Copper(II) complex with the tridentate ligand N,N-bis(2-ethyl-4-methyl- imidazol-5-ylmethyl)phenylethylamine (biaq). X-ray crystal structure and biological activity on bacillus subtilis of [Cu(biaq)Cl2].

Article abstract of DOI:10.1002/zaac.201300082

The ligand N,N-bis(2-ethyl-4-methyl-imidazol-5-ylmethyl)-1-phenylethylamine (biaq) was prepared by Mannich reaction of 2-ethyl-4-methyl-1H-imidazole, (R)-1-phenylethylamine and formaldehyde. It was characterized by FTIR and NMR spectroscopy. This ligand reacts with CuCl₂ to give a coordination compound with formula [Cu(biaq)Cl₂]. In this compound, biaq acts as a tridentate ligand with one tertiary amine and two azole nitrogen atoms coordinated to the central metal atom. The complex was characterized by FTIR spectroscopy, elemental analysis, and X-ray diffraction. The X-ray structure of [Cu(biaq)Cl₂] shows a pentacoordinate cooper(II) atom, where two chloride ions and the three nitrogen atoms of the biaq ligand are coordinated in a distorted square pyramidal arrangement. The biological activity of the complex [Cu(biaq)Cl₂] was tested using the Gram positive bacterium Bacillus subtilis as a model. Results revealed that the copper complex interfered with bacterial growth, inducing cell death, most probably by altering the primary structure of the chromosome, since a significant increase of the mutation frequency to rifampicin resistance was observed. Copyright

Full text of DOI:10.1002/zaac.201300082

ARTICLE
DOI: 10.1002/zaac.201300082
Copper(II) Complex with the Tridentate Ligand N,N-bis(2-ethyl-4-methyl-
imidazol-5-ylmethyl)phenylethylamine (biaq). X-ray Crystal Structure and
Biological Activity on Bacillus subtilis of [Cu(biaq)Cl2].
Ángeles Mendoza,[a] Guillermo Mendoza-Díaz,*[b] Mario Pedraza-Reyes,[c] and
Sylvain Bernès[d]
Keywords: Antibacterial properties; Copper; Imidazole derivatives; Mutagenic activity
Abstract. The ligand N,N-bis(2-ethyl-4-methyl-imidazol-5-ylmethyl)- ture of [Cu(biaq)Cl2] shows a pentacoordinate cooper(II) atom, where
1-phenylethylamine (biaq) was prepared by Mannich reaction of 2-
ethyl-4-methyl-1H-imidazole, (R)-1-phenylethylamine and formalde-
hyde. It was characterized by FTIR and NMR spectroscopy. This li-
gand reacts with CuCl2 to give a coordination compound with formula
two chloride ions and the three nitrogen atoms of the biaq ligand are
coordinated in a distorted square pyramidal arrangement. The bio-
logical activity of the complex [Cu(biaq)Cl2] was tested using the
Gram positive bacterium Bacillus subtilis as a model. Results revealed
[Cu(biaq)Cl2]. In this compound, biaq acts as a tridentate ligand with that the copper complex interfered with bacterial growth, inducing cell
one tertiary amine and two azole nitrogen atoms coordinated to the
central metal atom. The complex was characterized by FTIR spec-
troscopy, elemental analysis, and X-ray diffraction. The X-ray struc-
death, most probably by altering the primary structure of the chromo-
some, since a significant increase of the mutation frequency to rifampi-
cin resistance was observed.
many copper metalloproteins imidazole residues from histidine
appear to be coordinated to copper.[4,5] Therefore, the synthesis
of small complexes that could mimic the coordination environ-
ment, catalytic behavior or spectroscopic properties of such
metalloproteins, is currently a challenge for many groups. Se-
lected examples of ligands and complexes reported in the
literature related to the present work may be cited: The
linear tridentate ligand N,N-bis(2-ethyl-5-methylimidazol-4-yl-
methyl)aminopropane (biap) and the coordination compounds
of biap with CoII, NiII, ZnII,[6] FeIII,[7] and CuII[8] have been
reported. The copper complex of biap has been used success-
fully for modeling nitrite reductase.[9] Similar ligands as
N,N-bis(2-ethyl-5-methylimidazol-4-ylmethyl)aminomethane
(biam), N,N-bis(2-ethyl-5-methylimidazol-4-ylmethyl)amino-
ethanol (biae), and N,N-bis(2-ethyl-5-methylimidazol-4-yl-
methyl)amino-2-ethylpyridine (biapy) form complexes with
copper(II) featuring a distorted square pyramidal environ-
ment.[8] All the above mentioned ligands were prepared by a
Mannich condensation, a straightforward reaction using simple
reagents like formaldehyde, amines, and imidazoles.[6,9] in a
one pot procedure.
