Chiral manganese (IV) complexes derived from Schiff base ligands: Synthesis, characterization, in vitro cytotoxicity and DNA/BSA interaction

Article abstract of DOI:10.1016/j.jphotobiol.2015.11.005

Two new couples of chiral manganese (IV) complexes with Schiff-base ligands, Λ-[Mn(R-L¹)₂]·2(CH₃OH) (Λ-1) and Δ-[Mn(S-L¹)₂]·2(CH₃OH) (Δ-1), Λ-[Mn(R-L²)₂]·(H₂

Full text of DOI:10.1016/j.jphotobiol.2015.11.005

Chiral manganese (IV) complexes derived from Schiff base ligands: Synthesis,
characterization, in vitro cytotoxicity and DNA/BSA interaction
Zhen Li, Meiju Niu, Guoliang Chang, Changqiu Zhao
PII:
DOI:
Reference:
S1011-1344(15)00370-X
doi: 10.1016/j.jphotobiol.2015.11.005
JPB 10192
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3 August 2015
5 November 2015
6 November 2015
Please cite this article as: Zhen Li, Meiju Niu, Guoliang Chang, Changqiu Zhao,
Chiral manganese (IV) complexes derived from Schiff base ligands: Synthesis,
characterization, in vitro cytotoxicity and DNA/BSA interaction, (2015), doi:
10.1016/j.jphotobiol.2015.11.005
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ACCEPTED MANUSCRIPT
Chiral manganese (IV) complexes derived from Schiff base ligands:
synthesis, characterization, in vitro cytotoxicity and DNA/BSA
interaction
Zhen Li, Meiju Niu, Guoliang Chang, Changqiu Zhao.
Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology,
College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, Shandong
252059, China
ABSTRACT
Two new couples of chiral manganese (IV) complexes with Schiff-base ligands,
Λ-[Mn(R-L1)2]·2(CH3OH)
Λ-[Mn(R-L2)2]·(H2O)2 (Λ-2) and Δ-[Mn(S-L2)2]·(H2O)2 (Δ-2), {H2L1
(R/S)-(±)-1-[(1-hydroxymethyl-propylimino)-methyl]-naphthalen-2-ol,
H2L2
(Λ-1)
and
Δ-[Mn(S-L1)2]·2(CH3OH)
(Δ-1),
=
=
(R/S)-(±)-1-[(1-Hydroxymethyl-2-phenyl-ethylimino)-methyl]-naphthalen-2-ol} have
been synthesized, and fully characterized by elemental analyses, UV-Vis spectrum,
circular dichroism spectrum, FT-IR spectrum, mass spectrum, single crystal X-ray
diffraction (SXRD). The interaction of the four chiral Mn (IV) complexes with
CT-DNA and BSA were also investigated by various spectroscopic techniques
(UV-visible, fluorescence spectroscopic). The results show that the Δ-complexes
exhibit more efficient CT-DNA interaction with respect to the Λ-complexes. All the
complexes could quench the intrinsic fluorescence of BSA by a static quenching
process. In addition, the vitro cytotoxicity of these complexes toward four kinds of
cancerous cell lines (A549, HeLa, HL-60, and Caco-2) was assayed by the MTT
method, which exhibited to be selectively active against certain cell lines.
Keywords: Chiral Schiff-base; Manganese complexes; DNA binding; BSA
interaction; Cytotoxicity.
1. Introduction
Tel: +86 0635 8230665; Fax: +86 0635 8239121.
 Corresponding author
E-mail address: niumeiju@163.com.
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Nowadays, the cancer is the most of intractable disease, the morbidity and mortality
of which have been increased [1]. Trávníček et al. have synthesized the transition
metal complexes, which show highly cytotoxic [2]. Niu and coworkers have
successfully prepared manganese complexes that exhibit great anticancer activities.
Moreover, as one of the most primary bio-metals, manganese has become the subject
of fairly extensive research for the past couple of decades [3], especially higher valent
manganese. Manganese-containing compounds with pharmacological use include the
anticancer agent SC-52608 and the MRI contrast agent Teslascan [4]. Chiral
Schiff-base metal complexes have attracted much more attention because of its
biological and pharmacy importance [5–8]. Specially, the manganese complexes are
of considerable interests due to their important applications in the probe of chiral
structures for biological systems, the preparation of chiral catalysts and the
development of multifunctional materials [9, 10]. Moreover, it is generally counted
that chiral manganese complexes are less environmentally damaging than other
transition metal complexes [11]. However, until now, the reports for chiral amino
alcohols Schiff-base manganese (IV) complexes are relatively rare. Therefore, the
investigations on the synthesis, optical properties and biological activity of chiral
amino alcohols Schiff-base metal complexes are of paramount importance [12–14].
