Synthesis, structure, magnetism, and hydrolase and catecholase activity of a new trinuclear copper(II) complex

Article abstract of DOI:10.1016/j.ica.2015.06.023

In this paper we report the synthesis of the new multidentate N,O-donor ligand H₃L-2pyald = (N,N′-bis-(2-pyridylmethyl)-(2-hydroxy-3-carbonyl-5-methylbenzyl)-1,3-propanediamine-2-ol) and its first trinuclear copper(II) complex, [Cu₃(

Full text of DOI:10.1016/j.ica.2015.06.023

Inorganica Chimica Acta 435 (2015) 153–158
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, structure, magnetism, and hydrolase and catecholase activity
of a new trinuclear copper(II) complex
a
a,
⇑
a
a
Renata E.H.M.B. Osório , Ademir Neves , Tiago Pacheco Camargo , Sandro L. Mireski ,
a
b
c
d
Adailton J. Bortoluzzi , Eduardo E. Castellano , Wolfgang Haase , Zbigniew Tomkowicz
a
Laboratório de Bioinorgânica e Cristalografia (LABINC), Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil
Departamento de Física e Informática, Universidade de São Paulo, 13566-590 São Carlos, SP, Brazil
Institut für Physikalishe Chemie, Technische Universität Darmstadt, Petersenstraße 20, D-64287 Darmstadt, Germany
Institute of Physics, Reymonta 4, Jagiellonian University, PL-30-059 Krakow, Poland
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper we report the synthesis of the new multidentate N,O-donor ligand H L_2pyald =
3
Received 29 April 2015
Received in revised form 25 June 2015
Accepted 25 June 2015
0
(N,N -bis-(2-pyridylmethyl)-(2-hydroxy-3-carbonyl-5-methylbenzyl)-1,3-propanediamine-2-ol) and its
3 4 2
first trinuclear copper(II) complex, [Cu (L_2pyald)(l-OAc)](ClO ) (1). The structure of 1 was determined
by X-ray crystallography and variable-temperature magnetic data in the temperature range of 4–300 K
Available online 3 July 2015
À1
indicate weak antiferromagnetism (J1,2 ffi À1.0 cm ) between the Cu(1) and Cu(2) centers bridged by the
À1
l
{
-alkoxo and
Cu(1)(
catalytic activity towards the hydrolysis of the model substrate bis(2,4-dinitrophenyl)phosphate
2,4-BDNPP) as well as catechol oxidase activity since it efficiently catalyzes the oxidation of
l
-acetate groups and weak ferromagnetic coupling (J1,3 = J2,3 ffi + 4.0 cm ) involving the
Keywords:
Trinuclear Cu complex
l-phenoxo)Cu(3)} and {Cu(2)(l-phenoxo)Cu(3)} structural units. Complex 1 shows significant
II
Hydrolase and catecholase activity
Catalytic promiscuity
(
3
,5-di-tert-butylcatechol to 3,5-di-tert-butyl-o-benzoquinone by dioxygen. A comparison of the catalytic
2
+
0
0
2 2 3
efficiency of 1 with its dinuclear parent complex [Cu (H bbppnol)(l-OAc)] (H bbppnol = N,N ,N,N -
bis[(2-hydroxybenzyl)(2-methylpyridyl)]-1,3-propanediamine-2-ol) reveals that the hydrolase efficiency
(k
cat/K
M
) is 12 times higher for 1, while the catecholase efficiencies of these two compounds are similar.
Ó 2015 Elsevier B.V. All rights reserved.
1
. Introduction
Multimetallic complexes are of great interest due to the special
On the other hand, the ability of a given natural or non-natural
active site to catalyze more than one chemical transformation (cat-
alytic promiscuity) constitutes a very important property of many
enzymes, playing a natural role in evolution and, occasionally, in
the biosynthesis of secondary metabolites [5]. While several
enzymes displaying catalytic promiscuity have been the subject
of recently reported investigations [6], there are few examples of
synthetic analogues which exhibit such multifunctional activities
[7–11].
chemical and physical properties that result from the mutual inter-
action of two or more metal centers [1].
Trinuclear copper clusters with trigonal symmetry play a cen-
tral role in the fundamental steps of biological catalysis by ubiqui-
tous multicopper oxidases. The study of model complexes of these
systems should not only provide a better understanding of the bio-
logical molecules but also assist in the development of new low-
molecular-weight catalysts [2]. In fact, trinuclear copper(II) com-
plexes with trigonal symmetry have attracted attention as mag-
netic materials and systems mimicking trinuclear metalloenzyme
sites present in copper oxidases, such as ascorbate oxidase, ceru-
plasmin and laccase [3]. One of the forms of the copper oxidases
corresponds to an antiferromagnetically coupled trinuclear Cu(II)
cluster [4].
In this paper we present a trinuclear copper(II) complex
[Cu
3
(L_2pyald)(
L_2pyald (Chart 1) which is able to stabilize a triangular
3
structure containing Cu(II) centers with distinct coordination
4 2
l-OAc)](ClO ) – 1 with the novel symmetrical
ligand H
3
II
Cu
environments. It was found that complex 1 shows significant cat-
alytic activity toward the hydrolysis of the activated substrate
bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) as well as in the oxi-
dation of 3,5-di-tert-butylcatechol (3,5-DTBC), thus indicating cat-
alytic promiscuity.
