Synthesis, characterization, DNA-binding, and antioxidant activities of four copper(II) complexes containing N-(3-hydroxybenzyl)-amino amide ligands

Article abstract of DOI:10.1080/00958972.2012.754018

Four new substituted amino acid ligands, N-(3-hydroxybenzyl)-glycine acid (HL1), N-(3-hydroxybenzyl)- alanine acid (HL₂), N-(3-hydroxybenzyl)- phenylalanine acid (HL3), and N-(3-hydroxybenzyl)- leucine acid (HL ₄), were synthesized and characterized on the basis of 1H NMR, IR, ESI-MS, and elemental analyses. The crystal structures of their copper(II) complexes [Cu(L₁)₂].2H₂O (1), [Cu (L ₂)₂(H₂O)] (2), [Cu(L₃) ₂(CH₃OH)] (3), and [Cu(L4)2(H2O)].H2O (4) were determined by X-ray diffraction analysis. The ligands coordinate with copper(II) through secondary amine and carboxylate in all complexes. In 2, 3, and 4, additional water or methanol coordinates, completing a distorted tetragonal pyramidal coordination geometry around copper. Fluorescence titration spectra, electronic absorption titration spectra, and EB displacement indicate that all the complexes bind to CT-DNA. Intrinsic binding constants of the copper(II) complexes with CT-DNA are 1.32×10⁶M^-1, 4.32×10⁵M^-1, 5.00×10⁵M ^-1, and 5.70×10⁴M^-1 for 1, 2, 3, and 4, respectively. Antioxidant activities of the compounds have been investigated by spectrophotometric measurements. The results show that the Cu(II) complexes have similar superoxide dismutase activity to that of native Cu, Zn-SOD.

Full text of DOI:10.1080/00958972.2012.754018

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Synthesis, characterization, DNA-
binding, and antioxidant activities of
four copper(II) complexes containing N-
(3-hydroxybenzyl)-amino amide ligands
ZHI Li- HUA a , WU WEI- NA a , WANG YUAN a & SUN GUANG b
a Department of Physics and Chemistry, Henan Polytechnic
University, Jiaozuo, PR, China
b School of Materials Science and Engineering, Henan Polytechnic
University, Jiaozuo, PR, China
Accepted author version posted online: 28 Nov 2012.Published
online: 14 Jan 2013.
To cite this article: ZHI Li- HUA , WU WEI- NA , WANG YUAN & SUN GUANG (2013): Synthesis,
characterization, DNA-binding, and antioxidant activities of four copper(II) complexes containing N-
(3-hydroxybenzyl)-amino amide ligands, Journal of Coordination Chemistry, 66:2, 227-242
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Journal of Coordination Chemistry
Vol. 66, No. 2, 20 January 2013, 227–242
Synthesis, characterization, DNA-binding, and antioxidant
activities of four copper(II) complexes containing
N-(3-hydroxybenzyl)-amino amide ligands
ZHI LI- HUA†, WU WEI- NA†*, WANG YUAN† and SUN GUANG‡
†Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo, PR,
China; ‡School of Materials Science and Engineering, Henan Polytechnic University,
Jiaozuo, PR, China
(Received 20 May 2012; in final form 1 October 2012)
Four new substituted amino acid ligands, N-(3-hydroxybenzyl)-glycine acid (HL1), N-(3-hydroxy-
benzyl)-alanine acid (HL2), N-(3-hydroxybenzyl)-phenylalanine acid (HL3), and N-(3-hydroxyben-
zyl)-leucine acid (HL4), were synthesized and characterized on the basis of 1H NMR, IR, ESI-MS,
and elemental analyses. The crystal structures of their copper(II) complexes [Cu(L1)2]·2H2O (1), [Cu
(L2)2(H2O)] (2), [Cu(L3)2(CH3OH)] (3), and [Cu(L4)2(H2O)]·H2O (4) were determined by X-ray dif-
fraction analysis. The ligands coordinate with copper(II) through secondary amine and carboxylate in
all complexes. In 2, 3, and 4, additional water or methanol coordinates, completing a distorted tetrag-
onal pyramidal coordination geometry around copper. Fluorescence titration spectra, electronic
absorption titration spectra, and EB displacement indicate that all the complexes bind to CT-DNA.
Intrinsic binding constants of the copper(II) complexes with CT-DNA are 1.32 ꢀ 106 Mꢁ1
,
4.32 ꢀ 105 Mꢁ1, 5.00 ꢀ 105 Mꢁ1, and 5.70 ꢀ 104 Mꢁ1 for 1, 2, 3, and 4, respectively. Antioxidant
activities of the compounds have been investigated by spectrophotometric measurements. The results
show that the Cu(II) complexes have similar superoxide dismutase activity to that of native Cu,
Zn-SOD.
Keywords: Substituted amino acid ligand; Copper(II) complex; Crystal structure; DNA Binding;
Antioxidant activity
1. Introduction
There has been increasing focus on binding of small molecules to DNA, since DNA con-
tains all the genetic information for cellular functions [1–3]. DNA molecules are prone to
damage by interacting with some chemicals. This damage may change the replication of
DNA and inhibit the growth of tumor cells in living organisms, which is the basis of
designing more efficient antitumor drugs [4–6].
Amino acids are basic structural units of proteins and play central roles both as building
blocks of proteins and as intermediates in metabolism. Amino acid Schiff bases have been
used as ligands in coordination chemistry [7]. Several metal complexes of Schiff bases
derived from salicylaldehyde and amino acid [8–11] were reported; some have been
*Corresponding author. Email: wuwn08@hpu.edu.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online Ó 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.754018

