Synthesis, magnetostructural correlation, and catalytic promiscuity of unsymmetric dinuclear copper(II) complexes: Models for catechol oxidases and hydrolases

Article

pubs.acs.org/IC

Synthesis, Magnetostructural Correlation, and Catalytic Promiscuity

of Unsymmetric Dinuclear Copper(II) Complexes: Models for

Catechol Oxidases and Hydrolases

†

Renata E. H. M. B. Osor

́

io, Rosely A. Peralta, Adailton J. Bortoluzzi, Vicente R. de Almeida,

‡

§

,§

⊥

Bruno Szpoganicz, Franciele L. Fischer, Hernan

Karen Mary Mantovani, Dalva E. C. Ferreira, Willian R. Rocha, Wolfgang Haase,

́

Terenzi,* Antonio S. Mangrich,

⊥

¶

∥

,▽

#

,†

Zbigniew Tomkowicz,

Ademir dos Anjos, and Ademir Neves*

†

‡

Laborator

Departamento de Química, and Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de

Santa Catarina, 88040-900 Florianopolis, Santa Catarina, Brazil

Departamento de Química, Universidade Federal do Parana, 81531-970 Curitiba, Parana,

cias Exatas, Universidade Federal de Minas Gerais, Campus Universitar

1270-901 Pampulha, Belo Horizonte, Minas Gerais, Brazil

́ ̂ ́

io de Bioinorganica e Crystalografia (LABINC), Departamento de Química, Laboratorio de Equilíbrio Químico,

§

́

⊥

́

Brazil

¶

Departamento de Química, Instituto de Cien

̂

́

io Pampulha,

3

∥

Institut fu

̈

r Physikalishe Chemie, Technische Universitat

̈

Darmstadt, Petersenstraße 20, D-64287-Darmstadt, Germany

▽

Institute of Physics, Reymonta 4, Jagiellonian University, PL-30-059 Krakow, Poland

#

Universidade Estadual de Mato Grosso do Sul, 79950-000 Unidade Universitar

́

ia de Naviraí, Mato Grosso do Sul, Brazil

*

S Supporting Information

ABSTRACT: Herein, we report the synthesis and character-

ization, through elemental analysis, electronic spectroscopy,

electrochemistry, potentiometric titration, electron paramag-

netic resonance, and magnetochemistry, of two dinuclear

copper(II) complexes, using the unsymmetrical ligands N′,N′,

N-tris(2-pyridylmethyl)-N-(2-hydroxy-3,5-di-tert-butylbenzyl)-

1

,3-propanediamin-2-ol (L1) and N′,N′-bis(2-pyridylmethyl)-

N,N-(2-hydroxybenzyl)(2-hydroxy-3,5-di-tert-butylbenzyl)-

,3-propanediamin-2-ol (L2). The structures of the complexes

Cu (L1)(μ-OAc)](ClO ) ·(CH ) CHOH (1) and [Cu (L2)-

1

[

(

2

4 2

3 2

2

μ-OAc)](ClO )·H O·(CH ) CHOH (2) were determined

4

2

3 2

2+

by X-ray crystallography. The complex [Cu (L3)(μ-OAc)]

2

[

3; L3 = N-(2-hydroxybenzyl)-N′,N′,N-tris(2-pyridylmethyl)-1,3-propanediamin-2-ol] was included in this study for comparison

II

purposes only (Neves et al. Inorg. Chim. Acta 2005, 358, 1807−1822). Magnetic data show that the Cu centers in 1 and 2 are

−1

antiferromagnetically coupled and that the difference in the exchange coupling J found for these complexes (J = −4.3 cm for

−1

1

and J = −40.0 cm for 2) is a function of the Cu−O−Cu bridging angle. In addition, 1 and 2 were tested as catalysts in the

oxidation of the model substrate 3,5-di-tert-butylcatechol and can be considered as functional models for catechol oxidase.

Because these complexes possess labile sites in their structures and in solution they have a potential nucleophile constituted by a

II

terminal Cu -bound hydroxo group, their activity toward hydrolysis of the model substrate 2,4-bis(dinitrophenyl)phosphate and

II

DNA was also investigated. Double electrophilic activation of the phosphodiester by monodentate coordination to the Cu

center that contains the phenol group with tert-butyl substituents and hydrogen bonding of the protonated phenol with the

phosphate O atom are proposed to increase the hydrolase activity (K_a_s_s_.and k ) of 1 and 2 in comparison with that found for

cat.

complex 3. In fact, complexes 1 and 2 show both oxidoreductase and hydrolase/nuclease activities and can thus be regarded as

man-made models for studying catalytic promiscuity.

INTRODUCTION

respectively, have been used as appropriate starting points in

the development of specific classes of synthetic metal

complexes, known as synthetic hydrolases and/or chemical

■

Currently, much of the information regarding the role of metals

in natural systems is gained through comparative studies on

metalloenzymes and model metal complexes. For example,

dinuclear hydrolytic and oxidative metalloenzymes, such as

purple acid phosphatases (PAPs) and catechol oxidases (COs),

Received: August 27, 2011

Published: January 19, 2012

©

2012 American Chemical Society

1569

dx.doi.org/10.1021/ic201876k | Inorg. Chem. 2012, 51, 1569−1589

Inorganic Chemistry

Article

nucleases, when mimicking PAPs, ⁻¹³catecholases,¹^,¹⁴⁻³¹and

CO. Indeed, studies with model complexes have been

described, aiming to mimic the structural and/or functional

properties of these metalloenzymes, such as the intermetallic

distance, asymmetry, and geometry around each metal center,

with the presence of labile sites essential for binding of the

substrate and/or available nucleophiles to initiate the catalytic

process. In this way, through structural and spectroscopic

analysis, as well as detailed reactivity studies on model com-

pounds in solution, it is possible to propose the most probable

mechanism involved in the catalytic process under inves-

tigation. In some cases, such information can be very helpful in

clarifying the most probable mechanism associated with the

1

like activities of two dinuclear copper(II) model complexes.

The unsymmetric dinucleating ligands (L1 and L2) utilized in

the syntheses of complexes 1 and 2 and the L3 ligand are

shown in Chart 1. The design of the ligands containing phenol

Chart 1. Unsymmetric Dinucleating Ligands

4

,14,22

native enzyme.

In the active sites of numerous metalloenzymes, two adjacent

metal centers act cooperatively in the transformation of sub-

strate molecules. This is particularly true for biological oxidase

or oxygenase activity involving molecular oxygen (O ), in

2

which the metal ions serve to activate the kinetically inert O₂

and the combined redox power of the two metal ions is used to

mediate and to control the multielectron redox reactions. In

view of the great importance of oxidation reactions in industrial

and synthetic processes and of the ongoing search for new and

efficient oxidation catalysts, it is of paramount interest to elucidate

the basic functional principles that govern such bimetallic reactivity

groups with tert-butyl substituents was introduced based on the

32

6

2+

of natural enzymes.

structure and properties of [Cu (L3)(μ-OAc)] (3), in order

2

Dicopper sites play a pivotal role in biological oxygen

chemistry and, consequently, understanding the structural and

functional aspects of copper metalloenzymes is a subject of

intensive research. A prominent member of these copper

proteins is CO, which features a type 3 center with two proxi-

mate Cu atoms.

CO, also known as o-diphenol oxidase or polyphenol oxidase,

catalyzes the two-electron-transfer reaction during the oxida-

tion of a broad range of o-diphenols to the corresponding

to obtain dinuclear copper(II) complexes with distinct Lewis

acidities and, consequently, distinct catecholase- and hydrolase-

like activities. Interestingly, while complex 1 has been used as

an efficient electrochemical sensor for the determination of

38

hydroquinone in cosmetics, complex 2 has been employed as

a biomimetic sensor for catechin determination in green tea

39

samples.

EXPERIMENTAL SECTION

■

o-quinones by O . The resulting highly reactive quinones

2

The abbreviations used are as follows: L1 = N′,N′,N-tris(2-

pyridylmethyl)-N-(2-hydroxy-3,5-di-tert-butylbenzyl)-1,3-propanedia-

min-2-ol; L2 = N′,N′-bis(2-pyridylmethyl)-N,N-(2-hydroxybenzyl)(2-

hydroxy-3,5-di-tert-butylbenzyl)-1,3-propanediamin-2-ol; L3 = N-(2-

hydroxybenzyl)-N′,N′,N-tris(2-pyridylmethyl)-1,3-propanediamin-2-ol;

L4 = bis(2-hydroxybenzyl)-N-N′-bis(2-pyridylmethyl)-1,3-propanedia-

min-2-ol; 2,4-BDNPP = bis(2,4-dinitrophenyl)phosphate; 2,4-DNPP =

2,4-dinitrophenyl phosphate; 3,5-DTBC = 3,5 di-tert-butylcatechol.

Materials and Measurements. N′,N′,N-tris(2-pyridylmethyl)-1,3-

autopolymerize to form brown polyphenolic catechol melanins,

a process thought to protect the damaged plant from pathogens

3

3−36

or insects.

Although the high specificity of this reaction

is of great importance for the determination of hormonal

catecholamines (adrenaline, noradrenaline, and dopamine) in

medical diagnoses, little is known about the mechanism of COs,

beyond the availability of the three-dimensional structures of

a CO from sweet potato (ibCO), which has been determined

II

I

6

for the oxidized Cu −Cu state and the reduced Cu −Cu

propanediamin-2-ol, N,N-bis(2-pyridylmethyl)-1,3-propanediamin-

6

40

41

3

3−36

2

4

-ol, 2-(chloromethyl)-4,6-di-tert-butylphenol, 2,4-BDNPP, and 2,

42

form.

-DNPP were synthesized as previously described. Other reagents,

On the other hand, there is great interest in the development

of new dinuclear model complexes that are able to mimic the

catalytic properties of hydrolases/nucleases, a class of metal-

loenzymes that catalyze the hydrolysis of proteins, activated

materials, gases, and solvents of analytical or spectroscopic grade (in

the kinetic studies and characterization of complexes) were purchased

from commercial sources and used without further purification.

The plasmid pBSK II (2961 bp), used for DNA cleavage assays, was

purchased from Stratagene, transformed into DH5a Escherichia coli

competent cells, and amplified as previously described by Ausubel

1

−13

phosphodiesters, and DNA or RNA.

Although a great

number of natural hydrolases are known, the search for small

molecules able to cleave diester bonds in models and DNA

molecules has been attracting special attention in attempts to

identify the mechanism of the cleavage reactions, as well as

their applicability as synthetic restriction enzymes, conforma-

4

3

et al. The plasmid DNA was extracted from E. coli and purified using

4

Qiagen Plasmid Maxi Kit protocol.

Physical Measurements. Elemental analyses were performed on a

Carlo Erba E-1110 instrument. IR spectra were recorded on a Perkin-

Elmer model FTIR-2000 spectrometer, using KBr pellets, in the range

37

tional probes, and catalysts.

−1

of 4500−450 cm . Electronic absorption spectra in the 200−1200 nm

range were recorded on a Perkin-Elmer Lambda 19 spectropho-

tometer. Electron paramagnetic resonance (EPR) spectroscopy

analyses were performed in solid-state and frozen solutions at 77 K

on a Bruker EMX spectrophotometer operating at a frequency of

around 9.5 GHz (X-band), precisely determined, with a modulation

frequency of 100 kHz, amplitude modulated from 2.024 G, and

On the basis of the structural and physicochemical properties

of natural enzymes, the principles postulated by Sargeson and

1

2

co-workers in the design of nucleophilic catalysts, and the

concept of catalytic promiscuity for models recently described

1

5

by Neves et al., we describe herein the syntheses, structure,

physicochemical properties, and catecholase- and phosphatase-

1

570

dx.doi.org/10.1021/ic201876k | Inorg. Chem. 2012, 51, 1569−1589

Inorganic Chemistry

Article

1

microwave power of approximately 20 mW. H NMR analysis was

Initially, pH-dependent studies were carried out to determine the

pH value at which catecholase- or phosphatase-like activities reached a

maximum. The influence of the catecholase-like activity pH on the

reaction rate for oxidation of 3,5-DTBC catalyzed by complexes 1

and 2 was determined over the pH range of 5.5−9.0 at 25 °C. To a

quartz cell were added 100 μL of an aqueous solution ([B]final = 3.00 ×

carried out using a Bruker 200 MHz spectrometer with CDCl as the

solvent, at room temperature. Chemical shifts were referenced to

tetramethylsilane.

Electrochemical measurements were obtained using a Princeton

Applied Research model 273 potentiostat/galvanostat. Square-wave

voltammograms were obtained for the complexes in acetonitrile

solutions containing 0.1 mol·L tetrabutylammonium hexafluoro-

phosphate as the supporting electrolyte under an argon atmosphere.