⇑
Corresponding author. Tel.: +55 48 37216849; fax. +55 48 37216850.
E-mail address: ademir.neves@ufsc.br (A. Neves).
http://dx.doi.org/10.1016/j.ica.2015.06.023
020-1693/Ó 2015 Elsevier B.V. All rights reserved.
0

1
54
R.E.H.M.B. Osório et al. / Inorganica Chimica Acta 435 (2015) 153–158
criteria. H atoms of the coordinated water molecule were not
found from the Fourier difference map.
N
N
The hydrolase and catecholase-like activities of 1 were deter-
mined by measuring the hydrolysis reaction of the model substrate
bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) and the oxidation of
the substrate 3,5-di-tert-butylcatechol (3,5-DTBC), respectively, in
a Varian Cary 50 BIO UV–Vis spectrophotometer fitted with a ther-
mostated water-jacketed cell holder. The reactions were accompa-
nied by monitoring the increase in the 2,4-dinitrophenolate
N
N
OH
OH
OH
O
O
H3L_2pyald
À1
À1
Chart 1. Ligand H
3
_pyald.
characteristic absorption band at 400 nm (pH/e L mol cm
=
3.5/2125; 4.0/3408; 4.5/7182; 5.0/10078; 5.5/11405; 6.0/
1
2004; 6.5–10.0/12100) at 50 °C for the hydrolase-like activity
[
4
18] and the formation of 3,5-di-tert-butylquinone (3,5-DTBQ) at
À1
À1
00 nm (e = 1900 M cm ) for the catecholase-like activity at
2
. Experimental
2
5 °C. Less than 5% of the conversion of the substrate to the pro-
duct was monitored and the data were treated using the initial rate
method as previously described [9].
The materials 2,4-BDNPP = bis(2,4-dinitrophenyl)phosphate
12] and CMFF = 2-chloromethyl-4-methyl-6-formylphenol [13]
[
0
Synthesis of N,N -(2-pyridylmethyl)-1,3-propanediamine-2-ol
were synthesized as previously described. Other reagents, materi-
als, gases and solvents of analytical or spectroscopic grade were
purchased from commercial sources and used without purification.
Elemental analysis (CHN) was performed on a Carlo Erba
E-1110 analyzer. Electronic absorption spectra in the 300–
(
2py): 4 mL of 2-pyridinecarboxaldehyde (40 mmol) in methanol
was added slowly under continuous agitation, in an ice bath, to a
solution containing 2.0 g (20 mmol) of 2-hydroxy-1,3-propanedi-
amine in 30 mL of methanol. After 2 h of reaction the Schiff base
was reduced overnight by catalytic hydrogenation using Pd/C
1
000 nm range were recorded on a UV–Vis Varian Cary 50 BIO
(
5%). The catalyst was filtered and the solvent removed under
1
spectrophotometer. H NMR analysis was carried out using a
Bruker 200 MHz spectrometer with CDCl as the solvent, at room
reduced pressure giving a yellow oil. Yield: 5.35 g (88%). The com-
pound was obtained in high purity as confirmed by 1H NMR
3
temperature. Chemical shifts were referenced to tetramethylsilane.
Electrochemical measurements were carried out using a Bas
Epsilon potentiostat/galvanostat. Cyclic voltammograms were
obtained for the complex in acetonitrile solution containing
(
(
3
CDCl ), d (ppm): 2.4–2.7 (4H); 3.3 (1H); 3.7–3.9 (4H); 6.9–7.1
2H); 7.2–7.3 (2H); 7.4–7.6 (2H); 8.4 (2H).
0
Ligand
H
3
L_2pyald = (N,N -bis-(2-pyridylmethyl)-(2-hydroxy-
3
-carbonyl-5-methylbenzyl)-1,3-propanediamine-2-ol):
25 mL round-bottom flask, 2.71 g (9.95 mmol) of 2py was dis-
Cl . To this solution, 30 mL of a dichloro-
methane solution of 2-chloromethyl-4-methyl-6-formylphenol
3.68 g; 19.9 mmol) was added dropwise. The resulting mixture
was allowed to react for 12 h with stirring at room temperature.
The product was extracted with an aqueous solution of NaHCO
The organic layer was dried over Na SO , filtered
In
a
0
.1 M tetrabutylammonium hexafluorophosphate as the support-
1
ing electrolyte under an argon atmosphere. The electrochemical
cell employed was of a standard three-electrode configuration:
glassy carbon electrode (working), platinum wire (counter),
solved in 30 mL of CH
2
2
(
+
Ag/AgCl (reference). The Fc /Fc couple (E1/2 = 400 mV versus NHE)
was used as the internal standard [14].
3
Electrospray ionization mass spectrometry (ESI-MS) of 1 dis-
solved in an ultrapure acetonitrile solution (500 nM) was per-
formed using an amaZon X Ion Trap MS instrument (Bruker
Daltonics) with an ion spray source using electrospray ionization
in positive-ion mode. The ion source condition was an ion spray
voltage of 4500 V. Nitrogen was used as the nebulizing gas
(
5 Â 50 mL)
.
