment temperature was stabilized
–
at 25.00 ± 0.05ºC. Doubly distilled water in the presence of KMnO
4
was used to
prepare CH CN/H O (50:50 v/v) solutions. The electrode was calibrated using the
3
2
-
2
data obtained from a titration of a known volume of a standard 1.00 × 10 M HCl
solution with a standard 0.100 M KOH solution. The ionic strength was kept
-2
constant at 0.1 M by addition of KCl, and the solutions, containing 5 × 10 mmol
of complex, were titrated with 0.100 M standard CO -free KOH. Experiments
were performed in 50.00 mL of a CH CN/H O (50:50 v/v) solvent system, purged
2
3
2
with argon cleaned using two bubbling towers with 0.1 M KOH solutions. The
titration process was initiated around pH 3.0 by the addition of HCl. The
experiments were run at least in triplicate and were all analyzed using the BEST7
[
29] program. The species diagrams were obtained with SPE and SPEPLOT
programs [29] .
2
.2 Kinetic assays
The catalytic activity of 1 towards the activated phosphodiester 2,4-
bis(dinitrophenyl)phosphate (2,4-bdnpp) [30] was evaluated
spectrophotometrically on a Varian Cary50 Bio spectrophotometer, at 400 nm
based on the appearance of the 2,4-dinitrophenylphenolate ion at 25 °C. The effect
of pH on the hydrolytic cleavage of 2,4-bdnpp was monitored in the pH range
7

from 4.0 to 10.5. Reactions were performed using the following conditions: 1.50
-
mL freshly prepared aqueous buffer solution ([buffer]final 5.00×10 M), buffer:
2
MES (pH 4.00 to 6.50), HEPES (pH 7.00 to 8.50), CHES (pH 9.00 to 10.00) and
CAPS (pH 10.50) with controlled ionic strength (LiClO ) 0.10 M; 200 µL of an
4
-5
acetonitrile complex solution ([C]final=2.00×10 M) and 300 µL of acetonitrile
were added to a 1-cm-path-length cell. The reaction was initiated by the addition
-
3
of 1.00 mL of an acetonitrile substrate solution ([2,4-bdnpp]final=2.50×10 M) and
monitored between 2 and 5% of reaction at 25 °C. The kinetic experiments under
conditions of excess substrate were performed as follows: 1.5 mL of freshly
-
2
prepared aqueous buffer CHES solution (at pH 9.00), [buffer]final= 5.00×10 M,
-5
and 50 µL of an acetonitrile complex solution ([C]final= 1.24×10 M) were added
to a 1-cm-path-length cell. The reaction was initiated with the addition of 2,4-
-4
-3
bdnpp solution ([2,4-bdnpp]final=2.00×10 to 6.80×10 M). Correction for the
spontaneous hydrolysis of the 2,4-bdnpp was carried out by direct difference
using a reference cell under identical conditions without adding the catalyst. The
initial rate was obtained from the slope of the absorbance vs. time plot over the
first 5 min of the reaction. The conversion of the reaction rate units was carried
-1
-1
out using ε=12,100 M cm for 2,4-dnp and the initial concentration of the
complex [31] . A kinetic treatment using the Michaelis–Menten equation
approach was applied [1] . The deuterium isotopic effect on the hydrolytic process
was evaluated following two reactions with identical conditions (vide experiments
on the effect of pH) using CHES buffer (pH or pD 9.00, previously prepared), and
a 100-fold excess of substrate in comparison to the complex concentration. The
reactions were monitored at 400 nm and 25 °C.
8

2.3 Synthesis of HL unsymmetrical ligand.
The dinucleating ligand 2-[(4,7-diisopropyl-1,4,7-triazonan-1-yl)methyl]-
-methyl-6-{[(pyridin-2-ylmethyl)amino]methyl}phenol (HL) was synthesized by
4
reductive amination [32] as follows.
In a 125 mL round-bottom flask 2.57 g (7.11 mmol; 361.53 g mol ) of
mff [23] and 0.73 mL of 2-(aminomethyl)pyridine (0.77 g; 7.11 mmol;
-1
i
tacn pr
08.14 g mol ; 1.05 g mL ) were mixed with 50 mL of methanol/THF (50% v/v)
yielding a dark-yellowish solution which was stirred magnetically for 2 h. Imine
reduction was initiated by adding small portions of NaBH (0.27 g; 7.11 mmol;
7.82 g mol ) at room temperature where, after 1 h, the reaction turned a pale-
2
-1
-1
1
4
-1
3
yellow color. The pH was adjusted to 7 with HCl (2.0 M) and the solvent was
o
evaporated under reduced pressure at 40 C. To the remaining oil, 50 mL of
dichloromethane were added and the mixture was washed twice with 30 mL of a
saturated aqueous NaHCO
Na SO and the solvent was rotoevaporated under reduced pressure yielding a
pale-yellow oil. Yield: 96 % (3.09 g, 6.82 mmol; 453.67 g mol ). Anal. Calc. for
3
solution. The organic phase was dried over anhydrous
2
4
-1
-1
27 43 5
C H N O, FW = 453.66 g mol : C, 71.48; H, 9.55; N, 15.44. Found: C, 71.57;
-
1
H, 9.50; N, 15.50%. IR spectrum (KBr pellet, cm ): ν (O-H) 3431(br); ν (C-Haliph
)
)
)
2
1
7
962-2817(s); ν
s
(C=C) 1589 - 1478(m); δ
s
(C-Haliph) 1381(s); δ ass (C-Halif
381(m); δ (O-H) 1357(m); ν (C-Ophenol) 1246(m); ν
s
(C-N) 1116(m); δ (C-Har
, δ, ppm): 0.96 (d, 12H, CH
); 2.70 (t, 4H, CH ); 2.88 (hept, 2H, CHiPr); 2.98 (s, 4H,
); 3.84 (s, 2H, CH ); 3.92 (s, 2H, CH
1
55(m). H-NMR (400 MHz, CDCl
); 2.51 (s, 4H, CH
); 3.78 (s, 2H, CH
3
3
); 2.20 (s, 3H,
CH
3
2
2
CH
2
2
2
2
); 6.73 (s, 1H, CHar);
9

