Reactivity of dinuclear copper(II) complexes towards melanoma cells: Correlation with its stability, tyrosinase mimicking and nuclease activity

Article abstract of DOI:10.1016/j.jinorgbio.2015.05.007

In this work, the influence of two new dinuclear copper(II) complexes in the viability of melanoma cells (B16F10 and TM1MNG3) was investigated, with the aim of verifying possible correlations between their cytotoxicity and their structure. One of the comp

Full text of DOI:10.1016/j.jinorgbio.2015.05.007

Journal of Inorganic Biochemistry 149 (2015) 49–58
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Reactivity of dinuclear copper(II) complexes towards melanoma cells:
Correlation with its stability, tyrosinase mimicking and nuclease activity
Cléia Justino Nunes a, Beatriz Essenfelder Borges b, Lia Sumie Nakao b, Eugénie Peyroux c, Renaud Hardré c,
Bruno Faure c, Marius Réglier c, Michel Giorgi d, Marcela Bach Prieto e,
Carla Columbano Oliveira e, Ana M. Da Costa Ferreira a,
⁎
a
Departamento de Química, Instituto de Química, Universidade de São Paulo, 05508-000 São Paulo, SP, Brazil
Departamento de Patologia Básica, Universidade Federal do Paraná, 81531-990 Curitiba, PR, Brazil
b
c
Aix Marseille Université, Centrale Marseille, CNRS, ISM2 UMR 7313, 13397 Marseille, France
Aix Marseille Université, CNRS, Spectropole FR1739, 13397 Marseille, France
d
e
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, the influence of two new dinuclear copper(II) complexes in the viability of melanoma cells (B16F10
and TM1MNG3) was investigated, with the aim of verifying possible correlations between their cytotoxicity and
their structure. One of the complexes had a polydentate dinucleating amine–imine ligand (complex 2), and the
other a tridentate imine and a diamine-bridging ligand (complex 4). The analogous mononuclear copper(II) spe-
cies (complexes 1 and 3, respectively) were also prepared for comparative studies. Crystal structure determina-
tion of complex 2 indicated a square-based pyramidal geometry around each copper, coordinated to three N
atoms from the ligand and the remaining sites being occupied by either solvent molecules or counter-ions. Com-
plex 4 has a tetragonal geometry. Interactions of these complexes with human albumin protein (HSA) allowed an
estimation of their relative stabilities. Complementary studies of their reactivity towards DNA indicated that all of
them are able of causing significant oxidative damage, with single and double strand cleavages, in the presence of
hydrogen peroxide. However, nuclease activity of the dinuclear species was very similar and much higher than
that of the corresponding mononuclear compounds. Although complex 2, with a more flexible structure, exhibits
a much higher tyrosinase activity than complex 4, having a more rigid environment around the metal ion, both
complexes showed comparable cytotoxicity towards melanoma cells. Corresponding mononuclear complexes
showed to be remarkably less reactive as tyrosinase mimics as well as cytotoxic agents. Moreover, the dinuclear
complexes showed higher cytotoxicity towards more melanogenic cells. The obtained results indicated that the
structure of these species is decisive for its activity towards the malignant tumor cells tested.
Received 19 November 2014
Received in revised form 8 May 2015
Accepted 11 May 2015
Available online 19 May 2015
Keywords:
Dinuclear copper complexes
Tyrosinase mimics
EPR and CD spectroscopies
Cell viability
Melanomas
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
(L-dopa), leading to the formation of both forms of melanins, eumelanin
and pheomelanin, possibly involved in the development of malignant
Tyrosinase is a ubiquitous enzyme found in bacteria, fungi, animals
and humans, capable of catalyzing the oxidation of phenols and catechols
to the corresponding quinones [1]. This enzyme plays also a key role in
melanogenesis, besides related proteins (TRP-1 and TRP-2), leading to
melanins, which are biological polymers produced by melanocytes, and
that confer color to the skin, hair, and eyes. Melanins protect the skin
from harmful solar radiation, and the level of melanin in epidermis is pos-
itively correlated with the tyrosinase activity [2]. The synthesis of melanin
initiates by the action of tyrosinases on L-3,4-dihydroxyphenylalanine
melanomas [3]. Melanins can actually have a dual role, acting as a pro-
or as an antioxidant agent. A recent paper on novel properties of melanins
reported its capability of coordinating metal ions, such as copper, and
binding to DNA, with consequent promotion of DNA strand breaks, prob-
ably by a Fenton-type reaction [4]. Further, melanins can also impair the
access of repair enzymes to lesions, contributing to the perpetuation of
DNA damage. On the other hand, some previous studies presented results
on the stimulation of proliferation and differentiation of melanocytes by
melanotropin, a melanocyte-stimulating hormone [5]. This effect howev-
er could be overcome if the culture medium was frequently changed or
the concentration of tyrosinase decreased. The presence of an inhibitor
of growth in the culture media of actively melanizing melanoma
cells was reported as a function of tyrosinase activity level and tyro-
sine concentration, when both enzyme and substrate were added
exogenously [6].
⁎
Corresponding author at: Departamento de Química Fundamental, Instituto de
Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-000, São
Paulo, SP, Brazil. Tel.: +55 11 3091 9147; fax: +55 11 3815 5579.
E-mail address: amdcferr@iq.usp.br (A.M. Da Costa Ferreira).
http://dx.doi.org/10.1016/j.jinorgbio.2015.05.007
0162-0134/© 2015 Elsevier Inc. All rights reserved.

50
C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
Taking into account that copper ions in the active site of tyrosinase
2.2.3. Complex [Cu(N22)(H20)(CF3SO3)](CF3SO3) 1
could be responsible for both activities, formation of melanin and, pos-
sibly, stimulation of melanoma growth, a study to verify the influence of
tyrosinase mimics on the viability of melanoma cells was carried out.
