Chelating ability and biological activity of hesperetin Schiff base Elzbieta Lodyga-Chruscinska, Marzena Symonowicz, Anna Sykula, Anna Bujacz, Eugenio Garribba, Magdalena Rowinska-Zyrek, Stanislaw Oldziej, Elzbieta Klewicka, Magdalena Janicka, Karolina Krolewska, Marcin Cieslak, Katarzyna Brodowska, Longin Chruscinski PII: DOI: Reference:
S0162-0134(14)00291-8 doi: 10.1016/j.jinorgbio.2014.11.005 JIB 9621
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
31 July 2014 19 November 2014 20 November 2014
Please cite this article as: Elzbieta Lodyga-Chruscinska, Marzena Symonowicz, Anna Sykula, Anna Bujacz, Eugenio Garribba, Magdalena Rowinska-Zyrek, Stanislaw Oldziej, Elzbieta Klewicka, Magdalena Janicka, Karolina Krolewska, Marcin Cieslak, Katarzyna Brodowska, Longin Chruscinski, Chelating ability and biological activity of hesperetin Schiff base, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.11.005
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ACCEPTED MANUSCRIPT Chelating ability and biological activity of hesperetin Schiff base Elzbieta Lodyga-Chruscinskaa*, Marzena Symonowicza, Anna Sykulaa, Anna Bujacza,
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Eugenio Garribbab, Magdalena Rowinska-Zyrekc, Stanislaw Oldziejd, Elzbieta
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Klewickaa, Magdalena Janickae, Karolina Krolewskae, Marcin Cieslake, Katarzyna
a
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Brodowskaa, Longin Chruscinskif
Faculty of Biotechnology and Food Chemistry, Lodz University of Technology,
Dipartimento di Chimica e Farmacia, Centro Interdisciplinare per lo Sviluppo della Ricerca
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b
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Stefanowskiego Street 4/10, 90-924 Lodz, Poland
Biotecnologica e per lo Studio della Biodiversità della Sardegna, Università di Sassari, via
c
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Vienna 2, I-07100 Sassari, Italy Department of Chemistry, University of Wroclaw, F. Joliot-Curie Street 14,
d
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50-383 Wroclaw, Poland
Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of
e
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Gdansk, Kladki 24, 80-922 Gdansk, Poland
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza Street 112, 90-363 Lodz, Poland
f
*
Faculty of Process and Environmental Engineering, Wolczanska 175, 90-924 Lodz, Poland
Corresponding author: e-mail: [email protected] phone:+48(42)6313417; fax:+48(42)6362860
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ACCEPTED MANUSCRIPT Abstract Hydrazone hesperetin Schiff base (HHSB) N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-ylidene]amino]benzamide has been synthesized and its crystal structure
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was determined. This compound was used for the formation of Cu(II) complexes in solid state
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and in solution which were characterized using different spectroscopic methods. The analyses of potentiometric titration curves revealed that monomeric and dimeric complexes of Cu(II) are formed above pH 7. The ESI MS (electrospray ionization-mass spectrometry) spectra
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confirmed their formation. The EPR and UV-visible spectra evidenced the involvement of oxygen and nitrogen atoms in Cu(II) coordination. Hydrazone hesperetin Schiff base can
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show keto-enol tautomerism and coordinate Cu(II) in the keto (O, N, Oket) and in the enolate form (O, N, Oenol). The semi-empirical molecular orbital method PM6 and DFT (density
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functional theory) calculations have revealed that the more stable form of the dimeric complex is that one in which the ligand is present in the enol form. The CuHHSB complex has shown high efficiency in the cleavage of plasmid DNA in aqueous solution, indicating its
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potential as chemical nuclease. Studies on DNA interactions, antimicrobial and cytotoxic activities have been undertaken to gain more information on the biological significance of
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HHSB and copper(II)-HHSB chelate species.
complexes
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Keywords: flavonoids, hesperetin, Schiff bases, copper complexes, metal-ionophore
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ACCEPTED MANUSCRIPT 1. Introduction Among fruits, citrus varieties are important sources of polyphenolic compounds, which could be responsible for the health-promoting effects. Hesperetin is one of the
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flavonones that belong to flavonoid class of compounds, found in citrus fruits. Hesperetin has
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multiple biological and pharmacological activities, including antioxidant properties [1,2] inhibition of cancer development [3,4], the prevention and/or treatment of certain eye diseases/disorders such as diabetic retinopathy, diabetic macular edema and cataract [5] and
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many others. Most of the pharmacological actions of hesperetin originated from its binding to proteins or other macromolecules, and regulating their structures and functions [6,7]. Besides,
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it is demonstrated that the coordination of copper(II) ion with bioactive ligands can actually improve their biological activity, for example Cu(II) complexes with hesperetin, naringenin
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and apigenin have showed higher inhibitory rate than their free ligands against SGC-7901 and HepG2 cell lines [8]. It has been also found that hydrazone hesperetin Schiff base (HHSB) and its copper chelate can bind to DNA. The complex has exhibited deeper intercalation of
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DNA and higher oxidative activity than the ligand [9, 10]. Understanding of complexation processes of flavonoids and their derivatives is
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important for explanation of the role of metals in biological functions of these medically promising compounds. Complexes of HHSB showed 1:1 stoichiometry and metal bonding via
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[C(5)-O- -C(4)-N] donor atoms set in the solid state [10]. Up till now, no solution studies of equilibrium and structure of copper(II)–hesperetin Schiff base complexes involving different protonation state of the ligand have been undertaken. These studies are also indispensable since biological active substances act in biological compartments with different water contents. Some important information on coordination, acid–base and metal binding properties can be achieved only by solution studies. This paper reports results of investigation of system containing Cu(II) ions and N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-ylidene]amino]benzamide in DMSO/water mixture
by means
of
potentiometric titration, spectroscopic techniques and theoretical calculations. The use of different research techniques and the convergence of the results, despite the complexity of the system allows for precise and unambiguous definition of how coordination and interactions of bioligands proceed in solution. DNA interaction and cleavage studies were done using circular dichroism and gel electrophoresis technique, respectively. The influence of different scavengers of reactive oxygen species: DMSO, glycerol, KI, sodium azide, and the minor or major groove binders (DAPI (2-(4-amidinophenyl)-1H -indole-6-carboxamidine) or methyl green) on the DNA 3
ACCEPTED MANUSCRIPT cleavage process, has been also studied. The ability of HHSB and CuHHSB complex to induce the cleavage of pEGFP-C1 DNA has prompted us to investigate their cytotoxic activity against human cancer cells (HeLa (human cervix carcinoma) and K562 (leukemia))
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and HUVEC (human umbilical vein endothelial cells) as a classic model system.
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Various Schiff bases showed antibacterial activity against Gram-positive (S. aureus and L. monocytogenes) and Gram-negative bacteria (E. coli and Pseudomonas aeruginosa) [11-13]. Therefore, studies on antimicrobial activities have been undertaken as well to get more
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information on the biological significance of the HHSB interactions and the role of copper(II)
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ions.
2. Experimental Materials
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2.1.
The racemic hesperetin, N-benzoyl hydrazine, NaOH, KCl, CuCl2, and all other compounds were purchased from Sigma-Aldrich Co. All reagents were of analytical quality
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and were used without further purification.
2.1.1. Synthesis of N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4-
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ylidene]amino]benzamide.
The ligand (N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4ylidene]amino]benzamide - HHSB) has been prepared according to the reference [10]. Yield: 1
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0.5 g, 45%. FAB-MS (fast atom bombardment mass spectrometry): m/z = 421 [M+H]+. H NMR (DMSO-d6, 200 MHz), 2.60 (1H, dd, J =27.48, 13.78 Hz, 3(a)-H), 2.88 (1H, dd, J =
11.96, 12.1 Hz, 3(e)-H), 5.03 (1H, d, J = 9.5 Hz, 2-H), 5.84 (1H, d, J = 2.2 Hz, 6-H), 5.90 (1H, d, J = 2 Hz, 8-H), 6.89 (3H, m, 2’, 5’, 6’H), 7.48 (2H, dd, J = 1.7, 7.4 Hz, 3”, 5”H), 7.54 (2H, dd, J = 4.0, 7.2 Hz, 4”H), 7.88 (2H, d, J = 6.8 Hz, 2”, 6”H), 9.08 (1H, s, 3’-OH), 9.98 (1H, s, 7-OH), 11.09 (1H, s, –NH–C═O), 13.04 (1H, s, 5-OH). M.p. 265-267 C. Anal. Calc. for C23H20N2O6: C, 65.71; H, 4.79; N, 6.66. Found: C, 65.62; H, 4.88; N, 6.71%. IR νmax(cm1
): ν(C═O): 1644, ν(C═N): 1601. UV-visible λmax(nm): 257, 323. Mp 265,6 - 267°C.
2.1.2. Synthesis of N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4ylidene]amino]benzamide copper(II) [CuLH3·OAc]·H2O. The ligand (84 mg, 0.20 mmol) was dissolved in acetone (20 mL). After 5 min, Cu(OAc)2·H2O (48 mg, 0.24 mmol) was quickly added into the ligand solution and the solution was refluxed on a oil-bath for 10 h with stirring. A green precipitate, [CuLH3·OAc]·H2O, where LH3=HHSB is in keto-form with C(7)-OH, C(3’)-OH and nitrogen of the benzamide protonated, was separated from the solution by suction filtration and washed 4
ACCEPTED MANUSCRIPT several times with methanol and dried for 24 h in vacuo. In biological studies instead of [CuLH3·OAc]·H2O the CuHHSB abbreviation has been used. Anal. Calc. [CuLH3·OAc]·H2O : C, 53.62; H, 4.32; N, 5.00; Cu, 11.35. Found: C, 54.01; H,
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4.91; N, 5.56; Cu, 10,5 %. IR νmax(cm-1): ν(C═O): 1604, ν(C═N): 1589, νas(COO-): 1568, = 482.1 [M-H]+ (M without acetate and water molecule). 2.2.
Methods of analysis
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νs(COO-): 1364, ν(M–O): 443, ν(M–N): 418. UV-Vis λmax(nm): 261, 383, 680. ESI-MS: m/z
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Elemental analysis (C, H and N) was carried out on an EuroVector 3018 analyzer. The metal content of the complex was determined using atomic absorption spectrometer: AAS
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GBC 932 Plus (GBC Scientific Equipment Ltd, Australia) with copper hollow cathode lamp. The melting point of the ligand was determined by an Electrothermal 9200 microscopic melting point apparatus. The IR spectra were recorded employing a Nicolet 6700 (Thermo-
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Scientific) FT-IR spectrometer, in the 4500-500 cm-1 region. 1H NMR spectra were recorded on a Bruker AV200 200MH spectrometer in DMSO-d6 with TMS (tetramethylsilane) as
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internal standard. Mass spectra were performed on a Finnigan MAT 9 instrument. 2.2.1. X-ray analysis
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The HHSB compound was crystallized from DMF (N,N-dimethylformamide)/water system. The hot solution was cooled and put into the fridge. After three days, the crystals
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were separated from the solution and dried. The obtained crystals belong to the monoclinic system with the space group C2/c, with the cell dimensions: 24.114(5) ×9.780(2) × 23.055(4) and β=111.19(3). The diffraction data were collected at room temperature on the CAD4 diffractometer with the CuKα radiation and processed by SDP software [14]. The structure was solved and refined using ShelxS and ShelxL programs, respectively [15]. The asymmetric unit contains one HHSB molecule and two molecules from crystallization solvent: DMF and water. The DMF molecule is disordered and was modelled in two positions. The occupancy of the atoms in both split positions is in ratio 0.55 : 0.45. The model of disorder can be described as a swinging of molecule around axis passing through the atoms O1 and N1. To maintain similarity between DMF in both positions the geometrical and temperature restrains were applied; SIMU (temperature vibration parameters of neighbouring atoms are set to be similar) and ISOR (temperature vibration parameters of each atom are restrained to be approximately the same). The hydrogens bound to carbon were set geometrically and refined as riding, the hydrogen atoms connected with nitrogen and oxygens were found on the difference Fourier map and refined freely. The anisotropic thermal parameters were applied for all non hydrogen atoms and isotropic for hydrogens, equal 1.2 of thermal parameter of parental atoms, except 5
ACCEPTED MANUSCRIPT for hydrogens connected to heteroatoms in HHSB molecule, which were refined with free thermal parameters. The final discrepancy parameter R for 3860 reflections with I > 2σI was equal 0.0868 and for all independent reflections (5202) - 0.1085. The crystal structure of
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HHSB is deposited in CCDC under accession code 950104.