The electrochemical cell employed was of a standard three-electrode

3

−2

−1

10 mol·L ) of buffer [MES (pH 5.5−6.5) and TRIS (pH 7.0−9.0)],

−1

−5

100 μL of a methanolic complex solution ([C]final = 2.40 × 10

−1

mol·L ), and 3000 μL of air-saturated methanol. The reaction was

initiated with the addition of 100 μL of a methanolic substrate solution

^[³^,⁵^-^D^T^B^C^]final = 5.00 × 10 mol·L ) and monitored for 15 min.

The kinetic experiments under conditions of excess substrate were

−3

−1

(

configuration: platinum working electrode, platinum wire counter

electrode, Ag/AgCl reference electrode. The Fc /Fc couple (E = 400 mV

+

0

45

performed as follows: A total of 100 μL of aqueous buffer TRIS at pH

vs NHE) was used as the internal standard.

−

2

−1

8

^.⁵⁽^[^B^]final = 3.00 × 10 mol·L ), 100 μL of a methanolic complex

5

Electrospray ionization mass spectrometry (ESI-MS) of the

complexes dissolved in an ultrapure acetonitrile/water (1:1, v/v)

solution (500 μL) was analyzed using an amaZon X Ion Trap MS

instrument (Bruker Daltonics) with an ion spray source using

electrospray ionization in positive-ion mode. The ion source condition

was an ion spray voltage of 4500 V. Nitrogen was used as the

nebulizing gas (20 psi) and curtain gas (10 psi). The samples were

directly infused into the mass spectrometer at a flow rate of 300 μL/h.

The scan range was m/z 200−3000.

Magnetic Measurements and Theoretical Calculations of

Magnetic Coupling. Magnetic susceptibility data were obtained in

the temperature range of 2−300 K and in a magnetic field of 1000 Oe

with a SQUID magnetometer on slightly pressed polycrystalline

samples of complexes 1 and 2. Diamagnetic corrections were applied

−

−1

^s^o^l^u^tⁱ^oⁿ⁽^[^C^]final = 2.40 × 10 mol·L ), and air-saturated methanol

from 2860 to 3076 μL, to complete 3300 μL) were added to a 1-cm-

(

path-length cell at 25 °C. The reaction was initiated with the addition

of known volumes (from 24 to 240 μL) of a 3,5-DTBC solution ([3,5-

−3

−1

^D^T^B^C^]final = 1.20 × 10 −12.00 × 10 mol·L ). To take into account

the spontaneous oxidation of the substrate, correction was carried out

using a reference cell under identical conditions but without the

addition of the catalyst. The initial rate was obtained from the slope

of the absorbance versus time plot over the first 15 min of the

reaction. The Michaelis−Menten model was applied and the kinetic

parameters were obtained from nonlinear square fits using the pro-

gram Origin 6.0.

To detect the formation of hydrogen peroxide (or water) during the

catalytic reaction, we employed a modified version of the iodometric

4

6

in the usual manner with use of the tabulated Pascal’s constants.

The calculations described herein were performed at the density

functional theory (DFT) level using Lee, Yang, and Parr’s correlation

functional B3LYP, a hybrid functional including exact HF (Hartree−

Fock) exchange in the ratio proposed by Becke. All calculations

3

2

method. Reaction mixtures were prepared as in the kinetic

experiments. After 1 h of reaction, an equal volume of water was

added and the quinone formed was extracted three times with

dichloromethane. The aqueous layer was acidified with H SO (5.00 ×

47

48

2

4

−3

−1

4

9

10 mol·L ) to pH 2 to stop further oxidation, and 1 mL of a 10%

solution of KI and 3 drops of a 3% solution of ammonium molybdate

employed the TZVPP basis set of Weigend and Ahlrichs for copper,

described as 17s11p6d1f, with the full contraction scheme to 842111/

5

0

were added. In the presence of hydrogen peroxide, the reaction H O +

2 2

6

311/411/1, and the Dunning’s DZP basis set for all other elements,

−

+

2

I + 2H → 2H O + I occurs and, with an excess of iodide ions, the

2 2

which for carbon, oxygen, and nitrogen is described as 9s5p1d, with

the full contraction scheme to 6111/41/1, and hydrogen is described

as 4s1p contracted to 31/1.

Potentiometric Titration. Potentiometric studies were carried out

with a Corning-350 research pH meter fitted with blue-glass and

−

triiodide ion is formed according to the reaction I (aq) + I → I . The

2

3

reaction rate is slow but increases with increasing concentrations of acid,

and the addition of an ammonium molybdate solution renders the

−

reaction almost instantaneous. The formation of I₃can be monitored

spectrophotometrically because of the development of the characteristic

+

Ag/AgCl reference electrodes, calibrated to read −log [H ] directly,

−1

−1 32

band at 353 nm (ε = 26 000 L·mol ·cm ).

designated as the pH, in ethanol/water (70:30, v/v). Potentiometric

studies of complexes 1 and 2 were made in an ethanol/water mixture,

instead of acetonitrile/water (as in the hydrolase experiments),

because of the low solubility of the complexes in this media and

need of higher concentrations for titration experiments.

Kinetic experiments for hydrolysis of 2,4-BDNPP were performed

in aqueous/buffer media. Studies on the effects of the pH on the

hydrolysis reaction were performed in the pH range of 3.50−10.00

at 50 °C. Reactions were performed using the following conditions:

−1

A total of 1500 μL of an aqueous solution ([B]_f_i_n_a_l= 0.05 mol·L )

Equilibrium measurements were performed in a thermostatted cell,

purged with argon, containing 50.00 mL of the ethanol/water (70:30)

solution and 0.02 mmol of the complex. The temperature was 25.00 ±

.05 °C, and the experimental solutions were adjusted to 0.100

mol·L of ionic strength by the addition of KCl. Computations of

of buffer [MES (pH 3.50−6.50), HEPES (pH 7.00−8.50), and CHES

−1

(

pH 9.00−10.00)] (I = 0.05 mol·L LiClO ), 200 μL of an

4

−5

−1

acetonitrile complex solution ([C]

1

= 4.00 × 10 mol·L ), and

000 μL of acetonitrile were added to a 1-cm-path-length cell. The

final

0

−1

reaction was initiated with the addition of 300 μL of an acetonitrile

5

1

the triplicate results were carried out with the BEST7 program,

51

−3

−1

substrate solution ([2,4-BDNPP]_f_i_n_a_l= 5.00 × 10 mol·L ) and

monitored for 15 min.

51

and species diagrams were obtained with the SPE and SPEPLOT

programs.

Under conditions of excess substrate, 200 μL of an acetonitrile

Reactivity. Catecholase- and hydrolase-like activities of complexes

and 2 were determined through oxidation of the model substrate 3,5-

di-tert-butylcatechol (3,5-DTBC) and hydrolysis reaction of the model

−5

−1

solution of the complex ([C]_f_i_n_a_l= 4.00 × 10 mol·L ) was added to

1

500 μL of an aqueous buffer solution [HEPES (pH 8.00)] ([B]

.00 × 10 mol·L ) and acetonitrile (from 480 to 1520 μL, to

=

final

−2

−1

5

41

substrate 2,4-BDNPP, respectively, under excess substrate con-

ditions. The experiments were carried out, in triplicate, and monitored

on a Varian Cary50 Bio spectrophotometer connected to a PC

computer by following the increase in the 3,5-di-tert-butylquinone

complete 3300 μL). The reaction was initiated with the addition of

known volumes (80−1120 μL) of a 2,4-BDNPP solution ([2,4-

−

4

−3

−1

BDNPP] = 5.00 × 10 −7.00 × 10 mol·L ). Corrections for the

final

spontaneous hydrolysis of 2,4-BDNPP were accomplished by a direct

difference using a reference cell under identical experimental

conditions, without adding the catalyst.

−

1

−1

absorption band at 400 nm (ε = 1900 L·mol ·cm ) characteristic of

oxidation reactions. For the hydrolase-like activity, the increase in the

,4-dinitrophenolate characteristic absorption band at 400 nm (pH/ε

L·mol ·cm = (3.5/2125, 4.0/3408, 4.5/7182, 5.0/10 078, 5.5/11

2

In order to establish the number of molecules of substrate that

are hydrolyzed per molecule of complex, the reaction was monitored

−1

52

53

−1

4

05, 6.0/12 004; 6.5−10.0/12 100) was followed. Reactions were

at 445 nm (ε = 3600 L·mol ·cm ), under a 50-fold substrate ex-

3

−

−1

monitored to less than 5% conversion of the substrate, and the data

were treated using the initial rate method, in the first 15 min of the

reaction.

cess ([2,4-BDNPP]_f_i_n_a_l= 2.00 × 10 mol·L ), relative to the com-

5

−

−1

plex ([C]_f_i_n_a_l= 4.00 × 10 mol·L ), at pH 8.00 and 50 °C.

−

5

−1

The stoichiometric reactions ([C]_f_i_n_a_l= 4.00 × 10 mol·L and

1

571

dx.doi.org/10.1021/ic201876k | Inorg. Chem. 2012, 51, 1569−1589

Inorganic Chemistry

Article

²^,⁴^-^B^D^N^P^P^]final = 4.00 × 10⁻mol·L ) were also monitored (400 nm)

5

−1

[

Table 1. Crystallographic Data for Complexes 1 and 2

to investigate the possible hydrolysis of the monoester 2,4-DNPP,

one of the products from the hydrolysis reaction of the diester

1

2

,4-BDNPP, at pH 8.00 and 50 °C.

empirical formula

C H Cl Cu N O

C H ClCu N O

42 59 2 4 11

41

57

2

5

13

DNA Cleavage Activity. The ability of 1and 2 to cleave DNA was

_f_w₍_g_·_m_o_l−1₎

1025.90

293(2)

0.710 69

triclinic

958.46

examined by following the conversion of supercoiled plasmid DNA

FI) to the open-circular DNA (FII) and/or linear DNA (FIII) forms

using agarose gel electrophoresis to separate the cleavage products. In

temperature (K)

wavelength (Å)

cryst syst

293(2)

0.710 69

triclinic

(

−1

general, 330 ng of pBSK II (25 μmol·L in pb) in PIPES (pH 6.0 and

7

different concentrations of 1and 2 in CH CN (25% in reaction

volume) for 6 h at 37 °C. Thereafter, each reaction was quenched by

adding 5 μL of a loading buffer solution (0.01% bromophenol blue,

space group

P1

̅

P1

̅

.0), HEPES (pH 8.0), or CHES (pH 9.0) buffer was treated with

unit cell dimens

a = 15.041(1) Å

b = 18.556(1) Å

c = 20.663(1) Å

α = 66.485(6)°

β = 68.511(6)°

γ = 72.333(7)°

4836.2(5)

a = 11.287(1) Å

b = 12.001(1) Å

c = 18.518(5) Å

α = 95.935(5)°

β = 99.886(5)°

γ = 110.484(7)°

2278.3(7)

3

5

0% glycerol, and 250 mM EDTA, pH 8.0) and the sample subjected

−1

to electrophoresis on a 0.8% agarose gel containing 0.3 μg·mL of

−1

ethidium bromide in a 0.5TBE buffer (44.5 mmol·L TRIS, 44.5

−1

mmol·L boric acid, and 1 mmol·L EDTA) at 90 V for about 1.5 h.

The resulting gels were visualized and digitalized with a Gel Logic 200

gel documentation system (Carestream Health, USA). The proportion

of DNA in each band was quantified using KODAK Molecular

Imaging Software 5.0 (Carestream Health, USA). The proportion of

supercoiled DNA was corrected by a factor of 1.47 because the ability

of ethidium bromide to intercalate into this topoisomeric form is

_v_o_l_u_m_e₍_Å3₎

Z, calcd density

4/1.409

2/1.397

−3

(

g·cm )

3

cryst size (mm )

0.50 × 0.26 × 0.16

1.054

0.60 × 0.26 × 0.20

1.053

−1

abs coeff (mm )

F(000)

2136

1004

54

θ range for data

collection (deg)

2.17−25.10

2.14−25.07

decreased relative to the circular and linear forms.

To elucidate the mechanism involved in the DNA cleavage

performed by 1 and 2, different cleavage inhibitors were added to

the DNA prior to the complex. The reactive oxygen species (ROS)

scavengers dimethyl sulfoxide (DMSO; 10%), KI (500 μmol·L ),

superoxide dismutase (SOD; 20 units), and NaN (500 μmol·L )

were used in order to identify the presence of ROS during the DNA

scission event. In addition, assays in the presence of a copper(I)

stabilizer, bathocuproine (500 μmol·L ), the minor groove binder

distamycin (50 μmol·L ), and the major groove binder methyl green

index ranges

−17 ≤ h ≤ 16, −22 ≤ k ≤ −12 ≤ h ≤ 13, −14 ≤ k ≤

0

, −24 ≤ l ≤ 22

0, −21 ≤ l ≤ 22

8509/8089 [R(int) =

0.0178]

reflns collected/

unique

17 443/16 884 [R(int) =

0.0418]

−1

3

abs corrn

ψ scan

max and min

transmn

0.9523 and 0.8214

0.9712 and 0.8774

−1

refinement method full-matrix least squares on full-matrix least squares on

−1

2

F

−1

(

50 μmol·L ) were also performed. The plasmid DNA was pretreated

data/restraints/

param

_G_O_F_o_n_F2

16 884/289/1119

8089/108/579

with distamycin and methyl green for 30 min and treated with the

complex as described above. Furthermore, the influence of the ionic

strength on DNA cleavage was evaluated using several concentrations

1.022

1.050

final R indices [I > R1 = 0.0733, wR2 =

2σ(I)] 0.1912

R = 0.0404, R_w= 0.0999

−1

(0−200 mmol·L ) of sodium chloride (NaCl).