On the other hand, antibiotic rifampicin, a potent inhibitor
of prokaryotic transcription[10] has been used as a highly clini-
cally effective drug, particularly in the treatment of tuberculo-
sis. Mutations conferring rifampicin resistance (RifR) occur at
specific clusters within the gene encoding the β subunit of
the prokaryotic RNA polymerase. RifR RNA polymerases have
been extensively characterized in E. coli; these have altered
properties in transcription elongation and/or termination.[11] In
E. coli, RifR mutations are usually located in the central region
1 Introduction
Imidazole-based ligands have been matter of medical inter-
est because they may have some biological activities as anti-
bacterial and antiparasitic agents. Also, the development of
such ligands is related to the preparation of small molecules
that may mimic some copper-containing enzymes, and there-
fore may have some interesting catalytic effects. The interest
in having a better understanding of how central copper atoms
work in biological systems, in order to develop new metal-
based drugs, is still a growing topic. In particular, this interest
has been directed toward the synthesis of copper complexes
with imidazole derivatives. Some of these imidazole-based li-
gands, NL3 (L = imidazole[1–3]), have a tripodal shape. The
interest in this type of ligands comes from the fact that in
* Prof. Dr. G. Mendoza-Díaz
E-Mail: mendozag@ugto.mx
[a] Departamento de Química
División de Ciencias Naturales y Exactas
Campus Guanajuato, Universidad de Guanajuato
Noria Alta s/n
Guanajuato, Gto. 36050, Mexico
[b] Departamento de Ingenierías Química
Electrónica y biomédica, División de Ciencias e Ingenierías
Campus León, Universidad de Guanajuato
Loma del Bosque 103
Lomas del Campestre, León, Guanajuato. 37150, Mexico
[c] Departamento de Biología
División de Ciencias Naturales y Exactas
Campus Guanajuato, Universidad de Guanajuato
Noria Alta s/n
Guanajuato, Gto. 36050 Mexico
[d] DEP Facultad de Ciencias Químicas, UANL
Guerrero y Progreso s/n Col. Treviño
64570 Monterrey, NL, Mexico
Z. Anorg. Allg. Chem. 2013, 639, (8-9), 1455–1460
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1455

G. Mendoza-Díaz et al.
ARTICLE
of the β subunit polypeptide within clusters I, II, and III.[12]
RifR mutations can also occur near the N-terminus of the β
subunit.[13] RifR mutations have been shown to map as well in
the rpoB gene[14] of B. subtilis but the effect of these mutations
on transcription elongation and termination is less known. In-
deed, the effect conferred by RifR mutations on transcription
[C(ipso) Ph], 128.02 [C(meta) Ph], 127.88 [C(para) Ph], 146.42
[C(imz)].
2.2 Synthesis of Complex [Cu(biaq)Cl2]
Into a solution of CuCl2·2H2O (1 mmol) in H2O (2 mL), the ligand
elongation and termination in non-enteric bacteria warrants a biaq (1 mmol) dissolved in H2O (3 mL) was added. The mixture was
stirred for 5 h at room temperature. The solvent was evaporated under
reduced pressure. The solution was left to stand for a few minutes, and
the green precipitate, which was formed, was filtered off. The isolated
green product was washed with cold ethanol and dried in air. Yield:
59% (see Scheme 2). Elemental analysis for C22H31N5CuCl2: calcd. C
52.85; H 6.20; N 14.01%; found: C 53.17; H 6.41; N 11.90%. FT-IR
(KBr): ν˜ 3213 (br., w) 2965 (w) 2917 (w) 1597 (w) 1571 (w) 1485
(w) 1445 (w) 1380 (s) 1280 (w) 1207 (w) 1084 (vs) 1036 (vs) 965
(m) 766 (s) 703 (s) 614 (s) 589 (s) 545 (m) cm–1.
deeper investigation. For its ability to display different devel-
opmental programs including sporulation, competence and
adaptive mutagenesis, among others, B. subtilis requirements
during transcription may in some instances be different than
those described in E. coli.[15]
In this paper, we present the synthesis of a new imidazole-
based ligand, namely N,N-bis(2-ethyl-4-methyl-imidazol-5-yl-
methyl)-1-phenylethylamine (biaq) and its copper(II) complex.