6
.90 (s, 1H, CHar); 7.14 (t, 1H, CHar); 7.36 (d, 1H, CHar); 7.63 (t, 1H, CHar); 8.54
s, 1H, CHar). The spectra are available in the supporting information file (Figures
S2 and S3).
(
II II
2
.4 Synthesis of [Ni Ni (L)(µ-CH
3 2 2 4 2
COO) (OH )]BPh • H O (1)
Complex 1 was synthesized through the dropwise addition of a HL ligand
-
1
solution in acetonitrile (0.227 g; 0.5 mmol; 453.67 g mol ) to Ni(ClO
4
)
2
·6H
CN. The mixture was
kept under magnetic stirring, at 40 ºC, for 30 min and 0.123 g (1.5 mmol; 82.03 g
2
O
-1
(
0.366 g; 1.0 mmol; 365.69 g mol ) also dissolved in CH
3
-
1
-1
3 4
mol ) of CH COONa and 0.171 g (0.5 mmol; 342.22 g mol ) of NaBPh were
then added, resulting in a pale greenish solution. After a few minutes a green solid
was formed. The crude complex was filtered and doubly recrystallized from
acetonitrile to afford a green microcrystalline solid. Yield: 65% (0.339 g, 0.325
-
1
mmol) based on ligand. Anal. Calcd. for C55
5 2 7
H72BN Ni O , FW = 1043.39 g mol :
C, 63.31; H, 6.96; N, 6.71. Found: C, 62.91; H, 5.57; N, 6.36%. IR spectrum (Kbr
-1
pellet, cm ): ν (O-H) 3440(br); ν (N-H) 3247(s); ν (C-Har) 3059-3034(m); ν (C-
-
aliph) 2998-2851(m); ν (C=N and C=C) 1595(s) and 1472; ν (COO ) 1422(s); ν
H
(
(
C-N) 1307(w), ν (C-Oph) 1261(w); δ (C-Har) 735 - 707(m). Molar conductivity
CH CN, 1.0 mM) Λ = 108 µS.
3
M
3. Results and discussion
3.1. Synthesis and general characterization
The N-rich unsymmetrical HL ligand was synthesized in a one-pot
reductive amination process through the addition of 2-picolylamine to the
10

i
2
tacn Pr mff precursor (Figure S1, supporting information). The reaction was
monitored through thin layer chromatography (TLC) and the product was
1
characterized by IR and H-NMR spectroscopy (see experimental section for
further details).
Complex 1 was synthesized via the reaction of nickel(II) perchlorate and
the ligand HL in a 2:1 stoichiometric ratio using acetonitrile as the solvent. The
coordination reaction could be observed from the color change during the metal
addition. Sodium acetate was added in order to neutralize the solution,
deprotonate the HL ligand, and provide the µ-acetato bridging ligands. These
features have been observed for several other complexes previously described in
the literature [4] [6] [13] [15] [22] .
A variety of physical and chemical measurements were performed for 1,
both in the solid state and in the fluid solution. These methods include elemental
analysis, molar conductivity, infrared and electronic spectroscopy, square-wave
voltammetry and electrospray mass spectrometry. In order to demonstrate the
acid/base properties of the complex, which allows an understanding of the
phosphodiester catalytic cleavage, potentiometric titrations were also performed.
Although much effort has been made to isolate single crystals of 1, their fragility
and poor quality prevent analysis using x-ray techniques.
3.2. Infrared spectroscopy and molar conductivity
Infrared spectroscopy was essential for the preliminary characterization of
the ligand and its complex. A spectral overlay of the ligand HL and 1 is shown in
Figure 1.
11