2
4
and the solvent evaporated under reduced pressure, to give an oily
product which was then purified by silica gel column chromatog-
raphy with dichloromethane/methanol. Yield: 3.50 g (60%). 1
H
NMR (CDCl
7
3
), d (ppm): 2.2 (6H); 2.4–2.6 (4H); 3.6–4.0 (9H);
.0–7.1 (4H); 7.2–7.3 (4H); 7.4–7.5 (2H); 8.4 (2H); 10.1 (2H). ESI-
+
(
20 psi) and curtain gas (10 psi). The samples were directly infused
MS: m/z 569.28 [M] .
Synthesis of [Cu (L_2pyald)(
methanolic solution (20 mL) of 0.28 g of the ligand H
0.5 mmol), 0.30 g of Cu(OAc) O (1.5 mmol) previously dis-
ÁH
into the mass spectrometer at a flow rate of 180 L/h. The scan
l
3
l
-OAc)](ClO
4
)
2
– 1: To a yellow
L-2pyald
range was m/z 200–3000. The simulated spectrum was calculated
using the mMass software [15,16].
3
(
2
2
Magnetic data were obtained with a SQUID magnetometer
using a slightly pressed (by hand) polycrystalline sample of com-
plex 1. Susceptibility data were measured in the temperature range
of 4–300 K with a magnetic field of 1000 Oe. Magnetization data
were obtained in the field range up to 5 T at a temperature of
solved in 10 mL of methanol was added dropwise, under stirring
and the color immediately turned to dark green. Sodium perchlo-
rate (1.5 mmol) was then added to the reaction mixture and after
cooling the solution to room temperature a green microcrystalline
precipitate was formed which was filtered off. After recrystalliza-
tion in isopropanol/water (1:1) and slow evaporation of the sol-
vents, green crystals suitable for X-ray analysis were isolated.
2
.0 K. Diamagnetic corrections were applied in the usual manner
with the use of the tabulated Pascal’s constants [17].
green crystal of complex with dimension of
.476 Â 0.42 Â 0.01 mm was selected from a crystalline sample
A
1
Yield: 50%. Anal. Calc. for C35
5
H
39Cl
2
Cu
3 4
N O16: C, 40.68; H, 3.80; N,
3
+
0
.42. Found: C, 41.66; H, 4.34; N, 5.53%. ESI-MS: m/z 912.00 [M] .
and the crystallographic analysis was carried out with a Bruker
KAPPA-CCD diffractometer at room temperature. Intensities were
corrected for Lorentz and polarization effects. Gaussian absorption
correction was also applied to all measured intensities with maxi-
mum and minimum transmission factors of 0.6092 and 0.9822,
respectively. High values of Rint and Rsigma can be attributed to
the shape and quality of the crystals. The structure was solved by
direct methods and refined using the full-matrix least-squares on
3
. Results and discussion
The ligand H L_2pyald was obtained with sufficient purity and
3
yield for use in the synthesis of the trinuclear copper(II) complex.
The reaction scheme for the preparation of the symmetrical ligand
is depicted in Scheme 1. H L_2pyald was synthesized by typical
3
procedures starting from the central skeleton 1,3-diamine-
propane-2-ol condensed with pyridinecarboxaldehyde followed
2
F method. All non-hydrogen atoms were refined with anisotropic
displacement parameters. Hydrogen atoms bonded to C atoms
were placed at their idealized positions using standard geometric

R.E.H.M.B. Osório et al. / Inorganica Chimica Acta 435 (2015) 153–158
155
CH3OH, RT, 2h
H2 Pd/C 20h
NH
N
NH
H2N
NH2
+
2
OH
O
OH
N
N
Cl
O
N
N
N
N
OH
OH
OH
2
OH
O
O
CH Cl2
H3L_2pyald
Scheme 1. Synthesis of the ligand H
3
L_2pyald.
by reduction with 5% Pd/C and alkylation with 2-chloromethyl-4-
methyl-6-formylphenol [13].
Table 1
Main bond distances (Å) and angles (°) for complex 1.
Reaction of H
monohydrate (3 equiv.), in the presence of sodium perchlorate,
affords the stable complex [Cu (L_2pyald)( -OAc)](ClO – 1.
Recrystallization of 1 from a H O/isopropyl alcohol solution
afforded crystals suitable for X-ray crystallographic analysis.