Very recently, we have reported the synthesis, structure and biological activity of
the non-chiral transition metal complexes and dealt with seeking for low toxicity, high
efficiency of anticancer drugs and specific DNA target interaction [15–17]. Herein,
we reported the synthesis and characterization of H2L1, H2L2 and their manganese
complexes: Λ-1 (1), Δ-1 (2), Λ-2 (3) and Δ-2 (4). The conclusion reveals that chiral
complexes interactions to DNA/BSA are more strongly than that of non-chiral
complexes. In addition, these complexes appeared to be selectively active and IC50
values of them are smaller than that of the previous non-chiral complexes.
2 Experimental
2.1 Materials and Physical measurements
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All solvents and reagents were purchased commercially available and used without
further purification. The two ligands, H2L1, H2L2 had been synthesized in our
laboratory. IR spectra were recorded on Nicolet-5700 FT-IR spectrophotometer with
KBr pellets in the 4000-400 cm-1 region. UV-Vis spectra were performed on a
1
UV-2550 ultraviolet spectrophotometer. H NMR spectra were obtained on a Varian
Mercury Plus 400 MHz NMR spectrometer. Elemental analyses for C, H and N were
performed at a PE-2400-II apparatus. Electrospray ionization mass spectroscopic
(ESI-MS) analyses are performed with a Bruker microTOF-Q mass spectrometer
(Bruker Daltonics Inc., Billerica, MA), and the mass spectra are obtained in the
positive mode. Circular dichroism (CD) spectra measurements were measured on a
Jasco J-810 spectropolarimeter. Electronic absorption spectra were recorded using a
HP-8453A diode array spectrophotometer. Emission spectra were measured with
LS55 spectrofluorometer.
2.2 Synthesis of ligands
2.2.1 Synthesis of (R)-H2L1
The methanol solution (10 mL) of R-(-)-2-amino-1-butanol (0.446 g, 5 mmol) were
added to the methanol solution (10 mL) of 2-hydroxy-1-naphthaldehyde (0.861 g, 5
mmol), respectively. The mixture was refluxed for 4 h under the magnetic stirring and
the resultant yellow solution was evaporated to dryness using rotavapor and vacuum
pump. A schematic representation of ligand synthesis is given in Scheme 1. Then the
resulting yellow precipitate or oily liquids was dried in vacuum to give (R)-/(S)-H2L.
The synthetic procedures for other ligands were very similar as the (R)-H2L1 and
discussed together in follow.
(R)-H2L1 Yield: 89.0%; M.p.: 171–173℃; Anal. Calc. (%) for C15H17NO2 (Mr =
243.30): C, 74.05; H, 7.04; N, 5.76%. Found: C, 73.98; H, 6.98; N, 5.75%. Selected
1
IR (KBr pellet: cm-1): 3446 (s, O-H), 1625 (s, C=N), 1100 (s, Ar-O). H NMR (400
MHz, CDCl3) δ/ppm 8.62 (s, 1H, –CH=N), 7.69–6.62 (m, 6H, Ar–H), 3.84 (m, 1H,
CH), 3.70 (m, 2H, –CH2OH), 1.64 (m, 2H, –CH2CH3), 1.01 (t, 3H, –CH2–CH3).
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(R)-H2L2 Yield: 86.2%; M.p.: 181–183℃; Anal. Calc. (%) for C20H19NO2 (Mr =
305.37): C, 78.66; H, 6.27; N, 4.59%. Found: C, 78.05; H, 6.54; N, 4.32%. Selected
1
IR (KBr pellet: cm-1): 3442 (s, O-H), 1620 (s, C=N), 1107 (s, Ar-O). H NMR (400
MHz, CDCl3) δ/ppm 8.41 (s, 1H, –CH=N), 7.52–6.68 (m, 11H, Ar–H), 3.91 (m, 1H,
CH), 3.71 (m, 2H, –CH2OH), 3.01–2.92 (m, 2H, Ph–CH2–).
2.3 Syntheses of complexes 1–4
Four manganese (IV) complexes 1–4, reported here, were prepared according to the
following general procedure.The methanol solution of the respective chiral ligand (0.4
mmol) was added to Mn(OAc)2·4H2O (0.0490 g, 0.2 mmol). The mixture was allowed
to be stirred at room temperature for 5 h and then filtered. The dark brown crystals
suitable for X-ray data collection were obtained by slow evaporation of methanol
solution for two week.
Λ-[Mn(R-L1)2]·2(CH3OH) (1) Yield: 79.8%. M.p.: 167–168 ℃. Anal. Calc. (%)
for C32H38MnN2O6 (Mr = 601.58): C, 63.89; H, 6.37; N, 4.66 %. Found: C, 63.83; H,
6.32; N, 4.65 %. Selected IR (KBr pellet: cm-1): 1616 (s, C=N), 1093 (s, νAr-O), 549 (m,

Mn-O), 412 (m, Mn-N). ESI-MS, m/z: 328.33 [L1+Mn+CH3OH+H]+, 592.42
[2L1+2Mn+H]+, 614.17 [2L1+2Mn+Na]+. UV-Vis (CH3OH), λmax/nm: 246, 308nm,
394nm.