The rates of DNA cleavage (kobs⁾^b^y¹^aⁿ^d²^w^e^r^e^d^e^t^e^r^mⁱⁿ^e^d^b^y

R indices (all data) R1 = 0.2336, wR2 =

R = 0.0854, R_w= 0.1134

0.443 and −0.340

treating the plasmid DNA at different complex concentrations (0−200

0.2450

−1

μmol·L to 1 and 0−25 μmol·L to 2) for different intervals of time

0−6 h). The k values were calculated for each complex

largest diff peak and 0.942 and −0.676

−

3

(

hole (e·Å )

obs

concentration, assuming pseudo-first-order kinetics, and then analyzed

55

following the pseudo-Michaelis−Menten formalism. The reaction

conditions were the same as those described above.

Syntheses of the Ligands and Complexes 1 and 2. Detailed

experimental procedures for the preparation of the ligands L1 and L2

and their dinuclear perchlorate complexes 1 and 2, respectively, have

From 17 443 collected intensities, 16 884 are unique and just 7240 are

observed. Even so, the structure was solved, and two conformational

isomers of 1, four perchlorate counterions, and two isopropyl alcohol

solvate molecules were found in the asymmetric unit. All perchlorate

ions are disordered, in which O atoms occupy two alternative posi-

tions. Two tert-butyl groups are also disordered, with two partial

occupancies for each terminal C atom. All non-H atoms were refined

with anisotropic displacement parameters, except for disordered tert-

butyl groups and perchlorate counterions, which were refined

isotropically. H atoms of the isopropyl alcohol solvate were found

from Fourier difference map and treated with riding model.

Complex 2. The crystallographic data were acquired from a selected

prismatic blue crystal token from the crystalline sample. Non-H atoms

were refined with anisotropic displacement parameters. H atoms of the

phenol group and water molecules were found from a Fourier map

treated with a riding model. H atoms of the isopropyl alcohol solvate

could not be located from a difference Fourier map maybe because its

O atom was found to be disordered.

3

8,39

been reported elsewhere.

Caution! Perchlorate salts of metal complexes are potentially explosive

and therefore should be prepared in small quantities.

Single-Crystal X-ray Structure Determinations. Blue crystals

for complexes 1 and 2 were selected, and crystallographic analyses

were carried out with an Enraf-Nonius CAD4 diffractometer with

graphite-monochromated Mo Kα radiation (λ = 0.710 69 Å), at room

temperature. Cell parameters were determined from 25 carefully

centered reflections. Intensities were collected using the ω−2θ scan

technique. All data were corrected for Lorentz and polarization effects.

A semiempirical absorption correction based on the azimuthal scans

of seven appropriate reflections was also applied to the collected

intensities. The structures were solved by direct methods and refined

by full-matrix least-squares methods using the SIR97 and

SHELXL97 programs, respectively. H atoms attached to C atoms

were placed at their idealized positions, with C−H distances and U

values taken from the default settings of the refinement program. The

figures for the molecular structures were produced with the

5

6

57

RESULTS AND DISCUSSION

■

eq

Syntheses. The ligands L1 and L2 (Chart 1) were obtained

with sufficient purity and yields for use without further

purification in the synthesis of the dinuclear copper(II)

58

PLATON program. Further crystallographic information for these

complexes are described below and summarized in Table 1.

3

8,39

complexes 1 and 2.

The unsymmetrical dinuclea-

Complex 1. A prismatic blue crystal was selected for X-ray analysis.

The crystals of complex 1 showed very poor scattering power.

ting ligands presenting two potentially different tridentate

pendant arms allowed one to obtain the dinuclear

1

572

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[

(

Cu (L1)(μ-OAc)](ClO ) ·(CH ) CHOH (1) and [Cu (L2)-

Figure 1 shows the structure of the cation complex 1, which is

composed of one unsymmetric L1 ligand coordinated to both

Cu centers (Cu1 and Cu2). Each Cu center is five-coordinated

and is bridged by the alkoxo donor O atom of the ligand and by

an exogenous acetate ion.

2

4 2

3 2

2

μ-OAc)](ClO )·H O·(CH ) CHOH (2) complexes in which

4

2

3 2

II

the metal centers reside in distinct coordination environments

and geometries (vide infra). The perchlorate salts of 1 and 2

were obtained in good yields by reacting respectively the

ligands L1 and L2 with Cu(OAc) ·H O (1:2) in methanol

in the presence of a stoichiometric amount of NaClO ·3H O.

Recrystallization from a CH Cl /isopropyl alcohol (1:1)

solution of 1 and a isopropyl alcohol solution of 2 afforded

crystals of both complexes suitable for X-ray crystallographic

analysis.

X-ray Structural Characterization. Complex 1 crystallizes

as single blue crystals belonging to the triclinic crystal system

and space group P1. The asymmetric unit of this complex

̅

consists of two molecules of 1. Because the bond lengths and

angles and the geometry around the Cu centers are identical in

the two molecules (1 and 2), only the parameters of molecule 1

will be discussed here. An ORTEP view of the cation complex

is presented in Figure 1. The crystallographic data and the main

bond distances/angles are given in Tables 1 and 2, respectively.

The Cu1 center is five-coordinated, having a square-

2

59

pyramidal geometry as determined by the τ value (0.22).

4

2

The basal plane is composed of the amine N1 atom, the

pyridinic N22 atom, and the alkoxo O1 and acetate O51

bridging atoms. The protonated phenol O10 atom is in the

2

apical position of the square pyramid with Cu−OH

=

phenol

2

.470(6) Å and completes the N O coordination sphere

2 3

of Cu1.

The geometry around the Cu2 center (N O -donor set) is

3

2

59

best described as distorted trigonal-bypiramidal with τ = 0.87.

In the trigonal plane, the Cu2 atom is coordinated by the

pyridinic N32 and N42 atoms and the alkoxo bridging O1

atom. The amine N5 atom and the acetate bridging O52 atom

are in the pseudoaxial positions, completing the five-coordina-

tion sphere of Cu2 with an O52−Cu2−N5 bond angle of

II

1

76.0(3)°.

The bond distances and angles observed in 1 and 2 are

in agreement with those found in copper(II) complexes with a

μ-alkoxo bridge and nonequivalent geometries around the

8

,60

metallic centers, coordinated by similar N,O-donor groups.

8

The main difference between complexes 1 and 3 is the

presence of tert-butyl groups in 1, localized in the positions

ortho and para to the phenolic O atom, which should cause

8

an increase in the electron density over the Cu1 center. In

fact, the Cu1−O

distance in 1 is only slightly shorter

2.470(6) Å] than the corresponding distance observed in 3

phenol

[

8

2.504(10) Å].

Complex 2 crystallizes as prismatic blue crystals that belong

to the triclinic crystal system and space group P1. An ORTEP

̅

+

view of the cation complex [Cu (L2)(μ-OAc)] is presented in

2

Figure 2. The crystallographic data and the main bond

distances and angles are given in Tables 1 and 3, respectively.

5

8

Figure 1. ORTEP view of the cation complex [Cu (L1)-

2

2+

(μ-OAc)] −1.

Table 2. Main Bond Distances (Å) and Angles (deg) for

Complex 1

Cu1−O1

Cu1−N22

Cu1−O51

Cu1−N1

Cu1−O10

Cu1−Cu2

1.892(5)

1.948(7)

1.957(6)

2.025(6)

2.470(6)

3.3975(15)

Cu2−O52

Cu2−O1

Cu2−N42

Cu2−N32

Cu2−N5

1.922(7)

1.943 (5)

2.070(7)

2.078(9)

2.033(7)

O1−Cu1−N22

O1−Cu1−O51

N22−Cu1−O51

O1−Cu1−N1

N22−Cu1−N1

O51−Cu1−N1

O1−Cu1−O10

N22−Cu1−O10

O51−Cu1−O10

N1−Cu1−O10

O52−Cu2−O1

164.5(3)

96.0(3)

94.1(3)

86.4(2)

83.7(3)

177.5(3)

101.4(2)

90.2(2)

88.6(2)

98.9(3)

O52−Cu2−N5

O1−Cu2−N5

176.0(3)

84.4(3)

98.0(3)

121.3(3)

82.1(3)

95.5(3)

123.7(3)

80.8(3)

110.0(3)

124.7(3)

110.5(5)

5

8

Figure 2. ORTEP view of the cation complex [Cu (L2)-

2

O52−Cu2−N42

O1−Cu2−N42

N5−Cu2−N42

O52−Cu2−N32

O1−Cu2−N32

N5−Cu2−N32

N42−Cu2−N32

Cu1−O1−Cu2

C12−O10−Cu1

+

(

μ-OAc)] −2.

In complex 2, the Cu1 center is five-coordinated, showing a

square-pyramidal geometry as determined by the τ value

0.11). The phenolic O20 atom containing the tert-butyl

groups is protonated and is coordinated in the apical position of

the pyramid with Cu−OH_p_h_e_n_o_l= 2.455(2) Å in agreement

with a Jahn−Teller distortion observed in copper complexes.

59

(

1

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Table 3. Main Bond Distances (Å) and Angles (deg) for

Complex 2

Magnetic Properties. The variable-temperature magnetic

data for complexes 1 and 2 in the solid state, collected in the

temperature range of 2−300 K and in a magnetic field of 1000

Oe, are shown in Figure 3 and indicate a distinct degree of

Cu1−O1

Cu1−O10

Cu1−O51

Cu1−N1

Cu1−O20

Cu1−Cu2

1.897(2)

1.902(2)

1.977(2)

2.042(2)

2.455(2)

3.4837(6)

Cu2−O52

Cu2−O1

Cu2−N5

Cu2−N32

Cu2−N42

1.916(2)

1.937(2)

2.017(3)

2.092(3)

2.118(3)

II

coupling between the Cu centers in these complexes. For

complex 1, the effective magnetic moment (μ = 2.828-

eff

1/2

[

χ_m_o_lT] ) per 2Cu is approximately constant (∼2.70 μ ) in

B

the temperature range of 300−16 K and then decreases more

rapidly to 1.62 μ at 2 K. On the other hand, for complex 2, μ

B

eff

per 2Cu decreases gradually from 2.60 μ at 300 K to 2.3 μ at

B

O1−Cu1−O10

O1−Cu1−O51

O10−Cu1−O51

O1−Cu1−N1

O10−Cu1−N1

O51−Cu1−N1

O1−Cu1−O20

O10−Cu1−O20

O51−Cu1−O20

N1−Cu1−O20

O52−Cu2−O1

167.93(11)

92.77(9)

85.66(10)

85.54(9)

94.90(10)

174.50(11)

96.26(9)

95.80(9)

98.00(9)

87.39(9)

98.04(9)

O52−Cu2−N5

O1−Cu2−N5

177.14(10)

84.82(10)

96.63(12)

127.79(11)

81.53(11)

97.26(12)

117.52(11)

81.37(12)

109.80(11)

130.67(11)

126.5(2)

6

0 K and then decreases more rapidly to 1.63 μ at 2 K. The

B

data were fitted by the exact diagonalization of the spin

Hamiltonian

O52−Cu2−N32

O1−Cu2−N32

N5−Cu2−N32

O52−Cu2−N42

O1−Cu2−N42

N5−Cu2−N42

N42−Cu2−N32

Cu1−O1−Cu2

C12−O10−Cu1

/

= − J·S ·S + μ [(g ·S + g ·S ) + 2ρS]H

B

1 2

1 2 1 2

−

zJ′ S (S + S )

z 1z 2z

(1)

where S and S are spin operators of the copper dimer, S is a

1

2

copper monomer impurity spin, ρ is an impurity fraction, H is

the magnetic field, and the last term in the Hamiltonian

presents intermolecular interaction in the molecular-field

approximation. The values calculated for susceptibility were

powder-averaged. Fits obtained are shown in Figure 3. There

was essentially no improvement in the fit when the anisotropy

of the exchange and intermolecular interactions was taken into

account. In particular, it was not possible to obtain a good fit

for complex 1 with J > 0 by zJ′ < 0. The results obtained from

the fits are consistent with those obtained by applying the

formula of Bleaney and Bowers, modified with respect to the

This coordination mode is comparable to those observed in 1

and in complexes 3 and [Cu (L4)(μ-OAc)(H O) ] in which

2

+

2

8

the Cu−OH_p_h_e_n_o_l₆^d0ⁱ^s^t^aⁿ^c^e^s^a^r^e^r^e^s^p^e^c^tⁱ^v^e^l^y²^.⁴⁷⁰⁽⁶⁾^,²^.⁵⁰⁴⁽¹⁰⁾^,

and 2.433(10) Å.