The aim was to establish possible correlations between an observed
stimulation or inhibition in melanoma cells growth, and the structure
of some copper(II) complexes. Two new structurally related dinuclear
complexes, one with a polydentate dinucleating amine–imine ligand,
and the other with a tridentate imine and a diamine-bridging ligand,
were then prepared and investigated (complexes 2 and 4, shown in
Fig. 1). The corresponding mononuclear copper(II) species (complexes
1 and 3, respectively) were also isolated, and used in comparative stud-
ies. These complexes showed to be thermodynamically stable through
CD measurements, using HSA as copper competitive ligand. Additional-
ly, oxidative damage to DNA caused by these complexes, in the presence
of hydrogen peroxide, was verified in complementary studies.
Copper complex [Cu(N22)](CF3SO3)2 1 was prepared in good yields
(75%) by complexation of 1 eq. of copper(II) triflate with 1 eq. of N-
benzyl-N-bis(2-pyridylethyl)amine in methanol [10]. Calc. for C2 H23
N F S2O6Cu (FW = 679.11 g/mol): C, 40.68; H, 3.41; N, 6.19; Cu,
9.36%; Experim. values: C, 41.09; H, 3.14; N, 6.25; Cu, 9.22%. UV–vis
bands, λmax, nm (ε, 103 M−1 cm−1): 261 (13.5), 268 s (10.6), 684
(0.142).
2.2.4. Complex [Cu2(N22-22)(H2O)2(CF3SO3)2](CF3SO3)2 2
This complex was prepared according to a procedure previously de-
veloped [11,12]. To a methanolic solution (10 ml) of copper(II) triflate
(558 mg, 1.54 mmol), 487 mg (0.77 mmol) of the ligand N22-22 dis-
solved in 10 ml methanol was added, under stirring. The blue solution
obtained was maintained under stirring for 2 h, and after the addition
of some diethyl ether a precipitate was formed. The suspension was
filtrated, the precipitate washed with 50 mL ether, and after dried
gave 622 mg of a blue powder (64% yield). After a slow evaporation of
a methanolic solution of this compound, blue crystals suitable for crys-
tallographic analysis were isolated. Calc. for C44H44Cu2F6N6O6S2
(FW = 1392.22 g/mol): C, 40.68; H, 3.41; N, 6.19; Cu, 9.36%; Experim.
values: C, 41.25; H, 3.18; N, 6.23; Cu, 8.96%. ESI-MS m/z = 528.1, calcu-
lated for C44H44Cu2F6N6O6S2 = 528.07. UV–vis bands, λmax, nm (ε,
103 M−1 cm−1): 261 (45.7), 268 s (37.2), 684 (0.275).
2. Experimental
2.1. Materials
Most of the reagents (percent purity) used: 2-(acetyl)pyridine
(99%), 2,3-bis(pyridyl)pyrazine, histamine hydrochloride (99%),
isatin (98%), 2-(2-aminoethyl)pyridine (95%), copper(II) perchlorate
hexahydrated (98%) and copper(II) triflate (98%) were purchased from
Aldrich Chemical Co. Solvents ethanol (96%), ethanol absolute (99%),
methanol (99%), dichloromethane (99%) and acetone (99%), as well as
hydrochloric acid were from Merck Chemical Co. DNA sodium salt from
calf thymus (CT-DNA) and human serum albumin (HSA) were from
Sigma-Aldrich. All the solutions were prepared with deionized water
from a Millipore instrument.
2.2.5. Crystal structure determination and refinement
Crystal data for the complex [Cu2(N22-22)(H2O)2(CF3SO3)2]
(CF3SO3)2 2 together with the details of X-ray diffraction experiments
are reported in Tables 1 to 3. Measurements were made on a Bruker–
Nonius KappaCCD diffractometer with graphite monochromatized
Mo(Kα) radiation (λ = 0.71073 Å). The structures were solved by di-
rect methods and refinements, based on F2, made by full-matrix least-
squares techniques. The H atoms for the water molecule were placed
at idealized positions in order to optimize the H-bonds with the oxygen
atoms of the closest triflate counter-ions. The CIF file with data for
complex 2 has been deposited at the Cambridge Crystallographic Data
Centre, CCDC 1030475.
2.2. Syntheses of the ligands and corresponding complexes
2.2.1. Ligand 2,2′-bis(aminomethyl)diphenyl
2,2′-Bis(aminomethyl)biphenyl (dpam) was prepared in 2 steps by
a Gabriel's reaction from 2,2′-bis(bromomethyl)biphenyl [7] according
to a described procedure [8]. N-benzyl-N-bis(2-pyridylethyl)amine
(N22) was prepared by addition of benzylamine to an excess of 2-
vinylpyridine, according to a procedure reported in the literature [9].
2.2.6. Complex [Cu(apyhist)H2O](ClO4)2 3
The imine ligand apyhist, where apyhist = ligand obtained from
2-(acetyl)pyridine and histamine (see Fig. 1), was prepared according
to methods already described in previous works [13,14], through con-
densation reaction of suitable carbonyl and amine precursors, followed
by immediate in situ metallation. The formed imine copper(II) complex
2.2.2.
N2,N2,N2′,N2′-tetrakis[2-(2-pyridyl)ethyl][1-1′-biphenyl]-2,2′-
dimethylamine (N22-N22)
Table 1
To a solution of 2,2′-bis(aminomethyl)diphenyl (1.05 g, 5 mmol) in
anhydrous methanol (30 mL) maintained under argon, freshly distilled
2-vinylpyridine (6.42 g, 61 mmol) and glacial acetic acid (0.35 g,
5.8 mmol) were carefully added. After a 5-day reflux under argon, the
mixture was then extracted in dichloromethane, and washed with
NaOH (1 M, pH ≈ 12). Organic layers were dried over sodium sulfate,
filtered and evaporated under vacuum. The resulting yellow oil was
chromatographied on silica gel with CH2Cl2/MeOH (95/5) and finally
with CH2Cl2/MeOH/NH3 (90/10/1). Ligand N22-N22 was obtained as
pale yellow oil (1.3 g, 41%). MS (FAB+) [MS = mass spectrometry,
FAB = fast atomic bombardment]: m/z = 633,3692 [M + 1] (100);
C42H44N6 FW = 632.84 g/mol. 1H RMN (300 MHz, CDCl3) in δ (ppm):
8.46 (dd, J = 0.8Hz, J = 4.1Hz, 4H); 7.50 (dt, J = 1.7Hz, J = 7.7Hz,
4H); 7.32 (m, 2H); 7.19 (dd, J = 3.8Hz, J = 5.2Hz, 4H); 7.06 (dd, J =
4.9Hz, J = 7.5Hz, 4H); 6.99 (m, 6H); 3.38 (s, 4H); 2,78 (s, 8H). 13C
RMN (50 MHz, CDCl3) in δ (ppm): 160.46 (4C Py, where Py = pyridine);
148.84 (4CH Py); 140.51 (2C Ph); 137.34 (2C Ph); 135.93 (4CH Py);
129.40 (2CH Ph); 128.61 (2CH Ph); 126.98 (2CH Ph); 125.96 (2CH
Ph); 123.21 (4CH Py); 1120.81 (4CH Py); 55.49 (2CH2-Ph); 53.59
(4CH2-Py); 35.58 (4CH2-N).