Δ-[Mn(S-L1)2]·2(CH3OH) (2) Yield: 81.0%. M.p.: 167–169 ℃. Anal. Calc. (%)
for C32H38MnN2O6 (Mr = 601.58): C, 63.89; H, 6.37; N, 4.66 %. Found: C, 63.86; H,
6.35; N, 4.62 %. Selected IR (KBr pellet: cm-1): 1618 (s, C=N), 1092 (s, νAr-O), 548 (m,

Mn-O), 419 (m, Mn-N). ESI-MS, m/z: 328.33 [L1+Mn+CH3OH+H]+, 592.33
[2L1+2Mn+H]+, 614.25 [2L1+2Mn+Na]+. UV-Vis (CH3OH), λmax/nm: 248, 308nm,
396 nm.
Λ-[Mn(R-L2)2]·2(H2O) (3) Yield: 53.2%. M.p.: 179–182℃. Anal. Calc. (%) for
C40H38MnN2O6 (Mr = 697.66): C, 69.39; H, 5.40; N, 3.95 %. Found: C, 69.33; H,
5.35; N, 3.94 %. Selected IR (KBr pellet: cm-1): 1616 (s, C=N), 1081 (s, νAr-O), 538 (m,

Mn-O), 424 (m,  Mn-N). ESI-MS, m/z: 662.58 [2L2+Mn+H]+, 684.50 [2L2+Mn+Na]+.
UV-Vis (CH3OH), λmax/nm: 246, 311 nm.
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Δ-[Mn(S-L2)2]·2(H2O) (4) Yield: 57.7%. M.p.:178–180℃. Anal. Calc. (%) for
C40H38MnN2O6 (Mr = 697.66): C, 69.06; H, 5.22; N, 4.03 %. Found: C, 69.01; H,
5.19; N, 4.02 %. Selected IR (KBr pellet: cm-1): 1615 (s, C=N), 1082 (s, νAr-O), 543 (m,

Mn-O), 426 (m, Mn-N). ESI-MS, m/z: 662.50 [2L2+Mn+H]+, 684.50 [2L2+Mn+Na]+.
UV-Vis (CH3OH), λ max/nm: 248, 308 nm.
2.4 Crystal Data Collection and Refinement
Diffraction data for the title complexes were obtained on a Bruker Smart 1000
CCD diffractometer (graphite monochromized Mo Kα radiation, λ = 0.71073 Å) and
collected by the y-2θ scan technique at 298 (2) K. Empirical absorption corrections
were applied using the SADABS program [18]. The semiempirical absorption
correction was applied to the data. The structure was solved by direct methods by
SHELXS-2014 and refined against F2. All non-hydrogen atoms were refined with
anisotropic thermal parameters [19]. A summary of the crystallographic data and
refinement parameters for the complexes are provided in Table 1. Selected bond
lengths and angles are listed in Table S1.
2.5 DNA-binding studies
The concentration of DNA was determined by using UV-Vis absorbance and the
molar absorption coefficient (6600 M-1 cm-1) at 260 nm [20]. UV-Vis absorbance was
performed by varying the concentration of complex (10 μM) in 10 mM Tris-HCl/10
mM NaCl (pH = 7.4) buffer while making the CT-DNA concentrations constant from
0 to 14 μM. For the fluorescence quenching experiments, the EB solution was added
to the prepared buffer solution of CT-DNA for 2 h, and then added to the solution of
complex from 0 to 48 μM. All samples were excited at 258 nm, and emission spectra
were recorded at 540–700 nm.
2.6 Protein binding studies
The BSA binding experiments with manganese (IV) complexes were studied from
the fluorescence spectra in 10mM buffer solution recorded with an excitation at 280
nm. In the measurement of UV-Vis spectra, the concentration of BSA was kept at
1.0×10-4 M and the complex was kept at 2.0×10-3 M. The fluorescence spectra were
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measured at a scanning speed of 200 nm/min and slit width of 7 nm both the
excitation and emission monochromators. For synchronous fluorescence spectra, we
also used the same concentration of BSA and the complexes, and measured the
spectra at two different Δ λ values，but between the excitation and emission
wavelengths of BSA are different, such as 15 and 60 nm.