The tertiary amine N1 atom, the phenolate O10 atom, and

the O51 and O1 atoms from the acetate and alkoxo bridges,

respectively, compose the basal plane of the square pyramid.

^T^h^e^C^u⁻^Ophenolate [Cu1−O10 = 1.902(2) Å] bond distance

is similar to those observed for other phenolate complexes

containing Cu ,

65

presence of impurities. The parameters obtained are listed

II 61−64

II

and the other phenolic O20 atom

in Table 4, together with the data of other dinuclear [Cu (μ-

II

completes the five-coordinated environment around the Cu1

center.

alkoxo)(μ-OAc)Cu ] complexes described in the litera-

1

6,60,66

ture.

The X-ray structures of complexes 1 and 2 reveal that the

The coordination environment around the Cu2 center is

similar to that observed in 1 with a tendency toward a distorted

trigonal-bipyramidal geometry, as reflected by the structural

index parameter τ of 0.83. The Cu atom is coordinated to three

N and two O atoms: the pyridine N atoms (N32 and N42), the

tertiary amine N atom (N5), and the acetate and μ-alkoxo

O atoms (O52 and O1), respectively. Finally, with an increase

in the Cu···Cu distance in 2 [3.4837(6) Å] compared to 1

Cu1 centers have square-pyramidal geometry in which the

2

unpaired electron is in the d

magnetic orbital, while both

x −y

Cu2 centers have trigonal-bipyramidal geometry, with the

2

z

unpaired electron localized in the d magnetic orbital (Figures

1

and 2, respectively). Indeed, a great number of dinuclear

II

copper(II) complexes containing the structural [Cu (μ-

alkoxo)(μ-carboxylato)Cu ] unit have been structurally

II

1

6,23,26,67−69

and magnetically characterized.

However, with a

most of these complexes have been

obtained with symmetrical dinucleating alkoxo and phenoxo

8

,28,70,71

[

3.3975(15) Å], there is a concomitant opening of the Cu−

O(alkoxo)−Cu angle from 124.7(3)° in 1 to 130.67(11)° in 2.

few exceptions,

Figure 3. Temperature dependence of χ_M(◆) and μ_e_f_f(○) for complexes 1 (a) and 2 (b).

1

574

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Table 4. Magnetic and Structural Data for Complexes 1 and 2 and Other Complexes Described in the Literature

3

−1

J (cm⁻¹)

complex

g

TIP (cm ·mol )

% impurity

Cu···Cu (Å)

Cu−O−Cu (deg)

6

1

2

2.01

2.11

2.075

2.0

∼210 × 10⁻

−4.3

−40

−78

−76

43

6.9

7.5

3.0

1.5

3.397

3.483

3.511

3.517

3.159

3.081

3.239

3.286

3.400

124.7

∼350 × 10⁻

6

130.66

132.85

131.7

1

6

4

5

6

7

8

9

1

2.062

2.01

2.04

2.007

2.1

108.08

104.76

114.4

77.6

26

−6

40.0 × 10

60.0 × 10

−6

16.7

−25

114.3

6

0

−6

0

5.7

127.9

2

ligands, but even so, they revealed a significant range of

interesting structural and magnetic properties. For instance,

the d_zorbital associated with the trigonal-bipyramidal

2

geometry with the d

ground state of the square pyramid,

x −y

II

−1

it has been assumed that in the [Cu (μ-alkoxo)

thus giving rise to stronger antiferromagnetism (J = −40 cm )

in 2. Alternatively, it could be speculated that the acetate

bridging group is able to interact with the unpaired electron on

II

(

μ-carboxylato)Cu ] unit μ-acetate cannot promote coupling

II

72

between the Cu ions and that differences in the magnetic

behavior of these systems can be rationalized using the

ligand orbital complementary” concept.

II

each Cu ion by direct overlap. However, the weak

6

6,73,74

“

In addition, it

antiferromagnetic coupling observed for 1 indicates that the

contribution of the carboxylate group to the exchange mech-

anism should be minor compared with that of the μ-alkoxo

has been concluded that dinuclear copper(II) complexes

2

containing both d

and d magnetic ground states show

x −y

z

75

ferromagnetic coupling because of the orthogonality of the

ligand.

7

5

16

magnetic orbitals.

For the complexes described herein, the weak antiferromag-

Wei and co-workers have established that the magnitude

of the exchange integral in dinuclear copper(II) complexes con-

II

−1

II

netic coupling between the Cu centers in 1 (J = −4.3 cm ) is

taining the [Cu (μ-alkoxo)(μ-carboxylato)Cu ] structural unit

is a linear function of the Cu−O−Cu bridging angle; the ex-

change is ferromagnetic below 116.5° and antiferromagnetic

above this value. Interestingly, according to the modified plot pre-

sented by Wei et al. given in Figure 5 (θ vs J instead of θ vs 2J)

in agreement with the fact that the μ-alkoxo group can interact

with the unpaired electron in the d

2

orbital around Cu1, but

x −y

it does not overlap with the orbital having the unpaired electron

2

z

in the d orbital (Cu2), which is oriented toward the acetate

bridging group, as shown in Figure 4. On the other hand,

Figure 4. Schematic representation of the orientation of orbitals in

complexes 1 and 2.

Figure 5. Exchange integral (J) versus Cu−O−Cu (θ) bridge angle for

complex 2 shows a significantly higher antiferromagnetic

II

[

Cu (μ-alkoxo)(μ-OAc)Cu ] complexes. The numbers next to the

−1

II

interaction (J = −40 cm ) between the Cu centers compared

points represent the compounds. See references for complexes 4−10

in Table 4.

to complex 1, and such a phenomenon has not been observed

II

previously for [Cu (μ-alkoxo)(μ-carboxylato)Cu ] complexes

in which the magnetic ground states of the Cu1 and Cu2

and including 1 and 2, it becomes apparent that the difference

in the exchange coupling J found for these complexes is also a

function of the Cu−O−Cu bridging angle, as was already

2

centers are respectively d

and d . In fact, magnetic

x −y

z

interactions between the metal centers in dinuclear copper(II)

complexes are strongly structure-dependent, and in the present

study, precise structural and magnetic data for complexes 1 and

II

established for other [Cu (μ-alkoxo)(μ-carboxylato)Cu ] com-

16

plexes. Moreover, this conclusion is also supported by theore-

tical calculations for complex 2, which show that the computed

J value obtained from the fully optimized structure is in excel-

lent agreement with the experimental antiferromagnetic coupling

constant.

2

are available. On the basis of the structure of 2, it can be

concluded that the geometry around Cu1 is only slightly

distorted (τ = 0.11) from a square-pyramidal structure. Thus,

2

the dominant magnetic orbital of Cu1 appears to be the d

x −y

orbital. On the other hand, it is anticipated that distortion in

the coordination geometry around the Cu2 center (τ = 0.83)

and the greater bridging Cu−O−Cu angle (θ) of 130.66° in 2

compared to 124.7° in complex 1 could lead to some mixing of

Study of the Magnetic Coupling Constants J for 1 and

2 Obtained by Theoretical Calculations. The interaction

between two paramagnetic metallic centers can be quantified by

the sign and magnitude of the isotropic magnetic exchange

1

575

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Inorganic Chemistry

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coupling constant, J, based on the most commonly used spin

Hamiltonian, the so-called HDVV (Heisenberg−Dirac−

trends. For example, the values for the magnetic exchange

interaction between the two Cu ions calculated using the

II

76

van Vleck) spin Hamiltonian (eq 1a).

X-ray structure of complex 2 are −19.41, −9.70, and −19.28

−

1

(1) (2)

(3)

cm for J , J , and J , respectively. When the positions of

the H atoms are optimized, these values change to −30.66,

H_H_D_V_V= − JS₁S₂

(1a)

−1

where S and S denote the individual spin operators of the two

−15.33, and −30.44 cm . An excellent agreement between the

−1 −1

1

2

metallic centers. DFT, combined with the broken-symmetry

BS) approach, was adopted in our calculations of the magnetic

computed J = −38.55 cm and the experimental J = −40 cm

is obtained when the structure is fully optimized, which shows

the dependence of the coupling constant on the structural

parameters.

(

II

interaction between the Cu centers in complexes 1 and 2. In

fact, the BS formalism has been proven to be an efficient and

practical alternative to elucidate the magnetic exchange

The computed coupling constant of complex 1 calculated

77−80

II

couplings of dinuclear transition-metal systems.

using the X-ray structure showed that the two Cu ions are

The magnetic exchange coupling constant J was evaluated

using three different equations:

ferromagnetically coupled. This is in contrast with the

II

experimental findings, which suggest that the Cu centers are

−1

antiferromagnetically coupled (J = −4.3 cm ). Nevertheless,

when the J constant is determined using the fully optimized

structure, the coupling behavior is correctly described; however,

the results are not quantitatively correct. This disagreement

between calculated and experimental data of the magnetic

coupling constant of 1 can be explained by the fact that the

optimized structure does not faithfully represent the exper-

imental structure. Some optimized geometrical parameters for

compounds 1 and 2 are shown in Table 6.

E

− E

J⁽¹⁾= − ₍

HS

BS

2

S + S )

(2)

(3)

(4)

1

2

^EHS − E_B_S

_J₍2) ⁼⁻(

S + S )(S + S + 1)

1

2

1

2

^EHS − E

_J₍3) = −

BS

2

⟨S ⟩ − ⟨S ⟩

HS

BS

^w^h^e^r^e^EHS ^aⁿ^d^EBS are the energies of the high-spin (HS) and

BS configurations, respectively.

Table 6. Selected Geometrical Parameters for Complexes 1

and 2 Calculated Using the B3LYP Functional

a

All calculations reported here were performed using the

81

structural parameter

1

2

ORCA Quantum Chemical Program. To understand the struc-

tural effects on the computed coupling constant, we performed

three different types of calculations. First, we simply used the

crystallographic structures of complexes 1 and 2 and added H

atoms to complete the valences of the atoms. Second, we

optimized only the positions of the H atoms and allowed the

remainder of the molecular skeleton to be fixed. Third, we

considered the full system and optimized the structure with no

geometry or symmetry constraints. In the latter case, the

geometry was optimized considering only the low-spin (LS)

Cu1−Cu2

Cu1−O1

Cu2−O1

∠Cu1−O1−Cu2

3.530(3.397)

1.909(1.892)

1.971(1.943)

130.9(124.7)

3.560(3.484)

1.901(1.897)

2.018(1.937)

130.5(130.7)

a

Experimental X-ray values are given in parentheses. Distances are

given in angstroms and angles in degrees.

As can be seen in Table 6, there is a good agreement between

the calculated and experimental parameters for complex 2. In

general, the difference between the calculated and experimental

distances is less than 0.08 Å, while the difference in the Cu−

O−Cu angle is only 0.2°. However, for complex 1, the

difference between the calculated and experimental Cu−O−Cu

angles is 6°, thus distorting the structure and making the

calculated angle identical in both structures. This finding

suggests that the deviation of this angle is most probably

responsible for the disagreement between the computed and

experimentally observed coupling constants and indicates that

the μ-alkoxo bridge is directly involved in the coupling process.

Again, as discussed in the interpretation of the experimental

J values for complexes 1 and 2 (vide supra), it can be concluded

that the Cu−O−Cu angle probably plays the most important

role in determining the degree and sign of the magnetic

80

state because a previous study showed that a better agreement

between the magnetic susceptibility measurements and the

computed magnetic coupling constant is obtained when the LS

79

geometry is used. Neese and co-workers also noted that the

use of the LS geometry to compute J seems to be more

appropriate because the electron density of the LS state

corresponds to the electron density of the real antiferromag-

netic state. The magnetic coupling constants computed are

given in Table 5.

−1

Table 5. Magnetic Exchange Coupling Constants, J, in cm ,

Calculated for Complexes 1 and 2 Using the B3LYP

a

Functional

1

2

II

coupling in these [Cu (μ-alkoxo)(μ-carboxylato)Cu )]

complexes.