The copper complex crystalizes as single green crystals that
belong to the monoclinic crystal system, in the space group
1
P2 /c. An ORTEP view of the trinuclear cation complex is shown
3
L_2pyald in methanol with copper(II) acetate
Cu1–O3
Cu1–O1
Cu1–N12
Cu1–N11
Cu1–O31
Cu2–O3
Cu2–O2
Cu2–N22
1.864(3)
1.928(4)
1.960(4)
2.047(4)
2.565(4)
1.878(3)
1.937(3)
1.983(4)
2.049(4)
Cu2–O41
Cu3–O41
Cu3–O31
Cu3–O42
Cu3–O32
Cu1–Cu2
Cu2–Cu3
Cu1–Cu3
2.908(4)
1.886(4)
1.888(4)
1.921(4)
1.951(4)
3.3716(8)
4.0535(9)
3.7535(9)
2.485(5)
3
l
4 2
)
2
Cu2–N21
Cu2 O1W
O3–Cu1–O1
O3–Cu1–N12
O1–Cu1–N12
O3–Cu1–N11
O1–Cu1–N11
N12–Cu1–N11
O3–Cu1–O31
O1–Cu1–O31
N12–Cu1–O31
N11–Cu1–O31
O41–Cu3–O31
O41–Cu3–O42
O31–Cu3–O42
O41–Cu3–O32
O31–Cu3–O32
O42–Cu3–O32
Cu3–O41–Cu2
98.11(15)
160.88(16)
94.31(17)
85.42(14)
171.88(18)
84.21(16)
89.45(13)
87.63(17)
105.59(15)
85.09(13)
89.39(15)
93.60(17)
174.4(2)
O3–Cu2–O2
O3–Cu2–N22
O2–Cu2–N22
O3–Cu2–N21
O2–Cu2–N21
N22–Cu2–N21
O3–Cu2–O1W
O2–Cu2–O1W
N22–Cu2–O1W
N21–Cu2–O1W
O3–Cu2–O41
O2–Cu2–O41
N22–Cu2–O41
N21–Cu2–O41
O1W–Cu2–O41
Cu1–O3–Cu2
Cu3–O31–Cu1
98.84(15)
164.80(16)
94.67(17)
85.37(14)
163.14(17)
83.67(17)
84.32(15)
103.67(19)
85.76(16)
92.96(17)
80.09(12)
83.36(14)
108.52(14)
81.30(13)
163.75(13)
128.58(16)
114.04(16)
in Fig. 1. The crystallographic data and the main bond dis-
tances/angles are given in Table S1 and Table 1, respectively. The
resolution of the crystal structure revealed that 1 is composed of
3
a trinuclear core in which one symmetrical ligand H L_2pyald is
coordinated to two Cu(II) centers (Cu1 and Cu2) bridged by the
alkoxo donor O atom of the ligand and by an exogenous acetate
ion. The third Cu(II) center (Cu3) is tetra coordinated by four oxy-
gen atoms of the ligand H
The Cu1 center is pentacoordinated, showing a slightly dis-
torted square pyramidal geometry ( = 0.18), while the coordina-
3
L_2pyald.
s
tion around Cu2 is best described as a distorted octahedron with
the phenolate (O41) and water (O1w) oxygen atoms bound in
the axial positions. The equatorial plane of the two copper centers
consists of one amine and one pyridine nitrogen and one acetate
and one alkoxyde oxygen atom. A deprotonated oxygen atom
177.54(17)
93.05(17)
84.01(19)
113.7516
(
O31) of the phenol group in the axial position completes the
coordination sphere of the Cu1 center. The oxygen atoms of the
phenolate groups interact weakly with the Cu1 and Cu2 centers
since the observed Cu(II)–O distances are relatively long
sphere of the Cu3 center is composed of four oxygen atoms,
provided by two endogenous phenolates (O31 and O41) and two
aldehyde oxygen atoms of the ligand H L_2pyald (O32 and O42).
3
The O–Cu3–O bond angles close to 90° and 180° found for the
Cu3 center are in agreement with a square planar geometry.
Fig. 2 shows the magnetic data obtained for 1. They are pre-
sented in the form of the product of susceptibility multiplied by
(
Cu1–O31–2.565(4) Å and Cu2–O41–2.908(4) Å), as a result of
the Jahn–Teller effect displayed by the Cu(II) ion. The coordination
temperature (vT) plotted as a function of the temperature T and
of the magnetization M plotted as a function of the field H/T. The
former dependence was recorded in a field of 1000 Oe and the lat-
ter at temperatures of 2.0, 4.5 and 10 K. It can be observed that the
room temperature value for
compared with the predicted value for three free 1/2 spins of
.125 emu/mol K (for g = 2). It can also be seen in Fig. 2 that the
M value in the highest available field of 5 T at a temperature of
K is 2.48 , which may be compared with the value of 2.8
vT is 1.48 emu/mol K, which may be
1
2
l
B
l
B
expected for three free 1/2 spins (see the dot-dashed Brillouin
curve in Fig. 2). Magnetization curves plotted as a function of H/T
superimpose above 4.5 K, indicating simple paramagnetic behavior
above this temperature.
The lower value for the high field-low temperature magnetiza-
tion may be caused by zero field splitting due to the antisymmetric
3 4 2
Fig. 1. ORTEP view of the cation complex [Cu (L_2pyald)(l-OAc)](ClO ) – 1.