2.7 Cytotoxicity
In vitro cell culture studies are valuable tools for the screening of chemotherapy
agents and provide preliminary data for further relative studies. The cytotoxicity of
the complexes to cells was evaluated through the loss of cell viability using MTT
assay [21]. Cell lines of HL-60 were grown in RPMI-1640 medium and A549, Caco-2,
HeLa were grown in RPMI-DMEM medium, maintain culture at 37℃, 5% CO2 and
95% air in the CO2 incubator for 24h. Various concentrations of prepared complexes
were added to the cells and incubation continued for 48h. Then the MTT was
dissolved in medium and added to each well, and incubated for another 4h. The purple
formazan crystals were solubilized by the addition of 100 L DMSO. The absorbance
was measured at 570 nm by the ELISA reader. The values are the averages from at
least three independent experiments, which were measured as the percentage ratio of
the absorbance of the treated cells to the untreated controls. The IC50 values were
determined by the plot of percentage of cell inhibition versus concentration by
non-linear regression analysis.
3 Results and discussion
3.1 IR spectrum
The IR spectra of complexes show a strong band at 1616-1618 cm−1 assigned to the
υ(C=N) vibration, as compared to the spectra of the Schiff base ligand υ(C=N)
1623–1633 cm−1, which shifts lower frequency and supports the coordination of the
(C=N) group [22]. The strong absorption bands of Ar–O in the Ligand at 1100–1107
cm–1 locates at lower frequency for complexes, viz. 1093 cm–1 for 1, 1092 cm–1 for 2,
1081 cm–1 for 3 and 4, respectively, it could be considered that the deprotonated
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phenol O group has coordinated to the manganese ion. Additional support for the
formation of the complexes were provided by the existence of medium intensity bands
in the region 549–538 cm-1 and 412–424 cm-1 assigned to υ(Mn–N) and υ(Mn–O),
respectively [23].
3.2 UV-Vis spectrum
The UV–Vis spectra of the ligands and complexes are recorded in methanol
solution. The ligand 2 showed four main bands in the range 227–232 nm, 245–273 nm
305–316nm and 405–421 nm, respectively, which may be attributed to π–π* and n–π*
transitions of the aromatic rings and the non-bonding electrons on N atoms of imino
groups [24]. While complexes displays only three strong absorption bands at 222–238
nm, 241–273 nm and 302–335 nm as compared to the bands of the ligands. It does not
exhibit d–d transition due to its completely filled d10 electronic configuration [25].
3.3 Mass spectrum
Electrospray mass spectra of methanol solution of 1 showed an intense peak at
328.33 corresponding to the molecular fragments [L1+Mn+CH3OH+H]+. The
structure of complex 1 decomposes in polar solvents and the complex dissociates as
monomers. The complex 2 has retained its dimeric structure in MeOH. The intense
peaks of complex 2 are also observed at 328.33 for the molecular fragment
[L1+Mn+CH3OH+H]+. The remaining two complexes 3 and 4 have been intense
peaks at 662.58 and 662.50 for the identical molecular fragment [2L2+Mn+H]+. There
is no evidence for higher nuclearity fragments in solution.
3.4 Description of the structures
Complexes 1–4 are characterized by single-crystal X-ray crystallography. Since 1
and 2, 3 and 4 are two pair of enantiomers, they have the similar crystal structures.
Only the crystal structure features of complexes 1 and 3 are described in detail here.
3.4.1 Λ-[Mn(R-L1)2]·2(CH3OH) (1)
Single crystals of complex 1 suitable for X-ray crystallography were grown from
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methanol solutions by slow evaporation method. Molecular structure of complex 1
with the atom-numbering scheme is shown in Figure 1. Selected angles and bond
lengths are list in Table S1. It crystallizes in the chiral orthorhombic P21212 space
group and each asymmetric unit contains one mononuclear neutral manganese
complex and two free CH3OH solvent molecules. In complex 1, the coordination
geometry of the Mn center is octahedral, where four oxygen atoms from the R-H2L1.
Each ligand contains deprotonated phenolic and alcoholic groups along with one
imine nitrogen. Two oxygen atoms from alcoholic hydroxyl group and another
oxygen atom from the phenolic group are located at the basal coordination plane. The
apical position is occupied by two nitrogen atoms from the imine of adjacent R-H2L1
ligand, and the Mn–O and Mn–N bond lengths are very analogous in the range of
1.846(4)–1.965(4) Å. Moreover, The four oxygen atoms occupy the equatorial plane
and the axial angle N(1)–Mn(1)–N(1A) is 173.43 (17)°. The O–Mn–O and O–Mn–N
angles are from 84.69 (10)° to 95.31(10)°. The Mn–O–N bond distances vary from
1.850(2) Å to 1.962(3) Å and the trans-angles of 172.49(11)°–174.43(17)°, indicating
a marginal distortion in the geometry, are in agreement with those reported for Mn
complexes [26]. The mononuclear molecular are associated into 2–D supramolecular
network (Figure 2) through the C–H···π interactions between carbon atom C (11) and
the neighboring naphthalene ring.
3.4.2 Δ-[Mn(S-L1)2]·2(CH3OH) (2)
Since complex 2 and 1 as a pair of enantiomers, they have the similar crystal
structures (Figure 3). Therefore, the crystal structure of the complex 2 is no longer
described in detail here.