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2.2.2. Potentiometry
The protonation constants of the ligand (pKa) and the stability constants of Cu(II) complexes (log) were determined by pH-potentiometric titrations of 2.0 mL samples in
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DMSO /water mixtures (40%:60% v/v) due to slight solubility of HHSB in pure water (see Fig.S01, supplementary material). The ligand : metal molar ratio was 1:1, the concentrations
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of Cu(II) and the ligand were 110-3 M. Measurements were carried out at 298 K and at a constant ionic strength of 0.1 M KCl with a MOLSPIN pH meter (Molspin Ltd., Newcastle-
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upon-Tyne, UK) equipped with a digitally operated syringe (the Molspin DSI 0.250 ml) controlled by computer. The titrations were performed with a carbonate-free NaOH solution of known concentration (ca. 0.1 M) using a Russel CMAWL/S7 semi-micro combined
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electrode. The pH measuring circuit was calibrated with potassium hydrogenphthalate and phosphate buffers [16]. The conversion of the operational pH reading to the pcH value at
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DMSO /water mixture (40%:60% v/v) were accomplished by the Eq. 1 as given below
pH = α + 𝑆pc H + 𝑗H [H+] + 𝑗𝑂𝐻
Kw [H + ]
(1)
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The parameters α, S, jH, jOH and Kw were taken from the literature [17]. In order to find autoprotolysis constant of water, titrations were performed in a series of DMSO/water HCl solutions of known concentration containing 0.1 M KCl using 0.1 M NaOH solution. The autoprotolysis constant of water in this medium was determined from Eq. 2 pKap = (E’a - E’b)/k
where
E’a
and
E’b
are
Eocell+
(2)
k log10 aKCl + k log10 γH+ + ELJ and
Eocell
+ k log10 aKCl −
k log10 γOH− + ELJ, respectively. The procedure of the calculations was adopted from the literature [18]. The obtained pKap value 14.64 is in general agreement with those reported before (14.98 [18], 14.42 [19]). The difference may be due to other experimental conditions, including the use of a different electrolyte. The number of experimental points was 100-150 for each titration curve. The reproducibility of the titration points included in the evaluation was within 0.005 pH units in the whole pH range examined (2-11.5). Protonation constants of the ligand and the overall stability constants (pqr, where p, q and r represent the number of metal, ligand and proton, respectively) of the complexes, were evaluated by iterative non-liner least squares fit of potentiometric equilibrium curves through mass balance equations for all 6
ACCEPTED MANUSCRIPT the components expressed in term of known and unknown equilibrium constants using a computer program SUPERQUAD [20]. The value for sigma (the root mean squared weighted residual), after refinement of the stability constants, was 1, which signifies that the data have
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been fitted within experimental error. The equilibrium constants were obtained as averaged
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values of three titrations. 2.2.3. Spectroscopic analysis
Electronic absorption (UV-Vis) spectra were recorded with a Perkin-Elmer Lambda 11
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spectrophotometer using quartz cell with a path length of 1 cm.
Anisotropic X-band EPR spectra of frozen solutions were recorded at 100 K, using an
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X-band (9.15 GHz) Varian E-9 spectrometer after addition of ethylene glycol to ensure good glass formation. Copper(II) stock solution for EPR measurements were prepared from natural 63
Cu and 0.7%
65
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CuSO4·5H2O or 63CuSO4·5H2O to get better resolution of EPR spectra. Metallic 63Cu (99.3% Cu) was purchased from JV Isoflex, Moscow, Russia for this purpose and
converted into pentahydrate 63CuSO4·5H2O. The EPR parameters were read from the spectra
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(estimated uncertainties for A and g values are 1×10–4 cm–1 and 0.002, respectively, in the spectra of a single species). All spectroscopic measurements were performed at the maximum
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concentration of each species found in titrations. Circular dichroism (CD) spectra were recorded with a Jobin Yvon CD6
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spectropolarimeter at room temperature using quartz cell with a path length of 0.5 cm, 2nm bandwidth and 1-2s integration time. Spectra were corrected for buffer signal and 25-point smooting algorithm was performed with the Jobin Yvon CD6 Standard Analysis software. The CT DNA concentration used in the experiments was 9.4×10-5M. Hydrazone hesperetin Schiff base and its copper(II) complex concentrations varied from 1.88×10-5M to 9.4×10-5M. The samples were prepared in Tris-HCl buffer (5mM Tris-HCl, 50 mM NaCl, pH 7.2). The CD spectra of CT DNA were recorded at time zero (immediately after preparation of the sample) and then, after 15min, 1h, 2hrs and 20hrs incubation at 37°C with increasing concentrations of hydrazone hesperetin Schiff base and its copper(II) complex. High resolution mass spectra (ESI MS) were obtained on a BrukerQ-FTMS spectrometer (Bruker Daltonik, Bremen, Germany), equipped with Apollo II electrospray ionization source with ion funnel. The mass spectrometer was operated in the positive ion mode. The instrumental parameters were as follows: scan range m/z 300–1600, end plate offset-500 V, dry gas – nitrogen (4 L min-1), temperature 200 °C, ion energy 5 eV. Capillary voltage was optimized to the highest S/N ratio and it was 4500 V. The small changes of voltage (±500 V) did not significantly affect the optimized spectra. The samples (HHSB and 7
ACCEPTED MANUSCRIPT HHSB with CuCl2 in the ratio 1:1 and 1:2, the concentration of CHHSB = 1×104 M) were prepared in a 1:1 MeOH-H2O mixture. Variation of the solvent composition down to 5 % of MeOH did not change the species composition. The sample was infused at a flow rate of 3
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μL min-1. The instrument was calibrated externally with the Tunemix™ mixture (Bruker
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Daltonik, Germany) in quadratic regression mode. Data were processed by using the Bruker Compass Data Analysis 4.0 program. The mass accuracy for the calibration was better than 5 ppm, enabling together with the true isotopic pattern (using SigmaFit) an unambiguous
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confirmation of the elemental composition of the obtained complex. 2.2.4. Theoretical calculations
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Semi-empirical calculations were carried out using MOPAC2012 program [21]. The geometries of free ligand and all complexes were fully geometry optimized using semi-
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empirical molecular orbital method PM6 [22]. Moreover DFT calculations were carried out with the Gaussian 03 package [23] using the Becke gradient corrected exchange functional [24] and Lee-Yang-Parr correlation functional [25,26] with 6-31G* basis set. For all
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structures with two copper ions calculations were carried out using Restricted Hartree-Fock (RHF) approximation, assuming electronic state of the molecule as a singlet (compound with
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two copper ions possesses even number of electrons and formally is closed-shell system). All initial structures for calculations were built manually. DNA cleavage
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2.3.
Electrophoresis experiments were performed with pEGFP-C1 (4731 bp) DNA. The cleavage of pEGFP-C1 by Cu(II), ligand, e.i. HHSB and Cu(II)-HHSB systems, respectively, were accomplished by mixing in the order: 1 µL of 5 mM Tris-HCl (pH 7.5 containing 5 mM NaCl) buffer, varying concentrations (0; 0,025; 0,05; 0,1; 0,15; 0,2 mM) of CuCl2, HHSB and Cu(II)-HHSB complex, and 1 µL of pEGFP-C1 (0.25 µg/µL; 10 mM Tris-buffer, pH 8.0). After mixing, the DNA solutions were incubated at 37°C for 20hrs. The reactions were quenched by the addition of EDTA and bromphenol blue and the mixtures were analyzed by gel electrophoresis (0.5% agarose gel). In order to examine if hydroxyl radicals or reactive oxygen species were present, scavengers: DMSO (0.4M) and glycerol (0.4M) for OH·, KI for H2O2 and Na3N for singlet oxygen were added to give a final concentration of 10 mM before the complex addition. The bindings of HHSB and CuHHSB complex to minor or major groove of DNA were tested using DAPI and methyl green, respectively. Plasmid cleavage products were quantitated and analyzed with G-BOX Syngene system. The GeneTools software was used to complete gel documentation and analysis 2.4.
Cells and cytotoxicity assay 8
ACCEPTED MANUSCRIPT Human umbilical vein endothelial cells (HUVEC) were isolated from freshly collected umbilical cords as previously described [27], and cultured in plastic dishes coated with gelatin, in RPMI 1640 medium supplemented with 20% FBS (fetal bovine serum), 90 U/ml
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heparin, 150μg/ml ECGF (endothelial cell growth factor, Roche Diagnostics, Mannheim,
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Germany) and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin). 10x103 cells were seeded on each well on 96-well plate (Nunc).
The HeLa (human cervix carcinoma) and K562 (leukemia), cells were cultured in
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RPMI 1640 medium supplemented with antibiotics and 10 % fetal calf serum (HeLa, K562) in a 5 % CO2 - 95 % air atmosphere. 7×103 cells were seeded on each well on 96-well plate
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(Nunc). 24h later cells were exposed to the test compounds for additional 48 hours. Stock solutions of test compounds (HHSB and CuHHSB systems) were freshly prepared in DMSO.
compounds
tested
in
the
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Stock solutions (doxorubicyna) was freshly prepared in water. The final concentrations of the cell
cultures
were:
1mM,
1×10-1
mM,
1×10-2 mM, 1×10-3 mM, 1×10-4 mM, 1×10-5 mM. Each concentration was assayed in
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triplicate in each experiment, and all experiments were repeated at least two times. The concentration of DMSO in the cell culture medium was 1%. The values of IC50 (the
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concentration of test compound required to reduce the cell survival fraction to 50 % of the control) were calculated from dose-response curves and used as a measure of cellular
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sensitivity to a given treatment.
The cytotoxicity of all compounds was determined by the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described [28]. Briefly, after 72h of incubation with drugs, the cells were treated with the MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20 % SDS and 50 % DMF at pH 4.7 and absorbance was read at 570 and 650 nm on an ELISA (enzyme-linked immunosorbent assay)-PLATE READER (FLUOstar Omega). As a control (100 % viability), we used cells grown in the presence of vehicle (1% DMSO) only. As a control for doxorubicin (100 % viability), untreated cells were used. 2.5.
Antimicrobial activity In vitro antibacterial activity studies were carried out against three Gram-negative
strains from Enteriobacteriaceae family: Salmonella Enteritidis ATCC 13076, Salmonella Typhimurium, ATCC 14028, Escherichia coli ATCC 8739 and two Gram-positive strains Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111. The bacteria were activated on a Nutrient Agar (Merck) for 24 h at 37ºC (Salmonella sp., E.coli, S.aureus) and at 30ºC Listeria strain. The overnight cultures were inoculated in Nutrient Broth (Merck) 9
ACCEPTED MANUSCRIPT before use. The bacterial counts of the diluted cultures were corrected by adding isotonic NaCl solution to be within the range of 106 – 107 colony forming units per one ml (CFU ml-1). Samples of test compounds: HHSB and CuHHSB, were dissolved in DMSO to obtain
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concentration 5 mg mL-1 and were sterilized by filtration (filter pore width 0.2 m; Sartorius).
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Paper discs ( = 6 mm) were impregnated with 50 l of the samples and the solvent allowed to evaporate at room temperature in the dark. The diluted bacterial test culture (200 L) was spread on sterile Mueller-Hinton Agar (Merck) plates before placing the sample impregnated
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paper discs on the plates. As a negative control was used DMSO solution at the concentration of 20 mg mL-1 (this concentration of DMSO did not inhibit the growth of microorganisms)
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[29]. As positive control were used Kanamycin and Doxycycline (Oxoid) at the concentration of 30 g mL-1 each. The plates were incubated at 37ºC (Salmonella sp., E.coli, S.aureus) and
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at 30ºC Listeria strain. After 24 h the inhibition diameters were measured. As a result, the final taken into account (subtracted) the diameter of the disc. The experiments were repeated three times and results were expressed in average values.
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2.5.1. Growth of bacteria in the presence of the test compounds. To 0.895 mL of Nutrient Broth (Merck) was added 5.0 L (depending on the sample)
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CuHHSB or HHSB, or CuCl2 and 0.100 mL the bacterial suspension (1106 CFU mL-1). The control sample was a growth of bacteria without addition copper compounds. After 24h
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incubation the amount of bacteria were determined by the plate method. Assay was done in triplicate. The results were analyzed using one-way analysis of variance (ANOVA) p≤0.05. 2.5.2. Aggregation of bacterial strains The aggregation assay was performed according to Ahmadowa et al. [30] with some modifications. The overnight cultures of test strains (E.coli, Salmonella Typhimutium, S. aureus) were pelleted by centrifugation (7000 × g, 10 min., 20ºC), washed twice in sterile phosphate-buffered saline (PBS, Sigma, pH 7.4) and resuspended in the same solvent to achieve an OD of 0.5 at 600 nm. To 0.995 ml of the bacterial suspension was added 5.0 L (depending on the sample) CuHHSB or HHSB or CuCl2. The control sample was a suspension of bacteria in PBS. After 5 h incubation at 37ºC the absorbance of the upper part of cell suspension was measured at 600 nm. The percentage of aggregation was calculated as: aggregation (%) = [(A0 – A5)/A0]×100; where: A0 initial absorbance, A5 absorbance after 5 h. Assay was done in triplicate. The results were analyzed using one-way analysis of variance (ANOVA) p≤0.05. 2.5.3. Membrane permeabilisation by fluorescence microscopy 10
ACCEPTED MANUSCRIPT To determine the permeability of cell membranes fluorescent assay was performed LIVE/DEAD BacLight bacterial viability kit (L-7012). Bacterial suspensions of S.aureus, S. Typhimurium and E.coli were exposed to (depending on the sample) CuHHSB or HHSB, or
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CuCl2 within 20 min. and stained with a fluorescent cell viability kit. Overnight liquid
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bacteria culture were diluted to OD 0.5 - 1.0 McFarland standard. The stock solution of the studied chemical compounds (3L, 250 g mL-1 in DMSO) was added to bacterial culture (997 L). Equal volumes of component A and component B (LIVE/DEAD BacLight bacterial
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viability kit) were mixed thoroughly and the resulting solution (3L) was added to cultures (1000 L) treated with copper complex. The solution of the treated samples was incubated at
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room temperature (20-23ºC) for 15 min and protected from light. Then, the sample (5L) was mounted between a cover slip and POLYSINE SLIDES (Thermo Scientific, Gerhard Menzel
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GmbH). Fluorescence microscopy images of the treated bacterial suspension were obtained at 40 magnification with an excitation wavelength of 488 nm through emission filters (long pass >650 nm and band pass 505-550 nm) corresponding to the DNA-bound emission range
3. Results and discussion
The solid state structure of HHSB
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3.1.