Crystallographic data for complex [Cu2(N22-22)(H2O)2(CF3SO3)2](CF3SO3)2 2.
[CuII2(N22-22)(CF3SO3)4/3(H2O)8/3
]
Formula
Molecular weight
Crystal system
Space group
Z
C147H172Cu6F36N18O50S12
4440.99
Triclinic
P-1
2
a (Å)
b (Å)
c (Å)
α (°)
19.7799 (4)
22.6303 (8)
23.049 (1)
70.399 (4)
78.025 (2)
73.780 (1)
9258.4 (6)
1.593
β (°)
γ (°)
V (Å3)
ρ (g·cm−3
)
N° of measured refl.
N° of observed refl. (F2 N 4σ(F))
R (F2 N 4σ(F))
41,601
19,558
0.0806
wR (all reflections)
Gof
0.2774
0.998
1.022
−1.152
Δρ max (e Å−3
)
Δρ min (e Å−3
)

C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
51
Table 2
Selected distances (Å) and angles (°) around the copper atoms for complex 2.
Cu(1)–O(37) 2.218(5)
Cu(1)–O(38) 2.049(7)
Cu(1)–N(1) 2.113(5)
Cu(1)–N(2) 2.003(5)
Cu(1)–N(3) 2.006(7)
Cu(2)–O(1) 2.186(5)
Cu(2)–O(39) 2.077(4)
Cu(2)–N(4) 2.111(5)
Cu(2)–N(5) 1.998(7)
Cu(2)–N(6) 2.011(7)
Cu(3)–O(4) 2.242(5)
Cu(3)–O(40) 2.088(7)
Cu(3)–N(7) 2.111(7)
Cu(3)–N(8) 1.991(7)
Cu(3)–N(9) 1.977(6)
Cu(4)–O(7) 2.236(5)
Cu(4)–O(41) 2.055(4)
Cu(4)–N(10) 2.108(5)
Cu(4)–N(11) 1.990(7)
Cu(4)–N(12) 2.018(6)
Cu(5)–O(10) 2.265(7)
Cu(5)–O(42) 2.029(5)
Cu(5)–N(13) 2.114(5)
Cu(5)–N(14) 1.985(6)
Cu(5)–N(15) 1.999(7)
Cu(6)–O(43) 2.068(7)
Cu(6)–O(44) 2.214(5)
Cu(6)–N(16) 2.112(5)
Cu(6)–N(17) 2.015(7)
Cu(6)–N(18) 2.002(7)
O(37)–Cu(1)–O(38) 94.1(2)
O(1)–Cu(2)–O(39) 95.1(3)
O(1)–Cu(2)–N(4) 116.0(2)
O(1)–Cu(2)–N(5) 91.6(3)
O(1)–Cu(2)–N(6) 91.4(3)
O(39)–Cu(2)–N(4) 148.8(2)
O(39)–Cu(2)–N(5) 87.0(3)
O(39)–Cu(2)–N(6) 85.7(3)
N(4)–Cu(2)–N(5) 88.9(3)
N(4)–Cu(2)–N(6) 96.1(2)
N(5)–Cu(2)–N(6) 172.4(3)
O(7)–Cu(4)–O(41) 92.8(2)
O(7)–Cu(4)–N(10) 110.6(2)
O(7)–Cu(4)–N(11) 91.2(2)
O(7)–Cu(4)–N(12) 93.8(2)
O(41)–Cu(4)–N(10) 156.2(3)
O(41)–Cu(4)–N(11) 87.1(3)
O(41)–Cu(4)–N(12) 85.9(2)
N(10)–Cu(4)–N(11) 88.2(3)
N(10)–Cu(4)–N(12) 96.4(2)
N(11)–Cu(4)–N(12) 171.6(2)
O(43)–Cu(6)–O(44) 89.9(2)
O(43)–Cu(6)–N(16) 161.6(2)
O(43)–Cu(6)–N(17) 88.0(3)
O(43)–Cu(6)–N(18) 85.3(3)
O(44)–Cu(6)–N(16) 107.7(2)
O(44)–Cu(6)–N(17) 92.7(2)
O(44)–Cu(6)–N(18) 96.7(2)
N(16)–Cu(6)–N(17) 96.3(2)
N(16)–Cu(6)–N(18) 87.4(2)
N(17)–Cu(6)–N(18) 168.4(3)
O(37)–Cu(1)–N(1) 110.71(18)
O(37)–Cu(1)–N(2) 91.30(19)
O(37)–Cu(1)–N(3) 94.0(2)
O(38)–Cu(1)–N(1) 154.8(2)
O(38)–Cu(1)–N(2) 87.0(2)
O(38)–Cu(1)–N(3) 85.3(3)
N(1)–Cu(1)–N(2) 96.2(2)
N(1)–Cu(1)–N(3) 88.8(2)
N(2)–Cu(1)–N(3) 171.0(2)
O(4)–Cu(3)–O(40) 94.8(2)
O(4)–Cu(3)–N(7) 104.7(2)
O(4)–Cu(3)–N(8) 91.4(3)
O(4)–Cu(3)–N(9) 95.8(2)
O(40)–Cu(3)–N(7) 159.7(2)
O(40)–Cu(3)–N(8) 87.0(3)
O(40)–Cu(3)–N(9) 85.2(2)
N(7)–Cu(3)–N(8) 98.0(3)
N(7)–Cu(3)–N(9) 87.1(2)
N(8)–Cu(3)–N(9) 169.8(2)
O(10)–Cu(5)–O(42) 94.6(2)
O(10)–Cu(5)–N(13) 106.3(2)
O(10)–Cu(5)–N(14) 94.0(3)
O(10)–Cu(5)–N(15) 89.3(3)
O(42)–Cu(5)–N(13) 159.0(2)
O(42)–Cu(5)–N(14) 87.4(2)
O(42)–Cu(5)–N(15) 85.8(3)
N(13)–Cu(5)–N(14) 88.7(2)
N(13)–Cu(5)–N(15) 96.7(2)
N(14)–Cu(5)–N(15) 172.6(2)
was isolated as green crystals, as previously reported [15], at pH 7.5.