The X-ray crystal structure is reported, along with some bio-
logical properties tested in vitro in the Gram-positive bacte-
rium B. subtilis.
2.3 Crystal Structure of [Cu(biaq)Cl2]
Green crystals suitable for X-ray diffraction were obtained by crystalli-
zation from a saturated solution of [Cu(biaq)Cl2] in water at room
temperature. Diffraction data were collected with a Bruker P4 dif-
fractometer with Mo-Kα radiation (λ = 0.71073 Å) and θ/2θ scans.[17a]
The selected crystal was twinned in space group P21/n by pseudo-
merohedry, through a twofold rotation about [001] direct lattice direc-
tion, and refinement of fractional contributions of twin domains con-
verged to k1 = 0.801(2) and k2 = 0.199(2). Such a twinning is consis-
tent with features observed during data reduction and structure refine-
ment: the cell parameters emulate an orthorhombic symmetry, Wilson’s
statistics are abnormally low for a centrosymmetric space group (for
instance, |E2–1| = 0.776), and a significant drop for residual R1, from
0.15 to 0.07, was obtained after twinning was accounted for. The
methyl group bonded to the chiral center in the ligand, C20, is clearly
disordered over two positions, C211 and C212, with occupancies ½.
It is worth mentioning that a satisfactory refinement was only obtained
in space group P21/n, while strong correlations were observed when
attempting to refine in non-centrosymmetric space groups, P21 or Pn,
with two molecules in the asymmetric unit, along with expected symp-
toms for a wrong space group, like a Flack parameter close to 1/2. As
a consequence, regardless of twinning and positional disorder in the
crystal, the crystallized compound is a racemic mixture of complexes
with R and S biaq ligands. The structure was solved and refined using
SHELXS and SHELXL[17b] following standard procedures (Table 1).
Selected bond lengths, and angles are collected in Table 2. ORTEP-
like[17a] projection of the structure is given in Figure 1.
2 Experimental Section
Starting materials (R)-1-phenylethylamine, 2-ethyl-4-methyl-1H-imid-
azole (Sigma-Aldrich products), and CuCl2·2H2O (J.T. Baker products)
1
were used as provided. H NMR spectra were obtained with a VAR-
IAN Gemini 2000 (200 MHz) spectrometer. FTIR spectra were ob-
tained with a Perkin-Elmer spectrum100 spectrophotometer, using the
transmittance technique in the range 4000–400 cm–1 (KBr pellets).
2.1 Synthesis of N,N-bis(2-ethyl-4-methyl-imidazol-5-
ylmethyl)-1-phenylethylamine (biaq)
This ligand was prepared by a modification of the Mannich reaction.
First, 2-ethyl-4-methyl-5-hydroxymethyl-imidazole was prepared, fol-
lowed by two different procedures:
Method a: Into
a solution of 2-ethyl-4methyl-1H-imidazole
(4.5 mmol) in water (5 mL) was added conc. HCl (20 mmol) and form-
aldehyde (13.62 mmol). The mixture was refluxed for 48 h.[16] After
this time, solvent was removed until a yellow oil was formed. This
oil was dissolved in ethanol and the product crystallized upon slow
evaporation. Yield: 82%.
Method b: 2-Ethyl-4methyl-1H-imidazole (1.0 mmol) was mixed with
water (3 mmol) and formaldehyde (3 mmol) and KOH (1.5 mmol)
were added, to give a final pH of 12.5. The mixture was stirred and
maintained between 15 and 25 °C during 28 h. Afterwards the mixture
was dried by slow evaporation at room temperature until a white pre-
cipitate was obtained. The precipitate was washed with cold acetone
and dried at 45 °C. Yield: 98%.
Crystallographic data (excluding structure factors) for the structure in
this paper have been deposited with the Cambridge Crystallographic
Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK.
Copies of the data can be obtained free of charge on quoting the
depository number CCDC-923618 (Fax: +44-1223-336-033; E-Mail:
deposit@ccdc.cam.ac.uk, http://www.ccdc.cam.ac.uk)
Into a solution of (R)-1-phenylethylamine hydrochloride (1 mmol,
30% in H2O), 2-ethyl-4-methyl-5-hydroxymethyl-imidazole (2 mmol)
was added. This mixture was vigorously stirred for 8 d at room tem-
perature in a closed vessel. After this time, solvent was evaporated,
affording a yellow oil.