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of PI and SYTO9 respectively.
The central chroman bicyclic ring is almost flat and only C2 atom is standing out from
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the plane. Two hydroxyl groups, 5 and 7, are located in the same plane. The 3’-hydroxy-4’methoxy phenyl ring is twisted about 80° with respect to the chroman plane. The 4-ylideneamino-benzamide moiety is approximately coplanar with the chroman fragment. This fragment has keto-form in contrast to the preferable, more stable enol-form in solution. The conformation of the ligand is stabilized by bifurcated hydrogen bond from 5-hydroxyl group interacting with ylidene nitrogen and with carbonyl oxygen of the benzamide (Fig. 1). The crystal structure shows "host-guest" interactions in which the HHSB molecule is a host [31]. The main interaction of the host is π-stacking with symmetry related molecule of the ligand (Fig. 2). Fig. 1. Fig. 2. These hydrophobic contacts are supported by two hydrogen bonds created between 7hydroxyl group and hydrogen from amide group with benzamide carbonyl oxygen and methoxy oxygen from symmetry related molecules, respectively. The guest molecules, DMF and water are involved in three hydrogen bonds with HHSB. The water molecule serves as an acceptor for 3'-hydroxyl group and as a donor for carbonyl oxygen from DMF and for oxygen 11
ACCEPTED MANUSCRIPT from 7-hydroxyl group. The crystal structure of the ligand shows spatial arrangement of the potentially chelating atoms. 3.2.
Ionization constants of HHSB
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The acid dissociation (acid-ionization) constant, pKa is important parameter describing
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the extent of ionization of functional groups with respect to pH of biologically active substances. It can affect their solubility, dissolution rate, absorption across biological membranes, distribution to the site of action, renal elimination, metabolism, protein binding,
SC
and receptor interactions [32]. Knowledge of pKa values as a function of solvent composition is also useful in the application of reversed-phase HPLC for the separation of ionizable
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compounds [33]. Potentiometry is a very good tool to establish pKa value of compounds of pharmaceutical and biological interest because it does not require the presence of
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chromophore. When compound is sparingly soluble in water, the pKa determination is commonly done in organic/water mixtures.
DMSO/water mixtures are often used because they have lower polarity than pure
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water, but keeping a similar environment. The unique capability of dimethyl sulfoxide (DMSO) to penetrate living tissues without causing significant damage is most probably
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related to its relatively polar nature, its capacity to accept hydrogen bonds, and its relatively small and compact structure. This combination of properties results in the ability of DMSO to
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associate with water, proteins, carbohydrates, nucleic acid, ionic substances, and other constituents of living systems. One of the possible functions of DMSO in biological systems is its ability to replace some of the water molecules associated with the cellular constituents, or to affect the structure of the omnipresent water. DMSO is highly polar, very weakly acidic and fairly basic, while water is also highly polar but acidic and negligibly basic. DMSO can interact strongly and specifically with water through hydrogen bonds. So DMSO is miscible like alcohol with water in all proportions. Aqueous mixtures of DMSO have extraordinary penetration properties and can induce cell fusion and intensify cell permeability which is used in drug delivery systems [34]. In fact, this compound has found a variety of applications as pharmaceutical agent of low toxicity and also as an effective solvent [35]. Taking all this into account it can be assumed that the studies both physicochemical and biological are relevant. Therefore we have decided to carry out the investigation in DMSO/water mixtures in order to reveal properties of HHSB and its complexes with copper(II) ions. The ionization constants of hydrazone hesperetin Schiff base and overall stability constants of complexes formed in HHSB-Cu(II) system, were calculated via the potentiometric method in a mixture H2O/DMSO 60:40 v/v. The results are presented in 12
ACCEPTED MANUSCRIPT Table 1 and Table 2. The Yasuda–Shedlovsky extrapolation pKa+log[H2O]=a +b/ε was used to derive acid dissociation constants in aqueous solution ( pK aw ) (Fig. 3, Table 1). Terms a and b symbolize the intercept and the slope of the plot, respectively. It has been previously
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reported that when using organic solvent/aqueous mixtures with ε values greater than 50, the
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extrapolation to the purely aqueous domain is linear and produces relatively accurate pKa values [17]. Taking this into account pK aw values were evaluated using log 55.5 and 1/78.3,
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the logarithm of the molar concentration and the inverse of the dielectric constant of pure water, respectively.
HHSB can reveal a keto-enol tautomerism (Scheme 1), as other hydrazone Schiff
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bases [38,39]. Taking into account the tautomerism, the ligand has four possible acidic hydrogens. However, since the proton dissociation of imine nitrogen (-NH-) or enolic OH
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occurs in strongly basic conditions, which are outside of the experimental pH range of the methodology, it was not possible to determine all of them. Hence, only three ionization constants have been calculated and therefore the ligand is indicated as LH3. (a similar
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convention is accepted for oligopeptides where pKa of –NH– group is omitted in stoichiometric protonations of these ligands [40]). For comparison in the same experimental
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conditions the pKa values of hesperetin have been determined as well. Some differences in numerical values of pKa’s originate from different methods and conditions using to estimate
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them. According to the literature data [38,39] in hesperetin pKa1 corresponds to the ionization of the 7-OH group since the negative charge resulting from deprotonation of this group will be stabilized by conjugation. The next deprotonation step occurs at 3 ’-OH group in ring B and it corresponds to pKa2. The third and the last one proceeds on the 5-OH group which corresponds to pKa3. It must be the hardest to occur due to hydrogen bonding with the vicinal carbonyl group; therefore it can be registered at high pH range. One can expect the same pKa trend in hesperetin Schiff base. The values of pKa1 and pKa2 are similar to those found for hesperetin but the pKa3 is more acidic as compared to the unmodified flavonoid. It may be derived from an electron withdrawing effect of benzohydrazide group. This might induce diminishing electron density and lead to release the 5-OH hydrogen at lower pH. The decreased acidic strength characteristic of all HHSB forms present in DMSO-rich mixtures (Fig. 3) is probably in part a consequence of the increased solvent stabilization of the acidic species. This interaction is favored by the large dipole moment of the DMSO molecule [41]. In Fig. 4 the equilibrium distribution of different protonated species of HHSB is shown as a function of pcH calculated from the recorded potential values. As this figure shows, a 13
ACCEPTED MANUSCRIPT completely protonated LH3 is the main species in low pH whilst LH2-, LH2- and L3- dominate at alkaline range. As pH increases the LH3 dissociates and in neutral solution transforms into the LH2- form of the ligand. At pcH 9 the LH2- species coexists with LH2-, while in the range
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of 10 − 11 pcH the L3- is dominant.
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The absorption UV spectra of the ligand registered with increasing pH support a stepwise deprotonation. Significant changes in the spectral HHSB profile as a function of pcH have been attributed to the formation of the different protonated species found by
SC
potentiometry, whose light absorbing properties are different from one another. Thus, it can be assumed that four absorbing species are formed by the successive deprotonation of HHSB
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in positions 7-OH, 3’-OH, and 5-OH, respectively in pcH range of 2–12 (Fig. 5). The results of spectra in Fig. 5 indicate that the first and third deprotonation have a strong change in the
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spectral shape and intensity of HHSB, while the second deprotonation causes a less modification of the spectrum. The absorption bands appearing in the HHSB spectrum can be assigned on the basis of literature data on absorption spectra of Schiff bases [42]. The bands
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in the range of high energy, at λmax = 267 and λmax = 289 nm, are due to the excitation of the π electrons (π-π* transitions) of the aromatic rings. The third band at λmax = 324 or 340 nm could be assigned to the π-π* transition within the C=N group, while the longest wavelength
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band at λmax = 408 nm is due to an intramolecular charge transfer (CT) transition involving
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the whole molecule. From the results one can deduce that in the biological compartments with varying amount of water, the HHSB may exists in partially or fully protonated form depending on hydrogen ions concentration. 3.3.
Copper complexes of HHSB in DMSO/water mixture
3.3.1. Potentiometric and UV-VIS studies The HHSB possesses the donor atoms on adjacent A and C rings. It is the ylidene nitrogen, phenolic oxygen of 5-OH group and carbonyl oxygen of the benzamide as a metalchelating site. The overall stability constants of the complexes were determined via pH titration using the dissociation/ionization constants of the ligand. The best fittings of the titration curves were obtained using the species listed in Table 2. The species distribution curves of complexes are shown in Fig. 6. Fig. 6. It can be observed that the different protonated species appear one after the other with increasing pcH region. The complexation process starts above a pcH of 3. It is not surprisingly that in spite of the high pKa values of HHSB ionisable groups, the protons are displaced at much lower pH range in the presence of copper(II) ions. The same process was observed for 14
ACCEPTED MANUSCRIPT phenols and flavonoids under metal chelation [43]. The copper complexes with variously protonated forms of the ligand are formed. The formation of complexes in acid medium promotes the deprotonation of the ligand at the 5-OH group and the simultaneous copper binding to the nearest nitrogen situated in the position C(4) and two chelating rings are
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formed. The first CuLH2 species has the (O, N, Oket) donor atom set with the 7-OH and 3’OH fully protonated. The next CuLH can acquire two possible coordination modes: the (O, N, Oket) with the 7-OH group deprotonated and the 3’-OH protonated or the (O, N, O-enol)
respectively).
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Scheme 2.
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donor atom set with the 7-OH and 3’-OH groups protonated (Scheme 2 a) and b),
Acoording to the fitting of potentiometric data in the same pH range the CuLH can coexist
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with a dimeric Cu2L2H2 complex which is a minor species but it can be recognizable by EPR and ESI MS techniques (see the paragraphs 3.3.2, 3.3.3). In the high pH region, the CuL and CuLH-1 complexes can be found. The first complex can exist in the keto form with the 7-OH
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and 3’-OH groups deprotonated or in the enolate form with the 7-OH deprotonated and the 3’OH group still protonated. On the other hand, in the second one the only donor atom set could
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be the (O, N, O-enol) and the 7-OH and 3’-OH fully deprotonated. Complex formation can be seen in UV-Vis spectra (Fig. 7).
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Fig. 7.
According to the speciation diagram at pcH above 3 initially CuLH2 is formed. The [C(5)-O- C(4)-N] coordination mode, i.e. one oxygen and one nitrogen as donor atoms, is supported by the d-d electron transition energy corresponding to λmax = 640 nm which is similar to many other Cu(II) systems [44,45]. This species coexists in low pH with aqua copper(II) ions therefore λmax registered at 3.50 pH is slightly shifted to lower energy (see Fig. 7). With the increasing pcH the same donor atom set is also clearly seen in the other complexes. The hypsochromic shift of the band can be caused by both a specific coordination environment and the coexistence of different species. 3.3.2. EPR studies The Cu(II)-HHSB system with the ratio 1:1 was investigated in DMSO/water 40:60 v/v (Fig. S05). The anisotropic EPR spectra recorded in the system confirm the formation of the species [CuLHx] with x = 2, 1, 0, -1. The resonances of two species are observable with gz = 2.277, Az = 181.6104 cm1 and gz = 2.243, Az = 188.8104 cm1. The difference between these compounds is due to the deprotonation of the keto complex with the donor set (O, N, 15
ACCEPTED MANUSCRIPT Oket) or the enolate coordination (O, N, Oenol) (see Scheme 2a or b, respectively) which results in a decrease of gz and an increase of Az. The superhyperfine coupling with
14
N
nucleus is observable in the enol complex but not in the keto complex. To clarify the number
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of nitrogen atoms coordinated to copper, EPR spectra with 63CuSO4·5H2O were recorded; the
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comparison of the first parallel resonance in the spectra of the enol complex with natural and enriched (63Cu 99.7%) copper is reported in Fig. 8. It can be noticed that with
63
Cu isotope
only three resonances are detected with the expected intensity ratio of 1:1:1 due to the
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coupling with one 14N nucleus (I = 1). Therefore, the multiplet observed with natural copper is due to the overlap of the two triplets given by the coupling with
63
Cu and
65
Cu, which
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accounts for the experimental ratio of 1:3:3:2 between the signals (Fig. 9a). Fig. 9.
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The values of 14.5 and 15.3104 cm1 for A||N and AN are in good agreement with those reported in the literature [46-48]. The complete spectrum of the enol complex is shown in Fig. 9b. The asterisks indicate the resonances of the parallel and perpendicular region where
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the coupling with the nitrogen nucleus is observed. The decrease of the spectral intensity and the transition in the ΔM = 2 region above neutral pH support the formation of the dinuclear
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species with the stoichiometry Cu2L2H2 (Fig. S06). The theoretical calculations suggest the enolic coordination mode (O, N, Oenol).
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3.3.3. ESI MS spectra
Mass spectrometry showed that the ligand itself was pure, proved the formation of copper complexes and clearly confirmed the stoichiometry 1:1 and 2:2 of the species formed in HHSB-Cu(II) system (Fig. 10A). This finding is in the agreement with potentiometric and EPR results. The presence of CuL and Cu2L2 complexes is supported by the mono-charged m/z signals at 482.1 and 965.1, respectively. The signal attribution was confirmed not only by the comparison of the experimental mass of the clusters with their simulations (Fig. 10B, where the simulated elemental compositions are CuC23H19N2O6 for the for CuL and Cu2C46H36N4O6 for Cu2L2), but also in a method of the analysis of their isotopic pattern which is quite specific for copper complexes, due to the fact that this metal has two major naturally occurring isotopes, 63Cu and 65Cu. The spectra were measured at pH range from 3 to 9, and the differences between them are negligible; mass spectrometry is unable to detect different protonation states, due to the ionisation process itself, which involves the evaporation of solvent.