1
56
R.E.H.M.B. Osório et al. / Inorganica Chimica Acta 435 (2015) 153–158
2
2
H/T (Oe/K)
dx–y orbital (Cu1–O3 = 1.864(3) Å and Cu2–O3 = 1.878(3) Å) and
0
5000
10000 15000 20000 25000
thus the unpaired electrons can interact with the
l
-alkoxo group
3
2
1
0
À1
giving rise to weak antiferromagnetism (J1,2 ffi À1.0 cm ). On the
other hand, in 1, Cu3 lies in a slightly distorted square planar
geometry as can be observed from the bond angles around this
center and thus the unpaired electron is also in the dxÀy magnetic
orbital. However, in this case, this orbital is orthogonally oriented
1
1
0
0
.5
.0
.5
.0
2
χT exp
χT fit
2
in relation to the dxÀy orbitals of the Cu1 and Cu2 centers, leading
À1
to weak ferromagnetic coupling (J1,3 = J2,3 ffi + 4.0 cm ) between
the Cu3 and Cu1/Cu2 centers. For comparison, the weak antiferro-
M 10.0 K
M 4.5 K
M 2.0 K
fit
À1
magnetism (J = À1.0 cm ) between Cu(1) and Cu(2) falls in the
range of J values observed for many Cu(l-alkoxo)(l-Acetate)Cu
χ
complexes reported in the literature [22,23]. On the other hand,
À1
the weak ferromagnetic exchange (J1,3 = J2,3 ffi + 4.0 cm ) between
Brillouin
the Cu(3) and Cu(1)/Cu(2) centers are in agreement with the ferro-
magnetic coupling observed for open type dinuclear Cu(II) com-
0
50
100
150
200
250
300
plexes with a single
The redox properties of the complex [Cu
OAc)](ClO are given in Fig. S1 (see Supplementary Material)
and were investigated in acetonitrile by cyclic voltammetry which
revealed the presence of three irreversible waves with Fc /Fc as the
l-phenoxo bridging ligand [24].
3
(L_2pyald)(l-
T (K)
4 2
)
Fig. 2. Temperature dependence of the
and H/T dependence of magnetization M measured at 2, 4.5 and 10 K for the
complex [Cu (L_2pyald)( -OAc)](ClO – 1. Solid lines are fits. Dash-dot line is the
vT product measured in a field of 1000 Oe
+
3
l
4 2
)
internal standard. The first process at -360 mV (Epc) versus NHE is
assigned to the one-electron reduction of Cu
Brillouin function plotted for three uncoupled 1/2 spins.
II
II
I
3 2
/Cu Cu , while the
waves at À580 and À980 mV versus. NHE can be attributed to
II
I
II
I
II
I
I
and anisotropic exchange [19,20]. For the description of the mag-
netic behavior we used the Hamiltonian of the following form
the couples Cu
2 2 2 3
Cu /Cu Cu and Cu Cu /3Cu , respectively. In fact,
these values are in good agreement with the reduction potentials
observed for dinuclear copper(II) complexes containing a
bridge and phenolate/pyridine ligands [7,9,10].
l-alkoxo
3
3
X
X
~
~
H ¼ À2 ½ ~s Á Jij Á ~s
þ dij Á ð~s
Â~s
ފ þ lB ð~s
Á g Þ Á H;
j
i
j
i
i
The electronic spectrum of 1 (Fig. S2) in methanolic solution
and in the solid state exhibits transitions at max = 662 nm
i<j
i¼1
k
2
3
2
3
À1
À1
À1
À1
d
ijx
Jijx
0
0
0
J
0
0
0
Jijz
(
e
= 185 L mol cm ) and kmax = 510 nm (
e = 110 L mol cm )
where Jij
¼
ijy
and ~d ij ¼ 4
6
~d ij
7
typical of CuII d–d transitions and one band at 382 nm
4
5
y
À1
À1
~
(e
= 4934 L mol cm ), which can be attributed to
a
d
ij
z
ligand-to-metal charge transfer between the phenolates and cop-
per centers, in agreement with the X-ray structure characteriza-
tion. In acetonitrile solution, an additional shoulder was observed
at 330 nm, possibly associated with an intraligand transition
involving phenolate/pyridine ligands. In fact, these results together
In the above equations J ; Jijy ; J are the components of the iso-
ij
x
ij
z
tropic/anisotropic exchange when all these components are
equal/not equal, respectively. dijx ; dijy ; dijz are components of the
antisymmetric exchange.
To obtain the exchange parameters, the simultaneous fitting of
the values for the
formed using the PHI software [21]. The trials were initially carried
out with the lowest possibly number of parameters, i.e., assuming
that only the J12 interaction differs from zero, which was based on
the relatively long distances of Cu2–O31 (2.908 Å) and Cu1–O41
2.565 Å) in comparison with Cu1–O3 (1.877 Å) or Cu2–O3
1.864 Å). The decrease in the M values observed in high fields at
K was not reproduced.
In the second stage, the J23 interaction was taken into account.
To obtain a good fit it was necessary to assume the presence of ani-
sotropy for both the J12 and J23 interactions and antisymmetric
exchange for at least the J23 interaction. However, due to the large
number of parameters no unambiguous solution was obtained,
even though we used all three sets of magnetization data (2, 4.5
and 10 K) during each fitting. We could only confirm, in agreement
with expectations, that the average value for J12 is negative, of the
with the ESI-MS spectrum (Fig. S3), which shows a peak at
v
T product and the magnetization data was per-
+
m/z = 912.00 assigned to the [Cu
3
(L_2pyald)(l-OAc)(ClO
4
)] spe-
cies, suggest that the coordination of the ligand L_2pyald around
II
the Cu centers is maintained when the complex is dissolved under
these experimental conditions.