2.4 Biological Test
Organism and Growth Conditions: Strain B. subtilis PERM115 har-
1H NMR (D2O): δ = 1.36 (t) CH2–CH3, 1.68 (d) CH3, 2.21 (s) CH3, boring a recA-lacZ fusion integrated into the amyE locus was used in
2.91 (q) CH2–CH3, 4.02 (s) CH2, 4.59 (q) CH, 7.56 (s) Ph. 13C{1H} this study. The growth medium used routinely was Luria Bertani
NMR (D2O): δ = 7.40 [CH2CH3], 9.88 [CH3(imz)], 18.06 [CH2CH3], (LB)[18] supplemented with chloramphenicol (Cm) at a final concentra-
18.89 [CHCH3(amine)], 50.14 [CH(amine)], 51.14 [CH2(bridge)], tion of 5 μg·mL–1. Liquid cultures were incubated at 37 °C with vigor-
125.05 [C(imz)], 125.61 [C(ortho) Ph], 136.55 [C(imz)], 125.17 ous aeration.
1456
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 1455–1460

X-ray Crystal Structure and Biological Activity on Bacillus subtilis of [Cu(biaq)Cl2]
Analysis of Mutagenesis Induced by [Cu(biaq)Cl2]: A 20 mL B.
Table 1. Crystal and refinement data for [Cu(biaq)Cl2].
subtilis culture grown to an OD600nm of 0.5 was treated with the
[Cu(biaq)Cl2] complex at a final concentration of 0.031 mM. The cul-
ture was incubated for 24 h at 37 °C and shaken at 250 rpm. After this
period, cells were collected by centrifugation (4500 rpm over 10 min)
and the cell pellets re-suspended in 1 mL of Spizizen’s 1X salts. Mu-
tation frequencies were determined by plating aliquots of each culture
on six LB plates containing rifampicin at 5 μg·ml–1 concentration, as
well as plating aliquots of appropriate dilutions on the LB plates with-
out rifampicin. Rifampicin-resistant colonies (RifR) were counted after
24 h of incubation at 37 °C. A culture that was not treated with the
metal complex was used as a control to estimate the spontaneous mu-
tation frequency of the strain. Similar experiments were repeated using
the free ligand biaq instead of the biaq complex.
Formula
C22H31Cl2N5Cu
499.96
0.42ϫ0.40ϫ0.36
monoclinic
P21/n
8.6999(13)
Formula weight
Crystal size /mm3
Crystal system
Space group
a /
b /
c /
11.7224(19)
23.262(3)
β /°
90.541(12)
2372.2(6)
V /3
Z
4
2θ Range /°
3.48–50.00
1.400
1.165
Dc /g·cm–3
μ /mm–1
Transmission factors
Reflections collected
Independent reflections
Final R indices [I Ͼ 2 σ(I)]
Final R indices [all data]
Largest difference peak,
hole /e·Å–3
min = 0.552, max = 0.658
7435
4156 (Rint = 6.68%)
R1 = 7.14%, wR2 = 18.70%
R1 = 8.80%, wR2 = 20.45%
0.688, –0.778
Effect of [Cu(biaq)Cl2] on the Growth of B. subtilis as Determined
by Viable Counts: Strain B. subtilis PERM115 was propagated in LB
medium to an OD600nm of 0.5; at this point, the culture was split into
two equal subcultures, one of which was left untreated as a control,
while the other was supplemented with [Cu(biaq)Cl2] to a final con-
centration of 0.3125 mM; both cultures were re-incubated and shaken
at 37 °C. Cell samples collected from both cultures at 0.5, 1, and 1.5
h were processed to determine the fraction of surviving cells, as mea-
sured by viable counts. To this end, aliquots of appropriate dilutions
from each time point were plated on solid LB medium to determine
the number of colonies that grew after 24 h of incubation at 37 °C.
Goodness-of-fit on F2
S = 1.058
Table 2. Selected bond lengths /Å and angles /° of [Cu(biaq)Cl2].