16
ACCEPTED MANUSCRIPT 3.3.4. Theoretical calculations Coordination sphere of copper ions was based on the model structures for x-ray salicylaldehyde acetylhydrazone bonded to copper ion [49]. Initial structure was fully
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geometry optimized using PM6 semi-empirical method and using DFT method (B3LYP/6-
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31G*). Energy was minimized at B3LYP/6-31G* level of theory structure of Cu2L2H2 complex is shown in Fig. 11. Unlike in the salicylaldehyde acetylhydrazone [49] hydrazone hesperetin Schiff base does not form planar Cu2L2H2 complex. Highly distortion form
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planarity of coordination sphere of cooper ions is caused probably by steric repulsion between hydrogen atoms from phenyl ring of one ligand and O5 oxygen atom from the another ligand
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(see green arrows Fig. 11). Such a steric clash does not appear in the structures of salicylaldehyde acetylhydrazone complexed with copper ion [49] when dinuclear complex
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were shown to dominate in the broad range of pH. However in another studies when (E)-N’(2-oxy-3-methoxybenzylidene)benzohydrazide was used as a ligand, more complicated tetranuclear complexes (cuban-like) were observed [39]. Our results as well as Monfared et al.
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[39] clearly suggest that the presence of benzoic group attached to hydrazide moiety destabilizes the formation of planar complexes that includes two or more ligands because of
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steric repulsion.
The destabilization of Cu2L2H2 is clearly supported by results presented in Fig. 6
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where the concentration of dinuclear complex is very low as to compare to another species. DFT (B3LYP/6-31G*) and semi-empirical calculations (PM6) clearly show that in Cu2L2H2 complex the ligand exists in an enolic form (see Fig. 11). All attempts of geometry optimization models of Cu2L2H2 complex when ligand was in the keto form lead to the complex dissociation and the formation of two separate CuLH molecules (data not shown). Free ligand exists in the keto form (at least in acidic pH) as it was shown in X-ray studies presented in this paper (see Fig. 1). In comparison to the complex of salicylaldehyde acetylhydrazone with copper ions [49] the ligand exists in keto form. However, in the case of the complex of (E)-N’-(2-oxy-3-methoxybenzylidene) benzohydrazide with the copper(II) only enolic form of ligand is observed [39]. Keto form of the ligand is destabilized by the repulsion interaction between hydrogen atom from benzoic group and hydrazide hydrogen (place of such a repulsion interaction is marked in Fig. 11 by violet arrows). In the case of the free ligand (see Fig. 1) such a repulsion does not exist, however probably when the complex is formed the bond lengths as well as the valence angles change in a such way that coplanarity of benzoic group with hydrazide moiety is no longer possible for the keto-type ligand. 3.3.5. Circular dichroism study 17
ACCEPTED MANUSCRIPT The results of electronic absorption and luminescence studies supported that hesperetin Schiff base and its metal complexes can bind to DNA via intercalation mode [10]. But for understanding the mechanism of compounds binding to DNA and helical
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conformation of nucleic acids circular dichroism is an efficient spectroscopic technique to test
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effects of synthetic agents induced on DNA three-dimensional structure [50]. Analysis of the CD spectra is very useful in the investigation of morphological changes in double-stranded DNA caused by the interaction of small molecules. The negative
SC
band near 244 nm with the positive band near 275 nm can be used as a spectral discriminator for the B forms, right handed helicity duplexes. [51]. The positive band near 265 nm and the
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negative one near 236 nm have been ascribed to the exciton splitting of the π-π* transitions resulting from base stacking [52]. Any change in the base stacking pattern or the helicity of
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the strands is manifested by either a change in the band position, the intensity, or both. A simple electrostatic interaction or binding to the grooves has almost no or very slight effect on the band at 275 nm, while the same band undergoes significant variations in intensity due
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to intercalation by small molecules [53]. In Fig. 12 the spectra registered at increasing concentration of CuHHSB complex and the constant CDNA are presented. The CD spectrum of
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DNA undergoes some changes. The band at 275 nm is blue shifted to 269 nm, changed in the intensity and at the ratio of DNA/CuHHSB 4 a new band above 300 nm appears.
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Fig. 12.
The spectrum at the ratio of DNA/CuHHSB 9.4 is slightly different from that of DNA alone but after 20 hrs an impact of the complex on DNA is more significant. The comparison of the DNA-CuHHSB spectra with those of CuHHSB clearly indicates some interactions between DNA and the complex. The CuHHSB may be bound externally to the phosphate backbone or in the minor/major grooves or intercalated between DNA bases. On the base of the results it is difficult to indicate unequivocally an intercalative or covalent cross-linker binding mode of CuHHSB complex to DNA. It is supposed that combinations of all possibilities are feasible. 3.3.6. Cleavage of pEGFP-C1 DNA The biological activity of metal complexes is often related to their ability to cleave DNA. They are able to bind to DNA either specifically or sequence independent and cleave one or both strands by either a radical or a hydrolytic pathway, the latter similar to the one of natural nucleases [54-57]. The cleavage of plasmid DNA (4731 bp) by HHSB and CuHHSB was monitored by agarose gel electrophoresis. Strand cleavage of the naturally occurring supercoiled DNA (Form I SC) led either to an open circular relaxed form (Form II OC) upon single strand cleavage or to a linear form (Form III) upon double strand cleavage. The 18
ACCEPTED MANUSCRIPT different forms could be distinguished by agarose gel electrophoresis, as form I migrated faster than the other forms, while form II was the slowest of the three due to its relaxed structure (Fig. 13a) [58].
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Fig. 13
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To prevent DNA aggregation, the final concentration of CuHHSB in each experiment was never higher than 100 μM. The results presented in Fig. 13a show that the complex was able to cut DNA effectively under applied conditions in contrast to the ligand HHSB (Fig. 13b).
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The complex behaves as a chemical nuclease by nicking the DNA Form I into Form III. It is suggested that the CuHHSB compound may cut the DNA strand at two positions. Although
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the cleavage reaction promoted by the complex does not require additional external agents, we carefully investigated the effects of scavengers on the DNA cleavage ability of the
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complex to gain information if any oxidative cleavage occurs. Hydroxyl radical scavengers (DMSO, glycerol), hydrogen peroxide scavenger (KI) and a singlet oxygen scavenger (NaN3) were added into the samples under the same conditions. The electrophoresis gel results are
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shown in Lines 3-6 (Fig. 13c). The experiments reveal that hydroxyl radical and hydrogen peroxide inhibitors can’t prevent the DNA degradation, while the presence of NaN3 efficiently
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inhibits the DNA scission (Lane 6, Fig. 13c.). Hence, the singlet oxygen species might be involved in an oxidative cleavage pathway. It may indicate that the localized generation of
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singlet oxygen or singlet oxygen-like entity, possibly a copper-peroxide complex, rather than free hydroxyl radical probably plays a role in the CuHHSB -induced DNA strand breaks as it was observed with other copper complexes [59]. However, according to some authors this result should not be interpreted as an indication that 1O2 is also participating in the process; it is probably due to the affinity of sodium azide for transition metals [60]. Therefore, hydrolytic pathway can’t be ruled out as well. DNA cleavage experiments in the presence and absence of DAPI and methyl green, these recognized minor and major groove’s binders of DNA, indicated that CuHHSB is probably not groove binder (Fig. S07). Therefore, the obtained data may point out to its action as intercalator or covalent cross-linker. 3.3.7. Anticancer Activity Anticancer drugs may act specifically on particular types of cancer. Therefore, the anticancer activity of the HHSB ligand and the CuHHSB complex was tested against two different tumor cell lines: HeLa (cervix carcinoma) and K562 (leukemia). The cells were challenged with each of the compound, over a range of concentrations from 10nM to 1mM for 72h and cell viability was measured by MTT assay. Cells incubated with different concentration of doxorubicin served as a reference sample. 19
ACCEPTED MANUSCRIPT Both HHSB and CuHHSB complex decreased cell viability (Fig. S08), with IC50 values in the micromolar range (Table 3). They have lower cytotoxic activity than doxorubicin. The comparison of IC50 values indicates that CuHHSB complex exhibits greater
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inhibitory effect on investigated human cancer cell lines than HHSB. Therefore one can
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consider that the observed cytotoxicity results from the activity of the complex CuHHSB rather than the HHSB ligand only. Interestingly, published data point out that copper(II) ions have very low cytotoxicity against cancer cells (HeLa) [61]. The compound HHSB was the
SC
least toxic toward HUVEC cells and it was impossible to determine IC50 in our experimental settings. This result is very promising and it could be of interest for further investigation.
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3.3.8. Antimicrobial activity
For in vitro antimicrobial activity, the investigated compounds were tested against the
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bacteria: Salmonella Enteritidis ATCC 13076, Salmonella Typhimurium, ATCC 14028, Escherichia coli ATCC 8739 and two Gram-positive strains Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111. In Table 4, bacteria growth inhibition by
ED
CuHHSB complex at a concentration of 250 μg mL-1was observed for all tested bacteria. The values of the zones of inhibition of bacterial growth were comparable to the control, which
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was a solution of CuCl2 (250 μg mL-1).
Table 4
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A surprising result was obtained for S. Enteritidis, S. aureus and L. monocytogenes, whose growth was limited by the ligand. But the ligand did not inhibit the growth of the bacteria E. coli and S. Typhimurium. This phenomenon can be explained by individual lipophilic nature of the complexes. Such increased activity of the metal chelates can be explained on the basis of Overtone’s concept and Tweedy’s chelation theory [62]. According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only lipid soluble materials due to which liposolubility is considered to be an important factor that controls the antimicrobial activity. On chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of metal ion with donor groups [63,64]. Further, it increases the delocalization of the π electrons over the whole chelate ring and enhances the lipophilicity of the complex. This increased lipophilicity enhances the penetration of the complexes into cell lipid membrane [65]. They can disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism [66]. The variation in the activity of different complexes against different organisms depend either on the impermeability of the cells of the microbes or difference in ribosomes of microbial cells. The results have also 20
ACCEPTED MANUSCRIPT indicated that if Cu(II) ions have strong effect on bacteria growth inhibition (Escherichia coli, Salmonella Typhimurium) then the impact of the CuHHSB complex is also higher (no effect of the ligand is observed). This may mean that the HHSB can act as a transporter of Cu(II)
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ions (copper-ionophore) increasing in this way intracellular metal concentration and leading
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to a reduction of the bacterial growth. On the other hand when Cu(II) ions have lower effect on the activity of bacteria (Salmonella Enteritidis Staphylococcus aureus) then the impact of the HHSB and CuHHSB can be observed.
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Table 5 shows the results of the reducing growth of three selected test bacterial cultures: Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 8739 and
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Staphylococcus aureus ATCC 25923 after 24 hours. Growth strains of S. Typhimurium and E. coli was effectively inhibited by CuCl2. In the presence of the copper complex of the growth
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of S. Typhimurium was at the same level as innokulum (4.6 - 5.5 Log CFU mL-1). In the case of the bacterial count of E. coli after 24 hours of exposure to the complex remained at the same level (4.7-4.6 Log CFU mL-1). This shows the bacteriostatic effect of the copper
ED
complex on the bacteria E. coli and S. Typhimurium. Bacteria from genus S.aureus were able to grow in the presence of either CuCl2 or the complex.
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During the test of the permeability of cell membranes by fluorescent method (LIVE/DEAD BacLight bacterial viability kit) the ability of bacteria to form cell aggregates in
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the presence of both ligand and copper complex was observed (Fig. S09). Aggregation test was done for the three selected bacteria strains. The results are shown in Table 6. The aggregation of SalmonellaTyphimurium in the presence of CuCl2 was low (4.0 %). On the other hand, the aggregation ability of SalmonellaTyphimurium was the highest in the presence of the ligand (73%). The copper complex showed the aggregation similar to the control probe. In the case of Escherichia coli, the highest bacteria aggregation was observed in the presence of the ligand and the copper complex, respectively 73.7% and 75.2%. The aggregation of Staphylococcus aureus was different, the highest values of aggregation were observed in case of CuCl2 (53.7%) and CuHHSB (65.3%). The aggregation of Staphylococcus aureus in the presence of the ligand was low and comparable to that of the control probe. The results have indicated that the interactions of the compounds and microorganisms are individual. They depend on the pair: compound and organism. At this stage of our studies we can suppose that the compounds HHSB or CuHHSB may influence both surface proteins and enzymes regulated their presence. It is related to the differences in cell-wall structure of the Gram-positive (Staphylococcus aureus) and Gramnegative (Salmonella Typhimurium and Escherichia coli) bacteria. The differences in the 21
ACCEPTED MANUSCRIPT percentage of aggregation within a given bacteria group (Gram-negative for example, see Table 6) can be driven from subtle chemical differences to their wall fabric. One can suppose that fimbrial filament of the bacterial cell surface may play critical role in interactions with
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HHSB or CuHHSB and influences the aggregation of bacteria. Each fimbrial filament on the
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bacterial surface is composed of approximately 500 to 3,000 copies of a major fimbrial subunit protein [67]. The HHSB and CuHHSB may affect chemically these proteins, (e.g. by changing secondary structure) and this can reduce the virulence of bacteria.