Calc. for C12H16N4CuCl2O9 (FW = 494.73 g/mol): C, 29.14; H, 3.26; N,
11.33; Cu, 12.83%; Experim. values: C, 29.26; H, 3.50; N, 11.30; Cu,
12.63%. UV–vis bands, λmax, nm (ε, 103 M−1 cm−1): 208 (31.5), 277 s
(4.1), 285 (8.0), 295 s (5.8), 632 (0.084).
pellets (~20 mg complex/200 mg KBr), previously dried at 120 °C.
EPR spectra were recorded in a Bruker EMX instrument, working
at X-band (9.65 GHz frequency, 20 mW power, 100 kHz modulation
frequency). DPPH (α,α′-diphenyl-β-picrylhydrazyl) was used as
magnetic field calibrator (g = 2.0036), and measurements were carried
out with solid samples or frozen acetone- or acetonitrile-aqueous solu-
tions of the different complexes, in Wilmad quartz tubes (4 mm internal
diameter). Usually, 15 G modulation amplitude, 7.96 × 103 or 3.56 × 104
receiver gain, and 2 or 4 scans were standard conditions for the spectra re-
cording, at 77 K.
2.2.7. Complex [Cu2(apyhist)2dpam](ClO4)4 4
At first, the precursor complex 3 was prepared as described
above, followed by the addition of stoichiometric amounts of the
bridge-ligand 2,2′-bis(aminomethyl)diphenyl (dpam). After a while,
brownish-violet crystals of the resulting dinuclear species were collected,
washed with cold methanol, and dried in desiccator, under low pressure.
Calc. for C38H44N10Cu2Cl4O16 (FW = 1165.72 g/mol): C, 39.15; H, 3.80; N,
12.02; Cu, 10.90%; Experim. values: C, 39.45; H, 3.83; N, 11.48; Cu, 11.09%.
UV–vis bands, λmax, nm (ε, 103 M−1 cm−1): 277 s (16.0), 285 (18.0),
295 s (14.0), 510 (0.48), 664 (0.175).
2.4. Relative thermodynamic stability of the complexes
The relative stability of the complexes was estimated in titration
experiments, using HSA as competitive ligand, in similar procedures al-
ready reported [16]. CD spectra were recorded in a JASCO J-720 spectro-
polarimeter, using a quartz cuvette of 0.1 cm path length, at room
temperature, in the range 200–300 nm or 350–650 nm. The initial ex-
perimental HSA concentration was 500 or 700 μM, and the spectra
were registered in phosphate buffer (50 mM, pH 7.4) containing NaCl
0.1 M, in the absence or in the presence of increasing amounts of each
complex studied, up to 1:2 stoichiometric ratio. By considering the com-
petitive equilibria:
2.3. Physical methods
Elemental analyses of all the prepared compounds were performed
at Central Analítica of USP, using a CNH 2400 Perkin-Elmer instrument.
Analyses of copper were done by ICP-OES, in a Spectro instrument,
model Arcos-FHS12. Electronic spectra were recorded in a UV-1650PC
spectrophotometer from Shimadzu, using quartz cells with de 1.000 cm
optical length, and solutions of the diverse complexes in the range 10−4
to 10−3 M. Vibrational spectra in the infrared region were registered in
a FT-ABB Bomen instrument model MB, by diffuse reflectance, in
the range 400–4000 cm−1. The samples were prepared in KBr
½CuLꢀ⇄Cu þ L
1=K
½CuLꢀ
K
½CuðHSAÞꢀ
Cu þ HSA⇄½CuðHSAÞꢀ
½CuLꢀ þ HSA⇄½CuðHSAÞꢀ þ L
K ¼ K
=K
½CuðHSAÞꢀ ½CuLꢀ
Table 3
Geometric parameters reflecting the flexibility of complex 2.
Coordination type
Angle of the diphenyl (°, angle of the 2
planes around the selected bond)
Cu–Cu distance (Å)
τ parameter
(OTf, H20)(H20, H20)
(OTf, H20)(H20, H20)
(OTf, H20)(OTf, H20)
C21–C22
C105–C106
C63–C64
64.1
64.9
67.2
Cu1–Cu2
Cu5–Cu6
Cu3–Cu4
7.65
7.82
8.36
0.27/0.39
0.23/0.12
0.17/0.27

52
C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
Fig. 1. Structures of the studied copper(II) complexes.
and stoichiometric relations of the reagents, the K[CuL] for each complex
could be determined, since the corresponding constant for copper ion
inserted at N-terminal site of the protein is known. The reported value
for insertion of copper ions at the selective N-terminal metal binding
site of HSA is log K[Cu(HSA)] 12.0 [17]. Copper-aqua species was used in
control experiments, to obtain standard curve of CD band intensity vs.
[Cu(HSA)] formed (see Supplementary Information, Fig. S1).
TAE buffer (40 mM Tris–acetate, 1 mM EDTA) at 100 V, for 2 h. The elec-
trophoresis was performed in a Gibco Horizon 11–14 Gel Electrophoresis
System at 100 V.