<
< Please add Fig. 1 here, as a single-column picture >>
4000
3500
3000
2500
2000
1500
1000
500
-
1
cm
Figure 1. Overlay of the IR spectra for the HL (top) and 1 (bottom).
The infrared spectrum of 1 is mainly characterized by the skeletal ligand
vibrations and the presence of exogenous species (bridging ligands) and the
counterion. Despite the ligand deprotonation, the O-H stretching becomes more
-
1
evident in 1 (3440 cm ), indicating the presence of water molecules bonded to a
nickel center and acting as a crystallization solvent. This proposal was confirmed
by the elemental analysis and ESI-MS. The spectrum for the complex reveals a
-
1
sharp IR absorption peak at 3247 cm attributed to N-H stretching, which is not
resolved in the isolated ligand. This behavior could be associated with the ligand
12

-1
rigidity after the complexation. The increase in the absorption at 1595 cm in 1
strongly suggests the presence of the tetraphenylborate counterion, while the band
-1
broadening between 1472 and 1422 cm indicates the presence of the bridging µ-
-
OAc exogenous ligands. The intensity of the C-H out-of-plane angular
-
1
deformation (δCH) at 755 cm in HL also increased in 1, as a second indication of
-
the presence of the B(Ph)
4
counterion. A similar IR profile was observed by
Greatti and co-workers [14] .
An acetonitrile solution of 1 was analyzed via molar conductometry [33] ,
where the cationic character of the complex was confirmed by the 1:1 electrolyte
type (Λ
M
= 108 µS). These data are in full agreement with the results of the
elemental analysis, IR spectroscopy and electrospray mass spectrometry (vide
infra).
3.3. Electrospray ionization-mass spectrometry
Electrospray ionization-mass spectrometry (ESI-MS) studies were
3
performed on 1 in ultrapure CH CN (Figure 2) and both the positive and negative
modes were monitored. For the positive scan mode six peak clusters were
observed at mass to charge ratios (m/z) of 702.2, 686.2, 672.3 for monovalent
species and 313.6, 306.6 and 299.6 for divalent species. The peak cluster
assignments are shown in Table 1 and the isotopic distribution simulations are
shown in Fig. S4.
<
< Please add Fig. 2 here, as a single-column picture >>
13

1
00
(b)
80
60
40
20
0
(c)
(
d)
(a)
(
e)
(
f)
200
400
600
m/z (Da)
800
1000
Figure 2. ESI-MS spectrum (positive mode) of 1 in acetonitrile.
The cluster at 702.2 Da (species “a”, Table 1) is attributed to the cation
II
2
+
complex [Ni
3 2 2
(L)(µ-CH COO) (OH )] , which confirms the obtainment of the
structural unit aimed at for the cation complex during the synthetic procedure. The
base peak cluster at 686.2 Da (100%) can be attributed to the loss of the aqua
II
2
+
ligand in the softer nickel center, generating the moiety [Ni
(L)(µ-CH
3
2
COO) ]
II
2
+
OH)] at
(
species “b”, Table 1). Species “c” [Ni
(L)(µ-OH)(CH
3
CN)(CH CH
3
2
672.3 Da is comprised of the ligand L and both the nickel centers bonded through
a µ-hydroxo bridge. These data are in agreement with the results of the
potentiometric titrations and kinetic studies (vide infra). Acetonitrile and ethanol
molecules from the ion spray and acetate reduction, respectively, were also
observed as exogenous ligands. This behavior has been previously described by
II
2
2+
3
(L)(µ-CH COO)] ,
Piovezan and co-workers [13] . The dicationic complex [Ni
at 313.6 Da, (species “d”, Table 1) is formed when “b” releases one of the
intermetallic µ-acetato bridges into the ion spray. The cluster at 306.6 Da (species
14

I
“
2
e”, Table 1) is best interpreted as a dinuclear reduced Ni species where the
ligand is protonated (HL). The final mass fit is consistent with an ethanol
molecule coordinated to a nickel center. The peak cluster with a lower relative
intensity at 299.6 Da (species “f”, Table 1) can be attributed to the
I
2+
[
2 3
Ni (HL)(CH OH)] complex. This moiety is very similar to species “e” and is
also generated in the ion spray during the experiment.
When 1 was scanned in the negative mode using the ESI-technique (Fig.
S5) a single peak cluster was found at 319.2 Da (species “g”, Table 1). The mass
fit is in full agreement with the tetraphenylborate counterion, the presence of
which was also confirmed by elemental analysis and infrared spectroscopy (1595
-1
cm ).
<
< Please add Table 1 here, as a single-column picture >>
Table 1. ESI-MS data for complex 1 in CH3CN.
m/z (Da)
a = 702.2
b = 686.1
c = 672.3
d = 313.6
e = 306.6
f = 299.6
g = 319.2
Formula
Species
II
+
C
31
C
31
C
31
C
29
C
29
C
28
C
24
H
50
N
N
N
N
N
N
B
5
5
6
5
5
5
Ni
Ni
Ni
Ni
Ni
Ni
2
O
6
5
3
3
2
2
[Ni
[Ni
[Ni
[Ni
[Ni
[Ni
2
3 2 2
(L)(µ-CH COO) (OH )]
II
II
II
I
+
H
48
2
O
2
(L)(µ-CH
(L)(µ-OH)(CH
(L)(µ-CH COO)]
3
COO)
2
]
+
H
52
2
O
2
3
CN)(CH CH OH)]
3 2
2+
H
45
2
O
2
3
2+
H
47
2
O
2
(HL)(CH
3
CH OH)]
2
I
2+
OH)]
H
45
2
O
2
(HL)(CH
3
-
H
20
BPh
4
3.4. Electronic and redox properties
15