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4. Conclusions
In summary, the crystallographic characteristic of hydrazone hesperetin Schift base -
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HHSB and solution speciation of HHSB and Cu(II)-HHSB chelate have been made. Our results are the first report evaluating the coordinating ability of hydrazone hesperetin Schiff
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base towards copper(II) ion in solution. The crystal structure of the ligand shows spatial arrangement of the potentially chelating atoms. The protonation and overall stability constants of hydrazone hesperetin Schiff base and its copper complexes have been determined,
ED
respectively. The higher values of ionization constants found by the extrapolating Yasuda– Shedlovsky method for aqueous solution can be related to the increased solvent stabilization
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of the acidic species in DMSO-rich mixtures. This interaction is favored by the large dipole moment of the DMSO molecule. The copper(II) ions form complexes with different
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stoichiometry depending on the pH range. The chelation reaction in acid medium promotes the deprotonation of the ligand at the C(5)-OH group and the simultaneous copper binding to the nearest nitrogen situated in the C(4) position. The species with variously ionized ligand can acquire two possible coordination modes: (O, N, Oket) or (O, N, O-enol). Theoretical calculations have shown that the most stable dimer structure is that one containing the enol form of the ligand.
CD spectrum analysis indicates that the copper complex with HHSB alters the DNA structure as it induces the changes of the position and the intensity of the DNA band. The CuHHSB complex may partially intercalate into DNA base-pairs with an unclassical intercalative mode and exhibits efficient cleavage of supercoiled DNA (pEGFP-C1 DNA) in the absence of any external agents via oxidative/hydrolytic (or both) mechanism. In vitro studies revealed that hydrazone hesperetin Schiff base and the copper complex exhibit cytotoxic and antimicrobial activities. The CuHHSB complex has a greater cytotoxic activity against HeLa and K562 cells than the HHSB ligand. On the other hand, the ligand does not exhibit cytotoxicity toward HUVEC cells in the experimental conditions. Beside the mechanistic speculations, which need to be supported by further investigations, the main 22
ACCEPTED MANUSCRIPT feature of the present system is the high reactivity in the absence of cofactors which makes the CuHHSB complex a very promising candidate for biological applications in vivo, especially in the therapeutic area, and for all those applications which are not compatible with
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the use of cofactor activated DNA-cleaving agents.
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The results of antimicrobial activity lead to the conclusion that HHSB and CuHHSB display antimicrobial activities against tested strains of bacteria, especially S. aureus is more sensitive to CuHHSB in comparison to other bacteria however the synthesized compounds
SC
exhibit lower activity compared to that one of the control drugs. The HHSB and CuHHSB trigger the bacteria aggregation which can limit the virulence of the bacteria. The mechanism
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for the antimicrobial activities of these compounds needs further study and the experimental results above would provide basic data for pharmacological research of HHSB and its
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copper(II) complex.
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Acknowledgement
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Financial support of this work by Statute Funds No. I28/DzS/9184.
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SC
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AC CE
PT
ED
MA
NU
[66]
27
ACCEPTED MANUSCRIPT
Table 1. Ionization constants of hydrazone hesperetin Schiff base (HHSB) in (DMSO/H2O
RI P
T
40%/60% v/v as pKa and in aqueous solution as pK aw ) and hesperetin for comparison
Ligand
Species
LogβHnL
pKa1
SC
(standard deviations are in parentheses). (I = 0.1M (KCl), T = 298K).
pKa2
pKa3
w ( pK a2 )
w ( pK a3 )
7-OH
3’-OH
5-OH
7.65
9.39
10.58
(7.24)
(9.18)
(10.21)
7.63
8.33
11.29
This work
6.80
10.40
>11.50
[36]
6.67
8.76
11.54
[37]
LH2-
19.97(±0.02)
LH3
27.62(±0.02)
LH2-
ED
10.58(±0.01)
11.29(±0.02)
AC CE
Hesperetin
LH2-
PT
HHSB
MA
NU
w ( pK a1 )
LH2-
19.62(±0.03)
LH3
27.25(±0.03)
28
Ref.
This work
ACCEPTED MANUSCRIPT
Table 2. Stability constants (logβpqr) of copper(II) complexes with HHSB (standard deviations are in parentheses) and spectral parameters
logβpqr
UV-Vis
Coordination mode
US
Species
CR
IP
T
(I = 0.1 M (KCl), T = 298 K).
-1
MA N
λ/nm -1
(ε/M cm ) 640(22)
CuLH
19.85(±0.02)
625(23)
Cu2L2H2
43.55(±0.08)
CuL
9.68(±0.02)
CuLH-1
-1.71(±0.04)
O, N, Oket
Az / 104 cm1
H / Gaussa
2.277
181.6
37.2
2.243
188.8
38.2
O, N, Oket/O, N, Oenol O, N, Oenol
O, N, Oket/O, N, Oenol
AC
625(25)
gz
TE D
26.81(±0.02)
CE P
CuLH2
EPR
a
625(25)
O, N, Oenol
Linewidth of the first parallel hyperfine resonance measured on the complexes formed with 63Cu.
29
ACCEPTED MANUSCRIPT
Table 3. The IC50 values for HHSB and CuHHSB complex against all used cell lines calculated from the dose-response curves.
IP
T
Doxorubicin was used as a reference compound.
K562
HUVEC
IC50
IC50
10 µM ± 1.27
ND*
6 µM ± 0.76
8 µM
0,06 µM ± 0.011
0.03 µM
CR
HeLa
Compound
70 µM ± 6.65
CuHHSB
30 µM ± 2.7
Doxorubicin
1 µM ± 0.15
TE D
MA N
HHSB
US
IC50
*
AC
CE P
ND-not determined
30
ACCEPTED MANUSCRIPT
Table 4. Antibacterial activity of test compounds.
IP
T
Inhibition zone diameters (mm, mean value, n=3)
Concentration Salmonella
Escherichia
Staphylococcus
Listeria
Enteritidis
Typhimurium
coli
aureus
monocytogenes
7.30.91
10.60.47
11.00.0
5.30.94
0.00.0
0.00.0
7.32.35
9.02.00
MA N
US
Salmonella
250
7.71.24
HHSB
250
8.73.77
CuCl2
250
8.31.88
10.61.41
14.00.0
8.00.50
9.00.90
Doxycycline
30
20.01.80
19.51.41
26.50.47
29.00.90
36.02.30
Kanamycin
30
16.00.47
14.00.47
17.51.41
12.00.82
18.00.47
DMSO
20000
0.00.0
0.00.0
0.00.0
0.00.0
0.0
AC
TE D
CuHHSB
CE P
(μg mL-1)
CR
Sample
31
ACCEPTED MANUSCRIPT Table 5. Growth of selected bacteria in the presence of the test compounds after 24 hours incubation.
Salmonella
Staphylococcus
CuCl2A
2.90.04b
HHSB A
8.40.31c
CuHHSB A
5.30.11a
Control
9.20.18c
4.70.20a
6.40.03b
2.80.85b
7.90.05c
9.50.20c
6.60.59b
4.60.08a
9.60.07d
9.50.44c
coli
4.11.48a
PT
ED
concentration 250 g mL-1, SD – standard deviation, a,b,c significantly different, p≤0.05.
AC CE
A
NU
4.60.13a
MA
Inoculum
aureus
SC
Typhimurium
Escherichia
RI P
Sample
T
Growth [Log10 CFU mL-1] SD
32
ACCEPTED MANUSCRIPT
Table 6. Aggregation of selected bacteria in the presence of the test compounds. Aggregation [%] SD Sample
Staphylococcus
RI P
T
Salmonella
4.03.46a
HHSB A
73.03.89b
CuHHSBA
34.613.06c 21.31.3c
coli
53.73.19a
37.316.05a
27.610.17b
73.72.8b
65.39.45a
75.23.25a
16.32.04b
30.47.71b
PT
ED
concentration 250 g mL-1, SD – standard deviation, a,b,c significantly different, p≤0.05.
AC CE
A
MA
Control
NU
CuCl2A
aureus
SC
Typhimurium
Escherichia
33
ACCEPTED MANUSCRIPT Scheme/Figure Legends Scheme 1. Keto-enol tautomerism of N-[(E)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-
T
phenyl)chroman-4-ylidene]amino]benzamide (HHSB).
RI P
Scheme 2. Proposed structure for the keto (a) and enolate (b) coordination. The charges are omitted, X symbolises a solvent molecule, R ring B with C(3’)-OH protonated/deprotonated.
SC
Fig.1. The molecule HHSB, DMF and water in the asymmetric unit. The hydrogen bonds are shown by dash lines.
NU
Fig.2. The unit cell packing of the compound HHSB.
MA
Fig.3. Yasuda–Shedlovsky extrapolations of HHSB in DMSO–water mixtures (40, 50, 60, 70% v/v) at 25°C and an ionic strength of 0.1 M KCl. The values of the dielectric constants have been taken from Ref. [19].
ED
Fig. 4. Species distribution diagram for the HHSB system as a function of pcH. CL =1×10-3 M.
PT
Fig. 5. Electronic absorption (UV-Vis) spectra recorded in DMSO/water mixture (40%/60% v/v fraction) at different pcH corresponding for dominant species of HHSB;
AC CE
CL=1×10-5 M. The values of pcH are indicated above the arrows. Fig. 6. Species distribution curves for the system Cu(II)-HHSB. Ligand/metal = 1/1, CL= 110-3 M; CCu(II) = 1×10-3 M Fig. 7. Electronic absorption (UV-Vis) spectra recorded at different pcH; Cu(II)/L = 1/1; CCu(II) = 1×10-3 M; CL = 1×10-3 M (DMSO/Water: 40%/60% v/v fraction). Fig. 8. First parallel hyperfine resonance of the enol complex (Cu concentration 2102 M): a) system with natural copper (69.2% of 63Cu and 30.8% of 65Cu) and b) system with 63Cu (99.7% of
63
Cu). The triplet due to the superhyperfine coupling between the nuclei of
63
Cu and 65Cu with 14N nucleus are indicated.
Fig. 9. a) Origin of the multiplet in the first parallel hyperfine resonance measured with natural copper on the enol complex. b) Anisotropic X-band EPR spectrum recorded
34
ACCEPTED MANUSCRIPT at 100 K on 1:1 solid complex in the enol form dissolved in DMSO/water (40%/60% v/v). With an asterisk are indicated the resonances of the parallel and
T
perpendicular region where the coupling with the 14N nucleus is observed.
RI P
Fig. 10. A) ESI-MS spectra of the system containing copper and HHSB at the ratio 1/1. Cligand= 110-4 M, m/z ratio of all shown species = 1. B) The simulated isotopic
SC
patterns.
Fig. 11. Structure of Cu2L2H2 complex obtained from DFT (B3LYP/6-31G*) calculations.
NU
The copper(II) ions are shown in cyan, oxygen, carbon, nitrogen hydrogen atoms are
MA
shown in red, black, blue and white, respectively. Green arrows show the possible repulsive interactions that may destabilize the complex (see the text for details).
ED
Violet arrows show the places in molecule where the repulsive interactions could lead to a destabilization of the keto form of the ligand (see the text for details).
PT
Fig. 12. CD spectra recorded in DNA-Tris, DNA-CuHHSB-Tris and CuHHSB-Tris systems after: a) 15 min of incubation, b) 15 min of incubation in the larger scales of molar
AC CE
ellipticity and wavelength and c) 20 hrs of incubation. The concentrations of DNA 9.4×10-5M, CuHHSB 1×10-5M or 2.35×10-5M. Fig. 13 (a) Quantification of gel electrophoresis bands originating from SC, OC and linear DNA in cleavage experiments for different concentrations of CuHHSB. Line 1DNA (pEGFP-C1 0.25 µg/µL; 10 mM Tris-buffer, pH 7.0), line 2- DNA+ 25μM CuHHSB, line 3- DNA+50 μM CuHHSB, line 4- DNA+100 μM CuHHSB. (b) Quantification of gel electrophoresis bands originating from SC and OC DNA in cleavage experiments for different concentrations of HHSB. Line 1- DNA, line 2DNA+ 25μM, line 3- DNA+50 μM, line 4- DNA+100 μM, line 5- DNA+150 μM, line 6- DNA+200 μM. (c) Gel electrophoresis diagrams showing the cleavage of supercoiled pEGFP-C1 DNA (0.25 µg/µL; 10 mM Tris-buffer, pH 7.0) by CuHHSB
35
ACCEPTED MANUSCRIPT (100 µM): with/without scavengers including DMSO (0.4 M), glycerol (0.4 M), KI (10 mM) and NaN3 (10 mM) in Tris–HCl buffer at pH 7.0 and 37 °C with incubation
T
time of 20 h. Line 1 DNA control; line 2 DNA+CuHHSB; line 3 DNA + CuHHSB +
RI P
DMSO; line 4 DNA+CuHHSB+glycerol; line 5 DNA + CuHHSB+ KI; line 6
AC CE
PT
ED
MA
NU
SC
DNA+CuHHSB+NaN3.
36
ACCEPTED MANUSCRIPT
T
Scheme 1.
3'
O
4'
8
O
C
A
6
4
2 3
N NH 2" 3"
O
keto
OH
N
A
6
HHSB forms in solution
6"
4"
AC CE
PT
ED
5"
37
OH 3' 4'
2'
B
O
C
7
6'
5
OH
8
HO
5'
NU
7
B
MA
HO
CH3
SC
2'
RI P
OH
4
5'
2
6'
3
5
N 2"
HO
enol
3" 6"
4" 5"
O
CH3
ACCEPTED MANUSCRIPT
Scheme 2.
O-
T
O
X
Cu
RI P
X
O(H) O
Cu
O
N
O
O
R
AC CE
PT
ED
MA
(a)
38
O
N
R
Ph
NU
H
Ph
SC
N
N
(b)
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 1.
39
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 2.
40
ACCEPTED MANUSCRIPT
Fig. 3.