3.4.3 Λ-[Mn(R-L2)2]·2(H2O)·(3)
Molecular structure of complex 3 with the atom-numbering scheme is shown in
Figure 4. It crystallizes in the chiral orthorhombic P212121 space group and each
asymmetric unit contains one mononuclear neutral manganese molecules [27] and two
water solvent molecules. In complex 3, the manganese atom is six-coordinate with an
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octahedral geometry. Bond dimensions, with those of complex 3, are also in Table S1.
The two imine N(1) and N(2) atoms of the amino alcohol occupy the sites. Two
oxygen atoms from alcoholic hydroxyl group and another oxygen atom from the
phenolic group are located at the basal coordination plane. The naphthalene rings of
two ligands kept their original configurations, exhibiting no distortions. The apical
position is occupied by two nitrogen atoms from the imine of adjacent dianion of
R-H2L2 ligand, and the Mn–O and Mn–N bond lengths are very analogous in the
range of 1.831(4)–1.972(4) Å. Moreover, The four oxygen atoms occupy the
equatorial plane and the axial angle N(1)–Mn(1)–N(2) is 173.6(3)°. The O–Mn–O and
O–Mn–N angles are from 86.0(3)° to 93.0(3)°. The Mn–O–N bond distances vary
from 1.831(5) Å to 1.972(7) Å and the trans-angles of 170.1(3)°–173.6(3)°.
In the crystal structure, there are intermolecular C–H···O hydrogen bonding
interactions between complex and solvent molecules resulting in a supramolecular
hydrogen bonding network.
3.4.4 Δ-[Mn(S-L2)2]·2(H2O) (4)
Since complexes 4 and 3 are a pair of enantiomers, respectively, they have the
similar crystal structures. However, compound 4 contains two free water molecules
and one mononuclear neutral manganese complex (Figure 5). The complex 4 is
associated into 3–D supramolecular net in Figure S1.
3.5 Circular dichroism spectroscopy
The CD spectra have been utilized as a powerful tool for exploring the chiral aspect
of complexes and to provide valuable information about the chiral complexes [28].
The CD spectra of complex 1, 2, 3 and 4 in the buffer solution are shown in Figure S2
and S3. The CD spectrum of complex 1 (Λ-1) exhibits two strong positive Cotton
effect peaks at λ = 293, 387 nm, and negative dichroic signal centered at λ = 216, 240,
329 nm, while complex 2 (Δ-1) shows the Cotton effects of the opposite sign at the
same wavelengths. The CD spectrum of complex 3 (Λ-2) exhibits three positive peaks
at λ = 211, 293, 389 nm, and negative dichroic signal centered at λ = 262, 339 nm,
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while complex 4 (Δ-2) shows the Cotton effects of the opposite sign at the same
wavelengths. The above CD spectral changes preliminary study suggest that the
different matching of enantiomers of the chiral complexes [29]. The absolute
configuration at the metal center could be determined as Δ or Λ according to the two
ligands around manganese atom on the close examination of Flack parameters.
Moreover, adding the complex Λ-2 and complex Δ-2 in a 2:1 ratio exhibit the peaks
is doubles smaller than single complexes Λ-2 or Δ-2 (The complexes 1 and 2 are
shown in Figure S4). And adding the complexes Λ-2 and Δ-2 in a 1:1 ratio exhibit
little peak indicated the Cotton effect of one couple chiral complexes was likely to
counteract spin (Figure 6). Investigations on the circular dichroism spectroscopy
indicated that synthetic four manganese (IV) complexes are chiral compounds, and
complexes 1 and 2, 3 and 4 are enantiomers, respectively.
3.6 DNA binding studies
3.6.1 UV-Vis absorption studies
The absorption spectra of complex 2, in presence and absence of CT-DNA are
shown in Figure 7 while that of complexes 1, 3 and 4 are presented as Figure S5, S6
and S7, respectively. In the UV spectral changes, the complex 2 exhibited the intense
absorption band with maxima at 251 nm which could be attributed to intra-ligand (IL)
π/π* transitions. However, increasing concentrations of CT DNA (from 0 to 14 μM) to
a fixed concentration of complex exhibited an evident hyperchromism and along with
a slight red shift of 3 nm. ‘Hypochromic effect’ and ‘Hyperchromic effect’ are the
spectral features of CT-DNA concerning its double helix structure. Hyperchromism
results from the damage of the DNA double helix structure [30, 31]. From the
absorption titration data, the intrinsic binding constant (Kb) of the metal complexes
with CT-DNA was determined using the equation given as:
[DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/Kb(εb–εf)
To study quantitatively, the binding ability of complexes with CT-DNA, the
intrinsic binding constant Kb values determined as given in Table 2. The binding
constant Kb values follows the order Δ-2 > Δ-1 > Λ-2 > Λ-1. From the results of the
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binding constants, it was derived that the Mn (IV) complexes Δ-1 and Δ-2 revealed a
stronger binding affinity for DNA double helix which was slightly higher in
magnitude than complexes Λ-1 and Λ-2.