2.7. Cell treatment
B16F10 and TM1MNG3 were used as melanoma cell models. B16F10
is a well-known murine melanoma cell lineage, and TM1MNG3 is a
melan-a-derived melanoma clone, which has already shown to be sus-
ceptible to another copper(II) complex, [Cu(isaepy)]2+, related to the
present study [18]. The copper(II) complexes studied [Cu(N22)
(H2O)OTf]+, [Cu2(N22-22)(H2O)2(OTf)2]2+, [Cu(apyhist)H2O]2+, or
2.5. Kinetic experiments of tyrosinase activity
The catalytic activity of both dinuclear copper(II) complexes 2 and 4
in the oxidation of L-dopa was verified in phosphate buffer (50 mM,
pH = 7.00), at 25 °C. The activities of the corresponding mononuclear
compounds 1 and 3 were also measured for comparison. The experi-
mental conditions used in these experiments were: [L-dopa] =
8.0 mM, [CuL] in the range 1 to 400 μM; or [CuL] = 160 mM (for dinu-
clear species) and 320 mM (for mononuclear species), for [L-dopa] in
the range 4.0 to 8.0 mM. From curves of increasing absorbance versus
concentration, monitored at 475 nm, the pseudo-first- and second-
order kinetic constants were then determined from graphics of initial
rate of the corresponding dopaquinone formation vs. concentration of
the catalyst, or of the substrate.
2.6. DNA cleavage after incubation with the copper(II) complexes
Since in many cases DNA is an important target for potential antitu-
mor agents, the copper complexes studied had also their nuclease activ-
ity compared. Single and double DNA strand cleavages were monitored
by electrophoresis experiments, in agarose gel. The plasmid pBluescript II
(Stratagene) was purified using Qiagen plasmid purification kit (Qiagen).
Reaction mixtures (20 μL total volume) containing 240 ng of supercoiled
DNA (Form I), in 50 mM phosphate buffer (pH 7.4), in the presence or
absence of 120 μM of H2O2, with concentrations of copper(II) complexes
1–4, varying in the range 1 to 50 μM, were incubated at 37 °C for different
periods of time. After incubation, a quench buffer solution (4 μL, 15% Ficoll
(type 400) in water; 0,25% bromophenol blue) was added, and the final
solution was subjected to electrophoresis on an 1% agarose gel in 1×
Fig. 2. Ortep view of one of the three independent dinuclear copper complexes 2 in the
asymmetric unit.

C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
53
Table 4
Table 5
EPR parameters (g and hyperfine constant A values) of copper(II) complexes samples
in solid state, and in frozen acetonitrile-aqueous solution, at 77 K. The data for the aqua-
complex was included for comparison.
Relative stability constants of the copper(II) complexes studied, as determined in competitive
experiments towards HSA by CD spectroscopy. Copper-aqua species was used in control
experiments, to obtain standard curve.
Solid samples
g⊥ g//
Frozen solutions
Complex
log K[CuL]
12.4
Complexes
g⊥
g//
A//, 10−4 cm−1a
g///A//, cm
1
2
3
4
[Cu(N22)(H2O)OTf]+
[Cu2(N22-22)(H2O)2(OTf)2]2+
[Cu(apyhist)H2O]2+
[Cu2(apyhist2dpam)]4+
[Cu(HSA)]b
12.0
1
2
3
4
2.125 (giso
2.094 (giso
)
)
2.071
2.078
2.062
2.081
2.075
2.253
2.227
2.259
2.225
2.361
152
166
177
182
154
148
134
128
122
153
a
a
2.060
2.061
2.208
2.206
12.0
a
[Cu(H2O)4]2+
The main interaction in this case occurred at Cys34, a secondary site (Ref. [25]).
Copper(II) inserted at the primary N-terminal site of the protein (Ref. [17]).
b
A//(×10−4 cm−1) = 0.46686 × 10−4 g//A//(G).
a
[Cu2(apyhist)2(dpam)]4+ were used to treat the cells. Stock solutions
of these complexes (5 mM) were prepared in 10% DMSO and were
stored at −20 °C. These solutions were diluted in buffer to their
final concentrations immediately before the experiments. An aque-
ous DMSO solution, corresponding to the highest DMSO concentra-
tion (0.1%) in the complex solutions, was employed as a vehicle
control. Typically, the cells were plated and then treated with the
metal complexes for 24 h (or for the specified time) after they had
adhered to the plate (usually 4 h later). Experiments were carried
out at least in triplicates.
solution, for 3 h in a CO2 incubator. The cells were then washed, and
formazan crystals were extracted with 200 μL of DMSO. The absorbance
was read at 570 nm in a microplate reader (BioRad, Hercules, CA, USA).
Results are average values obtained from 7 (for B16F10) or 4 (for
TM1MNG3) independent experiments, considering the values obtained
for control as 100%.