T
pKa1 pKa2
14
RI P
pKa3
SC NU
12
11
10
0.0132
0.0134
0.0136
0.0138
1/
AC CE
PT
ED
9 0.0130
MA
pKa+log[H2O]
13
41
0.0140
0.0142
0.0144
ACCEPTED MANUSCRIPT
RI P
T
Fig. 4.
0.0010
LH3
0.0009
0.0007
LH
L
2-
NU
0.0006 0.0005
MA
0.0004 0.0003 0.0002
ED
0.0001 0.0000
7
PT
6
8
9
pc H
AC CE
concentration of species
0.0008
SC
-
LH2
42
10
11
3-
ACCEPTED MANUSCRIPT Fig. 5.
0.35 2-
LH3
-
LH2
0.25
L
8.35
3-
12.05
0.20
A
SC
0.15 0.10
0.00 300
350
400
NU
6.05 0.05
T
10.05
0.30
RI P
LH
450
AC CE
PT
ED
MA
[nm]
43
500
550
600
ACCEPTED MANUSCRIPT
CR
IP
T
Fig. 6.
0.0010
CuLH2
MA N
0.0008 0.0007 0.0006
US
LH3
Cu
2+
TE D
0.0005 0.0004
0.0002 0.0001 0.0000 3
4
5
CuLH-1
CuL
CuLH
Cu2L2H2
CE P
0.0003
AC
concentration of species
0.0009
LH
-
LH2 6
7
pc H
44
8
9
10
2-
free L 11
3-
ACCEPTED MANUSCRIPT Fig. 7.
0,3
8.50
RI P
0,2
T
5.50
SC
3.50
NU
0,1
500
600
MA
0,0
700
PT
ED
nm
AC CE
Absorbance
11.05
45
800
900
1000
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 8.
46
ACCEPTED MANUSCRIPT
T
Fig. 9.
NU
SC
RI P
a
63
Cu
0.31
0.69
0.31
0.31
1.00
1.00
b
AC CE
PT
Tot.
0.31
ED
65
0.69
MA
Cu
47
0.69
0.69
ACCEPTED MANUSCRIPT
Fig. 10
Cu: hespertin (L) = 2:1, pH 7
Intens. x10 9
Cu2L2 965.1
A)
965.1
CuL
T
482.1 482.1
RI P
1.5
SC
1.0
NU
0.5
421.1 321.8
902.2 836.3
0.0 200
400
600
800
1000
1200
Intens. x10 9
17_01_Marzena_K3_CID50_z ekstra Cu_000001.d: +MS
experimental
482.1
1.5
ED
1.0
484.1
0.5 483.1
485.1
0.0
PT
17_01_Marzena_K3_CID50_z ekstra Cu_000001.d: C23H19N2O6Cu, M ,482.05
482.1
2000
1500
1000
AC CE
B)
m/z
MA
CuL
1400
simulation
484.1
483.1
500
0
480
482
485.1
484
486
488
m/z
Cu2L2
Intens. x10 9
17_01_Marzena_K3_CID50_000001.d: +MS
965.1
963.1
experimental
1.5
1.0 964.1 966.1
967.1
0.5
968.1 969.1 0.0
17_01_Marzena_K3_CID50_000001.d: (C23H18N2O6)2HCu2, M ,963.10
2500
simulation 963.1
2000
965.1
1500
964.1
1000
966.1
500
967.1 968.1 969.1
0 960
962
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Fig. 11
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a)
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0 -1
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DNA DNA/CuHHSB = 9.4 DNA/CuHHSB = 4 -5 CCuHHSB=2.3510 M
-2 -3
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CCuHHSB=110 M
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-4 -5
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22 20 16 14 10 8 6
0
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270
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molar ellipticity [M cm
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b)
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molar ellipticity [M cm
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2
-4
250
275
300
325
350
375
400
wavelength [nm]
c)
-1 -1 molar ellipticity [M mcm ]
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DNA/CuHHSB = 9.4 1
0
DNA -1 DNA_20h DNA DNA:CuHHSB=9.4:1_20h
-2
-3 250
275
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Wavelength [nm]
50
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Fig. 13.
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GRAPHICAL ABSTRACT
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ACCEPTED MANUSCRIPT Highlights: Cu(II)-hesperetin Schiff base complex shows nuclease activity
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The complex distinctly suppresses cancer cell viability.
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HHSB and CuHHSB reveal some antimicrobial activities.
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ACCEPTED MANUSCRIPT Graphical Abstract (synopsis). Please provide a synopsis of 50 words maximum that illustrates your work submitted.
X-ray characteristic of novel hesperetin Schiff base is reported. Different
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species in copper(II)-hesperetin Schiff base-water-DMSO system are
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characterized. Hesperetin Schiff base and Cu(II) complex have revealed
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nucleolytic, antimicrobial and cytotoxic activities.
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S0162-0134(14)00291-8 doi: 10.1016/j.jinorgbio.2014.11.005 JIB 9621
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
31 July 2014 19 November 2014 20 November 2014
Please cite this article as: Elzbieta Lodyga-Chruscinska, Marzena Symonowicz, Anna Sykula, Anna Bujacz, Eugenio Garribba, Magdalena Rowinska-Zyrek, Stanislaw Oldziej, Elzbieta Klewicka, Magdalena Janicka, Karolina Krolewska, Marcin Cieslak, Katarzyna Brodowska, Longin Chruscinski, Chelating ability and biological activity of hesperetin Schiff base, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.11.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Chelating ability and biological activity of hesperetin Schiff base Elzbieta Lodyga-Chruscinskaa*, Marzena Symonowicza, Anna Sykulaa, Anna Bujacza,
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Eugenio Garribbab, Magdalena Rowinska-Zyrekc, Stanislaw Oldziejd, Elzbieta
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Klewickaa, Magdalena Janickae, Karolina Krolewskae, Marcin Cieslake, Katarzyna
a
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Brodowskaa, Longin Chruscinskif
Faculty of Biotechnology and Food Chemistry, Lodz University of Technology,
Dipartimento di Chimica e Farmacia, Centro Interdisciplinare per lo Sviluppo della Ricerca
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b
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Stefanowskiego Street 4/10, 90-924 Lodz, Poland
Biotecnologica e per lo Studio della Biodiversità della Sardegna, Università di Sassari, via
c
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Vienna 2, I-07100 Sassari, Italy Department of Chemistry, University of Wroclaw, F. Joliot-Curie Street 14,
d
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50-383 Wroclaw, Poland
Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of
e
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Gdansk, Kladki 24, 80-922 Gdansk, Poland
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza Street 112, 90-363 Lodz, Poland
f
*
Faculty of Process and Environmental Engineering, Wolczanska 175, 90-924 Lodz, Poland
Corresponding author: e-mail: [email protected] phone:+48(42)6313417; fax:+48(42)6362860
1
ACCEPTED MANUSCRIPT Abstract Hydrazone hesperetin Schiff base (HHSB) N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-ylidene]amino]benzamide has been synthesized and its crystal structure
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was determined. This compound was used for the formation of Cu(II) complexes in solid state
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and in solution which were characterized using different spectroscopic methods. The analyses of potentiometric titration curves revealed that monomeric and dimeric complexes of Cu(II) are formed above pH 7. The ESI MS (electrospray ionization-mass spectrometry) spectra
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confirmed their formation. The EPR and UV-visible spectra evidenced the involvement of oxygen and nitrogen atoms in Cu(II) coordination. Hydrazone hesperetin Schiff base can
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show keto-enol tautomerism and coordinate Cu(II) in the keto (O, N, Oket) and in the enolate form (O, N, Oenol). The semi-empirical molecular orbital method PM6 and DFT (density
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functional theory) calculations have revealed that the more stable form of the dimeric complex is that one in which the ligand is present in the enol form. The CuHHSB complex has shown high efficiency in the cleavage of plasmid DNA in aqueous solution, indicating its
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potential as chemical nuclease. Studies on DNA interactions, antimicrobial and cytotoxic activities have been undertaken to gain more information on the biological significance of
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HHSB and copper(II)-HHSB chelate species.
complexes
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Keywords: flavonoids, hesperetin, Schiff bases, copper complexes, metal-ionophore
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ACCEPTED MANUSCRIPT 1. Introduction Among fruits, citrus varieties are important sources of polyphenolic compounds, which could be responsible for the health-promoting effects. Hesperetin is one of the
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flavonones that belong to flavonoid class of compounds, found in citrus fruits. Hesperetin has
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multiple biological and pharmacological activities, including antioxidant properties [1,2] inhibition of cancer development [3,4], the prevention and/or treatment of certain eye diseases/disorders such as diabetic retinopathy, diabetic macular edema and cataract [5] and
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many others. Most of the pharmacological actions of hesperetin originated from its binding to proteins or other macromolecules, and regulating their structures and functions [6,7]. Besides,
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it is demonstrated that the coordination of copper(II) ion with bioactive ligands can actually improve their biological activity, for example Cu(II) complexes with hesperetin, naringenin
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and apigenin have showed higher inhibitory rate than their free ligands against SGC-7901 and HepG2 cell lines [8]. It has been also found that hydrazone hesperetin Schiff base (HHSB) and its copper chelate can bind to DNA. The complex has exhibited deeper intercalation of
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DNA and higher oxidative activity than the ligand [9, 10]. Understanding of complexation processes of flavonoids and their derivatives is
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important for explanation of the role of metals in biological functions of these medically promising compounds. Complexes of HHSB showed 1:1 stoichiometry and metal bonding via
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[C(5)-O- -C(4)-N] donor atoms set in the solid state [10]. Up till now, no solution studies of equilibrium and structure of copper(II)–hesperetin Schiff base complexes involving different protonation state of the ligand have been undertaken. These studies are also indispensable since biological active substances act in biological compartments with different water contents. Some important information on coordination, acid–base and metal binding properties can be achieved only by solution studies. This paper reports results of investigation of system containing Cu(II) ions and N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-ylidene]amino]benzamide in DMSO/water mixture
by means
of
potentiometric titration, spectroscopic techniques and theoretical calculations. The use of different research techniques and the convergence of the results, despite the complexity of the system allows for precise and unambiguous definition of how coordination and interactions of bioligands proceed in solution. DNA interaction and cleavage studies were done using circular dichroism and gel electrophoresis technique, respectively. The influence of different scavengers of reactive oxygen species: DMSO, glycerol, KI, sodium azide, and the minor or major groove binders (DAPI (2-(4-amidinophenyl)-1H -indole-6-carboxamidine) or methyl green) on the DNA 3
ACCEPTED MANUSCRIPT cleavage process, has been also studied. The ability of HHSB and CuHHSB complex to induce the cleavage of pEGFP-C1 DNA has prompted us to investigate their cytotoxic activity against human cancer cells (HeLa (human cervix carcinoma) and K562 (leukemia))
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and HUVEC (human umbilical vein endothelial cells) as a classic model system.
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Various Schiff bases showed antibacterial activity against Gram-positive (S. aureus and L. monocytogenes) and Gram-negative bacteria (E. coli and Pseudomonas aeruginosa) [11-13]. Therefore, studies on antimicrobial activities have been undertaken as well to get more
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information on the biological significance of the HHSB interactions and the role of copper(II)
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ions.
2. Experimental Materials
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2.1.
The racemic hesperetin, N-benzoyl hydrazine, NaOH, KCl, CuCl2, and all other compounds were purchased from Sigma-Aldrich Co. All reagents were of analytical quality
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and were used without further purification.
2.1.1. Synthesis of N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4-
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ylidene]amino]benzamide.
The ligand (N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4ylidene]amino]benzamide - HHSB) has been prepared according to the reference [10]. Yield: 1
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0.5 g, 45%. FAB-MS (fast atom bombardment mass spectrometry): m/z = 421 [M+H]+. H NMR (DMSO-d6, 200 MHz), 2.60 (1H, dd, J =27.48, 13.78 Hz, 3(a)-H), 2.88 (1H, dd, J =
11.96, 12.1 Hz, 3(e)-H), 5.03 (1H, d, J = 9.5 Hz, 2-H), 5.84 (1H, d, J = 2.2 Hz, 6-H), 5.90 (1H, d, J = 2 Hz, 8-H), 6.89 (3H, m, 2’, 5’, 6’H), 7.48 (2H, dd, J = 1.7, 7.4 Hz, 3”, 5”H), 7.54 (2H, dd, J = 4.0, 7.2 Hz, 4”H), 7.88 (2H, d, J = 6.8 Hz, 2”, 6”H), 9.08 (1H, s, 3’-OH), 9.98 (1H, s, 7-OH), 11.09 (1H, s, –NH–C═O), 13.04 (1H, s, 5-OH). M.p. 265-267 C. Anal. Calc. for C23H20N2O6: C, 65.71; H, 4.79; N, 6.66. Found: C, 65.62; H, 4.88; N, 6.71%. IR νmax(cm1
): ν(C═O): 1644, ν(C═N): 1601. UV-visible λmax(nm): 257, 323. Mp 265,6 - 267°C.
2.1.2. Synthesis of N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4ylidene]amino]benzamide copper(II) [CuLH3·OAc]·H2O. The ligand (84 mg, 0.20 mmol) was dissolved in acetone (20 mL). After 5 min, Cu(OAc)2·H2O (48 mg, 0.24 mmol) was quickly added into the ligand solution and the solution was refluxed on a oil-bath for 10 h with stirring. A green precipitate, [CuLH3·OAc]·H2O, where LH3=HHSB is in keto-form with C(7)-OH, C(3’)-OH and nitrogen of the benzamide protonated, was separated from the solution by suction filtration and washed 4
ACCEPTED MANUSCRIPT several times with methanol and dried for 24 h in vacuo. In biological studies instead of [CuLH3·OAc]·H2O the CuHHSB abbreviation has been used. Anal. Calc. [CuLH3·OAc]·H2O : C, 53.62; H, 4.32; N, 5.00; Cu, 11.35. Found: C, 54.01; H,
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4.91; N, 5.56; Cu, 10,5 %. IR νmax(cm-1): ν(C═O): 1604, ν(C═N): 1589, νas(COO-): 1568, = 482.1 [M-H]+ (M without acetate and water molecule). 2.2.