Cu1–N3
Cu1–N13
Cu1–N10
Cu1–Cl1
Cu1–Cl2
2.007(6)
2.019(6)
2.164(6)
2.255(2)
2.650(2)
N3–Cu1–N13
N3–Cu1–N10
N13–Cu1–N10
N3–Cu1–Cl1
N13–Cu1–Cl1
N10–Cu1–Cl1
N3–Cu1–Cl2
N13–Cu1–Cl2
N10–Cu1–Cl2
Cl1–Cu1–Cl2
160.2(2)
80.2(2)
80.9(2)
98.15(19)
98.91(18)
166.46(19)
92.99(18)
94.34(18)
94.91(17)
98.60(10)
Induction Experiments: A culture of B. subtilis PERM115 was grown
to an OD600nm of 0.5, and at this point, the culture was split into 7
subcultures of equal volumes. One culture was left untreated, another
one was supplemented with 1 μg·mL–1 of mitomycin-C, and the other
five were supplemented with the copper complex at the following final
concentrations: 0.03, 0.06, 0.12, 0.25, and 0.5 mM, respectively. The
different cultures were shaken at 250 rpm and 37 °C; samples of each
culture were collected at 1, 2, and 4 h and processed to determine the
OD600 and β-galactosidase activity as described previously.[19,20] The
β-galactosidase activity is reported in Miller units.[21,22]
3 Results and Discussion
3.1 Ligand Preparation
Ligand biaq was synthesized in two steps using a modifica-
tion of the Mannich reaction. First, 2-ethyl-4-methyl-5-hy-
droxymethyl-imidazole was prepared, either as described in
the literature[16] or, alternatively, using a basic media (see Ex-
perimental Section), giving an improved yield. Next, the reac-
tion of this intermediate with (R)-1-phenylethylamine hydro-
chloride was carried out (Scheme 1). The product was isolated
as an oil and the enantiomeric purity of the starting material
was lost ([α]D = 0). The racemic nature of biaq suggests that
during the synthesis some intermediate should be formed, in
which the methylene group is deprotonated, generating a
planar carbocation in water. The product is very soluble in
water, methanol, ethanol, and acetonitrile. The infrared spectra
show bands typical for imidazolic N–C bonds, and the mono-
substitution of an aromatic ring.
Figure 1. ORTEP-like[17a] view of complex [Cu(biaq)Cl2], with dis-
placement ellipsoids at 30% probability level for non-hydrogen atoms.
The potentially chiral center is C20, and the inset shows the positional
disorder for the methyl group bonded to C20. This methyl is disordered
over two sites, C211 and C212, with equal occupancies. In the inset
figure, hydrogen atoms are omitted, except for the disordered methyl
groups.
LD50 Determination: 30 mL of LB medium supplemented with Cm
were inoculated with 1 mL of an overnight culture of strain B. subtilis
PERM115. The culture was propagated at 37 °C in a metabolic stirrer
adjusted at 250 rpm until an optical density of 0.5 at 600 nm (OD600nm
= 0.5) was reached. 2 mL aliquots from this culture were indepen-
dently transferred to different assay tubes containing different concen-
trations of the [Cu(biaq)Cl2] complex. These tubes were incubated for
2 h, and afterwards the OD600nm of each culture was measured.
3.2 Synthesis and Crystal Structure of the Complex
[Cu(biaq)Cl2]
The synthesis of [Cu(biaq)Cl2] was realized in water, using
equimolar quantities of copper chloride and biaq ligand (see
Z. Anorg. Allg. Chem. 2013, 1455–1460
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1457

G. Mendoza-Díaz et al.
ARTICLE
Scheme 1. Synthesis of the ligand biaq.
Scheme 2). The complex was isolated as
a
green 3.3 [Cu(biaq)Cl2] Interferes with the Growth of Bacillus
powder, which is soluble in ethanol and water. Crystals of
[Cu(biaq)Cl2] were grown by slow evaporation of a water
solution of the complex.
subtilis
To analyze the biologic effects of [Cu(biaq)Cl2], different
amounts of this metal complex, in the range from 1.25 to
10 mM, were added to logarithmically growing cultures of
strain B. subtilis PERM115. Table 3 presents the results ob-
tained at different times of incubation. From these data it is
possible to conclude that even at concentrations as low as
1.25 mM, the complex exert an important effect on the bacteria
growing, making that cell culture develops slower and appar-
ently producing a 40% inhibition after 3 h of culture. As the
complex concentration is increased the inhibition of the growth
is bigger, but also it is observed that as incubation time in-
creases and concentration is higher, the complex interfere more
with bacterial growth, most probably by promoting cell death
(Table 3). Based on these results, apparent LD50 and LD75 of
[Cu(biaq)Cl2] on the growth of B. subtilis are ca. 1.25 mM and
2.5 mM, respectively.