228
L. ZHI et al.
proven to be efficient DNA cleavers [12,13] and as novel tumor chemotherapeutic and
tumor radio imaging agents [14]. Copper(II) complexes containing amino acids have
attracted attention for their biological and pharmaceutical activities and studied as models
for the behavior of copper enzymes. Some copper complexes with amino acid ligands were
reported to exhibit potent antitumor and artificial nuclease activities [15–17]. Recent
reports have also shown that amino acid/peptide based copper(II) complexes show efficient
DNA cleavage activity [18–20].
Four new Cu(II) complexes with amino acid ligands (figure 1) were synthesized and
characterized. DNA-binding properties of the complexes have been investigated by UV
absorption and fluorescence spectroscopy as well as their antioxidant activities in vitro.
These results help to understand the binding mode of this kind of complex to DNA, useful
to explore new anticancer agents and antioxidants, and to modify or detect bio-molecules.
2. Experimental
2.1. Materials and instrumentation
All chemicals were of analytical grade and used without purification unless otherwise
noted. All measurements involving interactions of the four metal complexes with CT-DNA
were carried out in doubly distilled water buffer. UV–Vis spectrometry was employed to
check the solution concentration of CT-DNA (ɛ = 6600 Mꢁ1 cmꢁ1 at 260 nm) and the purity
(A260:A280 > 1.80) in the buffer. UV–Vis absorption spectra were recorded using a
Purkinje General TU-1901 spectrophotometer and fluorescence emission spectra were
recorded using a Varian CARY Eclipse spectrofluorophotometer. Melting points were
determined on an XT4-100ꢀ microscopic melting point apparatus (Beijing Electrooptical
Instrument Factory, China). Elemental analyses for C, H, and N were carried out on an
Elemental Vario EL analyzer. IR spectra (m = 4000–400 cmꢁ1) were determined by the KBr
Figure 1. The structure of the ligands.

Synthesis and Characterization
229
1
pressed disk method on a Bruker V70 FT-IR spectrometer. H NMR spectra were recorded
with a Bruker AV400 NMR instrument using appropriate deuterated solvents. ESI mass
spectra were recorded on a Finnigan MAT LCQ mass spectrometer using the syringe pump
method. Antioxidant activities were tested on a 722 N spectrophotometer (Shanghai
Analytical Instrument Factory, China).
2.2. Synthesis of the ligands
All ligands were synthesized by reducing the Schiff base formed from the corresponding
amino acid and 3-hydroxy benzaldehyde via NaBH4 according to the literature [21].
HL1: Yield: 66%. m.p. 215–216 °C. Anal. Calcd for C9H11NO3: C, 59.66; H, 6.12; N,
7.73. Found: C, 59.37; H, 5.86; N, 7.48%. ESI-MS Calcd. for C9H11NO3: 181.07. Found:
1
182.1. H NMR (D2O) δ 1.93 (s, 1H, ꢁNH), 3.63 (s, 2H, ꢁCH2ꢁ), 4.22 (s, 1H, ꢁOH),
6.98–7.06 (m, 3H, CꢁH, phen), 7.36–7.41 (m, 1H, CꢁH, phen). IR (KBr, cmꢁ1): υ(NH)
3104; υ(CH2) 2889; υ(COOꢁ) 1605, 1382; υ(OH) 3416.
HL2: Yield: 62%. m.p. 245–247 °C. Anal. Calcd for C10H13NO3: C, 61.53; H, 6.71; N,
7.18. Found: C, 61.25; H, 6.43; N, 6.9%. ESI-MS Calcd. for C10H13NO3: 195.09. Found:
1
196.1. H NMR (D2O) δ 1.51 (s, 3H, CH3), 1.93 (s, 1H, NH), 3.72 (p, 1H, CH), 4.22 (d,
2H, CH2),7.04 (m, 3H, CH, phen), 7.38(t, 1H, CH, phen). IR (KBr, cmꢁ1): υ(NH) 3150;
υ(CH2) 2990; υ(COOꢁ) 1611, 1414; υ(OH) 3433.
HL3: Yield: 58%. m.p. 229–231 °C. Anal. Calcd for C16H17NO3: C, 70.83; H, 6.32; N,
5.16. Found: C, 70.56; H, 6.14; N, 4.88%. ESI-MS Calcd. for C16H17NO3: 271.12. Found:
1
272.2. H NMR (MeOD) δ 1.88 (s, 1H, NH), 3.02–3.27 (m, 2H, CH2), 3.66–3.69 (d, 2H,
CH2), 3.89–3.99 (t, 1H, CH), 4.8 (s, 1H, OH), 6.75–6.78 (t, 3H, CH, phen), 7.14–7.18 (t,
1H, CH, phen), 7.21–7.27 (m, 5H, CH, phen). IR (KBr, cmꢁ1): υ(NH) 3088; υ(CH2)
2949; υ(COOꢁ) 1632, 1390; υ(OH) 3180.
HL4: Yield: 68%. m.p. 179–183 °C. Anal. Calcd for C13H19NO3: C, 65.8; H, 8.07; N,
5.9. Found: C, 65.57; H, 8.26; N, 5.64%. ESI-MS Calcd. for C13H19NO3: 237.29. Found:
1
238.3. H NMR (D2O) δ 0.62–0.75 (m, 6H, CH3), 1.37–1.49 (m, 3H, CH, CH2), 1.69 (s,
1H, NH), 3.29–3.52 (t, 1H, CH), 3.81–3.97 (d, 2H, CH), 6.75–6.78 (m, 3H, CH, phen),
7.06–7.14 (t, 1H, CH, phen) cm. IR (KBr, cmꢁ1): υ(NH) 3061; υ(CH2) 2960; υ(COOꢁ)
1610, 1408; υ(OH) 3443.
2.3. Synthesis of the complexes
All complexes were synthesized using a similar method according to the literature [21]. 1
and 2 were obtained from water solutions. However, 3 and 4 were obtained from MeOH/
H2O (2:3) solution.
1: Yield: 82%. Anal. Calcd for C18H24CuN2O8: C, 47.00; H, 5.26; N, 6.09. Found: C,
46.72; H, 4.97; N, 5.83%. IR (KBr, cmꢁ1): υ(NH) 2970; υ(CH2) 2939; υ(COO-) 1593,
1402; υ(H2O and/or OH) 3423, 3269.
2: Yield: 78%. Anal. Calcd for C20H26CuN2O7: C, 51.11; H, 5.58; N, 5.96. Found: C,
50.84; H, 5.30; N, 5.68%. IR (KBr, cmꢁ1): υ(NH) 3076; υ(CH2) 2966; υ(COOꢁ) 1604,
1479; υ(H2O and/or OH) 3425, 3215.