Kinetic experiments were carried out to assess the ability of
complex 1 to hydrolyze the substrate bis(2,4-dinitrophenyl)phos-
phate (2,4-BDNPP). In order to determine the optimum pH, the
(
(
2
dependence of the reaction rate was evaluated in CH
1:1) within the pH range of 4.00–10.00. As can be seen in
Fig. S4, the optimum pH is around 8.0 and sigmoidal fitting of
the initial rate (V ) versus pH curve in the pH range of 4.0–8.0
reveals a kinetic pK
value of ꢀ6.9, which can be attributed to
3 2
CN:H O
(
0
a
II
the deprotonation of a Cu -bound water molecule to generate
the initiating nucleophile [9,25]. It is important to note that under
these experimental conditions dissociation of the acetate bridge
with replacement by two water molecules is expected, as previ-
ously reported in the literature [9]. Thus, the small decrease in
reactivity at pH > 8.0 (see Fig. S4) most probably originates from
À1
order of À1 cm , and the average value for J23 is positive (ferro-
À1
magnetic coupling), of the order of 4 cm . The exact values for
the parameters from one exemplary fit are ðJ ; J12y ; J12z Þ = (À3.7,
II
12
x
the formation of a further Cu -OH group in which the leaving ten-
À1
À1
À3.7, 3.4) cm
;
ðdijx ; dijy ; dijz Þ = (À1.5, À1.5, 2.8) cm
;
-
dency of the OH ion is lower, even though a more concentrated
À1
-
ðJ ; J23y ; J23z Þ = (2.8, 0.2, 8.5) cm ; ðd23x ; d23y ; d23z Þ fixed at (0, 0,
2
3
x
OH solution can increase the spontaneous hydrolysis [13].
0
); g
1
= g
2
= 2.24, g
3
= 2.16.
The dependence of the reaction rate on the concentration of the
diester 2,4-BDNPP by complex 1 was investigated at pH 8.0, reveal-
ing saturation behavior with a higher concentration of the sub-
strate (Fig. 3). The data were treated using the Michaelis–Menten
From the X-ray structure of 1 it can be concluded that the
geometries around Cu(1) and Cu(2) are, respectively, distorted
square pyramidal and octahedral with significant tetragonal elon-
gation toward the Cu1–O31 and Cu2–O41 bonds. Therefore, in both
copper centers, the dominant magnetic orbital appears to be the
À8
À1 À1
model and the parameters
V
max = 2.54 Â 10 mol L
s ,
À3
À1
À4 À1
K
M
= 2.61 Â 10 mol L and kcat = 9.76 Â 10
s
were obtained

R.E.H.M.B. Osório et al. / Inorganica Chimica Acta 435 (2015) 153–158
157
-8
-8
-8
-8
-9
-9
-9
-9
1.6x10
1.4x10
1.2x10
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
-7
1
.2x10
-7
1
.0x10
-8
8.0x10
-8
6
4
2
.0x10
-8
.0x10
-8
.0x10
0.000
0.001
0.002
0.003
0.004
0.005
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
-1
[
BDNPP] (mol.L )
-1
[3,5-DTBC] (mol.L )
Fig. 3. Dependence of the initial rate on the 2,4-BDNPP concentration for the
Fig. 4. Dependence of the reaction rates on the 3,5-DTBC concentration for the
hydrolysis reaction catalyzed by complex 1. Conditions: solution 1:1 CH
3
CN/H
2
O;
À5
À1
À1
oxidation reaction catalyzed by complex
1
in
a
methanol/water solution.
[
complex] = 2.6 Â 10 mol L
;
[buffer] = 0.05 mol L
;
HEPES
(pH 8.0);
À5
À1
À2
À1
À1
À3
À1
Conditions: [complex] = 1.2 Â 10 mol L
;
[buffer] = 3.0 Â 10 mol L
; HEPES
I = 0.05 mol L LiClO
4
; [2,4-BDNPP] = (0.5–8.0) Â 10 mol L at 50 °C.
À4
À1
(
pH 9.0); [3,5-DTBC] = (1.5–12.0) Â 10 mol L at 25 °C.
Michaelis–Menten approach it is possible to characterize the
from a nonlinear least-squares fit. A comparison of these kinetic
parameters with those previously reported for the dinuclear
À1
kinetic behavior using the parameters:
k
s
cat = 0.0223 s
,
À3
À1
À1 À1
K
M
= 1.51 Â 10 mol L
and kcat/K
M
= 14.86 M
. A compar-
[
2 2 4 2 2
Cu (H bbppnol)(l-OAc)](ClO ) complex (H bbppnol corresponds
2
+
2 2
ison with the [Cu (H bbppnol)(l-OAc)] complex [27] reveals that
to the L_2pyald ligand without the carbonyl function) reveals that
À1 À1
the catalytic constant kcat is approximately 3 times higher for 1
1
is about 12 times more efficient (E = kcat/K
M
= 0.37 L mol
s )
while Kass ffi 1/K
M
is almost double for the dinuclear complex,
resulting in similar catalytic efficiencies (kcat/K ) for the two
M
than the dinuclear complex [26]. Indeed the higher catalytic effi-
ciency of 1 is a direct consequence of the catalytic constant kcat
À5 À1
systems.