Scheme 2. Synthesis of the complex [Cu(biaq)Cl2].
[Cu(biaq)Cl2] is a mononuclear complex, in which the cop-
per ion displays a distorted square pyramidal N3Cl2 environ-
ment, with an Addison τ parameter[23] of 0.10. The copper ion
is slightly above (0.201 Å) the least-squares basal plane pass-
ing through N13, N3, N10, and Cl1 (Figure 1). The Cu–N dis-
tances (see Table 2) of 2.007(6) and 2.019(6) Å for the imid-
azole nitrogen atoms and 2.164(6) Å for the amine nitrogen
atom are in the normal range.[9,24] The apical Cu–Cl bond
length of 2.650(2) Å is longer than the basal Cu–Cl distance
of 2.255(2) Å due to the Jahn-Teller effect. The three-bonds
bite-angles of the ligand induce coordination angles in the
basal plane to be smaller than 90°, i.e. 80.2(2)° for N3–Cu1–
N10 and 80.9(2)° for N13–Cu1–N10, while angles involving
basal chloride Cl1 are larger than 90° (see Table 2).
Table 3. Effect of different amounts of the [Cu(biaq)Cl2] complex on
the growth of strain B. subtilis PERM115 at different periods of incu-
bation.
Growing /%
Incubation time /h [Cu(biaq)Cl2] concentration /mM
0
1.25
2.5
5
10
1
2
3
100
100
100
40.3
68
62
29
25
8
15
23
8
10
23
9
Interestingly, two very similar mononuclear complexes,
[Cu(biap)Cl2][8] and [Cu(biam)Cl2]·MeOH were synthesized
and characterized by single-crystal X-ray diffraction. Also in
these two complexes the copper ion is five-coordinated with
an N3Cl2 donor set, and with the N donor sites of the tridentate
ligands belonging to the base of the polyhedron of coordina-
tion. These two previously reported complexes present a coor-
dination arrangement close to that observed for [Cu(biaq)Cl2],
To investigate whether the metal complex affected growth
by promoting cell death, a B. subtilis culture growing logarith-
mically was treated for different periods of time with the metal
complex at a concentration of 0.03125 mM, and the fraction
of surviving cells was determined by viable counts in compari-
son with an untreated control culture. As shown in Figure 2,
after 1.5 h of incubation the metal complex dramatically re-
duced the amount of viable cells. Taken together, these results
strongly suggest that [Cu(biaq)Cl2] not only interferes with
bacterial growth but also promotes cell death. In agreement
with the former notion, a microscopic analysis of cells grown
in the presence of 0.03125 mM of the copper complex revealed
the formation of cell filaments (Figure 3), a phenomenon usu-
ally associated with the inhibition of replication and/or cellular
division of bacterial cells.[25]
the Addison
τ parameters being 0.13 and 0.08 for
[Cu(biap)Cl2] and [Cu(biam)Cl2]·MeOH, respectively.
The X-ray diffraction study shows that the complex is crys-
tallized as a racemic mixture, as reflected by the centrosym-
metric character of space group P21/n (see Experimental Sec-
tion 2.3). However, the amine group was introduced in ligand
biaq using an enantiomerically pure starting material, (R)-(+)-
α-methylbenzylamine. We thus assume that racemization oc-
curred at some point during the synthesis of the complex, or,
alternatively, during its crystallization for the preparation of
single crystals. More studies will be necessary in order to re-
solve this issue.
1458
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 1455–1460

X-ray Crystal Structure and Biological Activity on Bacillus subtilis of [Cu(biaq)Cl2]
Figure 2. Effect of [Cu(biaq)Cl2] on the growth of B. subtilis strain
measured as viable count. Grey columns are for negative control cul-
tures, while black columns are for cultures treated with [Cu(biaq)Cl2]
0.0312 mM.
Figure 4. Effect of [Cu(biaq)Cl2] on the growth (A) and induction
of expression of a reporter recA-lacZ fusion (B) in strain B. subtilis
PERM115. (L = biaq).