The electronic absorption spectrum for 1 recorded in acetonitrile solution
is shown in Figure 3. As observed, all of the bands have low molar absorption
coefficients, indicating ligand field (d-d) transitions, except that at 303 nm. In
general, nickel(II) complexes in octahedral environments show three spin-allowed
d-d transitions in the ranges 1250-900, 670-520, and 500-370 nm, with low molar
-
absorptivity coefficients (< 30 mol L cm ) [34]
1
-1
-
1
-1
Two broad absorption bands are observed at 621 nm (ε = 20 mol L cm )
-
1
-1
3
3
and 1005 nm (ε = 27 mol L cm ), which can be assigned to the A2g → T1g (F)
3
3
and A2g → T1g (F) transitions, respectively. A weak band is also observed at 790
-
1
-1
3
nm (ε = 9 mol L cm ), which is ascribed to the spin-forbidden transition A2g
→
1
g
E (D). In addition to these transitions, another band is observed at 298 nm (ε =
-
1
-1
611 mol L cm ), which can be attributed to a charge-transfer transition
3
involving orbitals from nickel(II) and the pyridine ligand, as previously observed
for other correlated complexes previously described in the literature [13] [15] [35]
[
36]
<
< Please add Figure 3 here, as a single-column picture >>
0.5
0.4
0.3
0.2
0.1
0.0
0
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
.0
270
285
300
315
330
345
λ (nm)
500
600
700
800
900 1000 1100 1200 1300
λ (nm)
16

-2
Figure 3. Electronic spectrum of complex 1 in acetonitrile solution (1.0×10 mol
-
1
-4
L ) and inset (1.0×10 mol L ).
-1
The electrochemical behavior of 1 was determined by square-wave
-1
4 6
voltammetry in acetonitrile (0.1 mol L n-Bu NPF as supporting electrolyte).
The best electrochemical response of 1 was obtained employing a gold working-
electrode with a cathodic-direction scan. Table 2 shows the electrochemical data
collected and the process attributions for 1 and other compounds described in the
literature.
<
< Please add Table 2 here, as a single-column picture >>
Table 2. Selected electrochemical data and process attributions for complex 1 and
other complexes described in the literature.
Complex
Potential (V) vs NHE Attribution
Ref.
II/II
II/I
1
-1.29
1.66
Ni → Ni
This study
II/I
I/I
-
Ni → Ni
II/II
II/III
1
.00
.40
Ni → Ni
II/III
Ni
III/III
1
→ Ni
II/II
II/I
A
-1.27
1.64
Ni → Ni
[13]
II/I
I/I
-
Ni → Ni
II/II
II/I
B
C
-1.41
1.04
Ni → Ni
[37]
[14]
II/II
II/III
Ni → Ni
II/III
Ni
III/III
1
.20
→ Ni
17