which is approximately 30 times the value of 3.4 Â 10
s
Finally, accumulation of H
by means of the molybdate-accelerated I assay, which indicates
that reoxidation of the copper(I) species back to the active
2
O
2
during turnover was confirmed
observed for the dinuclear complex. This is most probably due to
the higher nucleophilic reactivity of the Cu -bound OH group in
(pK = 6.9 in 1 versus 5.7 in [Cu (H bbppnol)(l-OAc)] ). In addi-
a 2 2
À3
II
À
2+
1
copper(II) species occurs with a 1:1 (O
and the concomitant formation of hydrogen peroxide. Thus, it
can be concluded that 1 and [Cu (H bbppnol)(
-OAc)]2+ most
2
:3,5-DTBC) stoichiometry
tion, this kcat corresponds to an acceleration of ꢀ4000 times with
respect to the uncatalyzed reaction [12].
2
2
l
In order to assess the possible hydrolysis of the monoester
probably catalyze the oxidation of 3,5-DTBC through a similar
mechanism [27]. In brief, bidentate binding of the diphenol sub-
strate is followed by an intramolecular electron-transfer reaction
with concomitant release of the o-quinone as the rate-determining
2
,4-dinitrophenylphosphate (2,4-DNPP), one of the products
formed from the hydrolysis of the diester 2,4-BDNPP, the stoichio-
metric reaction between complex 1 and the substrate 2,4-BDNPP
was monitored. It was observed that over 3 days at 50 °C, only
I
I
step. In the presence of dioxygen, the Cu Cu center is immediately
1
.2 equiv. of 2,4-dinitrophenolate is released, which indicates only
oxidized back to the original active site with the formation of H
completing the catalytic cycle.
2 2
O ,
diesterase activity for [Cu3(L_2pyald)( -OAc)](ClO ) .
l
4 2
À3
Furthermore, a hydrolysis reaction of 2,4-BDNPP (2.0 Â 10
À1
In summary, we synthesized and fully characterized the new
mol L ) promoted by complex 1 at 445 nm, pH 8.0 and 50 °C
revealed that over 24 h the complex was able to hydrolyze 3 mole-
cules of substrate, indicating that the active species is regenerated.
II
trinuclear Cu complex [Cu
has a dinuclear Cu(1)( -alkoxo)Cu(2) core similar to that observed
for its dinuclear parent complex [Cu (H bbppnol)(
3 4 2
(L_2pyald)(l-OAc)](ClO ) (1) which
l
l-
H D
The measured deuterium kinetic isotope effect k /k of 1.40 sug-
2
2
2
+
OAc)] [23,25]. The complex displays interesting magnetic proper-
gests that no proton transfer is involved in the rate-limiting step
and thus supports an intramolecular nucleophilic attack of a termi-
nal Cu-bound hydroxide on the phosphodiester which is monoden-
ties which reveal antiferromagnetism between the Cu(1) and Cu(2)
centers within the Cu(1)(l-alkoxo)Cu(2) unit and ferromagnetism
between the Cu(1)Cu(3) and Cu(2)Cu(3) centers, in good agree-
ment with the X-ray structural data. Finally, 1 shows significant
catecholase activity and is also able to cleave the phosphate diester
bis(2,4-dinitrophenyl)phosphate with a catalytic activity (kcat) 30
II
tate bonded to the adjacent Cu center in 1 [13,18].
The catecholase-like activity of the trinuclear copper(II) com-
plex was determined by the catalytic oxidation of 3,5 di-tert-butyl-
catechol (3,5-DTBC). The pH dependence of the catalytic activity
between pH 5.50 and 9.00 shows a sigmoidal-shaped profile
times higher than that of the dinuclear [Cu
2
(H
2
bbppnol)(l-
2
+
OAc)] complex, due to the higher nucleophilic reactivity of the
(
Fig. S5) and reveals an optimum value of pH 9.0. This value is
II
À
Cu -bound OH group in 1.
ꢀ
1.0 pH unit higher than that found for the dinuclear
+
[
Cu
2
(H
2
bbppnol)(
l
-OAc)]2 complex [26], again in agreement with
II
the lower Lewis acidity of the Cu centers within the Cu(1)(
l
-
alkoxo)Cu(2) unit in 1. To take into account the auto-oxidation of
the substrate, the same solution without the catalyst was used as
an internal reference. Saturation kinetics were obtained at the pH
value (9.0) where complex 1 showed greatest activity in the 3,5-
Acknowledgements
The authors are grateful for grants awarded to support this
research from CNPq, FINEP, CAPES-STINT and INCT-Catálise
(Brazil).
0
DTBC oxidation. The graph of initial rates (V ) versus. 3,5-DTBC
concentrations shows a saturation profile (Fig. 4). Applying the

1
58
R.E.H.M.B. Osório et al. / Inorganica Chimica Acta 435 (2015) 153–158
[
[
11] M.C.B. Oliveira, D. Mazera, M. Scarpellini, P.C. Severino, A. Neves, H. Terenzi,
Inorg. Chem. 48 (2009) 2711.