3.6.2 Fluorescence quenching studies
The emission spectra of the EB-DNA system with increasing the concentration of
the Mn (IV) complex 2 is shown in Figure 8 (respective spectra of complexes 1, 3 and
4 are presented as Figure S8, S9 and S10), and the experimental data are given in
Table 2. The intensity of the spectra band obviously decreases along with the increase
of concentration of the complex. The result clearly indicates that the EB molecules are
displaced from their DNA binding sites which are replaced by the complexes under
investigation. In addition, the experimental quenching constants and binding constants
of the Mn (IV) complexes 1–4 indicated that the interaction of those with DNA should
be strong binding mode [32]. The Stern–Volmer quenching constant was calculated
for each metal complex using the equation given as:
I0/I=1 + Ksqr
The calculated value of Ksq for complex 2 is higher than rest of the complexes.
Moreover, Δ-complexes showed stronger binding with DNA than their Λ-complexes
in all the complexes, suggesting that the DNA which is essentially chiral in nature
indeed show stereochemical preference for binding with external chiral molecules.
These results are in consonance with the electronic absorption titration studies given
earlier in the section.
3.7 BSA-binding studies
3.7.1 Fluorescence quenching of BSA by complexes 1–4
The fluorescence of protein is due to intrinsic characteristics of tyrosine, tryptophan
residues. Fluorescence quenching refers to the decreasing of the fluorophore induced
by the environmental alteration around the fluorophore, which can reveal the nature of
BSA binding reaction [33]. The interaction of BSA with the complex 3 on the
fluorescence emission spectrum was given in Figure 9 (for complexes 1, 2 and 4, see
Figure S11, S12 and S13). A significant decrease of the fluorescence intensity occurs
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with a red shift at 347 nm, upon the addition of the Mn (IV) complex to the solution
of BSA, has been observed respectively. These results suggest the formation of
complex-BSA system [34]. The fluorescence quenching is described by Stem-Volmer
relation:
I0/I = 1 + KSV [Q]
The KSV values obtained from the plot of [Q] versus I0/I are found to be 2.44×104,
4.44×104, 8.32×104 and 9.14×104 M-1 for the complexes 1, 2, 3 and 4, respectively, in
Table 2. If it is assumed that the binding of complexes with BSA occurs at equilibrium,
the equilibrium binding constant can be analyzed according to the Scatchard equation
(Figure 10):
log((I0–I)/I) = logKb + n log[Q]
The values of Kb and n are listed in Table 2. These values of n are approximately
equal to 1, suggest that there is only one binding site for these complexes on the BSA
molecule. Moreover, the results indicated that Δ-complexes interact with BSA more
strongly than Λ-complexes. This result is consistent with the above CT-DNA binding
studies.
3.7.2 UV-Vis absorption measurements of BSA by metal complexes
UV-Vis absorption spectra could be performed to differentiate these two quenching
types, dynamic quenching and static quenching [35, 36]. The representative spectra of
the BSA-metal complex 2 (Figure 11, for complexes 1, 3 and 4, see Figure S14, S15
and S16) displays the absorption occurs with a red shift at 278 nm. This result may
suggest that there exists a static interaction between the complex and BSA due to the
formation of a complex-BSA ground state system, which has been found in other
reported samples. In other words, the fluorescent quenching may be ascribed to static
quenching.
3.7.3 Characteristic of synchronous fluorescence spectra
In order to study the structural changes of BSA in the presence of the Mn (IV)
complexes, we measured synchronous fluorescence spectra with the addition of
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complexes 1–4. If the Δ λ value is 15 nm, the synchronous fluorescence of BSA is
characteristic of a tyrosine residue and large Δ λ values such as 60 nm is characteristic
of tryptophan [37]. The fluorescence intensity of emission corresponding to
tryptophan was reflected to decrease with a bathochromic shift of emission
wavelength when increasing the concentration of the complexes. And the tyrosine
fluorescence emission also showed a decrease in the intensity, but with a blue shift of
emission wavelength. The spectrum of complex 2 is given in Figure 12 (for
complexes 1, 3 and 4, see Figures S17 S18 and S19). These results indicate that the
metal complexes increase the polarity around the tryptophan residues and also the
hydrophobicity around the tyrosine residues is strengthened. To show that in
synchronous fluorescence spectroscopy confirmed the effective binding of the
complexes with the BSA.