Figure 3. Microscopic analysis of B. subtilis PERM115 cultures. (A)
Control culture growth on LB for 12 h. (B) Culture growth on LB
supplemented with 0.0312 mM of [Cu(biaq)Cl2] during 12 h. Aliquots
of 10 μL from each culture were observed and photographed using a
Zeiss Axioskop microscope, with 100X objective.
3.4 [Cu(biaq)Cl2] do not Induce the Bacterial SOS
Response
To investigate if the genetic material is the molecular target
of [Cu(biaq)Cl2], we first explored whether this metal complex
induces the expression of the reporter recA-lacZ fusion in-
serted in the genome of strain B. subtilis PERM115. Several
lines of evidence have demonstrated that treatment of this
strain with DNA damaging factors promotes the SOS response
that results in induction of the recA-lacZ fusion.[19,20,26] As
shown in Figure 4, concentrations ranging from 0.03 to
0.5 mM were unable to induce the expression of the reporter
recA-lacZ fusion. In contrast, the N-alkylating agent mitomy-
cin-C (10 μg·mL–1) dramatically increased the transcription of
the recA-directed lacZ fusion. These results demonstrate that
despite interfering with microbial growth, the metal complex
did not generate on the bacterial chromosome the type of dam-
ages that activate the SOS response in B. subtilis.
Figure 5. Effect of [Cu(biaq)Cl2] on the mutagenesis frequency to RifR
of strain B. subtilis PERM115.
if [Cu(biaq)Cl2] actually possesses DNA-damaging properties.
To this end, we determined the mutation frequencies to rifam-
picin resistance (RifR) of B. subtilis cells treated with sublethal
concentrations (0.031, 0.62, 0.125, 0.25, 0.5, and 1.0 mM) of
[Cu(biaq)Cl2]. The results of this experiment revealed that
concentrations in the range 0.031–0.25 mM significantly in-
creased the RifR mutation frequency in comparison with the
non-treated strain (Figure 5). Although concentrations above
2.5 mM did not promote mutagenesis, this effect must be prob-
ably related to the cytotoxic effect of the complex at such high
3.5 [Cu(biaq)Cl2] Promotes Mutagenesis in B. subtilis
As mentioned above, [Cu(biaq)Cl2] failed to induce the SOS concentrations. As a whole, these results strongly suggest that
response in B. subtilis PERM115. However, as demonstrated sublethal amounts of [Cu(biaq)Cl2] modify the primary struc-
in this work, this metal complex significantly inhibits bacterial ture of bacterial DNA, promoting mutagenesis. To further sup-
growth. Therefore, we used a different approach to investigate port this claim, the experiment of mutation frequencies to RifR
Z. Anorg. Allg. Chem. 2013, 1455–1460
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1459

G. Mendoza-Díaz et al.
ARTICLE
[3] K. D. Karlin, J. C. Hayes, S. Juen, J. P. Hutchison, J. Zubieta,
Inorg. Chem. 1982, 21, 4106.
[4] W. Kaim, J. Rall, Angew. Chem. Int. Ed. Engl. 1996, 35, 43.
[5] J. M. Gauss, P. R. Harrowell, M. M. Valerie, A. Norris, H. C.
Freeman, J. Mol. Biol. 1986, 192, 361.
[6] E. Bouwman, B. Douziech, L. Gutierrez-Soto, M. Beretta, W. L.
Driessen, J. Reedijk, G. Mendoza-Díaz, Inorg. Chim. Acta 2000,
304, 250.
[7] M. Ch. Mimmi, F. Minnichè, H. Kooijman, A. L. Spek, S. T. War-
zeska, E. Bouwman, Inorg. Chim. Acta 2002, 340, 197–200.
[8] W. L. Driessen, D. Rehorst, J. Reedijk, I. Mutikainen, U. Turp-
einen, Inorg. Chim. Acta 2003, 355, 64.
[9] M. Beretta, E. Bouwman, L. Casella, B. Douziech, W. L.
Driessen, L. Gutierrez-Soto, E. Manzani, J. Reedijk, Inorg. Chim.
Acta 2000, 310, 41.
[10] W. Wehrli, F. Knusel, K. Schmid, M. Staehelin, Proc. Natl. Acad.
Sci. USA 1968, 65, 667–673.
[11] D. J. Jin, M. Cashel, D. I. Friedman, Y. Nakamaru, W. A. Walter,
C. A. Gross, J. Mol. Biol. 1988, 204, 247–261.
Figure 6. Frequency of production of colonies resistant to rifampicin
of cultures of strain B. subtilis PERM115 supplemented or not with
the complex [Cu(biaq)Cl2] or the ligand biaq.