Methods of analysis
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νs(COO-): 1364, ν(M–O): 443, ν(M–N): 418. UV-Vis λmax(nm): 261, 383, 680. ESI-MS: m/z
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Elemental analysis (C, H and N) was carried out on an EuroVector 3018 analyzer. The metal content of the complex was determined using atomic absorption spectrometer: AAS
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GBC 932 Plus (GBC Scientific Equipment Ltd, Australia) with copper hollow cathode lamp. The melting point of the ligand was determined by an Electrothermal 9200 microscopic melting point apparatus. The IR spectra were recorded employing a Nicolet 6700 (Thermo-
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Scientific) FT-IR spectrometer, in the 4500-500 cm-1 region. 1H NMR spectra were recorded on a Bruker AV200 200MH spectrometer in DMSO-d6 with TMS (tetramethylsilane) as
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internal standard. Mass spectra were performed on a Finnigan MAT 9 instrument. 2.2.1. X-ray analysis
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The HHSB compound was crystallized from DMF (N,N-dimethylformamide)/water system. The hot solution was cooled and put into the fridge. After three days, the crystals
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were separated from the solution and dried. The obtained crystals belong to the monoclinic system with the space group C2/c, with the cell dimensions: 24.114(5) ×9.780(2) × 23.055(4) and β=111.19(3). The diffraction data were collected at room temperature on the CAD4 diffractometer with the CuKα radiation and processed by SDP software [14]. The structure was solved and refined using ShelxS and ShelxL programs, respectively [15]. The asymmetric unit contains one HHSB molecule and two molecules from crystallization solvent: DMF and water. The DMF molecule is disordered and was modelled in two positions. The occupancy of the atoms in both split positions is in ratio 0.55 : 0.45. The model of disorder can be described as a swinging of molecule around axis passing through the atoms O1 and N1. To maintain similarity between DMF in both positions the geometrical and temperature restrains were applied; SIMU (temperature vibration parameters of neighbouring atoms are set to be similar) and ISOR (temperature vibration parameters of each atom are restrained to be approximately the same). The hydrogens bound to carbon were set geometrically and refined as riding, the hydrogen atoms connected with nitrogen and oxygens were found on the difference Fourier map and refined freely. The anisotropic thermal parameters were applied for all non hydrogen atoms and isotropic for hydrogens, equal 1.2 of thermal parameter of parental atoms, except 5
ACCEPTED MANUSCRIPT for hydrogens connected to heteroatoms in HHSB molecule, which were refined with free thermal parameters. The final discrepancy parameter R for 3860 reflections with I > 2σI was equal 0.0868 and for all independent reflections (5202) - 0.1085. The crystal structure of
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HHSB is deposited in CCDC under accession code 950104.
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2.2.2. Potentiometry
The protonation constants of the ligand (pKa) and the stability constants of Cu(II) complexes (log) were determined by pH-potentiometric titrations of 2.0 mL samples in
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DMSO /water mixtures (40%:60% v/v) due to slight solubility of HHSB in pure water (see Fig.S01, supplementary material). The ligand : metal molar ratio was 1:1, the concentrations
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of Cu(II) and the ligand were 110-3 M. Measurements were carried out at 298 K and at a constant ionic strength of 0.1 M KCl with a MOLSPIN pH meter (Molspin Ltd., Newcastle-
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upon-Tyne, UK) equipped with a digitally operated syringe (the Molspin DSI 0.250 ml) controlled by computer. The titrations were performed with a carbonate-free NaOH solution of known concentration (ca. 0.1 M) using a Russel CMAWL/S7 semi-micro combined
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electrode. The pH measuring circuit was calibrated with potassium hydrogenphthalate and phosphate buffers [16]. The conversion of the operational pH reading to the pcH value at
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DMSO /water mixture (40%:60% v/v) were accomplished by the Eq. 1 as given below
pH = α + 𝑆pc H + 𝑗H [H+] + 𝑗𝑂𝐻
Kw [H + ]
(1)
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The parameters α, S, jH, jOH and Kw were taken from the literature [17]. In order to find autoprotolysis constant of water, titrations were performed in a series of DMSO/water HCl solutions of known concentration containing 0.1 M KCl using 0.1 M NaOH solution. The autoprotolysis constant of water in this medium was determined from Eq. 2 pKap = (E’a - E’b)/k
where
E’a
and
E’b
are
Eocell+
(2)
k log10 aKCl + k log10 γH+ + ELJ and
Eocell
+ k log10 aKCl −
k log10 γOH− + ELJ, respectively. The procedure of the calculations was adopted from the literature [18]. The obtained pKap value 14.64 is in general agreement with those reported before (14.98 [18], 14.42 [19]). The difference may be due to other experimental conditions, including the use of a different electrolyte. The number of experimental points was 100-150 for each titration curve. The reproducibility of the titration points included in the evaluation was within 0.005 pH units in the whole pH range examined (2-11.5). Protonation constants of the ligand and the overall stability constants (pqr, where p, q and r represent the number of metal, ligand and proton, respectively) of the complexes, were evaluated by iterative non-liner least squares fit of potentiometric equilibrium curves through mass balance equations for all 6
ACCEPTED MANUSCRIPT the components expressed in term of known and unknown equilibrium constants using a computer program SUPERQUAD [20]. The value for sigma (the root mean squared weighted residual), after refinement of the stability constants, was 1, which signifies that the data have
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been fitted within experimental error. The equilibrium constants were obtained as averaged
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values of three titrations. 2.2.3. Spectroscopic analysis
Electronic absorption (UV-Vis) spectra were recorded with a Perkin-Elmer Lambda 11
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spectrophotometer using quartz cell with a path length of 1 cm.
Anisotropic X-band EPR spectra of frozen solutions were recorded at 100 K, using an
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X-band (9.15 GHz) Varian E-9 spectrometer after addition of ethylene glycol to ensure good glass formation. Copper(II) stock solution for EPR measurements were prepared from natural 63
Cu and 0.7%
65
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CuSO4·5H2O or 63CuSO4·5H2O to get better resolution of EPR spectra. Metallic 63Cu (99.3% Cu) was purchased from JV Isoflex, Moscow, Russia for this purpose and
converted into pentahydrate 63CuSO4·5H2O. The EPR parameters were read from the spectra
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(estimated uncertainties for A and g values are 1×10–4 cm–1 and 0.002, respectively, in the spectra of a single species). All spectroscopic measurements were performed at the maximum
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concentration of each species found in titrations. Circular dichroism (CD) spectra were recorded with a Jobin Yvon CD6
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spectropolarimeter at room temperature using quartz cell with a path length of 0.5 cm, 2nm bandwidth and 1-2s integration time. Spectra were corrected for buffer signal and 25-point smooting algorithm was performed with the Jobin Yvon CD6 Standard Analysis software. The CT DNA concentration used in the experiments was 9.4×10-5M. Hydrazone hesperetin Schiff base and its copper(II) complex concentrations varied from 1.88×10-5M to 9.4×10-5M. The samples were prepared in Tris-HCl buffer (5mM Tris-HCl, 50 mM NaCl, pH 7.2). The CD spectra of CT DNA were recorded at time zero (immediately after preparation of the sample) and then, after 15min, 1h, 2hrs and 20hrs incubation at 37°C with increasing concentrations of hydrazone hesperetin Schiff base and its copper(II) complex. High resolution mass spectra (ESI MS) were obtained on a BrukerQ-FTMS spectrometer (Bruker Daltonik, Bremen, Germany), equipped with Apollo II electrospray ionization source with ion funnel. The mass spectrometer was operated in the positive ion mode. The instrumental parameters were as follows: scan range m/z 300–1600, end plate offset-500 V, dry gas – nitrogen (4 L min-1), temperature 200 °C, ion energy 5 eV. Capillary voltage was optimized to the highest S/N ratio and it was 4500 V. The small changes of voltage (±500 V) did not significantly affect the optimized spectra. The samples (HHSB and 7
ACCEPTED MANUSCRIPT HHSB with CuCl2 in the ratio 1:1 and 1:2, the concentration of CHHSB = 1×104 M) were prepared in a 1:1 MeOH-H2O mixture. Variation of the solvent composition down to 5 % of MeOH did not change the species composition. The sample was infused at a flow rate of 3
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μL min-1. The instrument was calibrated externally with the Tunemix™ mixture (Bruker
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Daltonik, Germany) in quadratic regression mode. Data were processed by using the Bruker Compass Data Analysis 4.0 program. The mass accuracy for the calibration was better than 5 ppm, enabling together with the true isotopic pattern (using SigmaFit) an unambiguous
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confirmation of the elemental composition of the obtained complex. 2.2.4. Theoretical calculations
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Semi-empirical calculations were carried out using MOPAC2012 program [21]. The geometries of free ligand and all complexes were fully geometry optimized using semi-
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empirical molecular orbital method PM6 [22]. Moreover DFT calculations were carried out with the Gaussian 03 package [23] using the Becke gradient corrected exchange functional [24] and Lee-Yang-Parr correlation functional [25,26] with 6-31G* basis set. For all
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structures with two copper ions calculations were carried out using Restricted Hartree-Fock (RHF) approximation, assuming electronic state of the molecule as a singlet (compound with
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two copper ions possesses even number of electrons and formally is closed-shell system). All initial structures for calculations were built manually. DNA cleavage
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2.3.
Electrophoresis experiments were performed with pEGFP-C1 (4731 bp) DNA. The cleavage of pEGFP-C1 by Cu(II), ligand, e.i. HHSB and Cu(II)-HHSB systems, respectively, were accomplished by mixing in the order: 1 µL of 5 mM Tris-HCl (pH 7.5 containing 5 mM NaCl) buffer, varying concentrations (0; 0,025; 0,05; 0,1; 0,15; 0,2 mM) of CuCl2, HHSB and Cu(II)-HHSB complex, and 1 µL of pEGFP-C1 (0.25 µg/µL; 10 mM Tris-buffer, pH 8.0). After mixing, the DNA solutions were incubated at 37°C for 20hrs. The reactions were quenched by the addition of EDTA and bromphenol blue and the mixtures were analyzed by gel electrophoresis (0.5% agarose gel). In order to examine if hydroxyl radicals or reactive oxygen species were present, scavengers: DMSO (0.4M) and glycerol (0.4M) for OH·, KI for H2O2 and Na3N for singlet oxygen were added to give a final concentration of 10 mM before the complex addition. The bindings of HHSB and CuHHSB complex to minor or major groove of DNA were tested using DAPI and methyl green, respectively. Plasmid cleavage products were quantitated and analyzed with G-BOX Syngene system. The GeneTools software was used to complete gel documentation and analysis 2.4.
Cells and cytotoxicity assay 8
ACCEPTED MANUSCRIPT Human umbilical vein endothelial cells (HUVEC) were isolated from freshly collected umbilical cords as previously described [27], and cultured in plastic dishes coated with gelatin, in RPMI 1640 medium supplemented with 20% FBS (fetal bovine serum), 90 U/ml
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heparin, 150μg/ml ECGF (endothelial cell growth factor, Roche Diagnostics, Mannheim,
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The HeLa (human cervix carcinoma) and K562 (leukemia), cells were cultured in
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RPMI 1640 medium supplemented with antibiotics and 10 % fetal calf serum (HeLa, K562) in a 5 % CO2 - 95 % air atmosphere. 7×103 cells were seeded on each well on 96-well plate
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(Nunc). 24h later cells were exposed to the test compounds for additional 48 hours. Stock solutions of test compounds (HHSB and CuHHSB systems) were freshly prepared in DMSO.
compounds
tested
in
the
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Stock solutions (doxorubicyna) was freshly prepared in water. The final concentrations of the cell
cultures
were:
1mM,
1×10-1
mM,
1×10-2 mM, 1×10-3 mM, 1×10-4 mM, 1×10-5 mM. Each concentration was assayed in
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triplicate in each experiment, and all experiments were repeated at least two times. The concentration of DMSO in the cell culture medium was 1%. The values of IC50 (the
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concentration of test compound required to reduce the cell survival fraction to 50 % of the control) were calculated from dose-response curves and used as a measure of cellular
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sensitivity to a given treatment.
The cytotoxicity of all compounds was determined by the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described [28]. Briefly, after 72h of incubation with drugs, the cells were treated with the MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20 % SDS and 50 % DMF at pH 4.7 and absorbance was read at 570 and 650 nm on an ELISA (enzyme-linked immunosorbent assay)-PLATE READER (FLUOstar Omega). As a control (100 % viability), we used cells grown in the presence of vehicle (1% DMSO) only. As a control for doxorubicin (100 % viability), untreated cells were used. 2.5.