12] C.A. Bunton, S.J. Farber, J. Org. Chem. 34 (1969) 767.
Appendix A. Supplementary material
Crystallographic data for the structure reported in this paper
[13] P. Karsten, A. Neves, A.J. Bortoluzzi, M. Lanznaster, V. Drago, Inorg. Chem. 41
(2002) 4624.
[
[
(
complex 1) have been deposited with the Cambridge
14] R.R. Gagne, C.A. Koval, G.C. Lisensky, Inorg. Chem. 19 (1980) 2854.
15] M. Strohalm, M. Hassman, B. Košata, M. Kodí cˇ ek, Rapid Commun. Mass
Spectrom. 22 (2008) 905.
Crystallographic Data Centre as supplementary publication no.
CCDC 1049454. Copies of the data can be obtained free of charge
from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; tel:
[16] M. Strohalm, D. Kavan, P. Novák, M. Voln y´ , Anal. Chem. 82 (2010) 4648.
[
[
17] C.J. O’Connor, Prog. Inorg. Chem. 29 (1982) 203.
(
+44) 1223-336-408; fax: (+44) 1223-336-003; e-mail: deposit
18] R.A. Peralta, A.J. Bortoluzzi, B. de Souza, R. Jovito, F.R. Xavier, R.A.A. Couto, A.
Casellato, F. Nome, A. Dick, L.R. Gahan, G. Schenk, G.R. Hanson, F.C.S. de Paula,
E.C. Pereira-Maia, S.P. Machado, P.C. Severino, C. Pich, T. Bortolotto, H. Terenzi,
E.E. Castellano, A. Neves, M.J. Riley, Inorg. Chem. 49 (2010) 11421.
19] J. Yoon, L.M. Mirica, T.D.P. Stack, E. Solomon, J. Am. Chem. Soc. 126 (2004)
@
ccdc.cam.ac.uk; website www.ccdc.cam.ac.uk). Supplementary
data associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.ica.2015.06.023.
[
[
[
[
[
12586.
20] S. Ferrer, F. Lloret, E. Pardo, J.M. Clemente-Juan, M. Liu-Gonzáles, S. García-
Granda, Inorg. Chem. 51 (2012) 985.
21] N.F. Chilton, R.P. Anderson, L.D. Turner, A. Soncini, K.S. Murray, J. Comput.
Chem. 34 (2013) 1164.
References
[
[
1] S.L. Ma, W.X. Zhu, S. Gao, Q.L. Guo, M.Q. Xu, Eur. J. Inorg. Chem. (2004) 1311.
2] P. Mateus, R. Delgado, F. Lloret, J. Cano, P. Brandão, V. Félix, Chem. Eur. J. 17
22] C.H. Weng, S.C. Cheng, H.M. Wei, H.H. Wei, C.J. Lee, Inorg. Chim. Acta 359
(
2011) 11193.
(
2006) 2029.
[
[
3] A. Messerschmidt, R. Huber, Eur. J. Biochem. 187 (1990) 341.
4] M.J. Kobylka, J. Janczak, T. Lis, T. Kowalik-Jankowska, J. Klak, M. Pietruska, J.
Lisowski, Dalton Trans. 41 (2012) 1503.
23] A. Neves, L.M. Rossi, I. Vencato, V. Drago, W. Haase, R. Werner, Inorg. Chim.
Acta 281 (1998) 111.
24] R.C. Holz, J.M. Brink, F.T. Gobena, C.J. O’Connor, Inorg. Chem. 33 (1994) 6086.
25] R.A. Peralta, A. Neves, A.J. Bortoluzzi, A. dos Anjos, F.R. Xavier, B. Szpoganicz, H.
Terenzi, M.C.B. Oliveira, E.E. Castellano, G.R. Friedermann, A.S. Mangrich, M.A.
Novak, J. Inorg. Biochem. 100 (2006) 992.
[
[
[
[
[
5] P.J. O’Brien, Chem. Rev. 106 (2006) 720.
6] R.J. Kazlauskas, Curr. Opin. Chem. Biol. 2 (2005) 195.
7] N.A. Rey, A. Neves, A.J. Bortoluzzi, C.T. Pich, H. Terenzi, Inorg. Chem. 46 (2007)
3
48.
[
[
26] L.M. Rossi, A. Neves, A.J. Bortoluzzi, R. Hörner, B. Szpoganicz, H. Terenzi, A.S.
Mangrich, E.C. Pereira-Maia, E.E. Castellano, W. Haase, Inorg. Chim. Acta 358
[
[
8] K.M. Deck, T.A. Tseng, J.N. Burstyn, Inorg. Chem. 41 (2002) 669.
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)
(
2005) 1807.
27] A. Neves, L.M. Rossi, A.J. Bortoluzzi, B. Szpoganicz, C. Wiezbicki, E. Schwingel,
W. Haase, S. Ostrovsky, Inorg. Chem. 41 (2002) 1788.
1
569.
[
10] A. Neves, A.J. Bortoluzzi, R. Jovito, R.A. Peralta, B. de Souza, B. Szpoganicz, A.C.
Youssef, 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.