[12] K. Severinov, M. Soushko, A. Goldfarb, V. Nikiforov, J. Biol.
Chem. 1993, 268, 14820–14825.
[13] K. Severinov, M. Soushko, A. Goldfarb, V. Nikiforov, Mol. Gen.
Genet. 1994, 244, 120–126.
was repeated by quadruplicate using a concentration of
0.031 mM of the complex, since, as shown in Figure 5, this
concentration optimized mutagenesis in B. subtilis.
As shown in Figure 6, 0.031 mM of [Cu(biaq)Cl2] statistic- [14] K. J. Boor, M. L. Duncan, C. W. Price, J. Biol. Chem. 1995, 270,
20329–20336.
ally increased around eight times the mutation frequency to
[15] E. A. Robleto, H. A. Martin, A. M. Pepper, M. Pedraza-Reyes,
RifR with respect to the untreated strain control. It is worth
Gene Regulation of Sporulation in Bacillus subtilis, in: Bacterial
Spores: Current Research and Applications (Ed.: E. Abel-Santos),
Caister Academic Press, Wymondham, UK, 2012, p. 9–18.
noting that the free ligand biaq, at the same concentration did
not statistically increase its RifR mutation frequency in com-
[16] U. Kempe, T. Dockner, A. Frank, H. Karn, US Patent 4278801,
July 14, 1981.
[17] a) Siemens, XSCAnS (release 2.21) Users Manual, Siemens Ana-
lytical X-ray Instruments Inc., Madison, WI, USA, 1996; b) G. M.
Sheldrick, Acta Crystallogr., Sect. A 2008, 64, 112–122.
parison with the untreated control. Together, these results are
a strong evidence that [Cu(biaq)Cl2] uses the bacterial chromo-
some as a target, producing a damage that should interfere
with replication, although the kind of damage involved in the
interference mechanism does not initiate any SOS response. [18] J. H. Miller, Experiments in Molecular Genetics, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1972.
[19] N. Ramírez-Ramírez, G. Mendoza-Díaz, F. Gutiérrez-Corona, M.
Pedraza-Reyes, J. Biol. Inorg. Chem. 1998, 3, 188–194.
[20] N. Ramírez-Ramírez, G. Mendoza-Díaz, M. Pedraza-Reyes, Bi-
We thus assume that alterations induced by [Cu(biaq)Cl2] on
the rpoB gene, which in turn promote RifR mutagenesis, are
independent of the RecA protein.
oinorg. Chem. Appl. 2003, 1, 25–34.
[21] J. H. Miller, Experiments in Molecular Genetics, Cold Spring
Harbor Laboratory Press, New York, 1972.
[22] M. I. Ramírez, F. X. Castellanos Juárez, R. E. Yasbin, M. Ped-
4 Conclusions
The results presented in this report clearly indicate that the
tested CuII complex featuring a cis-dichlorido group and a tri-
dentate imidazole derivative may have interesting biological
activity, which could allow to design alternative chemothera-
peutic drugs.
raza-Reyes, J. Bacteriol. 2004, 186, 1050–1059.
[23] A. W. Addison, J. N. Rao, J. Reedijk, J. van Rijn, G. C. Ver-
schoor, J. Chem. Soc. Dalton Trans. 1984, 1349.
[24] C. A. Salata, M. T. Youinou, C. J. Burrows, Inorg. Chem. 1991,
30, 3454.
[25] Bacillus subtilis and Other Gram-Positive Bacteria; Biochemistry
Physiology and Molecular Genetics (Ed.: A. L. Sonenshein),
American Society for Microbiology, Washington, DC. 1993.
[26] E. Y. Bivián-Castro, M. G. López, M. Pedraza-Reyes, S. Bernès,
G. Mendoza-Díaz, Bioinorg. Chem. Appl. 2009, DOI:10.1155/
2009/603651.
References
[1] K. J. Oberhausen, R. J. O’Brien, J. F. Richardson, R. M. Bu-
chanan, Inorg. Chim. Acta 1990, 173, 145.
[2] H. Oka, Y. Nakao, S. Suzuki, W. Mori, I. Kinoshita, S. Ooi, A.
Ichimura, Inorg. Chim. Acta 1989, 165, 111.
Received: February 10, 2013
Published Online: May 29, 2013
1460
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 1455–1460