230
L. ZHI et al.
3: Yield: 87%. Anal. Calcd for C33H36CuN2O7: C, 62.30; H, 5.70; N, 4.40. Found: C,
62.05; H, 5.44; N, 4.12%. IR (KBr, cmꢁ1): υ(NH) 3028; υ(CH2) 2929; υ(COOꢁ) 1649,
1427; υ(OH) 3279, 3223.
4: Yield: 75%. Anal. Calcd for C26H40CuN2O8: C, 54.58; H, 7.05; N, 4.90. Found: C,
54.61; H, 7.27; N, 4.73%. IR (KBr, cmꢁ1): υ(NH) 3271; υ(CH2) 2960; υ(COOꢁ) 1616,
1465; υ(OH) 3421, 3298.
2.4. X-ray Crystallography
The crystals were mounted on a Bruker SMART APEX II CCD diffractometer equipped
with a graphite monochromated Mo-Kα radiation (k = 0.071073 nm) by using /–x scan
mode. SMART [22] was used to collect the intensity data, SAINT for integration of the
intensity [22], SADABS [23] for absorption correction, and SHELXTL [24] for structure
solution and refinements on F2. A summary of the crystal data is given in table 1. Selected
bond angles and distances are listed in table 2. The details of the hydrogen bond parame-
ters are given in table 3.
2.5. Spectroscopic studies on DNA interaction
2.5.1. Electronic absorption spectra. Electronic absorption titration of copper(II) com-
plexes in aqueous buffer solution (50 mM NaCl–5 mM Tris-HCl, pH 7.1) were performed
at a fixed complex concentration (10 μM) while gradually increasing the concentration of
CT-DNA. Absorption data were analyzed to evaluate the intrinsic binding constant Kb,
which can be determined from equation (1) [25],
½DNAꢂ=ðea ꢁ ef Þ ¼ ½DNAꢂ=ðeb ꢁ ef Þ þ 1=½Kbðeb ꢁ ef Þꢂ
ð1Þ
where, [DNA] is the concentration of DNA in base pairs; ɛa, ɛf, and ɛb are the apparent
extinction coefficient (Aobsd/[compound]), the extinction coefficient for the free compound
and the extinction coefficient for the compound in the fully bound form, respectively. A
plot of [DNA]/(ɛb ꢁ ɛf) vs. [DNA] gave a slope of 1/(ɛb ꢁ ɛf) and an intercept equal to
[Kb(ɛb ꢁ ɛf)]ꢁ1; Kb is the ratio of the slope to the intercept.
2.5.2. Fluorescence spectra. Fluorescence spectral titration. For fluorescence measure-
ments, fixed concentrations (10 μM) of the complexes were titrated with increasing
amounts of CT-DNA. The excitation and emission wavelengths were 275.2 and 302.96 for
1 and 274 and 302 for 2, 3, and 4. Excitation and emission slits were set at 5 nm. Experi-
ments were conducted at room temperature in a buffer containing 5 mM Tris-HCl (pH 7.1)
and 50 mM NaCl.
EB–DNA competition experiment. Further support for the binding mode of the four com-
plexes to DNA is given through emission quenching experiments. A 2 mL solution of
30 μM CT-DNA and 2 μM EB was titrated by small aliquots of concentrated compound
solutions until the drop in fluorescence intensity (Ex = 500 nm, Em = 520.0–720.0 nm)
attained saturation. Measurements were made at constant room temperature in Tris-HCl
buffer solution. Quenching data were analyzed according to the Stern–Volmer equation (2)
which could be used to determine the fluorescent quenching mechanism [26]:

Synthesis and Characterization
231

232
L. ZHI et al.
Table 2. Selected bond lengths (Å) and angles (°) for 1–4.
1
Cu(1)–O(1)і
1.933(2)
2.011(3)
84.50(10)
180.0(2)
Cu(1)–O(1)
Cu(1)–N(1)
1.933(2)
2.011(3)
84.50(10)
115.65(18)
O(1)і–Cu(1)–O(1)
O(1)і–Cu(1)–N(1)
O(1)–Cu(1)-N(1)і
180.00(10)
95.50(10)
95.50(10)
і
Cu(1)–N(1)
O(1)–Cu(1)–N(1)
O(1)і–Cu(1)–N(1)і
C(1)–O(1)–Cu(1)
N(1)–Cu(1)–N(1)і
2
Cu(1)–O(2)
Cu(1)–N(2)
1.9274(14)
2.0239(15)
85.00(6)
84.56(6)
97.25(6)
Cu(1)–O(5)
Cu(1)–O(7)
1.9423(13)
2.2834(14)
96.53(6)
167.14(6)
96.35(6)
Cu(1)–N(1)
2.0211(15)
171.54(6)
92.12(6)
90.84(6)
96.22(6)
O(2)–Cu(1)–O(5)
O(2)–Cu(1)–N(2)
O(2)–Cu(1)–O(7)
N(2)–Cu(1)–O(7)
C(20)–O(5)–Cu(1)
C(10)–O(2)–Cu(1)
O(2)–Cu(1)–N(1)
O(5)–Cu(1)–N(2)
O(5)–Cu(1)–O(7)
C(8)–N(1)–Cu(1)
C(17)–N(2)–Cu(1)
O(5)–Cu(1)–N(1)
N(1)–Cu(1)–N(2)
N(1)–Cu(1)–O(7)
C(7)–N(1)–Cu(1)
C(18)–N(2)–Cu(1)
107.52(11)
109.35(12)
113.68(12)
108.56(11)
115.82(12)
116.05(13)
3
Cu(1)–O(5)
Cu(1)–N(1)
1.9157(19)
2.0219(19)
93.17(8)
95.72(8)
93.05(9)
Cu(1)–O(2)
Cu(1)–O(7)
1.9464(18)
2.295(2)
83.44(9)
156.15(9)
104.48(9)
108.12(15)
105.73(15)
Cu(1)–N(2)
2.0077(19)
172.75(9)
84.79(8)
94.00(8)
99.37(9)
O(5)–Cu(1)–O(2)
O(5)–Cu(1)–N(1)
O(5)–Cu(1)–O(7)
N(1)–Cu(1)–O(7)
C(8)–N(1)–Cu(1)
O(5)–Cu(1)–N(2)
O(2)–Cu(1)–N(1)
O(2)–Cu(1)–O(7)
C(25)–O(2)–Cu(1)
C(24)–N(2)–Cu(1)
O(2)–Cu(1)–N(2)
N(2)–Cu(1)–N(1)
N(2)–Cu(1)–O(7)
C(9)–O(5)–Cu(1)
C(23)–N(2)–Cu(1)
115.67(17)
108.31(15)
106.44(15)
4
Cu(1)–O(2)
Cu(1)–N(1)
O(2)–Cu(1)–N(2)
O(5)–Cu(1)–N(1)
O(5)–Cu(1)–O(7)
1.903(3)
2.017(4)
93.31(14)
95.52(16)
93.39(18)
Cu(1)–O(5)
Cu(1)–O(7)
O(5)–Cu(1)–N(2)
N(2)–Cu(1)–N(1)
N(2)–Cu(1)–O(7)
1.940(4)
2.271(4)
83.44(16)
156.34(17)
102.98(17)
Cu(1)–N(2)
2.005(4)
174.09(17)
85.47(16)
92.15(17)
100.68(18)
O(2)–Cu(1)–O(5)
O(2)–Cu(1)–N(1)
O(2)–Cu(1)–O(7)
N(1)–Cu(1)–O(7)
Symmetry codes: (i) ꢁx + 1; ꢁy, ꢁz
Table 3. Main intra and intermolecular hydrogen bonds for 1–4 in this study (Å).
D–Hꢃ ꢃ ꢃA
1
d(D–H)
d(Hꢃ ꢃ ꢃA)
d(Dꢃ ꢃ ꢃA)
\ðDHAÞ
Symmetry
O(3)–H(3C)ꢃ ꢃ ꢃO(1W)
N(1)–H(1A)ꢃ ꢃ ꢃO(3)
O(1W)–H(1WA)ꢃ ꢃ ꢃO(2)
O(1W)–H(1WB)ꢃ ꢃ ꢃO(2)
2
0.85
0.91
0.849(10)
0.850(10)
1.87
2.41
2.00(2)
1.922(14)
2.672(3)
3.293(5)
2.794(3)
2.763(3)
157.7
163.7
156(4)
170(4)
ꢁx + 1,ꢁy,ꢁz + 1
ꢁx + 1,ꢁy,ꢁz + 1
x, yꢁ1, z
ꢁx + 2, ꢁy, ꢁz
N(1)–H(1B)ꢃ ꢃ ꢃO(6)
O(4)–H(4A)ꢃ ꢃ ꢃO(6)
O(1)–H(1A)ꢃ ꢃ ꢃO(3)
O(7)–H(7C)ꢃ ꢃ ꢃO(3)
O(7)–H(7D)ꢃ ꢃ ꢃO(4)
3
0.90
0.82
0.82
0.82
0.818(15)
2.09
1.84
1.98
1.97
1.974(15)
2.974(2)
2.650(2)
2.763(2)
2.767(2)
2.773(2)
165.4
169.4
159.5
162.3
165(3)
ꢁx + 1, y + 1/2, ꢁz + 3/2
x ꢁ 1/2, ꢁy + 1/2, ꢁz + 2
x + 1/2, ꢁy + 3/2, ꢁz + 2
ꢁx, y ꢁ 1/2, ꢁz + 3/2
ꢁx + 1/2, ꢁy + 1, z ꢁ 1/2
O(1)–H(1A)ꢃ ꢃ ꢃO(6)
O(4)–H(4A)ꢃ ꢃ ꢃO(2)
O(7)–H(7C)ꢃ ꢃ ꢃO(3)
4
0.82
0.82
0.85
1.88
1.91
1.86
2.693(3)
2.721(3)
2.658(3)
172.5
167.3
155.8
x + 1/2, ꢁy + 1/2, ꢁz + 2
ꢁx + 2, y + 1/2, ꢁz + 3/2
x ꢁ 1, y, z
O(1)–H(1B)ꢃ ꢃ ꢃO(4)
O(4)–H(4A)ꢃ ꢃ ꢃO(3)
O(8)–H(8B)ꢃ ꢃ ꢃO(6)
0.82
0.82
0.89(2)
1.91
1.75
1.87(5)
2.734(5)
2.567(6)
2.723(8)
178.2
178.2
161(12)
x + 1, y + 1, z
x ꢁ 1, y, z