3.8 Cytotoxicity
The inhibition effects of complexes 1, 2, 3 and 4 against the four cell lines at a
concentration of 25.0 μM are listed in Figure 13. The IC50 values against four cell
lines are shown in Table 3. Coordination of metal ions and optical activity were
essential for cytotoxicity of novel complexes. Two pairs of free ligands of IC50 values
were much higher than the values of corresponding complexes for the coordination of
metal ions play a vital role in. The complex 2 is conspicuous in displaying the
prominent cytotoxicity against the tested cell lines. While the cytotoxicity of these
chiral complexes showed inconsistent with previous result about enantiomer, thus the
complexes are selective towards different cancerous cells. All the above results reveal
that the complex 2 is higher than others, which is consistent with the high ability of 2
to bind to DNA and causes a conformational change on DNA.
As shown in Figure 13, after the tumor cells were incubated with 25.0 μM test
complexes for 48 h, each complex exhibited different inhibition effect against the four
cell lines, which further showed various cytotoxicity of these chiral manganese (IV)
complexes against the tested cell lines even though complexes 1 and 2, 3 and 4
possess similar structure. Such phenomenon is difficult to explain. In fact, except for
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cisplatin-derivatives, there are relatively a large number of studies mechanistic
information on how metal antitumour drugs function. However, it is clear that
different configuration could work through different routes that lead to different
cellular responses.
4 Conclusions
The use of the chiral Schiff-base ligand with manganese salts has afforded two new
couples of enantiomers chiral mononuclear complexes. Single crystal X-ray
diffraction studies reveal a distorted octahedral geometry around the metal centers for
all complexes. Their interactions with CT-DNA and BSA were investigated using
UV-visible, fluorescence and synchronous fluorescence spectroscopic methods. The
results show that all complexes could quench the intrinsic fluorescence of BSA in a
static quenching process, and the Δ absolute configuration of the Mn complexes
exhibited more efficient DNA interaction with respect to the Λ. The vitro cytotoxic
effect of the four complexes on selected cancerous cell lines exhibited substantial
cytotoxic activity. Also, chiral Mn complexes are more potent than previous
researched non-chiral complexes.
Abbreviations
CD
Circular dichroism
ESI-MS
CT-DNA
BSA
Electrospray ionization mass spectroscopic
Calf thymus DNA
Bovine serum albumin
EB
Ethidium bromide
MTT
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide
Acknowledgment
We acknowledge the financial support of the Natural Science Foundation of
Shandong Province (No. ZR2013BM017)
Appendix A. Supplementary data
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CCDC No. 1033336, 1033335, 1033334 and 1033337 for complexes 1, 2, 3 and 4.
These data can be obtained free of charge from the Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
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Table Captions
Table 1 Crystallographic data and structure refinement parameters for Schiff base
complexes 1, 2, 3 and 4.
Table 2 Comparison of interaction study results between complexes 1–4 on CT-DNA
and protein.
Table 3 IC50 (μM) of all complexes against A549, HeLa, HL-60, and Caco-2 for 48 h
treatment.
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Table 1 Crystallographic data and structure refinement parameters for Schiff base complexes 1, 2,
3 and 4.
Compounds
Formular
1
2
3
4
C32H38MnN2O6 C32H38MnN2O6 C40H38MnN2O6 C40H38MnN2O6
Crystal system
Space group
Orthorhombic
P21212
Orthorhombic
P21212
601.58
15.3349(17)
8.7392(9)
10.6613(11)
90
Orthorhombic
P212121
697.66
10.2974(8)
10.5687(9)
33.451(2)
90
Orthorhombic
P212121
697.66
10.3634(12)
10.6264(13)
33.582(3)
90
Formular weight 601.58
a, Å
15.4668(15)
b, Å
8.8333(7)
10.7394(8)
90
c, Å
α,deg
β,deg
90
90
90
90
γ,deg
90
90
90
90
V，Å3
1467.2(2)
2
1428.8(3)
2
3640.5(5)
4
3698.2(7)
4
Z
T, K
298(2)
0.71073
1.362
298(2)
0.71073
1.398
298(2)
0.71073
1.273
298(2)
0.71073
1.253
λ, Å
ρ，g·cm-3
Rint
0.0635
0.0447
0.0649
0.0884
0.0999
0.1332
0.0865
0.1247
0.1922
0.2378
0.52/-0.56
0.0696
0.0876
0.1473
0.2027
0.2277
0.40/-0.52
0.0953
0.0643
0.1489
0.1421
0.1676
0.42/-0.21
R1 [I>2σ(I)]
R1 (all data)
wR2 [I>2σ(I)],
wR2 (all data)
residual electron 0.32/-0.46
density
Flack
0.01(2)
0.01(7)
0.03(4)
-0.03(3)
Table 2 Comparison of interaction study results between complexes 1–4 on CT-DNA
and protein.