II/II
II/III
D
0.73
.94
Ni → Ni
[14]
II/III
Ni
III/III
0
→ Ni
II
2
III
II
A = [Ni
II
(HBPPAMFF)(µ-OBz)
COO) (OH )]ClO
2
(OH
; D = [Ni
2
)]BPh
II
4
; B = [Fe Ni (bpbpmp)(µ-OAc)
2 4
]ClO ; C =
[
Ni
2
(L1)(µ-CH
3
2
2
4
2
(L2)(µ-CH COO) (CH CN)]BPh
3
2
3
4
The square-wave voltammogram (Fig. S6) reveals four irreversible
electrochemical processes. Despite all efforts employed to improve the data
acquisition, the cathodic waves (-1.29 V and -1.66 V versus NHE) are poorly
resolved and the potentials can be attributed to the one-electron transfer processes
II
II
II
I
I
I
Ni Ni → Ni Ni → Ni Ni , respectively. These data are indicative of the inability
II
of the ligand to fully stabilize the Ni Ni and Ni Ni reduced species, although
I
I
I
II
these values are in good agreement with the compound [Ni
OBz) (OH )]BPh
coworkers [13] and similar to [FeNi(bpbpmp)(µ-OAc)
2
(HBPPAMFF)(µ-
2
2
4
(A) previously described in the literature by Piovezan and
]ClO (B) [37] .
2
4
The electrochemical response in the anodic region was found to be better
than that in the cathodic region. Two waves were detected at 1.00 V and 1.40 V
versus NHE and assigned to the irreversible stepwise one-electron oxidation
II
II
II
III
III
III
couples Ni Ni → Ni Ni → Ni Ni . This redox behavior is also displayed by
other dinuclear nickel(II) complexes described by Greatti and colaborators [14] .
II
II
II
III
Interestingly, the Eox1 (Ni Ni →Ni Ni ) for 1 (1.00 V) is comparable to
II
2
+
that for compound C – [Ni
(L1)(µ-CH
3 2 2
COO) (OH )] (1.04 V versus NHE)
where L1 is an unsymmetrical ligand containing three pyridine pendant arms [14]
.
This feature reveals that these nickel centers most probably have an equivalent
II
III
III
III
O
4 2
N -donnor set environment. When the Eox2 (Ni Ni →Ni Ni ) of 1 is
compared with correlated compounds described by Greatti and coworkers a trend
18

can be observed: D (0.94 V)
<
C (1.20 V) < 1 (1.40 V). These results can be
-coordination environment of each
interpreted as reflecting the ability of the N
3
O
3
ligand to stabilize the oxidized species formed. In fact, better electron-density
donors have lower oxidation potentials: D (pyridyl and N-methyl-imidazolyl
i
groups), C (two pyridyl groups) and 1 ( Pr
2
-tacn).
3.5. Potentiometric titrations
In order to detect the presence of metal-bonded water molecules when
complex 1 is in an aqueous solution, potentiometric titration experiments were
carried out. This study can be carried out since the carboxylate-bridging ligands
are labile in acetonitrile/water solutions [22] .
Experimentally, the titrations were performed in triplicate in a mixture of
acetonitrile/water (50% v/v) with a constant ionic strength (0.1 M KCl), and they
showed the neutralization of 3 mols of KOH per mol of complex in the pH range
2
to 12. Fitting the data with the BEST7 software [29] resulted in three discernible
pK values: 5.14, 6.21 and 8.34. The corresponding species distribution diagram is
a
shown in Figure 4, while the proposed deprotonation/protonation equilibrium
steps can be seen in Chart 2.
<
< Please add Figure 4 here, as a single-column picture >>
19

A
D
1
00
C
80
60
40
20
0
B
2
4
6
8
10
12
pH
Figure 4. Species distribution curves of 1 as a function of pH. Experimental
-1
3 2
conditions: CH CN/H O 50% v/v, I = 0.1 mol L (KCl).
The protonation equilibria can be interpreted in terms of the dissociation of
the bridging-carboxylate groups (Chart 2) which in aqueous medium is in line
with its well-documented lability, which is facilitated by increasing the pH of the
solution [5] [22] .
Firstly, when 1 is dissolved in CH
is spontaneously released leading to the {(H
structure A) yielding one equivalent of acetic acid. The pKa1 (5.14) corresponds
to the titration of this acid equivalent previously generated. After that, a release of
3
2
CN/H O at pH ~ 3.0 one acetate group
II
II
2
O)Ni (µ-OAc)Ni (OH
2
)} species
(
II
II
2 2
the second carboxylate group to yield the {(H O)Ni Ni (OH )} species occurs,
where no exogenous bridging ligand is coordinated to the metal centers (structure
B) and it reaches its maximum at pH 5.7 (63.1%). It has been proposed that upon
the dissociation of the carboxylate groups, water molecules occupy the remaining
coordination positions of the metal ions [5] [22] . It should be noted that the
II
II
{
Ni (µ-OAc)Ni } species with one carboxylate is present and no metal-bonded
water molecules could be detected in the ESI-MS experiment (m/z = 313.6 Da,
20

Table 1). This feature could be interpreted as a lability parameter for water
molecules and carboxylate anions bonded to the metal centers. Deprotonation of a
metal-bonded water molecule generates the µ-OH bridge according to pKa2 = 6.21,
II
II
yielding the species {(H
2
O)Ni (µ-OH)Ni (OH
2
)} (structure C) with a maximum
at pH 7.30 (85.3%). This pK
a
value is in full agreement with similar dinuclear
nickel(II) complexes described in the literature with compatible chemical
environments [13] [14] . Once again, through the ESI-MS experiment, the µ-OH
bridge could be found in solution at m/z = 672.3 Da, corresponding to the moiety
II
2
+
OH)] . In the last step of the equilibrium
[
Ni
(L)(µ-OH)(CH
3
CN)(CH
3
CH
2
II
process, pKa3 (8.34) is assigned to the deprotonation of the Ni -bound aqua ligand
to yield structure D (Chart 2), which is predominant at pH values higher than 11.0
and in good agreement with published data on several complexes containing the
2
Ni-OH moiety [5] [13] [15] [22] [37] ]. Indeed D is assigned as being the
catalytically active species in the hydrolysis of diester bonds (vide infra).
<
< Please add Chart 2 here, as a double-column picture >>
3 2
Chart 2. Proposed equilibrium of chemical species in CH CN/H O (50% v/v), for
complex 1.
21