Synthesis and Characterization
233
F0=F ¼ Kq½Qꢂ þ 1
ð2Þ
where, F0 and F are the fluorescence intensity in the absence and presence of drug at [Q]
concentration, respectively; Kq is the quenching constant and [Q] is the quencher
concentration. Plots of F0/F vs. [Q] appear to be linear and Kq depends on temperature.
2.6. Antioxidant measurements
2.6.1. Hydroxyl radical scavenging assay. Hydroxyl radicals (OH·) in aqueous media
were generated through the Fenton system [27]. Complexes 1–4 and HL3 were dissolved
in DMF, whereas HL1, HL2, and HL4 were dissolved in H2O. The 5 ml assay mixture con-
tained the following reagents: safranin (1 mL, 114 μM), EDTA-Fe(II) (1 mL, 0.945 mM),
H2O2 (1 mL, 3%), the tested compound (1–10 μM), and a phosphate buffer (2 mL, pH
7.4). The assay mixtures were incubated at 37 °C for 30 min in a water bath, after which
the absorbance was measured at 520 nm. All the tests were run in triplicate and are
expressed as the mean standard deviation (SD). The suppression ratio for OH· was
calculated from the following expression:
Suppression ratio ð%Þ ¼ ðAi ꢁ A0Þ=ðAc ꢁ A0Þ ꢀ 100%
ð3Þ
where, Ai = the absorbance in the presence of the tested compound; A0 = the absorbance in
the absence of the tested compound; and Ac = the absorbance in the absence of the tested
compound, EDTA–Fe(II).
2.6.2. Superoxide radical scavenging assay. Superoxide radicals (Oꢁ2 ꢀ) were generated
in vitro by a nonenzymatic system and determined spectrophotometrically by nitro blue tet-
razolium (NBT) photoreduction, with a little modification to the method adopted elsewhere
[28]. The amount of Oꢁ2 ꢀ and suppression ratio for Oꢁ2 ꢀ can be calculated by measuring the
absorbance at 560 nm. The tested complexes 1–4 and HL3 were dissolved in DMF,
whereas HL1, HL2, and HL4 were dissolved in H2O. Solutions of VitB2 and NBT were
prepared avoiding light. The assay mixture, in a total volume of 5 ml, contained
MET (1 mL, 50 mM), NBT (1 mL, 499.5 μM), VitB2 (1 mL, 33 μM), the tested compound
(1–10 μM), and a phosphate buffer (2 mL, pH 7.8). The assay mixtures were incubated at
30 °C for 10 min in a water bath avoiding light, and then, illuminated by a fluorescent
lamp for 10 min. After which, the absorbance of the samples (Ai) was measured at 560 nm.
The sample without the tested compound was used as control and its absorbance was A0.
All experimental results were expressed as the mean SD of triplicate determinations. The
suppression ratio for Oꢁ2 ꢀ was calculated from the following expression:
suppression ratio ð%Þ ¼ ðA0 ꢁ AiÞ=A0 ꢀ 100%
ð4Þ
Drug activity was expressed as the 50% inhibitory concentration (IC50). IC50 values
were calculated from regression lines where x was the tested compound concentration in
μM and y was percent inhibition of the tested compounds.