A1

A2

A3

A1

A2

A3

J⁽¹⁾

6.96

3.48

6.94

0.47

−19.51

−9.76

−19.39

−19.41

−9.70

−19.28

−30.66

−15.33

−30.44

−39.08

−19.54

−38.55

Table 7 shows the spin density (ρ) values for both complexes

in their HS and BS states. Only the spin densities at the metallic

atoms, the O atom of the μ-alkoxo bridge, and the μ-caboxylate

atoms are quoted. The positive and negative signs indicate α

and β spin densities, respectively. As can be seen, the spin

density at the Cu1 center remains constant at around 0.6 for

both complexes, in their HS and BS spin states, compared to

the formal electron counting value of 1.0. In the BS state, the

spin density at the Cu2 center, as expected, is inverted, and the

magnitude of the β spin density is almost equivalent to those of

J⁽²⁾

0.23

0.47

J⁽³⁾

J ^e^x^p^.

−4.3

−40

a

A1: using the X-ray structures of 1 and 2. A2: using the X-ray

structures of 1 and 2 and optimizing only the positions of the H atoms.

A3: using the fully optimized geometry.

As can be seen, the exchange coupling constants calculated

using the different approaches shown in eqs 2−4 show similar

1

576

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Table 7. Mulliken Spin Densities for Complexes 1 and 2

mechanism, for compounds 1 and 2. For both complexes, only

one exchange pathway, with reasonable overlap, is found. From

inspection of the figure, we can see that the exchange pathway

can be described as a σ/π pathway involving the in-plane alkoxo

p orbitals and the metal d orbitals. As can be seen, the p orbitals

of the O atoms of the carboxylate group overlap with the d

orbitals of the metal sites. However, there is no participation of

the p orbitals of the C atoms of the carboxylate group, which

makes this group an inefficient mediator of the superexechange

interactions and, thus, the contribution of the carboxylate group

to the exchange mechanism is minor compared with the

μ-alkoxo ligand. These results indicate that the exchange

interaction in the coumpounds studied is mediated preferen-

tially by the superexchange mechanism via the O atoms of the

μ-alkoxo bridge.

atom

1

2

HS State

0.596

Cu1

Cu2

O1

0.578

0.621

0.618

0.131

0.130

C53

O51

O52

−0.006

0.055

−0.007

0.081

0.115

0.093

BS State

0.588

Cu1

Cu2

O1

0.580

−0.615

0.037

−0.610

−0.007

0.004

C53

O51

O52

−0.001

0.078

0.054

Electronic Spectroscopy, EPR, and Electrochemical

Properties. The electronic spectrum of 1 in an acetoni-

trile solution (Figure 7, solid line) shows a band centered at

−0.113

−0.092

the α spin densities computed in the HS state. The bridging O

atom also has a small α spin density in the HS state, which is

reduced in the BS state, indicating that the bridge is involved in

the magnetic coupling mechanism. It is noteworthy that the O

atom of the μ-caboxylate ligand has a small α spin density in the

HS state, which is inverted at O52 in the BS state, suggesting

that the carboxylate group may be involved in the coupling

mechanism. However, the spin density at the C atom of the

carboxylate group is negligible, which, in turn, avoids overlap

with the p orbitals of the O atoms.

−

1

−1

6

98 nm (ε = 169 L·mol ·cm ) and one at 890 nm (ε = 194

−

1

−1

II

L·mol ·cm ), which are typical of Cu d−d transitions.

A further band at 482 nm (ε = 225 L·mol ·cm ) can be

−

1

−1

attributed to a ligand-to-metal charge transfer (LMCT) be-

II

tween the phenolate and the Cu ions. This suggests that in

solution an equilibrium between the protonated and depro-

tonated coordinated phenols is most probably taking place. It

is important to mention here that the reflectance spectrum of 1

in the solid state does not show this band.

On the other hand, complex 2, in an acetonitrile solution

(Figure 7, dotted line), shows only one wide band centered at

8

2,83

Figure 6 shows plots of the corresponding orbitals

of

−1

II

the magnetic electrons, involved in the exchange coupling

700 nm (ε = 370 L·mol ·cm ), typical of Cu d−d transitions,

Figure 6. Magnetic orbitals of compounds 1 and 2 (the orbital number is quoted).

1

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consistent with a singlet−triplet splitting (interaction between

metallic centers) with weak antiferromagmetic coupling, and

thus the spectra of both complexes, including the difference in

intensity, are in full agreement with the magnetic coupling

constants J obtained by magnetic measurements (vide supra).

In addition, the microcrystalline EPR spectra of 1 and 2 are

II

similar to those observed for other dinuclear Cu Cu

II

complexes in which the Cu centers are weakly antiferromag-

8,28,84

netically or ferromagnetically coupled.

Nevertheless, it is

important to mention here that, for the few described examples

II

of dinuclear Cu systems containing the unpaired electron in

2

both d

and d magnetic orbitals, EPR spectra do not show

x −y

z

75

2

the proposed d ground state for copper.

z

The electrochemical behavior of the compounds was studied

using square-wave voltammetry. The square-wave voltam-

mograms for 1 and 2 are shown in Figure S1 in the Supporting

Information. A solution of 1 in acetonitrile displays a quasi-

Figure 7. Electronic spectra of 1 (solid line) and 2 (dotted line).

Concentrations: 1 (1.0 × 10 mol·L ) and 2 (3.0 × 10 mol·L ) in

acetonitrile.

−3

−1

−3

−1

reversible process at E = −388 mV vs NHE, corresponding

−

1

−1

pc1

and another one at 446 nm (ε = 900 L·mol ·cm ) attributed

II

I

to the Cu Cu /Cu Cu redox couple, and another irreversible

II

to a LMCT of the type between equatorial phenolate to Cu

process at E = −788 mV vs NHE, originating from the

pc2

ions, which is in full agreement with X-ray structure

characterization. In fact, the spectrum of 2 is very similar to

that obtained for 1, but with the band observed in the 850−

50 nm range being less defined for 2. The reflectance

spectrum of 2 in the solid state is very similar to that observed

II

I

Cu Cu /Cu Cu process. The square-wave voltammogram of 2

shows a similar behavior, with cathodic processes at E_p_c₁

=

−

560 mV and E_p_c₂= −1083 mV vs NHE, corresponding to the

9

same reduction processes. The reduction potentials observed

for complexes 1 and 2 are in full agreement with those reported

in acetonitrile, indicating that the coordination environment

around the Cu centers is maintained when complex 2 is

22

II

in the literature for complex 3 but with E_p_cvalues being

anodically shifted (Table 8). Complex 3 is structurally similar

dissolved in solution.

The X-band EPR spectra of solid samples of 1 and 2 at 77 K

are shown in Figure 8. Complex 1 shows a typical axial

Table 8. Electrochemical Data for Complexes 1−3

spectrum with four well-defined lines (g > g > 2.0), and the

∥

⊥

complex

E_p_c₁mV vs NHE

E_p_c₂mV vs NHE

ΔE₍₁_,₂₎mV vs NHE

following parameters were obtained from the simulated

1

2

−388

−560

−890

−788

−1083

−1110

400

523

220

−4

−1

spectrum: g = 2.265, g = 2.056, A = 175 × 10 cm , and

A_⊥= 22 × 10 cm .

∥

⊥

∥

−4

−1

22

3

The spectrum also shows an additional signal, although not

well-defined, at the half-field transition (1500 G), which can be

to complex 1, without tert-butyl groups localized in the

positions ortho and para to the phenolic donor O atom.

In fact, negative reduction potential is a tendency observed

for dinuclear copper(II) complexes with μ-alkoxo bridge and

phenolate ligands because of the electron density at the metal

assigned to the forbidden transition ΔM = ±2. These data

s

II

suggest that the two Cu ions are antiferromagnetically

coupled, as demonstrated through magnetic measurements.

Similarly, under identical conditions, the spectrum of

complex 2 shows a less defined fine structure for ΔM = ±1,

s

17,22

centers originating from the donor O atoms.

and the following parameters were obtained from the simulated

−

4

−1

Solution Studies: Potentiometric Titration, ESI-MS,

and EPR. In order to establish the catalytically relevant species

for catecholase-like activity and the hydrolysis of diester bonds,

potentiometric titration, EPR, and ESI-MS studies were carried

spectrum: g = 2.245, g = 2.07, A = 191 × 10 cm , and A =

1

∥

⊥

∥

⊥

5 × 10⁻cm . However, in complex 2, there is a stronger

4

−1

Cu−Cu interaction, evidenced by a well-defined and intense

signal at the half-field transition g ≅ 4.3. Such behavior is

Figure 8. X-band EPR spectra for complexes 1 (a) and 2 (b) in the solid state at 77 K: , experimental points; ---, simulated curve. Inset: EPR

spectrum showing the ΔM = ±2 transition.

s

1

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Figure 9. Species distribution curves of complexes 1 (left) and 2 (right) as a function of the pH, at 25 °C and I = 0.05 mol·L⁻¹(KCl).

Scheme 1. Deprotonation/Protonation Equilibrium Steps for Complex 1

Scheme 2. Deprotonation/Protonation Equilibrium Steps for Complex 2

out in ethanol/water or CH CN/water solution. It is important

0.10. When 1 and 2 are dissolved in aqueous solution (pH 3.0),

3

to mention here that, although no potentiometric titration in a

for both complexes, dissociation of the acetate bridge with

CH CN/water solution could be carried out for 1 and 2

replacement by two water molecules to generate species H C

3

(

precipitation of the complexes), pK values obtained for other

and H C for 1 and 2, respectively, is expected and strongly

a

4

dinuclear complexes, in water/ethanol (30:70), are only ∼0.15 pH

supported by the ESI-MS data (vide infra). Thus, pK_a₁is

attributed to the protonation/deprotonation equilibrium of the

released acetate/acetic acid group for complexes 1 and 2.

52

units lower than those in water/CH CN (50:50).

3

Potentiometric titration of complexes 1 and 2 (Figures 9

and S2 in the Supporting Information) was carried out in

For complex 1, H C has a water molecule coordinated to one

2

II

ethanol/water (70:30), in order to compare the pK values

Cu ion and a hydroxide ion coordinated to the other Cu ion;

HC represents the complex with two hydroxide ions

coordinated to the dinuclear centers, and C can be attributed

to deprotonation of the terminal phenol group that is bound

to the Cu1 center, as observed in the X-ray structure of 1.

In addition, the assignment of this species is in full agreement

with that determined through spectrophotometric titration of

1 under identical experimental conditions (Figure S3 in the

Supporting Information).

a

obtained by potentiometric titration with those obtained from

the kinetic experiments (hydrolase- and catecholase-like

activities of 1 and 2).

In ethanol/water (70:30), neutralization of 4 mol of

KOH per 1 mol of complex 1 and 5 mol of KOH per 1 mol

of complex 2, in the pH range of 3.00−12.00, was obtained,

and the deprotonation/protonation equilibrium steps and

corresponding pK values are proposed in Schemes 1 and 2,

a

respectively.

The pK values for complex 2 (4.49, 5.50, and 8.25) are

a

The pK values found for complex 1 are 4.32 ± 0.14, 6.13 ±

.19, 8.24 ± 0.11, and 9.43 ± 0.19, and those for complex 2 are

.49 ± 0.16, 4.70 ± 0.18, 5.50 ± 0.20, 8.25 ± 0.13, and 10.18 ±

attributed, respectively, to dissociation of the acetate bridge, a

a

II

0

4

water molecule coordinated to one Cu ion, and a water

II

molecule coordinated to the other Cu ion. These values are in

1

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agreement with the protonation/deprotonation equilibrium

described by Neves et al. for dinuclear copper(II) complexes

reveals that when the complex is dissolved in a CH CN/

3

22

water (1:1) buffer solution at pH 7.0, the g /A = 118 ratio is

∥ ∥

containing μ-alkoxo and terminal phenol/phenolate ligands.

significantly decreased compared to that of the solid-state

spectrum (g /A = 129), a strong indication that the ligand

Assignment of the pK values 6.13 and 5.50, respectively, for 1

a

∥

II

−

and 2, to deprotonation of the water molecule bound to the

field around the Cu centers is increased when OH groups

are coordinated. Indeed, these results are in full agreement

with the potentiometric data, which suggests that, under

II

Cu center containing the protonated phenol ligands is in

agreement with the higher Lewis acidity of this center

II

compared to the other Cu ions in the dinuclear complexes.

these experimental conditions, [(OH)Cu Cu (OH )] (H C in

2 2

In fact, this hypothesis is supported by the averaged Cu1−

Scheme 1) should be the main species present in solution.

II

N,O/Cu2−N,O bond distances around the Cu ions, which are

In addition, the spectrum of 1 shows a signal at the half-field

́

II

respectively 2.058/2.009 and 2.054/2.016 Å for 1 and 2. On the

transition (g ≅ 4.3), characteristic of dinuclear Cu Cu species

other hand, it seems that the same argument is not apparent for

the difference of ∼0.6 pH units observed between the

corresponding dissociation constants in 1 and 2 because the

averaged Cu1−N,O bond distances are similar in the two

complexes. However, the electrochemical data (Table 9) are in

with a weak antiferromagnetic coupling. Thus, it is reasonable

II

to conclude that in solution the dinuclear [(OH)Cu (μ-

II

alkoxo)Cu (OH )] structural unit is present as the catalytic

2

species (vide infra). The EPR results for complex 2 (Figure S6,

bottom, in the Supporting Information) are similar to

those presented for 1 and will not be discussed further in the

manuscript.