3.6. Phosphatase-like activity
The catalytic activity of complex 1 was evaluated in the hydrolysis
reaction of the activated substrate 2,4-bdnpp [30] and monitored
-1
-1
-
spectrophotometrically at 400 nm (ε = 12,100 M cm of the 2,4-DNP anion) at
5°C, as described elsewhere [5] [22] The hydrolysis reaction was investigated in
the pH range 4.00-10.5 and, as shown in Figure 5, the initial rates (v ) versus pH
plot for complex 1 has a sigmoidal shape. This curve was fitted by the Boltzmann
model and a kinetic pK of 8.10 was obtained. This value is in good agreement
2
0
a
with the pKa3 obtained from the potentiometric titrations (8.34) and with values
reported for other dinuclear nickel(II) complexes in the literature (Table 3).
<
< Please add Table 3 here, as a doubled-column picture >>
22

III/II
Table 3. pK
a
values for the generation of selected species: {(H
2
O)M (µ-OH)Ni(OH )} (µ-
2
III/II
OH exogenous bridge) and {(H O)M (µ-OH)Ni(OH)} (terminal nucleophile) of complex 1
2
and other nickel(II) complexes described in the literature.
[13]
[37]
B
[14]
[14]
1
A
C
D
Species generated
pK
a
potentiometric/kinetic
- / - 5.98/ -
III/II
{
(H
2
O)M (µ-OH)Ni(OH
2
)}
6.21/ -
6.19/ -
6.65/ -
III/II
O)M (µ-OH)Ni(OH)} 8.34/8.10
{(H
2
9.75/ -
8.61/ 8.30 9.70/9.20 8.20/7.80
II
III
II
II
2
A = [Ni
CH COO)
2
(HBPPAMFF)(µ-OBz)
2
(OH
2
)]BPh
4
; B = [Fe Ni (bpbpmp)(µ-OAc)
2
]ClO
4
; C = [Ni
(L1)(µ-
II
3
(OH )]ClO
2 2
4
; D = [Ni
2
(L2)(µ-CH
3
COO) (CH CN)]BPh
2 3 4
a
The agreement between the kinetic and the potentiometric pK values
reinforces the proposal that deprotonation of a metal-coordinated water molecule
II
is necessary to generate the catalytically active aqua-hydroxo {(H
2
O)Ni (µ-
II
OH)Ni (OH)} species (vide Chart 2). Data obtained from these experiments also
revealed that v
0
is highly influenced by alkaline pH, since for an increase in the
pH from 4.0 to 8.0 the reaction rate increases by a factor of 135.
<
< Please add Figure 5 here, as a single-column picture >>
23

-
7
7
8
8
8
8
1
1
8
6
4
2
.2x10
-
.0x10
-
.0x10
-
.0x10
-
.0x10
-
.0x10
0.0
3
4
5
6
7
8
9
10
11
12
pH
0
Figure 5. Dependence of the reaction rates (v ) on the pH for 1 under the
-
CN 50% solution, [complex] = 2.0×10 M, [2,4-
5
following conditions: H
2
O/CH
3
-
3
-2
BDNPP] = 2.5×10 M, [buffer] = 5.0×10 M (MES, HEPES, CHES, CAPS); I =
4
0.1 M (LiClO ).
The effect of the diester 2,4-bdnpp concentration on the hydrolysis rate
promoted by complex 1 was also investigated, and the experiments were carried
out at pH 9.00 and 25 °C (Figure 6). As can be observed, the dependence of the
0
initial reaction rate (v ) on the 2,4-bdnpp concentration plots shows a linearity at
low substrate concentrations and a saturation curve is obtained as the
concentration increases, which suggest the formation of an intermediate complex-
substrate. In view of this behavior, the data were treated using the Michaelis–
Menten model and the kinetic parameters were obtained from a nonlinear least-
squares fit. The complete kinetic data for 1 and similar compounds described in
the literature by Piovezan [13] and Greatti [14] are listed in Table 4.
<
< Please add Figure 6 here, as a single-column picture >>
24