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3. Results and discussion
3.1. Structures of the copper complexes
As shown in figure 2, the asymmetric unit of 1 contains one-half of [Cu(L1)2] and a lattice
water, with Cu(II) on a twofold rotational axis. Copper coordinates to two nitrogens and
two carboxylate oxygens from two Lꢁ1 anions, giving a distorted square planar geometry
with a N2O2 donor set. The Cu–N and Cu–O bond distances are 1.933 and 2.011 Å,
respectively. The bond angle of O(1)–Cu(1)–N(1) is 84.50 (10)°.
The structures of 2, 3, and 4 are similar. Unlike 1, Cu2+ in 2, 3, and 4 coordinates to two
nitrogens from two ligand anions and three oxygens, two of which are from two ligand
anions and the other is from water, methanol and water, respectively. The coordination
geometry was estimated from τ values varying from 0 for an idealized square pyramid to 1
for an idealized trigonal bipyramid [29]. The τ values of 2, 3, and 4 are 0.0733, 0.2767, and
0.2985, respectively. (N(1)–Cu(1)–N(2) and O(2)–Cu(1)–O(5) in 2, 3, and 4 are 167.14(6)
and 171.54(6), 156.15(9) and 172.75(9)°, and 156.34(17) and 174.09(17), respectively. Cu2+
in 2, 3, and 4 possess coordination geometry closer to distorted square-pyramidal rather than
distorted trigonal-bipyramidal. In the five-coordinate structure, the four basal positions are
occupied by N(1), N(2), O(2), and O(5). The coordination sphere at the apical position is
completed by O7 of water in 2 and 4, but methanol in 3. In 4, the location occupied by C11
is disordered, as C11 and C11a, and both are 0.5. In crystals of 1–4, there are complicated
intermolecular and intramolecular O–Hꢃ ꢃ ꢃO interactions, constructing 3D frameworks.
3.2. Interactions with CT-DNA
3.2.1. Electronic absorption spectroscopy. Electronic absorption spectroscopy is one of
the most useful techniques in examining the binding mode of DNA with metal complexes
Figure 2. Molecular structures of 1–4. C–H hydrogens are omitted for clarity.