3. Results and discussion
All the copper(II) complexes studied were obtained in good yields and
high purity, as attested by elemental analysis data (see Experimental sec-
tion), and corroborated by spectroscopic data. For complexes 1 and 2, the
corresponding ligands N22 and N22-N22 were previously prepared and
then metallated. Complex 2 crystallized as three independent dinuclear
copper complexes in an asymmetric unit. In each complex the copper
are penta-coordinated in a square-based pyramidal geometry more or
less distorted: the Addison τ parameter [19] ranges from 0.12 Å to
2.8. Cellular viability of B16F10 and TM1MNG3 melanoma cells in the
presence of copper(II) complexes
Cells were plated at a density of 104 cells/well in a 96-well micro-
plate. Twenty-four hours after treatment with the metal complexes,
the cells were washed and incubated with 200 μL of 0.5 mg/mL MTT
[CuL]:[HSA] ratio
[CuL]:[HSA] ratio
A
B
a) 1:16
b) 1:12
c) 1:10
a)1:17
b)1:10
c)1:5
[Cu2(N22-22)]2+ dinuclear
[Cu(N22)(H2O)OTf]+
10
d) 1:7
e) 1:5
f) 1:3
g) 1:1
h) 1.5:1
i) 2:1
d)1:3
e)1:2.5
f)1:2
10
i
i
g)1:1
h
h
g
h)1.5:1
i)2:1
HSA
g
f
d
c
f
HSA
e
0
a
a
a
0
HSA
a
HSA
e
f
g
b
c
h
i
f,g,h,i
350
400
450
500
550
600
650
350
400
450
500
550
600
650
λ, nm
λ, nm
[CuL]:[HSA] ratio
[CuL]:[HSA] ratio
C
D
[Cu(apyhist)]2+
HSA
HSA
a)1 :20
b)1 :15
c)1:12
d)1:10
e)1:7
f)1:5
g)1:3
h)1:1
i)2:1
[Cu2(apyhist)2dpam]4+
a)1:20
b)1:15
c)1:12
d)1:10
e)1:7
f)1:5
g)1:3
h)1:1
i)2:1
20
10
0
20
i
10
0
h
g
a
i
h
g
a
a
a
a
h
i
g
g
h
h
-10
-10
i
i
350
400
450
500
550
600
650
350
400
450
500
550
600
650
λ, nm
λ, nm
Fig. 3. CD spectra registered in the visible region of HSA solutions (0.700 mM) titrated with the different copper(II) complexes: (A) [Cu(N22)(H2O)OTf]+ 1; (B) [Cu2(N22-
22)(H2O)2(OTf)2]2+ 2; (C) [Cu(apyhist)H2O]2+ 3 and (D) [Cu2(apyhist)2dpam]4+, in phosphate buffer (50 mM, pH 7.4), at room temperature.

54
C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
0.39 Å. Each copper is coordinated to the three nitrogen atoms of the
pyridylalkylamin moieties of the ligand, the remaining sites being occu-
pied by either two water molecules or one water molecule and one
trifluoromethane sulfonate counter-ion (Fig. 2). The flexibility of the
diphenyl spacer of the ligand allows a wide variation for the Cu–Cu dis-
tances, which are equal to 7.650 Å, 7.817 Å and 8.363 Å for each complex,
respectively.
3.2. Tyrosinase activity
The catalytic activities of all the studied complexes were verified,
using L-dopa as the substrate to be oxidized. Comparative curves of ab-
sorbance increasing values, attributed to the formation of quinone prod-
uct, are shown in Fig. 4A. Typical curves with a saturation effect
(Michaelis–Menten mechanism) attested that as expected the dinuclear
complexes are more active as tyrosinase mimics than the analogous
mononuclear species, although all of them are much less active than
the tyrosinase protein [26]. Also, complex 2 with a more flexible ligand,
is much more effective than complex 4. The dependence of initial L-dopa
Complex 3 synthesis has been already described [15]. The correspond-
ing dinuclear species 4 was obtained from this complex 3, by adding
stoichiometric amounts of the bridge-ligand, dpam (see Fig. 1). The char-
acterization of all these complexes was provided by different spectroscop-
ic techniques. Characteristic bands of the metal coordinated species,
related to internal ligand, charge transfer or d–d transitions were ob-
served by electronic spectroscopy, as specified in the Experimental sec-
tion. Particularly, d–d bands were observed at 684 nm for complexes 1
A
0.24
[Cu2(N22-22)(H2O)2(OTf)2]2+
0.20
and 2, with absorptivity values 0.142 × 103 and 0.275 × 103 M−1 cm−1
,
respectively. For complex 3, this band occurred at 632 nm (ε =
0.084 × 103 M−1 cm−1), and for complex 4 at 664 nm (ε = 0.175 ×
103 M−1 cm−1). EPR spectra in frozen solution, indicated that the envi-
ronment around copper ion is not very different in all of them, with spec-
troscopic parameters, g and A values consistent with a pentacoordinated
(in the case of complexes 1 and 2), or a tetragonal configuration showing
some tetrahedral distortion (for complexes 3 and 4), as shown in Table 4.
Considering the quotient g///A// determined from EPR data, usually used
to evaluate this distortion in copper centers [20], the dinuclear
species show a more tetragonal structure than the corresponding
mononuclear ones, in both cases. Complex 4 showed the more pla-
nar environment around the metal ion in this series, as expected,
since its imine tridentate ligand provide a more rigid coordination
structure around the copper ion.
0.16
0.12
[Cu(N22)(H2O)OTf]+
0.08
[Cu2(apyhist)2dpam]4+
0.04
[Cu(apyhist)H2O]2+
0.00
0
100
200
300
400
500
600
Time, s
B
3.0x10-4
2.0x10-4
1.0x10-4
0.0
[Cu(apyhist)]2+
[Cu(N22)(H2O)OTf]+
3.1. Estimation of relative stability of the complexes by CD
By considering competitive equilibria of copper ions coordinated to
each ligand in the studied complexes, or inserted at N-terminal site of
HSA, it was achieved an estimation of the apparent stability of com-
plexes 1 and 2, according to procedures already described [16]. Titra-
tions of a protein solution with each copper(II) complex, monitored
through CD spectra at the characteristic band at 564 nm, attributed to
the insertion of a copper ion at the selective N-terminal metal binding
site of the protein [17], were carried out with this purpose. In Fig. 3,
the corresponding titration curves are shown. The determined values
for log K[CuL] were based on the rising of this band, up to 1:1 stoi-
chiometric ratio [HSA]:[CuL], and they are of the same order as log
K[Cu(HSA)] reported in the literature [17,21], as shown in Table 5. An ad-
ditional raising band around 700 nm was observed for both complexes,
when excess of copper (up to 1:2) was added, attributed to a second
copper ion insertion, probably at a multimetal binding site, described
as composed by His67, His247, Asn99 and Asp249 [22]. Calibration
curve was provided by similar titration experiments of HSA with the
aqua–copper(II) complex (see Fig. S1).
In contrast, complexes 3 and 4 interacted only at a secondary
metal binding site of HSA, monitored by a growing band at
370 nm, and identified as the Cys34 pocket [23,24]. It has been pre-
viously observed that even when this Cys34 is blocked, after previ-
ous treatment of the protein with N-ethyl-maleimide, complex 3
does not insert copper at the primary usual metal binding site of
the protein [25]. Therefore, complex 4 presented here the same be-
havior as complex 3, attesting an interaction involving probably mo-
lecular recognition of the imine ligand apyhist at the Cys34 site of
the protein [25].