-7
-8
-8
-8
-8
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
0.0
-
3
-3
1.50x10 3.00x10 4.50x10 6.00x10 7.50x10
-3
-3
-3
0
.00
-
1
[
2,4-bdnpp] (mol L )
Figure 6. Dependence of the initial reaction rate (v
0
) on the 2,4-bdnpp
concentration for the hydrolysis reaction promoted by complex 1. Conditions:
-
5
-1
-3
complex] = 1.24×10 mol L ; [buffer] = 5.0×10 mol L (CHES, pH = 9.00); I
-1
[
-
2
-1
=
4 2 3
5.0×10 mol L (LiClO ) in H O/CH CN (50% v/v) at 25 °C.
<
< Please add Table 4 here, as a doubled-column picture >>
Table 4. Kinetic parameters for the 2,4-bdnpp hydrolysis promoted by 1 and other correlated dinickel(II)
o
complexes described in the literature at pH 9.00 and 25 C.
-
1
-1
-1
(mol L )
-1
-1
-1 -1
assoc (L mol ) E (L mol s )
Complex
v
max (mol L s
K
M
k
cat (s )
K
f
-
7
-3
-2
4
4
4
4
1
1.33×10
5.37×10
0.58×10
32.8×10
3.44×10
1.57×10
1.19×10
5.67×10
1.26×10
5.37×10
3.43×10
291
637
840
176
3.70
34.2
28.8
68.1
3.25×10
13.8×10
8.83×10
99.5×10
[13]
[14]
[14]
II
-7
-7
-7
-3
-3
-3
-2
-2
-2
A
C
D
38.6×10
III
II
II
2
A = [Ni
D = [Ni
2
(HBPPAMFF)(µ-OBz)
2
(OH
2
)]BPh
4
; B = [Fe Ni (bpbpmp)(µ-OAc)
. Kassoc = 1/K
2
]ClO ; C = [Ni
4
(L1)(µ-CH
3 2 2 4
COO) (OH )]ClO ;
II
2
(L2)(µ-CH
3
COO) (CH
2
3
-
CN)]BPh
4
o
M
(association constant); E = kcat/K
M
(catalytic efficiency); f = kcat/knc
7
-1
catalytic factor), where 3.88×10 s at 25 C (uncatalyzed reaction constant) [30] .
(
25

The kcat value calculated for 1 lies in the range of values found for
complexes A, C and D previously reported for the same substrate [13] [14]
-1
-1
However, 1 is 18 times less efficient (E/L mol s ) than compound D, where the
dinucleating ligand has two imidazolyl metal-donor groups attached. It should be
noted that imidazole groups are present in proteins/enzymes in biological media.
When these biomimetic groups are replaced with pyridines (dinucleating ligands
for compounds A and C) the catalytic efficiency for both is approximately half
that of D. Finally, during the synthesis of the ligand HL in this study two
pyridines were substituted with the triazacyclonone macrocyclic ring, which
explains the decrease in the E value, suggesting an influence from the ligand
electron density imposed on the nickel centers by the N-donor aromatic rings.
In order to establish whether the nucleophilic attack on the phosphorus
atom was via the terminal hydroxide ion or a general base catalysis, the isotopic
effect was evaluated in the hydrolysis of 2,4-bdnpp in D
. According to Burstyn and co-workers [38] , when 0.80 < (k
indicates that there is no proton transfer involved in the reaction limiting step, and
suggests an intramolecular nucleophilic attack mechanism [39] . The k /k ratio
2
O catalyzed by complex
1
H
/k ) < 1.50 this
D
H
D
obtained for 1 was close to 0.95, which corroborates the presence of a hydrolysis
reaction proceeding through an intramolecular mechanism, in which the
phosphorus atom undergoes a nucleophilic attack promoted by the coordinated
hydroxide ion.
Conclusions and Outlook. – We synthesized a novel unsymmetrical dinucleating
ligand containing a pyridine and tacn-derivative pendant arms via the route herein
26

described. This ligand seems to be suitable for obtaining the di-nickel(II) complex
1), as indicated by its physical and chemical properties observed through
(
elemental analysis, molar electrical conductivity, IR and UV-visible spectroscopy,
ESI-Mass spectrometry and electrochemistry. Potentiometric titrations confirmed
II
the acid/base properties of 1 in acetonitrile/water solution. The {(HO)Ni (µ-
II
OH)Ni (OH
2
)} core is compatible with a catalytically-active species in the
biomimetics previously reported in the literature. Complex 1 was able to catalyze
the hydrolysis of the model substrate 2,4-bdnpp and, when compared with other
similar complexes described in the literature, a trend could be noted, i.e., higher
catalytic efficiency is observed for compounds which have aromatic N-donor
moieties, such as imidazole and pyridine groups. Further studies with 1 on the
catalytic cleavage of nucleic acids (DNA) to generate new potential drugs for
technological applications, e.g., as anticancer agents, will be the subject of future
reports.
Acknowledgement
The authors are grateful to CNPq, CAPES/Stint, INCT-Catálise, and
FAPESC for financial support.
REFERENCES
th
1] D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry. 6 ed.
[
[
(
2012), New York: W. H. Freeman & Co, 1100 pp.
2] G.K. Schroeder, C. Lad, P. Wyman, N.H. Williams, R. Wolfenden, Proc.
Natl. Acad. Sci. U.S.A. 103 (2006) 4052.
27