Synthesis and Characterization
235
[2]. Intercalative binding usually results in hypochromism and red shift because of the
strong stacking interaction between an aromatic chromophore and the base pairs of DNA.
The extent of spectral change is related to the strength of binding [30]. Electronic absorp-
tion spectra of the ligand and its Cu(II) complexes in the absence and presence of
CT-DNA (at a constant concentration of the compounds) were obtained, (figure 3). With
increasing DNA concentration, absorption at 274 nm of 1–4 represents hypochromism of
4.89, 14.30, 13.92, and 43.34%, respectively. The hypochromism observed for the π–π⁄
transitions indicate strong binding of the complexes to DNA. The intrinsic binding con-
stant Kb of 1–4 was 1.32 ꢀ 106, 4.32 ꢀ 105, 5.00 ꢀ 105, and 5.70 ꢀ 104 Mꢁ1, respectively,
which is not in agreement with the hypochromism degree. The Kb values are lower than
that reported for the classical intercalator (for ethidium bromide Kb = 1.4 ꢀ 106 Mꢁ1 and
[Ru(phen)DPPZ] with binding constants of 106–107 Mꢁ1) [31–34]. However, they are
higher than some reported complexes as shown in table 4. Our results suggest an intimate
association of the compounds with CT-DNA and indicate binding strengths of the
complexes that follow the order of 1 > 3 > 2 > 4.
3.2.2. Fluorescence spectroscopy. Fixed amounts (10 μM) of 1–4 were titrated with
increasing amounts of CT-DNA; fluorescence titration spectra of the complexes in the
absence and presence of CT-DNA are given in figure 4. Compared to the complexes alone,
the fluorescence intensity of the complexes is quenched steadily with increasing concentra-
tion of CT-DNA. The quenching of luminescence of the complex by DNA may be attrib-
uted to photo-electron transfer from the guanine base of DNA to the excited MLCT state
of the complex [39–44]. These changes also proved that there were conjugation functions
between CT-DNA and the compounds [45, 46].
3.2.3. Competitive DNA-binding studies with EB. In order to further investigate the
interaction mode between the complexes and CT-DNA, fluorescence titration experiments
were performed. Fluorescence titration experiments, especially ethidium bromide (EtBr)
fluorescence competitive experiments, have been widely used to characterize the interaction
of complexes with DNA by following the changes in fluorescence intensity of the com-
plexes. The intrinsic fluorescence intensity of EB is low and that of DNA in Tris-HCl buffer
is also not high due to the quenching by solvent; fluorescence intensity of EB is enhanced
on addition of DNA, because EB intercalates into DNA. The enhanced fluorescence can be
quenched when there is a second molecule that can replace DNA-bound EB [47].
As depicted in figure 5, for 1–4 the fluorescence intensity of EB at 601 nm shows remark-
able decrease with increasing concentration of the complexes, indicating that some EB mole-
cules are released from EB–DNA after exchange with 1–4, quenching fluorescence of EB.
We assume the reduction of the emission intensity of EB on increasing complex concentra-
tion could be due to displacement of DNA-bound EB by the copper(II) complexes [48].
Such quenched fluorescence behavior of EB bound to DNA is also found in other copper
complexes [49]. Kq values of 1–4 are 1.516 ꢀ 105, 1.913 ꢀ 105, 1.436 ꢀ 105, and
1.481 ꢀ105 Mꢁ1, respectively, higher than those of some copper(II) complexes, such as
[Cu2(oxpep)phen]ClO4·(Kq = 9.75 ꢀ 104 Mꢁ1
)
[50], [Cu2(dmeo) (N3)2]n·(Kq = 1.85 ꢀ
103 Mꢁ1) [51], and [Cu2L(OAc)(CH3OH)]·CH3OH·(Kq = 4.89 ꢀ 102 Mꢁ1) [52]. The data for
Kq are in the order 2 > 1 > 4 > 3, which is not in agreement with the UV–Vis spectroscopy
results. Phenolic hydroxyl groups that can bind to nucleotides or/and the sugar–phosphate

236
L. ZHI et al.
Figure 3. (a), (b), (c), and (d) are the electronic spectra of 1–4 (10 μM) in the presence of increasing CT-DNA,
respectively. Arrow shows the absorbance changes upon increasing DNA concentration. Inset: Plot of [DNA]/
(ɛb ꢁ ɛf) vs. [DNA] for the titration of the Cu(II) complexes with CT-DNA.

Synthesis and Characterization
237
Ref.
Table 4. Kb values of some complexes.
Complex
Kb (Mꢁ1
)
[Cu(II)(La)(B)]
3.32 ꢀ 105
1.55 ꢀ 104
4.87 ꢀ 103
4.91 ꢀ103
[35]
[36]
[37]
[7]
CuLb
2.7 ꢀ 104
[Cu(Lc)(bpy)Cl]
[Cu(Ld)phen]CH3OH·0.5H2O
[Cu2(Le)2(H2O)]
[38]
H2La = salicylidene tryptophan, B = 1,10-phenanthroline.
H2Lb = Schiff base derived from condensation of 5-nitro-ovanillin and diaminoethane.
HLc = (E)-3-(2-hydroxyphenylimino)-N-o-tolylbutanamide.
H2Ld = (E)-2-((2-hydroxynaphthalen-1-yl)methyleneamino)-4-methylpentanoic acid.
H2Le = (E)-2-(2-hydroxybenzylideneamino)-4-(methylthio)butanoic acid.
Figure 4. (a), (b), (c), and (d) are emission spectra of 1–4 (10 μM) in the presence of increasing CT-DNA,
respectively. Arrow shows the emission intensity changes upon increasing DNA concentration.
backbone of DNA through hydrogen bonds may play roles in the EB–DNA quenching tests.
However, other weak interactions such as hydrophobic, van der Waals, and electrostatic
forces may not be excluded. The interaction mechanism is not only determined by complex
formation but also by weak interactions [53].
3.3. Hydroxyl radical scavenging activity
Antioxidant activities of the ligands and their Cu(II) complexes were investigated. The
inhibitory effect of the tested compounds on OH˙ is concentration related and the

238
L. ZHI et al.
Figure 5. (a) Emission spectra of EB–DNA system in the presence of 1. Inset: Stern–Volmer plot of the
fluorescence titration data of 1. (b) The emission spectra of EB–DNA system in the presence of 2. Inset: Stern–
Volmer plot of the fluorescence titration data of 2. (c) The emission spectra of EB–DNA system in the presence
of 3. Inset: Stern–Volmer plot of the fluorescence titration data of 3. (d) The emission spectra of EB–DNA system
in the presence of 4. Inset: Stern–Volmer plot of the fluorescence titration data of 4. Arrows show the emission
intensity changes upon increasing concentration.