0
1
2
3
4
[Copper complex], 10-4 mol/L
C
[Cu2(N22-22)(H2O)2(OTf)2]2+
[Cu2(apyhist)2dpam]4+
8.0x10-3
6.0x10-3
4.0x10-3
2.0x10-3
0.0
0
1
2
3
4
[Copper complex], 10-4 mol/L
Fig. 4. A) Comparative curves (Absorbance vs. time) of catalytic activity of studied
copper(II) complexes in the oxidation of L-dopa (8.0 mM), at concentrations 160 μM (dinu-
clear species) and 320 μM (mononuclear species). Reaction at 25 °C, in phosphate buffer
(50 mM, pH = 7.0), monitored by dopaquinone formation, at 475 nm; B) Dependence of
L-dopa initial oxidation rate on increasing concentrations of complexes, [Cu(N22)(H2O)OTf]+
1 and [Cu(apyhist)H2O]2+ 3; C) Dependence of L-dopa initial oxidation rate on increasing
concentrations of complexes, [Cu2(N22-22)(H2O)2(OTf)2]2+ 2 and [Cu2(apyhist)2dpam]4+
4, with [L-Dopa] = 8.0 mM, in phosphate buffer (50 mM, pH 7.0), at 25 °C.
Complementary measurements, monitoring the CD band at 208 nm
attributed to the α-helix content of the protein, indicated that none of
the complexes was able to disturb the protein secondary structure,
modifying significantly its ellipticity (see Fig. S2).

C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
55
Table 6
species 2 and 4 an induction period was observed (Fig. 4C). Considering
also a dependence on the concentration of the substrate, a second-order
rate constant was calculated for each of these complexes, displayed at
Table 6. In the case of complex 4 two steps were observed, probably
due to the equilibrium of this dinuclear species with the analogous
mononuclear one, since the estimated constant for a second step cata-
lyzed by complex 4 is very close to the corresponding value for the anal-
ogous mononuclear complex 3.
Determined values for kinetic constants in the oxidation of L-dopa, catalyzed by the
copper(II) complexes studied. Reaction at 25 °C, in phosphate buffer (50 mM, pH = 7.00).
Copper complex
Pseudo-first order
kinetic constant,
kobs, s−1
Second order kinetic
constant, k, 102 M−1 s−1
[Cu(N22)(H2O)OTf]+ 1
0.68
20.4
0.156
1.91 (step 1)
0.235 (step 2)
(108 9.1)b
0.85
25.5
0.195
2.38
0.294
[Cu2(N22-22)(H2O)2(OTf)2]2+ 2
[Cu(apyhist)H2O]2+ 3
[Cu2(apyhist)2dpam]4+ 4
3.3. Nuclease activity
Tyrosinase, from mushrooma
a
Since DNA is usually an important target for compounds exhibiting
potential antitumor properties, the possible activity of the studied com-
plexes as synthetic nucleases was also monitored. In the absence of an
added oxidant or reducing agent, no significant changes in DNA were
verified by electrophoresis experiments. However, in the presence of
hydrogen peroxide, single and double cleavages leading to form II and
Ref. [26].
kcat.
b
oxidation rates with increasing concentrations of each complex indicat-
ed a pseudo-first order reaction, only at very low concentrations of the
mononuclear complexes, as shown in Fig. 4B. For both the dinuclear
A
Form II
Form III
Form I
B
Form II
Form III
Form I
Fig. 5. Agarose gel of plasmidial DNA pBluescript II KS(+/−) incubated with hydrogen peroxide, and A) complex [Cu(N22)(H2O)OTf]+ 1 or B) complex [Cu2(N22-22)(H2O)2(OTf)2]2+ 2,
for 10, 20 and 40 min, at 37 °C.

56
C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
linear DNA form III were observed, in a process dependent on the con-
centration of the copper species, in the range 10–100 μM, and almost
independent of the incubation time (up to 40 min). The reactivity of com-
plexes 1 and 2 can be compared in Fig. 5. The mononuclear species
[Cu(N22)]+ 1 was not active up to 40 min, at 10 μM, although it was
able to cause single and double cleavages at 50 and 100 μM (Fig. 5A).
On the other hand, complex [Cu2(N22-22)]2+ 2 showed to be very active,
even at low concentration (10 μM). At higher concentrations (50 or
100 μM), a complete degradation of the nucleic acid seems to be occur-
ring, as shown by a continuous band at agarose gel, and complete deple-
tion of form I (Fig. 5B).
complexes regarding the corresponding mononuclear ones, for both
pairs of compounds tested.
3.4. Cell viability of melanomas in the presence of the complexes
Exposure of TM1MNG3 (Fig. 8A) and B16F10 cells (Fig. 8B) to copper
complexes for 24 h led to a dose-dependent cytotoxicity, as determined
by MTT assay. Complexes 2 and 4 presented the highest toxicities in
both melanoma cell lineages. IC50 values (95% confidence interval)
were estimated by performing a nonlinear fit of normalized response
versus log dose with variable slope using GraphPad Prism software, ver-
sion 6. For the dinuclear complexes 2 and 4 these values were 16.0 and
15.4 μM towards B16F10 cells, or 63.5 and 93.3 μM for TM1MNG3 cells,
respectively. In contrast, complexes 1 and 3 were much less toxic for
both cell lines. For complex 1 an IC50 (95% CI) value of 54.5 μM was cal-
culated towards B16F10 cells. However, for complex 3 its corresponding
IC50 values could not be reliably estimated, being too high (see Fig. 8) for
both cell lines, in a concentration range beyond its solubility. A complete
For complex [Cu(apyhist)]2+ 3, a more significant nuclease activity
was observed, in comparison to complex 1, dependent on the concentra-
tion used, as displayed in Fig. 6A. Finally, complex [Cu2(apyhist)2dpam]4+
4 showed a remarkable capacity of cleaving DNA, leading to form II and
form III, with quite complete depletion of form I, after 20 min incubation,
at 50 μM concentration (see Fig. 6B). For better comparison of all the com-
plexes, the ratio of obtained DNA (formII)/DNA (Form I) in each case is
shown in Fig. 7. These results pointed to a higher activity of dinuclear
A
Form II
Form III
Form I
B
Form II
Form III
Form I
Fig. 6. Agarose gel of plasmidial DNA pBluescript II KS(+/−) and A) complex [Cu(apyhist)H2O]2+ 3 or B) complex [Cu2(apyhist)2dpam]4+ 4 incubated with hydrogen peroxide for 10, 20
and 40 min, at 37 °C.