[
[
[
3] M.A. Zenkova, N.G. Beloglazova, in: M.A. Zenkova (Ed.), Nucleic Acids
and Molecular Biology: Artificial Nucleases, vol. 13, Springer, Berlin, 2004.
4] D.Desbouis, I.P. Troitsky, M.J. Belousoff, L. Spiccia, B. Graham, Coord.
Chem. Rev. 256 (2012) 897.
5] N.Mitic, S.J. Smith, A. Neves, L.W. Guddat, L.R. Gahan, G. Schenck, Chem.
Rev. 106 (2006) 3338.
[
[
6] A.L. Gavrilova, B. Bosnich, Chem. Rev. 104 (2004) 349.
7] B.de Souza, R. Heying, A.J. Bortoluzzi, J.B. Domingos, A. Neves, J. Mol.
Cat. A:Chem. 397 (2015) 76.
[
8] V.R. Almeida, F.R. Xavier, R.E.H.M.B. Osório, L.M. Bessa, E.L. Schilling,
T.G. Costa, T. Bortolotto, A. Cavalett, F.A.V. Castro, F. Vilhena, O.C. Alves,
H. Terenzi, E.C.A. Eleuthério, M.D. Pereira, W. Haase, Z. Tomkowicz, B.
Szpoganicz, A.J. Bortoluzzi, A. Neves, Dalton Trans. 42 (2013) 7059.
9] R.E.H.M.B. Osório, R.A. Peralta, A.J. Bortoluzzi, V.R. de Almeida, B.
Szpoganicz, F.L. Fischer, H. Terenzi, A.S. Mangrich, K.M. Mantovani,
D.E.C. Ferreira, W.R. Rocha, W. Haase, Z. Tomkowicz, A. dos Anjos, A.
Neves, Inorg. Chem. 51 (2012) 1569.
[
[
[
10] N.A. Rey, A. Neves, A.J. Bortoluzzi, W. Haase, Z. Tomkowicz, Dalton
Trans. 41 (2012) 7196.
11] R.A. Peralta, A.J. Bortoluzzi, B. Szpogznicz, T.A.S. Brandão, E.E.
Castellano, M.B. de Oliveira, P.C. Severino, H. Terenzi, A. Neves, J. Phys.
Org. Chem. 23 (2010) 1000.
28

[
[
[
12] N.A. Rey, A. Neves, P.P. Silva, F.C.S. Paula, J.N. Silveira, F.V. Botelho,
L.Q. Vieira, C.T. Pich, H. Terenzi, E.C. Pereira-Maia, J. Inorg. Biochem. 103
(
2009) 1323.
13] C. Piovezan, J.M.R. Silva, A. Neves, A.J. Bortoluzzi, W. Haase, Z.
Tomkowicz, E.E. Castellano, T.C.S. Hough, L.M. Rossi, Inorg. Chem. 51
(
2012) 6104.
14] A. Greatti, M. Scarpellini, R.A. Peralta, A. Casellato, A.J. Bortoluzzi, F.R.
Xavier, R. Jovito, M.A. de Brito, B. Szpoganicz, Z. Tomkowicz, M. Rams,
W. Haase, A. Neves, Inorg. Chem. 47 (2008) 1107.
[
[
15] A. Greatti, M.A. de Brito, A.J. Bortoluzzi, A.S. Ceccato, J. Mol. Struct.
6
88 (2004) 185.
16] T.P. Camargo, F.F. Maia, C. Chaves, B. de Souza, A.J. Bortoluzzi, N.
Castilho, T. Bortolotto, H. Terenzi, E.E. Castellano, W. Haase, Z.
Tomkowicz , R.A. Peralta, A. Neves, J. Inorg. Biochem. 146 (2015), 77.
17] S.J. Smith, R.A. Peralta, R. Jovito, A. Horn, Jr., A.J. Bortoluzzi, C.J.
Noble, G.R. Hanson, R. Stranger, V. Jayaratne, G. Cavigliasso, L.R. Gahan,
G. Schenk, O.R. Nascimento, A. Cavalett, T. Bortolotto, G. Razzera, H.
Terenzi, A. Neves, M.J. Riley, Inorg. Chem. 51 (2012) 2065.
[
[
[
18] A. Neves, A.J. Bortoluzzi, R. Jovito, R.A. Peralta, B. de Souza, B.
Szpoganicz, A.C. Joussef, H. Terenzi, P.C. Severino, F.L. Fischer, G. Schenk,
M.J. Riley, S.J. Smith, L.R. Gahan, J. Braz. Chem. Soc. 21 (2010) 1201.
19] A.C. Franzoi, R.A. Peralta, A. Neves, I. Cruz Vieira, Talanta 78 (2009)
2
21.
29