Synthesis and Characterization
239
suppression ratio increases with increasing concentration of the sample (figure 6). The IC50
values of the ligands cannot be read, while those of 1–4 are 4.61, 4.40, 2.92, and 4.42 μM,
respectively. Hydroxyl radical scavenging effects of the Cu(II) complexes are much higher
than those of the ligands, possibly from larger conjugated metal complexes reacting with
OH˙ to form larger stable macromolecular radicals [15]. The order of the suppression ratio
for OH˙ is 3 > 2 > 4 > 1. Usually, mannitol is employed as a standard since it is known to
selectively inhibit OH˙ radical. The IC50 values of the complexes are far less than that of
mannitol (10.34 μM) [54], however, slightly higher than that of other active compounds
and some reported Cu(II) complexes as shown in table 5.
3.4. Superoxide radical scavenging activity
The ligands cannot scavenge Oꢁ2 ꢀ in the tested concentration range. However, the com-
plexes can scavenge Oꢁ2 ꢀ in a concentration-dependent manner (figure 7). IC50 values of
1–4 are 0.04, 0.24, 0.18, and 0.22 μM, respectively, indicating that Cu(II) plays a role in
scavenging superoxide. These compounds show higher superoxide dismutase activity than
standard antioxidants like vitamin C (IC50 = 852 μM) or nitroxide Tempo (IC50 = 60
3 μM), which has been recently used in biological systems for its capacity to mimic
superoxide dismutase [15]. SOD data for the complexes are compiled in table 6 along with
SOD activities of similar complexes containing copper. Complexes 1–4 show higher SOD
activity than other similar complexes, but slightly lower than that of native Cu, Zn-SOD
[55]. The Cu(II) complexes show similar superoxide dismutase activity comparable with
that of native Cu, Zn-SOD. The order of the suppression ratio for Oꢁ2 ꢀ is 1 > 3 > 4 > 2 at
different concentrations, but similar at 0.5 μM. Superoxide radical scavenging activity of 1
is best, probably due to the relatively low coordination number of copper in 1. The order
of the suppression ratio for Oꢁ2 ꢀ is notably different from the order of suppression ratio for
OH˙, suggesting different mechanisms between scavenging or inhibiting OH˙ and Oꢁ2 ꢀ,
which should be further studied.
Figure 6. Plots of the hydroxyl radical scavenging effect (%) for ligands and Cu(II) complexes.

240
L. ZHI et al.
Table 5. Hydroxy radical scavenging activity of some copper(II) complexes.
Complex
IC50 (μM)
Ref.
Mannitol
10.34
4.6
5.5
<2.5
4.61
4.40
2.92
4.42
[54]
[15]
[15]
27[27]
[Cu4(phen)6(Lf)(H2O)2](ClO4)6·3H2O
[Cu2(phen)2(Lf)(H2O)2](ClO4)2·2.5H2O
[Cu(H3Lg)]
1
2
3
4
Present work
Present work
Present work
Present work
H2Lf = N,N′-(p-xylylene)di-alanine acid. phen = 1,10-phenanthroline.
H5Lg = naringenin-2-hydroxy benzoyl hydrazone.
Figure 7. Plots of superoxide radical scavenging effect (%) for Cu(II) complexes.
Table 6. Superoxide dismutase activity of some copper(II) complexes.
Complex
IC50 (μM)
Ref.
Native SOD
0.04
105
[55]
[56]
[57]
[58]
[59]
[Cu(Lh)(bipy)](ClO4)2
Cu(Li)2(Lj)2 (1)
0.81
9.24
1.42
0.04
0.24
0.18
0.22
[Cu2(μ-Lk)4(ClO4)2](ClO4)2·2MeOH
[Cu2(Ll)(Lm)](ClO4)3·H2O
1
2
3
4
Present work
Present work
Present work
Present work
Lh = N,N,N′,N′′,N′′-pentamethyldiethylenetriamine.
HLi = aspirinato.
Lj = 2-methylimidazole
Lk = 6-(3-fluorobenzylamino)purine.
HLl = 6, 20-bis(1H-imidazol-4-ylmethyl)-3, 6, 9, 17, 20, 23-hexaazatricyclo [24,3,1,111,15] hentriaconta-1(29), 11,
13, 15(31), 26(30), 27-hexaene.
Lm = imidazole.

Synthesis and Characterization
241
4. Conclusions
Four copper(II) complexes with amino acid ligands were prepared and characterized by
single-crystal X-ray crystallography. The Cu(II) complexes bind to CT-DNA through inter-
calation with binding constants of 105–106 Mꢁ1, and the Cu(II) complexes present stronger
affinities to DNA than ligands, showing potential as potential anticancer drugs. The
complexes show similar superoxide dismutase activities to that of native Cu, Zn-SOD.
Further research is needed to better determine the relationship between structures and
activities.
Acknowledgement
This work was supported by the National Science Foundation of China (21001040).
Supplementary data
CCDC 842993–842995 and 873438 contain the supplementary crystallographic data for 1, 2, 3, and
4, respectively. These data can be obtained free of charge from The Cambridge Crystallographic Data
Center via www.ccdc.cam.ac.uk/data_request/cif.
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