C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
57
[Cu(apyhist)]2+
A
A
[Cu(N22)(H2
O)OTf)]+
140
[Cu2(apyhist)2(dpam)]4+
120
100
80
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
[Cu2(N22-22)(H2O)2OTf)2]2+
[Cu(N22-
60
40
20
0
20 40 60 80 100 120 14 160
O2
[Copper complex], µM
M]
M]
M]
M]
H2
μ
[10
μ
[10
μ
μ
[50
DNA
[50
[Cu(apyhist)]2+
[Cu2(apyh+ist)2(dpam)]4+
[Cu(N22)]
140
120
100
80
B
[Cu(N22-22)]2+
B
[Cu(apyhist)]2+
[Cu2(apyhist)2dpam]4+
1 .5
1 .0
0 .5
0 .0
60
40
20
0
20 40 60 80 100 120 140 160
[Copper complex], µM
Fig. 8. A) Cellular viability assay of TM1MNG3 melanomas after incubation at 37 °C for 24 h
with different concentrations of all copper(II) complexes studied; B) Cellular viability
assay of B16F10 melanomas after incubation at 37 °C, for 24 h, with different concentra-
tions of all copper(II) complexes studied.
O2
M]
M]
M]
M]
H2
μ
[10
μ
[10
μ
[50
μ
[50
DNA
Fig. 7. Corresponding [DNA form II]/[DNA form I] ratio, quantified by using program ImageJ
1.46, for all the copper complexes studied: A) complexes 1 and 2; B) complexes 3 and 4.
Lanes: control, plasmidial DNA (36 ng/μL); DNA + H2O2 (125 μM); concentrations of
[CuL], as indicated.
A significant inhibition of melanoma viability was observed for both
lineage B16F10 and TM1MNG3 cells incubated with each one of the di-
nuclear complexes, with IC50 values in the range 20–100 μM, although
complex 2 exhibited a much higher tyrosinase activity than complex
4. This complex 4 is probably in equilibrium with the corresponding
mononuclear complex 3 during the catalytic cycle, since two steps
were observed in the catalyzed oxidation of L-dopa, with the second
step rate constant very close to that verified in the reaction catalyzed
by complex 3. The corresponding mononuclear complexes showed to
be remarkably less reactive both as inhibitors of melanoma prolifera-
tion, and as tyrosinase mimics. Further, the dinuclear species exhibited
higher activity as nuclease agents than the corresponding mononuclear
compounds, being able of causing single and double DNA strand cleav-
ages, at concentrations of 10–50 μM, in the presence of hydrogen perox-
ide. For the corresponding mononuclear species double cleavage was
observed only at higher concentrations, in the range 50–100 μM.
Those results indicated that the dinuclear structure of such copper
complexes is decisive for its inhibition of melanoma cells proliferation
as well as for their tyrosinase mimicking, and efficient nuclease activity.
A possible explanation for the observed higher activity of dinuclear spe-
cies could be an influence of its structure in the melanogenesis process, si-
multaneously occurring in melanoma cells. Indeed, heavily melanogenic
cells as those of B16F10 lineage were shown to be more susceptible to
the dinuclear species than TM1MNG3 cells with negligible content of
melanin. Besides DNA, these dinuclear complexes could have another in-
tracellular target. A better elucidation of the mechanism of action of such
table with the corresponding calculated values is available at Supple-
mentary Information.
These findings indicate that the dinuclear complexes present a
more intense cytotoxic effect than their corresponding mononuclear
complexes. Moreover, the dinuclear species were much more reactive to-
wards B16F10 cells (Fig. 8B) than to TM1MNG3 ones (Fig. 8A), especially
at low concentrations (b50 μM). These two types of cells differ remark-
ably on its melanin content. B16F10 cells are heavily melanogenic, while
TM1MNG3 cells have no detectable melanin, as verified in our previous
work [18]. Therefore, the melanin level in B16F10 cells seems to make
them more susceptible to the dinuclear complexes toxicity.
4. Conclusions
All the studied copper(II) complexes showed good thermodynamic
stability, in comparison to copper ion inserted at HSA, as attested by
CD measurements. Complexes 1 and 2 exhibited the expected com-
petitive behavior between the polydentate ligand and the selective
N-terminal site of the protein in coordinating the metal ion [17].
However, complex 4 displayed a predominant interaction at Cys34
site, already reported to complex 3 [25] containing the same ligand.
None of the complexes was able to modify noticeably the α-helix per-
centage of the protein.

58
C.J. Nunes et al. / Journal of Inorganic Biochemistry 149 (2015) 49–58
complexes regarding the viability of melanoma cells requires further
studies, actually in development in our lab.
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circular dichroism
CT-DNA calf thymus DNA
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Financial support by the Brazilian agencies FAPESP (grants 2011/
50318-1 and 2014/09367-7), CNPq (grant 302606/2010-6), INCT Redox
Processes in Biomedicine-Redoxoma (Programa Institutos Nacionais
de Ciência e Tecnologia, CNPq/FAPESP/MCT, Proc. 573530/2008-4), the
NAP-Redoxoma (Pro-Reitoria de Pesquisa-USP, Proc. 11.1.9352.1.8),
CEPID-Redoxoma (grant 2013/07937-8), and Project CAPESP-USP/
COFECUB (grant 2008.1.818.46.0) is gratefully acknowledged.
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Supplementary data to this article can be found online at http://dx.
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