Table 9. Kinetic Parameters for the Oxidation Reaction of

II

3

,5-DTBC Catalyzed by Complexes 1−3 at pH 8.50 and

Finally, for both complexes, the formation of a Cu (μ-

II

25 °C

OH)Cu is disfavored most probably because of the rigid five-

II

membered rings formed by coordination of the Cu ions to the

V

k

K_a_s_s_.

k

/K

M

6

max

cat.

(s )

cat.

−1 −1

complex (mol·L ·s ) K_M₍_m_o_l_·_L−1₎

−

1

−1

1,3-diaminopropan-2-ol moiety. Detailed studies would

(L·mol ) (M ·s )

require EPR measurements at other frequencies, which is

outside the scope of the present work.

Reactivity. Kinetic Studies for the Catecholase-Like

Activity. The catecholase-like activity of the dinuclear copper-

7

−3

1

2

3

6.17 × 10⁻

3.47 × 10

0.025

0.051

0.0078

288.18

105.93

1000.0

7.41

5.47

8.10

1.24 × 10⁻

6

9.44 × 10

−3

a

−7

−4

1.90 × 10

9.50 × 10

a

2+ 22

3

represents the complex [Cu (L3)(μ−OAc)] .

2

(

II) complexes was determined by the catalytic oxidation of 3,5-

DTBC. This substrate is most widely used because of its low

redox potential, which facilitates oxidation to quinone, and its

bulky substituents, which make further oxidation reactions,

full agreement with these results and reveal that the reduction

potentials for 2 are anodically shifted by 172 and 295 mV

compared to those for 1, thus confirming the higher Lewis

85

such as ring opening, slower.

II

acidity of both Cu centers in complex 2. Finally, H C and C

3

The kinetic studies of the oxidation of 3,5-DTBC by

complexes 1 and 2 were carried out by the method of initial

rates by monitoring the increase in the characteristic quinone

represent the species resulting from deprotonation of the

equatorial and axial phenol groups, respectively. These species

were confirmed through spectrophotometric titration of 2

under experimental conditions identical with those employed in

the potentiometric tritration experiments (Figure S3 in the

Supporting Information).

(

3,5-DTBQ) absorption band, which is sufficiently stable and

displays a strong absorption band at 400 nm (ε = 1900

−1

L·mol ·cm ), whose appearance was monitored by UV/vis

spectroscopy over time. It was found that under anaerobic

conditions only 1 equiv of 3,5-DTBQ is formed, and when

oxygen is bubbled into the solution, the catalytic activity is

regenerated, indicating that the catalyst is still active and that

oxygen must participate directly in the catalytic cycle of the

oxidation reaction, acting as a thermodynamic driving force by

reoxidizing any copper(I) species and regenerating them to the

active copper(II) species. Therefore, in all cases, the solvent was

ESI-MS measurements for 1 and 2 were recorded in

CH CN/water (50:50), the same solvent conditions as those

3

employed for kinetic assays. For complex 1, three main group

of peaks are observed at mass-to-charge (m/z) ratios of 864.19,

7

64.25, and 382.12 (Figure S4 in the Supporting Information).

II

I

The peak at m/z 764.25 can be attributed to [Cu Cu (L1)-

+

II

(

CH CN)(OH )] , indicating reduction of the Cu center

3 2

coordinated by the soft side of the ligand ion under the

saturated with O before the kinetic experiments. To correct for

2

conditions of the ESI-MS, while the peak at m/z 382.12

the effect of the spontaneous reaction, the same solution was used

without adding the catalyst as an internal reference.

II

2+

corresponds to the dication [Cu Cu (L1)(CH CN)(OH )] .

3

2

II

The peak at m/z 864.19 is assigned to the [Cu Cu (L1)-

CH CN)(OH )(ClO )] species. On the other hand, when

The dependence of the oxidation reaction catalyzed by

complexes 1 and 2 on the pH was investigated within the range

of 5.5−9.0 to determine the pH value at which the catecholase-

like activity is at a maximum. For both complexes, a sigmoidal-

shaped profile was obtained, as seen in Figure S7 in the

Supporting Information. The data were fitted using a Boltzman

model, and a sigmoidal fit of the curves revealed kinetic pK_a

values of 8.0 ± 0.1 for complex 1 and 7.7 ± 0.1 for complex 2,

+

(

3 2 4

complex 2 is submitted to ESI-MS, a prominent peak at

m/z 779.25 can be observed (Figure S5 in the Supporting

II

I

Information). This peak is assigned to the [Cu Cu (L2)-

+

II

(

CH CN)(OH ] species (reduction of one Cu center) in

3 2

which CH CN and H O molecules are probably added to the

3

2

II

Cu centers during the ionization process as with complex 1. It

is important to emphasize that, for both complexes, there is no

observation of acetato-bridged species under these conditions,

in full agreement with the proposed protonation/deprotonation

equilibrium from potentiometric titration (vide supra).

which are in relative good agreement with the pK values

a

obtained from the potentiometric titrations; pK = 8.24 for 1

a

and pK = 8.25 for 2. Indeed, these results suggest that

a

II

deprotonation of a second water molecule coordinated to a Cu

EPR spectra for complexes 1 and 2 were obtained in

center in complexes 1 and 2 occurs to generate the active

dihydroxo species (HC in Schemes 1 and 2) for the oxidation

reaction of 3,5-DTBC. As was previously postulated for other

frozen CH CN/water (50:50) solutions, pH 7.0 at 77 K. The

3

spectrum of 1 (Figure S6, top, in the Supporting Information)

1

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2

dinuclear copper(II) complexes described in the literature,

it seems that the presence of bulky tert-butyl groups localized in

the positions ortho and para to the phenolic donor O atom in 1

and 2 is responsible for the significant decrease in K_a_s_s_.

compared to 3, and thus the formation of the intermediate

substrate/complex would be inhibited by steric constraints

caused by the ligand under kinetic conditions.

the pH dependence of the catalytic activity is most probably

due to the fact that the Cu -coordinated hydroxo groups are

II

able to interact with catechol, promoting deprotonation of the

substrate and its bridging coordination to the metal centers.

The activity of the enzyme is observed in the pH range of 5−8

(

optimum at pH 7.8), with the loss of activity below pH 4.0 and

With respect to the catalytic turnover number (kcat.), we

found that the reactivity in the oxidation of 3,5-DTBC increases

in the order 3 < 1 < 2. From the electrochemical data given in

Table 8, one can observe that there is a correlation between kcat.

and the reduction potential values of complexes 1−3. The more

3

5

above pH 9.0.

Saturation kinetics were obtained at pH 8.5. The graph of the

initial reaction rates (V ) versus 3,5-DTBC concentrations

shows a saturation profile (Figure 10). This dependence of the

0

anodically shifted the individual E values, the higher the

pc

catalytic turnover of the complex. Thus, as expected, complex

2

, which is the strongest oxidant within the series of catalysts,

shows the highest k , while complex 3 has the lowest

cat.

activity. In addition, we also found a good correlation

between the electrochemical parameter [(ΔE) = E_p_c₁

−

1

,2

E

] and the catalytic efficiency (k /K ) of complexes 1−3,

pc2

cat.

M

with complex 3 being the most efficient and complex 2, which

has the highest k , being the least efficient in the conversion

cat.

of 3,5-DTBC to the corresponding o-quinone. In fact, as was

2

previously emphasized by these authors, (ΔE) is an

1

,2

important parameter because it represents how readily the

metal centers accept the electron pair from the substrate in

the oxidation reaction, while the correlation between k /K_M

cat.

and (ΔE)₁supports the mechanistic idea that a one-electron

,2

intermediate is disfavored in this two-electron reaction. Thus,

one can conclude that despite the higher catalytic turnover of

complex 2 it represents the less efficient catalyst in the

oxidation of 3,5-DTBC, which can be attributed to its higher

(ΔE)₁_,₂within the series of complexes 1−3.

Figure 10. Dependence of the reaction rates on the 3,5-DTBC

concentration for the oxidation reaction catalyzed by complexes 1

The accumulation of H O during turnover was confirmed

2

(

■

) and 2 (●), in a methanol/water (32:1 v/v) solution.

−

by means of the molybdate-accelerated I₃assay (75% of

the expected amount was detected), which indicates that

reoxidation of the copper(I) species back to the active

−

5

−1

Conditions: [complex] = 2.40 × 10 mol·L ; [3,5-DTBC] =

(

−

3

−1

−2

−1

1.20−12.00) × 10 mol·L ; [buffer] = 3.30 × 10 mol·L (TRIS,

pH 8.50) at 25 °C.

copper(II) species occurs with a 1:1 O /3,5-DTBC stoichi-

2

ometry and concomitant formation of hydrogen peroxide.

The release of H O is mechanistically distinct from the

native COs.

rate on the substrate concentration suggests that the oxidation

reaction occurs with the formation of an intermediate

substrate/complex. The Michaelis−Menten model was applied,

and the kinetic parameters were obtained from nonlinear

2

8

6

Finally, the catecholase-like activity was also studied in the

absence of oxygen. The reaction was carried out under identical

kinetic conditions, observed over 60 h at 25 °C, and only

square fits using the program Origin 6.0. The parameters V_m_a_x

K_M, and k_c_a_t_.are shown in Table 9.

,

1

equiv of quinone was found, which demonstrated that oxygen

As can be observed in Table 9, complex 1 is more effective in

must participate in the catalytic cycle of the oxidation reaction

the conversion of the substrate 3,5-DTBC to the corresponding

I

II

by reoxidizing any Cu back to the active Cu species.

−

1 −1

o-quinone, with a catalytic efficiency of 7.41 L·mol ·s

In summary, the combined experimental data support a

mechanistic model for the oxidation reaction of 3,5-DTBC

catalyzed by 1 and 2, as given in Figure 11. At pH 8.5, in the

first step of the reaction, we propose that the dihydroxo

−1 −1

compared to 5.47 L·mol ·s found for complex 2. This is a

−1

reflection of its association constant (K_a_s_s_.= 288.2 L·mol ),

which is around 3 times higher in comparison to that observed

3

−1

for complex 2. Interestingly, the value of K_a_s_s_.= 10 L·mol

previously reported for 3 is around 4 and 10 times higher than

those observed for 1 and 2, respectively. It is important to

emphasize that complex 3 corresponds to complex 1 without

substituents at the phenolic donor atoms and complexes 1 and

II

[

(OH)Cu Cu (OH)] complex is able to interact with catechol,

promoting deprotonation of the substrate and coordination to

II

the Cu centers and forming the adduct complex/substrate.

Next, an intramolecular electron-transfer reaction, as the rate-

determining step, results in oxidation of the catechol substrate

2

have respectively 2+ and 1+ formal charges. Indeed, several

II

to the corresponding o-quinone and reduction of the Cu

I

factors must be considered in assessing the difference in the

oxidation activities of complexes 1−3, such as the association

affinity, mode of interaction of substrate, electrochemical

centers to Cu . The Cu Cu complex in the presence of O is

2

immediately oxidized back to the original form, hydrogen

peroxide is formed, and the catalytic cycle is completed.

It is important to note that the release of H O observed in

22

properties, exogenous donors, and steric match. Thus, the

difference in the association constants for complexes 1 and 2

can be attributed to the different formal charges in these

catalysts, with K_a_s_s_.being around 3 times higher for 1, under

identical experimental conditions (pH 8.5). On the other hand,

2

the oxidation of 3,5-DTBC by 1 and 2 is mechanistically

86

distinct from the type III dinuclear enzyme COs. On the

other hand, the proposed mechanism (Figure 11) resembles

that of the copper-loaded form of S100B in which the catalytic

1

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Figure 11. Proposed mechanism for the oxidation of 3,5-DTBC catalyzed by complex 1. The mechanism proposed for 2 is similar.

two-electron oxidation of catechols is initiated by two strongly

The studies on the hydrolysis rate as a function of the 2,4-

II

coupled Cu centers and the release of H O .

BDNPP concentration in reactions catalyzed by complexes 1

and 2 were carried out at pH 8.0, where both complexes

showed the highest activity in the catalysis (Figure 12). Initially,

the cleavage rate increases linearly with an increase in the 2,4-

BDNPP concentration, but it deviates gradually from linearity

and tends toward a saturation curve at higher concentrations of

the substrate. The data were treated using the Michaelis−

Menten model, and the parameters V , K , and k were

2

Kinetic Studies for the Hydrolase-Like Activity. Kinetic

experiments were carried out to assess the ability of complexes

1

and 2 to catalyze hydrolysis of the substrate 2,4-BDNPP.