Complex
DNA-binding
Protein-binding
Ksq
Kb(M-1)
KSV(M-1)
Kb(M-1)
n
2.18
4.20
2.96
4.07
2.96×104
4.20×104
3.09×104
4.43×104
2.44×104
4.44×104
8.32×104
9.14×104
6.46×107
5.48×107
2.30×107
4.33×107
0.87
1.64
1.51
1.56
1
2
3
4
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Table 3 IC50 (μM) of all complexes against A549, HeLa, HL-60, and Caco-2 for 48 h
treatment.
A549
HeLa
HL-60
Caco-2
Complex
24.15±1.38 14.09±1.15 13.68±1.13 17.60±1.24
22.07±1.34 12.04±1.21 11.16±1.06 20.07±1.30
44.19±1.64 15.11±1.18 13.40±1.22 25.57±1.41
36.96±1.57 13.91±1.14 12.09±1.08 21.06±1.32
1
2
3
4
H2L1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2L2
[Mn(OAC)2]·4H2O
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Figure Captions
Scheme 1 Synthesis of chiral complexes 1–4.
Figure 1 Molecular structures of complex 1 (Thermal ellipsoids are shown at 30% probability
level. Symmetry code: –x+2, –y+1, z).
Figure 2 View of the 2–D supramolecular network.
Figure 3 Molecular structures of complex 2. (Thermal ellipsoids are shown at 30% probability
level. Symmetry code: –x+2, –y+2, z).
Figure 4 Molecular structures of complex 3. (Thermal ellipsoids are shown at 30% probability
level).
Figure 5 Molecular structures of complex 4. (Thermal ellipsoids are shown at 30% probability
level).
Figure 6 CD spectra of chiral Schiff base complexes 3 and 4.
Figure 7 UV-Vis absorption spectra of complex 2 (10 μM) in the absence and presence of
increasing amounts of DNA (0–14 μM). Arrow shows the absorbance changes upon increasing
DNA concentration.
Figure 8 Effects of complex 2 on the fluorescent spectra of EB–DNA system (λex= 258 nm);
CDNA= 20 μM: CEB= 2 μM. From 1 to 8 CVOL = 0–42 μM Inset: plot of I0/I vs r (r = CVOL/CDNA
)
for complexes 1–4.
Figure 9 Emission spectra of BSA (1.0 μM; λex= 280 nm) as a function of concentration of the
complex 3 (0–14 μM). Arrow indicates the effect of metal complexes on the fluorescence emission
of BSA.
Figure 10 Plot of log[(I0–I)/I] vs. log[Q].
Figure 11 UV-Vis absorption spectra of BSA in the absence and presence of increasing amounts
of the complex 2. CBSA =10 μM, [Complex] = 0 and 10 μM.
Figure 12 Synchronous spectra of BSA as a function of concentration of the complex 2 with
wavelength difference of Δλ = 15 nm and Δλ = 60 nm
Figure 13 Inhibition [%] of complexes 1, 2, 3 and 4 [dose level of 25.0 μM] against human tumor
cells.
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Scheme 1 Synthesis of chiral complexes 1–4
Figure 1 Molecular structures of complex 1 (Thermal ellipsoids are shown at 30% probability
level. Symmetry code: –x+2, –y+1, z).
Figure 2 View of the 2–D supramolecular network.
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Figure 3 Molecular structures of complex 2. (Thermal ellipsoids are shown at 30% probability
level. Symmetry code: –x+2, –y+2, z).
Figure 4 Molecular structures of complex 3. (Thermal ellipsoids are shown at 30% probability
level).
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Figure 5 Molecular structures of complex 4. (Thermal ellipsoids are shown at 30% probability
level).
Figure 6 CD spectra of chiral Schiff base complexes 3 and 4.
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Figure 7 UV-Vis absorption spectra of complex 2 (10 μM) in the absence and presence of
increasing amounts of DNA (0–14 μM). Arrow shows the absorbance changes upon increasing
DNA concentration.
Figure 8 Effects of complex 2 on the fluorescent spectra of EB–DNA system (λex=258 nm);
CDNA=20 μM: CEB=2 μM. From 1 to 8 CVOL= 0–42 μM Inset: plot of I0/I vs r (r = CVOL/CDNA) for
complexes 1–4.
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Figure 9 Emission spectra of BSA (1.0 μM; λex= 280 nm) as a function of concentration of the
complex 3 (0–14 μM). Arrow indicates the effect of metal complexes on the fluorescence emission
of BSA.
Figure 10 Plot of log[(I0–I)/I] vs. log[Q].
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Figure 11 UV-Vis absorption spectra of BSA in the absence and presence of increasing amounts
of the complex 2. CBSA = 10 μM, [Complex] = 0 and 10 μM.
Figure 12 Synchronous spectra of BSA as a function of concentration of the complex 2 with
wavelength difference of Δλ = 15 nm and Δλ = 60 nm.
Figure 13 Inhibition [%] of complexes 1, 2, 3 and 4 [dose level of 25.0 μM] against human tumor
cells.
29