Under excess substrate conditions, the experiments were

performed in triplicate and spectrophotometrically monitored

−1

at 400 nm (pH/ε L·mol ·cm = 3.5/2125, 4.0/3408, 4.5/

5

2

7

1

182, 5.0/10 078, 5.5/11 405, 6.0/12 004; 6.5−10.0/12

max

M

cat.

5

3

−1

−1 53

obtained by nonlinear least-squares fits. The kinetic parameters

for the hydrolysis of 2,4-BDNPP promoted by complexes 1 and

are described in Table 10, together with the data for 3, where

it can be observed that complex 2 is most effective in the

00) and/or 445 nm (ε = 3600 L·mol ·cm ), using a

Varian Cary 50 Bio UV/vis spectrophotometer coupled to a

thermostatic bath. Reactions were monitored to less than 5%

conversion of 2,4-BDNPP to 2,4-DNP, and the data were

treated using the initial rate method.

The activity of complexes 1 and 2 in the cleavage of 2,4-

BDNPP is strongly influenced by the pH of the reaction

mixture and reveals sigmoidal-shape profiles (Figure S8 in the

Supporting Information). The data were fitted using a

2

conversion of substrate to products, with a turnover rate that is

1

3

329 times faster than that of the uncatalyzed reaction (k

=

unc

.88 × 10⁻

7

s

−1

at pH 8.0 at 50 °C).

41

A comparison of the kinetic parameters obtained fo₆r

complexes 1 and 2 with those previously described for 3

reveals that the presence of the electron-donor tert-butyl groups

in complexes 1 and 2 caused significant changes in the

Boltzman model, resulting in pK = 6.36 ± 0.1 for complex 1

a

and pK = 7.45 ± 0.1 for complex 2. In fact, the kinetic pK =

6

a

hydrolytic cleavage of the substrate 2,4-BDNPP. As expected,

6.36 is in good agreement with the value found in the

II

according to the pK values for the equilibrium [(OH )Cu (μ-

a

2

potentiometric titration for complex 1 (pK = 6.13). However,

II

a

alkoxo)Cu (H O)] ↔ [(OH)Cu (μ-alkoxo)Cu (H O)], in

2

for complex 2, the pK = 7.45 is around 2.0 pH units higher

a

which the catalytically active species is generated, complex 2,

than that calculated from the potentiometric experiments

which has the highest pK (7.45), is the most active in the

a

(

pK = 5.50). A plausible explanation for this experimental

a

hydrolysis of the diester, while complex 3 has the lowest

observation is based on the strong interaction of the substrate

,4-DBNPP with catalyst 2 due to the formation of strong

catalytic turnover rate because it has the lowest pK (6.2) and

a

2

II

−

thus the lowest nucleophilic reactivity of the Cu -bound OH

group. A similar trend has recently been observed for a series of

hydrogen bonds between the entering substrate and the

protonated phenol ligand (vide infra) that in some way affects

III

II

dinuclear mixed-valence [Fe (μ-OH)Zn (L-R)] complexes in

which the catalytic turnover k_c_a_t_.for the hydrolysis of 2,4-

II

the Lewis acidity of the Cu centers, which are much more

antiferromagnetically coupled in 2 compared to 1, although

such an interpretation should be considered only speculative at

present because no further experimental and theorethical

evidence is available at present. Therefore, deprotonation of a

BDNPP has a linear dependence on the pK values (LFER)

a

III

attributed to deprotonation of the terminal Fe -bound water

52

for distinct substituents in the ligand L-R.

Concerning the affinity of the substrate 2,4-BDNPP with the

catalysts 1−3 to generate the intermediate substrate/complex,

it is observed that the reactions follow a trend similar to that

II

Cu -coordinated water molecule may occur at a higher pH to

II

generate the catalytically active species [(OH)Cu (μ-alkoxo)-

II

Cu (H O)] (species H C in Scheme 2) in complex 2.

observed for k , with 2 showing the highest association

2

cat.

1

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Figure 12. Dependence of the initial reaction rate on the 2,4-BDNPP concentration for the hydrolysis reaction promoted by complexes 1 (a) and 2

−5

−1

(

b). Conditions: solution 1:1 CH CN/H O; [complex] = 4.0 × 10 mol·L ; [buffer] = 0.05 mol·L HEPES (pH 8.0); I = 0.05 mol·L LiClO₄;

3

2

−

3

−1

[

2,4-BDNPP] = (0.5−8.0) × 10 mol·L at 50 °C.

Table 10. Kinetic Parameters for the Hydrolysis Reaction of 2,4-BDNPP, Catalyzed by Complexes 1 and 2

−1

K_M(mol·L⁻¹)

k_c_a_t_.(s⁻¹)

K_a_s_s_.(L·mol⁻¹)

−1 −1

complex

V_m_a_x(mol·L ·s )

k_c_a_t_./K_M

k_c_a_t_./k_u_n_c(M ·s )

1.19 × 10⁻

8

3.82 × 10

−3

2.97 × 10

−4

261.78

512.82

103.30

0.08

0.26

0.02

767

1329

86

1

2

2.06 × 10⁻

8

1.95 × 10

−3

5.16 × 10

−4

a

−8

−3

−4

3

0.9 × 10

9.68 × 10

1.80 × 10

a

2+

6

3

represents the complex [Cu (Hbtppnol)(OAc)] .

2

−

3

−1

−5

constant K_a_s_s_.and complex 3 the lowest. However, if we

consider the formal charges of the complexes, the reverse

behavior would be expected under identical experimental

conditions. We tentatively explain these results by taking into

account the possibility of strong hydrogen-bond formation

between the entering substrate and the protonated phenol

ligands in complexes 1 and 2 (vide supra) that should also

contribute to stabilization of the transition state in these

(2.0 × 10 mol·L ) promoted by complexes 1 and 2 (4.0 × 10

−1

mol·L ) at 445 nm, pH 8.0, and 50 °C was also monitored.

These data revealed that over 24 h the complexes were able to

hydrolyze 5 and 20 molecules of substrate for complexes 1 and 2,

respectively. Furthermore, the measured deuterium kinetic

isotope effect k /k of 0.89 for 1 and 0.80 for 2 suggests

that no proton transfer is involved in the rate-limiting step and

thus supports an intramolecular nucleophilic attack.

On the basis of the X-ray structures, solution studies, and

kinetic data, the mechanism shown in Figure 13 is proposed for

the hydrolysis reaction of the phosphate diester 2,4-BDNPP by

complexes 1 and 2. The dependence of the reaction rate on the

pH indicates that the active species in the hydrolysis reaction is

the aquo/hydroxo form of the complexes [(OH)Cu (μ-alkoxo)-

Cu (H O)], in agreement with the potentiometric titration

results, which is formed when a proton is removed from one of

the Cu -bound water molecules in the complexes. This species

possesses a labile coordination site (H O) that permits the

binding of a substrate molecule to a metallic center and a metal-

coordinated nucleophile group (OH ) available for intramolecular

attack. We propose double electrophilic activation of the

89

H

D

hydrolysis reactions. The pK = 7.45 obtained from kinetic

a

II

measurements for 2 (assigned to dissociation of the Cu -bound

H O in the substrate/complex intermediate), which is ∼2.0 pH

2

units higher than that calculated from the potentiometric

experiments, strongly corroborates this assignment, and thus

complex 2 shows a catalytic efficiency that is 10 times higher

than that observed for complex 3. A double electrophilic

activation of the phosphodiester by coordination to the metal

center and hydrogen bonding to the ammonium-functionalized

ligand in a mononuclear copper(II) complex has been

postulated to accelerate hydrolysis with a greater efficiency

compared to related systems that do not contain such a

II

2

II

2

−

87

III

II

functional group. The dinuclear Fe Mn sweet potato PAP

has considerably higher substrate turnover rates than other

PAPs, and this has been interpreted in terms of Glu365 and

Asp158 acting as proton donors for the leaving groups, at least

II

phosphodiester by monodentate coordination to the Cu center

that contains the phenol group with tert-butyl substituents and

hydrogen bonding of the protonated phenol with the phosphate

^O^a^t^o^m^.^T^hⁱ^sⁱⁿ^c^r^e^a^s^e^s^t^h^e^Kass. and k values in comparison

with those found for complex 3. In the next step, an intra-

molecular nucleophilic attack by the cis-oriented Cu OH

8

at low pH.

In order to assess the possible hydrolysis of the monoester 2,4-

DNPP, one of the products formed from the hydrolysis of the

diester 2,4-BDNPP, stoichiometric reactions between complexes

cat.

6

II

1

and 2 and the substrate 2,4-BDNPP were monitored. It was

group leads to the release of 2,4-dinitrophenolate and bridging

coordination of the monoester 2,4-DNPP. Finally, the μ-1,3-

coordinated monoester intermediate undergoes substitution by

two water molecules and regenerates the active site for the next

catalytic cycle. Under stoichiometric conditions, the release of

observed that, for both complexes, over 7 days at 50 °C, only 1

equiv of 2,4-dinitrophenolate is released, which indicates

only diesterase activity of 1 and 2. Because our main goal was

to obtain an effective catalyst, a hydrolysis reaction of 2,4-BDNPP

1

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Figure 13. Proposed mechanism for hydrolysis of 2,4-BDNPP catalyzed by complex 1. The mechanism proposed for 2 is similar.

Figure 14. Plot of % plasmid DNA forms (FI and FII) after incubation with a concentration of 20 μM 1 (A) and 2 (B) at different pH values for 6 h

−1

at 37 °C. Different buffers (25 mmol·L ) were used according to the pH: PIPES (pH 6.0 and 7.0), HEPES (pH 8.0), and CHES (pH 9.0).

−

1

only 1 equiv of 2,4-dinitrophenolate was observed, which suggests

the formation of a stable Cu(μ-2,4-DNPP)Cu complex.

(20 μmol·L ) at 37 °C, which indicates that the cleavage

rate increases with an increase in the pH value, with maximum

activity at pH 9.0.

DNA Cleavage Activity. Complexes 1 and 2 were assayed

in the cleavage of plasmid DNA (pBSK II, 30 μmol·L⁻bp) to

assess their nuclease activity. DNA cleavage was measured by the

conversion of supercoiled DNA (FI) to the open-circular (FII)

and/or linear (FIII) forms. Free Cu ions were not observed to

cleave supercoiled DNA under the conditions of this study (6 h of

incubation at 37 °C; Figure S9 in the Supporting Information).

Initially, the DNA cleavage activity of 1 and 2 was analyzed within

a broad pH range (6.0−9.0). Figure 14 presents a pH-dependent

profile for DNA cleavage promoted by both complexes

1

The effect of the complex concentration on the cleavage of

DNA was investigated under the optimum pH conditions.

Figure 15 shows that both complexes were able to cleave DNA

at all concentrations assayed (10−50 μmol·L ). The amount of

cleaved DNA increased with an increase in the concentration of

the complexes. After 6 h of incubation at 37 °C, a 50 μmol·L⁻

solution of 1 and 2 converted ∼70% and 84%, respectively, of

supercoiled DNA (FI) to open-circular DNA (FII). Complex 2

also promoted the formation of 15.3% linear DNA (FIII).

−1

1

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−

1

Figure 15. Agarose gel electrophoresis of plasmid DNA cleavage by 1 (A) and 2 (B) at different concentrations and at optimum pH [25 mmol·L

−1

CHES (pH 9.0)]. Lanes 1 and 7: DNA control. Lanes 2−6: DNA + 1 (10, 20, 30, 40, and 50 μmol·L , respectively). Lanes 8−12: DNA + 2 (10, 20,

−1

3

0, 40, and 50 μmol·L , respectively). Incubation: 6 h at 37 °C.

−1

Figure 16. Cleavage of pBSK II (25 μmol·L bp) by complex 1 under aerobic (oxygen atmosphere) and anaerobic (argon atmosphere) conditions

−

1

−1

at pH 8.0 (25 mmol·L HEPES buffer) and 9.0 (25 mmol·L CHES buffer). Lanes 1, 5, 9, and 13: DNA control. Lanes 2 and 6: DNA + 1

−1

(

40 μmol·L ). Lanes 3 and 7: DNA + 1 (100 μmol·L ). Lanes 10 and 14: DNA + 1 (20 μmol·L ). Lanes 11 and 15: DNA + 1 (40 μmol·L ).

1

−

Lanes 4, 8, 12, and 16: 100 μM Fe-EDTA + 10 mmol·L DTT. Incubation: 6 h at 37 °C.

−1

Figure 17. Cleavage of pBSK II (25 μmol·L bp) by complex 2 under aerobic (oxygen atmosphere) and anaerobic (argon atmosphere) conditions

−

1

−1

at pH 8.0 (25 mmol·L HEPES buffer) and 9.0 (25 mmol·L CHES buffer). Lanes 1, 5, 9, and 13: DNA control. Lanes 2 and 6: DNA + 2

−1

(

30 μmol·L ). Lanes 3 and 7: DNA + 2 (40 μmol·L ). Lanes 10 and 14: DNA + 2 (10 μmol·L ). Lanes 11 and 15: DNA + 2 (20 μmol·L ). Lanes 4,

1

−

−1

8

, 12, and 16: 100 μmol·L Fe-EDTA + 10 mmol·L DTT. Incubation: 6 h at 37 °C.

In order to determine the influence of O on the DNA cleavage

model substrate 2,4-BDNPP. Consequently, at pH 9.0, the

2

process, an aerobic versus anaerobic (argon atmosphere)

experiment was performed at pH 8.0 and 9.0. It was observed

that DNA cleavage promoted by 1 was inhibited in an argon

atmosphere at both pH values (Figure 16). However, the

inhibition was substantially greater at pH 9.0 than pH 8.0,

indicating a major oxidative mechanism at pH 9.0 and a major

hydrolytic mechanism at pH 8.0. According to the potentiom-

etric titration species distribution curves (see the Potentiometric

Titration section), at pH 8.0, there is a higher percentage of the

hydrolytic activity of 1 declines because the percentage of aquo/

hydroxo species decreases considerably, but this is the optimum

pH, probably because of the fact that the oxidative activity

increases. Concerning complex 2, a similar behavior was observed

in an argon atmosphere because the nuclease activity also showed

a greater decrease at pH 9.0 than at pH 8.0, suggesting a higher

hydrolytic activity at pH 8.0, where the aquo/hydroxo form of the

complex is also the active species in catalysis. However, at pH 9.0,

the inhibition was only partial, indicating a mixed cleavage

mechanism involving hydrolysis and oxidation of plasmid DNA in

similar proportions (see Figure 17). Thus, at the optimum pH, the

oxidative activity is higher for complex 1, as observed in oxidation

II

aquo/hydroxo form of the complex [(OH)Cu (μ-alkoxo)-

II

Cu (H O)]. Thus, this species is most probably associated with

2

the hydrolysis of plasmid DNA, as was observed in catalysis of the

1

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Figure 18. DNA cleavage of plasmid DNA pBSK II (25 μmol·L⁻¹in bp) by 1 (40 μmol·L ) (A) and 2 (10 μmol·L ) (B) for 6 h at 37 °C with

−1

different cleavage inhibitors: DMSO (10% v/v), KI (500 μmol·L ), SOD (20 units), and NaN (500 μmol·L ).

3

of the model substrate 3,5-DTBC, and the hydrolytic activity is

more pronounced for complex 2, as obtained in hydrolysis of the

model substrate 2,4-BDNPP (see the Reactivity section).

In addition, the ROS involved in the oxidative DNA cleavage

process was investigated by the use of different potential

inhibitors (see Figure 18). The DNA cleavage promoted by

1

was inhibited in the presence of DMSO and KI, which

suggests that hydroxyl radicals and hydrogen peroxide play

an important role in the cleavage mechanism pathway

(Figure 20A). In contrast, the nuclease activity of 2 was attenuated

in the presence of DMSO and SOD, indicating participation of the

hydroxyl and superoxide radicals in the scission event (Figure 20B).

Furthermore, in the presence of the copper(I) chelator bath-

ocuproine, the activity of 1 and 2 was significantly reduced

Figure 20. Influence of DNA groove binders distamycin and methyl

−1

green (50 μmol·L ) on the DNA cleavage activity of 1 (40 μmol·L )

−1

II

I

(A) and 2 (10 μmol·L ) (B). Reactions were performed in 25 mmol·L

CHES (pH 9.0) and incubated for 6 h at 37 °C.

(Figure 19), demonstrating that the reduction of Cu to Cu is an

important step in the cleavage process.

The effect of the ionic strength on the nuclease activity of

both complexes was determined by observing the effect of

NaCl on the DNA cleavage reaction. Figure 21 shows that the

process of cleavage by both complexes was sensitive to a change

in the ionic strength. The extent of DNA cleavage decreased

from more than 90% to a maximum of 30% and 50% for 1

and 2, respectively, when the ionic strength increased from

−1

2

0 to 200 mmol·L . These results suggest that electrostatic

interactions contribute to the DNA cleavage because the DNA

molecule is negatively charged and complexes 1 and 2 present a

positive net charge.

These results led us to propose a mechanistic pathway

through which the complexes cleave the plasmid DNA at

optimum pH. Complex 1 approaches plasmid DNA via electro-

static interactions with the phosphate backbone of DNA, and

−

1

Figure 19. Effect of bathocuproine (500 μmol·L ) on the plasmid

II

I

−1

the Cu sites are reduced to Cu . This reduction can be

attributed to electron transfer between the complex and DNA

DNA cleavage by 1 (40 μmol·L ) (A) and 2 (10 μmol·L ) (B).

Reactions were performed in 25 mmol·L CHES (pH 9.0) and

incubated for 6 h at 37 °C.

−1

9

0

in solution. O then reacts with the copper(I) complex,

2

•

generating OH , which attacks the DNA. Complex 2, in

Moreover, to study the binding of the complexes to DNA,

assays were performed using the minor groove binder

distamycin and the major groove binder methyl green. Figure

contrast, interacts with DNA via electrostatic attraction and

binds to the minor groove. The oxidative cleavage occurs as

•

−

described for complex 1, but the radicals OH and O are

2

0A shows the results obtained with 1. No apparent inhibition

generated as ROS. In a second pathway, the binding of the

aquo/hydroxo form of the complex at the minor groove

promotes the hydrolysis of DNA.

Kinetic measurements were carried out to monitor the

decrease in the amount of supercoiled DNA at different

reaction times and complex concentrations. The plots of k_o_b_s

values versus concentrations of both complexes are shown in

of the DNA cleavage activity was observed, indicating that

interaction of this complex with plasmid DNA does not occur

through the grooves. However, the DNA damage produced by

2

was partially inhibited in the presence of distamycin (see

Figure 20B). This finding indicates that complex 2 interacts

through the minor groove and then cleaves the plasmid DNA.

1

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−1

Figure 21. Effect of the ionic strength on supercoiled plasmid DNA cleavage by 1 (80 μmol·L ) (A) and 2 (10 μmol·L ) (B). Reaction conditions:

−1

25 mmol.L CHES buffer (pH 9.0). Incubation: 6 h at 37 °C.

Figure 22. Plot of k values versus concentrations of complexes 1 (A) and 2 (B), showing saturation behavior. Reactions were performed at pH 9.0

obs

−1

(

25 mmol·L CHES buffer) with 300 ng (25 μmol·L bp) of plasmid pBSK II and different concentrations of the complexes. Incubations were

performed at 37 °C, and aliquots were removed at different time intervals (0−6 h).

6

7

Figure 22. The pseudo-Michaelis−Menten kinetic parameters

are listed in Table 11.

reactivity, with 3.4 × 10 - and 1.5 × 10 -fold rate enhance-

ments, respectively. Compared to the previously published

complex 3 (k_o_b_s= 0.022 h ; half-life = 30.91 h, rate enhan-

cement of DNA cleavage of 6.2 × 10 at 50 °C), we clearly

observe that 1 and 2 are more efficient, even at the lower

temperature used in this study.

The catalytic efficiency reveals that 2 (2.3 × 10 h ·M )

is ∼23 times more active as a chemical nuclease than 1

(1.0 × 10 h ·M ) and the estimated supercoiled DNA half-

life times in the presence of 1 and 2 are ∼5.8 and ∼1.3 h,

respectively. The higher activity of complex 2 compared to

complex 1 is probably due to the fact that 2 interacts more

strongly with DNA (through electrostatic attractions and

binding at the minor grooves) and can thus promote oxidation

and hydrolysis in greater proportions.

In summary, as observed in hydrolysis of the model substrate 2,4-

BDNPP, the presence of electron donor tert-butyl groups localized

in positions ortho and para to the phenolic donor O atom in 1 and

2 caused significant changes in the plasmid DNA cleavage compared

to other analogous dinuclear copper(II) complexes found in the

−1

5

Table 11. Kinetic Parameters of 1 and 2 in DNA Cleavage

under Pseudo-Michaelis−Menten Conditions

a

b

d

k

K_M

k

_c_a_t_./K

t₁_/₂

4

−1 −1

cat.

M

−

1

−1

c

complex

(h )

(mol·L )

(h ·mol ·L)

k_c_a_t_./k_u_n_c

(h)

₁_.₂_×₁₀−

4

3

₃_.₄_×₁₀6

1

2

0.12

0.55

1.0 × 10

2.3 × 10

5.8

1.3

3

−1 −1

₂_.₄_×₁₀−

5

4

₁_.₅_×₁₀7

a

b

Catalytic constant (i.e., V

at the saturation level). Catalytic

max

c

efficiency. Rate enhancement compared to uncatalyzed cleavage of

double-stranded DNA (k_u_n_c= 3.6 × 10 h ). Supercoiled DNA half-

−8

−1

d

life time (t₁_/₂= −ln(0.5)/k_c_a_t_.).

As is well-known, the first-order hydrolysis rate constant for a

phosphodiester bond of double-stranded DNA is estimated to

be 3.6 × 10⁻h . Reported rate enhancements for copper(II)

8

−1

6

8

55,90

complexes are in the range of 1.1 × 10 −2.3 × 10 -fold.

−1

The k values of 0.12 and 0.55 h obtained for 1 and 2,

cat.

respectively, indicate that these complexes exhibit high cleavage

1

587

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Inorganic Chemistry

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6

,91,92

literature,

2

because of stronger interaction of the catalysts 1 and

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(

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CONCLUSIONS

́

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Schenk, G. Chem. Rev. 2006, 106, 3338−3363.

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II

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Haase, W. Inorg. Chim. Acta 2005, 358, 1807−1822.

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that the greater bridging Cu−O−Cu angle (θ) of 130.66° in 2

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(10) Young, M. J.; Wahnon, D.; Hynes, R. C.; Chin, J. J. Am. Chem.

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(

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the most active because it is the strongest oxidant compared to 1

and 3. In addition, the complexes were shown to efficiently

catalyze hydrolysis of the diester model substrate 2,4-bis-

2

(

3

(

5

(

dinitrophenyl)phosphate and to cleave plasmid DNA under

mild pH and temperature conditions in the following order of

reactivity: 2 > 1 > 3. Herein, we proposed that double

electrophilic activation of the phosphodiester 2,4-BDNPP by

II

monodentate coordination to the Cu center that contains

(

the phenol group with tert-butyl substituents and hydrogen

bonding of the protonated phenol with the phosphate O atom

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increase the hydrolase activity (K_a_s_s_.and k ) of 1 and 2 in

cat.

comparison with that found for complex 3.

(

́

18) Selmeczi, K.; Reglier, M.; Giorgi, M.; Speier, G. Coord. Chem.

ASSOCIATED CONTENT

■

Rev. 2003, 245, 191−201.

*

S

Supporting Information

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Inorg. Chem. 2002, 41, 3983−3989.

X-ray crystallographic data in CIF format and Figures S1−S9.

This material is available free of charge via the Internet at

http://pubs.acs.org. Crystallographic data (without structure

factors) for the structure(s) reported in this paper have also

been deposited with the Cambridge Crystallographic Data

Centre as CCDC 802096 for complex 1 and CCDC 802100 for

complex 2. Copies of the data can be obtained free of charge

from the CCDC (12 Union Road, Cambridge CB2 1EZ, U.K.;

tel (+44) 1223-336-408; fax (+44) 1223-336-003; e-mail

deposit@ccdc.cam.ac.uk; web site www.ccdc.cam.ac.uk).

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(

́ ́

kanyi, L.; Korecz, L.;

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necker, B.;

AUTHOR INFORMATION

■

̈

Corresponding Author

(

*

E-mail: ademir@qmc.ufsc.br. Tel.: +55 4837219219. Fax: +55

837219711.

4

̈

ken, M.; Moller, N.; Rompel, A.; Krebs, B.

(

ACKNOWLEDGMENTS

■

The authors are grateful for grants awarded to support this

research from CNPq, FAPESC, CAPES-PROCAD, and INCT-

3

20, 12−21.

(29) Torelli, S.; Belle, C.; Gautier-Luneau, I.; Pierre, J.-L.; Saint-

Catal

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ise (Brazil), BMBF/IB (Germany), and NN202103238

Aman, E.; Latour, J. M.; Le Pape, L.; Luneau, D. Inorg. Chem. 2000, 39,

3526−3536.

(Poland). We thank Juraj Pelikan for some magnetic experiments.

1

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Synthesis, magnetostructural correlation, and catalytic promiscuity of unsymmetric dinuclear copper(II) complexes: Models for catechol oxidases and hydrolases

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