Antimicrobial activity In vitro antibacterial activity studies were carried out against three Gram-negative
strains from Enteriobacteriaceae family: Salmonella Enteritidis ATCC 13076, Salmonella Typhimurium, ATCC 14028, Escherichia coli ATCC 8739 and two Gram-positive strains Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111. The bacteria were activated on a Nutrient Agar (Merck) for 24 h at 37ºC (Salmonella sp., E.coli, S.aureus) and at 30ºC Listeria strain. The overnight cultures were inoculated in Nutrient Broth (Merck) 9
ACCEPTED MANUSCRIPT before use. The bacterial counts of the diluted cultures were corrected by adding isotonic NaCl solution to be within the range of 106 – 107 colony forming units per one ml (CFU ml-1). Samples of test compounds: HHSB and CuHHSB, were dissolved in DMSO to obtain
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concentration 5 mg mL-1 and were sterilized by filtration (filter pore width 0.2 m; Sartorius).
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Paper discs ( = 6 mm) were impregnated with 50 l of the samples and the solvent allowed to evaporate at room temperature in the dark. The diluted bacterial test culture (200 L) was spread on sterile Mueller-Hinton Agar (Merck) plates before placing the sample impregnated
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paper discs on the plates. As a negative control was used DMSO solution at the concentration of 20 mg mL-1 (this concentration of DMSO did not inhibit the growth of microorganisms)
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[29]. As positive control were used Kanamycin and Doxycycline (Oxoid) at the concentration of 30 g mL-1 each. The plates were incubated at 37ºC (Salmonella sp., E.coli, S.aureus) and
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at 30ºC Listeria strain. After 24 h the inhibition diameters were measured. As a result, the final taken into account (subtracted) the diameter of the disc. The experiments were repeated three times and results were expressed in average values.
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2.5.1. Growth of bacteria in the presence of the test compounds. To 0.895 mL of Nutrient Broth (Merck) was added 5.0 L (depending on the sample)
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CuHHSB or HHSB, or CuCl2 and 0.100 mL the bacterial suspension (1106 CFU mL-1). The control sample was a growth of bacteria without addition copper compounds. After 24h
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incubation the amount of bacteria were determined by the plate method. Assay was done in triplicate. The results were analyzed using one-way analysis of variance (ANOVA) p≤0.05. 2.5.2. Aggregation of bacterial strains The aggregation assay was performed according to Ahmadowa et al. [30] with some modifications. The overnight cultures of test strains (E.coli, Salmonella Typhimutium, S. aureus) were pelleted by centrifugation (7000 × g, 10 min., 20ºC), washed twice in sterile phosphate-buffered saline (PBS, Sigma, pH 7.4) and resuspended in the same solvent to achieve an OD of 0.5 at 600 nm. To 0.995 ml of the bacterial suspension was added 5.0 L (depending on the sample) CuHHSB or HHSB or CuCl2. The control sample was a suspension of bacteria in PBS. After 5 h incubation at 37ºC the absorbance of the upper part of cell suspension was measured at 600 nm. The percentage of aggregation was calculated as: aggregation (%) = [(A0 – A5)/A0]×100; where: A0 initial absorbance, A5 absorbance after 5 h. Assay was done in triplicate. The results were analyzed using one-way analysis of variance (ANOVA) p≤0.05. 2.5.3. Membrane permeabilisation by fluorescence microscopy 10
ACCEPTED MANUSCRIPT To determine the permeability of cell membranes fluorescent assay was performed LIVE/DEAD BacLight bacterial viability kit (L-7012). Bacterial suspensions of S.aureus, S. Typhimurium and E.coli were exposed to (depending on the sample) CuHHSB or HHSB, or
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CuCl2 within 20 min. and stained with a fluorescent cell viability kit. Overnight liquid
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bacteria culture were diluted to OD 0.5 - 1.0 McFarland standard. The stock solution of the studied chemical compounds (3L, 250 g mL-1 in DMSO) was added to bacterial culture (997 L). Equal volumes of component A and component B (LIVE/DEAD BacLight bacterial
SC
viability kit) were mixed thoroughly and the resulting solution (3L) was added to cultures (1000 L) treated with copper complex. The solution of the treated samples was incubated at
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room temperature (20-23ºC) for 15 min and protected from light. Then, the sample (5L) was mounted between a cover slip and POLYSINE SLIDES (Thermo Scientific, Gerhard Menzel
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GmbH). Fluorescence microscopy images of the treated bacterial suspension were obtained at 40 magnification with an excitation wavelength of 488 nm through emission filters (long pass >650 nm and band pass 505-550 nm) corresponding to the DNA-bound emission range
3. Results and discussion
The solid state structure of HHSB
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3.1.
ED
of PI and SYTO9 respectively.
The central chroman bicyclic ring is almost flat and only C2 atom is standing out from
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the plane. Two hydroxyl groups, 5 and 7, are located in the same plane. The 3’-hydroxy-4’methoxy phenyl ring is twisted about 80° with respect to the chroman plane. The 4-ylideneamino-benzamide moiety is approximately coplanar with the chroman fragment. This fragment has keto-form in contrast to the preferable, more stable enol-form in solution. The conformation of the ligand is stabilized by bifurcated hydrogen bond from 5-hydroxyl group interacting with ylidene nitrogen and with carbonyl oxygen of the benzamide (Fig. 1). The crystal structure shows "host-guest" interactions in which the HHSB molecule is a host [31]. The main interaction of the host is π-stacking with symmetry related molecule of the ligand (Fig. 2). Fig. 1. Fig. 2. These hydrophobic contacts are supported by two hydrogen bonds created between 7hydroxyl group and hydrogen from amide group with benzamide carbonyl oxygen and methoxy oxygen from symmetry related molecules, respectively. The guest molecules, DMF and water are involved in three hydrogen bonds with HHSB. The water molecule serves as an acceptor for 3'-hydroxyl group and as a donor for carbonyl oxygen from DMF and for oxygen 11
ACCEPTED MANUSCRIPT from 7-hydroxyl group. The crystal structure of the ligand shows spatial arrangement of the potentially chelating atoms. 3.2.
Ionization constants of HHSB
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The acid dissociation (acid-ionization) constant, pKa is important parameter describing
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the extent of ionization of functional groups with respect to pH of biologically active substances. It can affect their solubility, dissolution rate, absorption across biological membranes, distribution to the site of action, renal elimination, metabolism, protein binding,
SC
and receptor interactions [32]. Knowledge of pKa values as a function of solvent composition is also useful in the application of reversed-phase HPLC for the separation of ionizable
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compounds [33]. Potentiometry is a very good tool to establish pKa value of compounds of pharmaceutical and biological interest because it does not require the presence of
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chromophore. When compound is sparingly soluble in water, the pKa determination is commonly done in organic/water mixtures.
DMSO/water mixtures are often used because they have lower polarity than pure
ED
water, but keeping a similar environment. The unique capability of dimethyl sulfoxide (DMSO) to penetrate living tissues without causing significant damage is most probably
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related to its relatively polar nature, its capacity to accept hydrogen bonds, and its relatively small and compact structure. This combination of properties results in the ability of DMSO to
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associate with water, proteins, carbohydrates, nucleic acid, ionic substances, and other constituents of living systems. One of the possible functions of DMSO in biological systems is its ability to replace some of the water molecules associated with the cellular constituents, or to affect the structure of the omnipresent water. DMSO is highly polar, very weakly acidic and fairly basic, while water is also highly polar but acidic and negligibly basic. DMSO can interact strongly and specifically with water through hydrogen bonds. So DMSO is miscible like alcohol with water in all proportions. Aqueous mixtures of DMSO have extraordinary penetration properties and can induce cell fusion and intensify cell permeability which is used in drug delivery systems [34]. In fact, this compound has found a variety of applications as pharmaceutical agent of low toxicity and also as an effective solvent [35]. Taking all this into account it can be assumed that the studies both physicochemical and biological are relevant. Therefore we have decided to carry out the investigation in DMSO/water mixtures in order to reveal properties of HHSB and its complexes with copper(II) ions. The ionization constants of hydrazone hesperetin Schiff base and overall stability constants of complexes formed in HHSB-Cu(II) system, were calculated via the potentiometric method in a mixture H2O/DMSO 60:40 v/v. The results are presented in 12
ACCEPTED MANUSCRIPT Table 1 and Table 2. The Yasuda–Shedlovsky extrapolation pKa+log[H2O]=a +b/ε was used to derive acid dissociation constants in aqueous solution ( pK aw ) (Fig. 3, Table 1). Terms a and b symbolize the intercept and the slope of the plot, respectively. It has been previously
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reported that when using organic solvent/aqueous mixtures with ε values greater than 50, the
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extrapolation to the purely aqueous domain is linear and produces relatively accurate pKa values [17]. Taking this into account pK aw values were evaluated using log 55.5 and 1/78.3,
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the logarithm of the molar concentration and the inverse of the dielectric constant of pure water, respectively.
HHSB can reveal a keto-enol tautomerism (Scheme 1), as other hydrazone Schiff
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bases [38,39]. Taking into account the tautomerism, the ligand has four possible acidic hydrogens. However, since the proton dissociation of imine nitrogen (-NH-) or enolic OH
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occurs in strongly basic conditions, which are outside of the experimental pH range of the methodology, it was not possible to determine all of them. Hence, only three ionization constants have been calculated and therefore the ligand is indicated as LH3. (a similar
ED
convention is accepted for oligopeptides where pKa of –NH– group is omitted in stoichiometric protonations of these ligands [40]). For comparison in the same experimental
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conditions the pKa values of hesperetin have been determined as well. Some differences in numerical values of pKa’s originate from different methods and conditions using to estimate
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them. According to the literature data [38,39] in hesperetin pKa1 corresponds to the ionization of the 7-OH group since the negative charge resulting from deprotonation of this group will be stabilized by conjugation. The next deprotonation step occurs at 3 ’-OH group in ring B and it corresponds to pKa2. The third and the last one proceeds on the 5-OH group which corresponds to pKa3. It must be the hardest to occur due to hydrogen bonding with the vicinal carbonyl group; therefore it can be registered at high pH range. One can expect the same pKa trend in hesperetin Schiff base. The values of pKa1 and pKa2 are similar to those found for hesperetin but the pKa3 is more acidic as compared to the unmodified flavonoid. It may be derived from an electron withdrawing effect of benzohydrazide group. This might induce diminishing electron density and lead to release the 5-OH hydrogen at lower pH. The decreased acidic strength characteristic of all HHSB forms present in DMSO-rich mixtures (Fig. 3) is probably in part a consequence of the increased solvent stabilization of the acidic species. This interaction is favored by the large dipole moment of the DMSO molecule [41]. In Fig. 4 the equilibrium distribution of different protonated species of HHSB is shown as a function of pcH calculated from the recorded potential values. As this figure shows, a 13
ACCEPTED MANUSCRIPT completely protonated LH3 is the main species in low pH whilst LH2-, LH2- and L3- dominate at alkaline range. As pH increases the LH3 dissociates and in neutral solution transforms into the LH2- form of the ligand. At pcH 9 the LH2- species coexists with LH2-, while in the range
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of 10 − 11 pcH the L3- is dominant.
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The absorption UV spectra of the ligand registered with increasing pH support a stepwise deprotonation. Significant changes in the spectral HHSB profile as a function of pcH have been attributed to the formation of the different protonated species found by
SC
potentiometry, whose light absorbing properties are different from one another. Thus, it can be assumed that four absorbing species are formed by the successive deprotonation of HHSB
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in positions 7-OH, 3’-OH, and 5-OH, respectively in pcH range of 2–12 (Fig. 5). The results of spectra in Fig. 5 indicate that the first and third deprotonation have a strong change in the
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spectral shape and intensity of HHSB, while the second deprotonation causes a less modification of the spectrum. The absorption bands appearing in the HHSB spectrum can be assigned on the basis of literature data on absorption spectra of Schiff bases [42]. The bands
ED
in the range of high energy, at λmax = 267 and λmax = 289 nm, are due to the excitation of the π electrons (π-π* transitions) of the aromatic rings. The third band at λmax = 324 or 340 nm could be assigned to the π-π* transition within the C=N group, while the longest wavelength
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band at λmax = 408 nm is due to an intramolecular charge transfer (CT) transition involving
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the whole molecule. From the results one can deduce that in the biological compartments with varying amount of water, the HHSB may exists in partially or fully protonated form depending on hydrogen ions concentration. 3.3.
Copper complexes of HHSB in DMSO/water mixture
3.3.1. Potentiometric and UV-VIS studies The HHSB possesses the donor atoms on adjacent A and C rings. It is the ylidene nitrogen, phenolic oxygen of 5-OH group and carbonyl oxygen of the benzamide as a metalchelating site. The overall stability constants of the complexes were determined via pH titration using the dissociation/ionization constants of the ligand. The best fittings of the titration curves were obtained using the species listed in Table 2. The species distribution curves of complexes are shown in Fig. 6. Fig. 6. It can be observed that the different protonated species appear one after the other with increasing pcH region. The complexation process starts above a pcH of 3. It is not surprisingly that in spite of the high pKa values of HHSB ionisable groups, the protons are displaced at much lower pH range in the presence of copper(II) ions. The same process was observed for 14
ACCEPTED MANUSCRIPT phenols and flavonoids under metal chelation [43]. The copper complexes with variously protonated forms of the ligand are formed. The formation of complexes in acid medium promotes the deprotonation of the ligand at the 5-OH group and the simultaneous copper binding to the nearest nitrogen situated in the position C(4) and two chelating rings are
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formed. The first CuLH2 species has the (O, N, Oket) donor atom set with the 7-OH and 3’OH fully protonated. The next CuLH can acquire two possible coordination modes: the (O, N, Oket) with the 7-OH group deprotonated and the 3’-OH protonated or the (O, N, O-enol)
respectively).
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Scheme 2.
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donor atom set with the 7-OH and 3’-OH groups protonated (Scheme 2 a) and b),
Acoording to the fitting of potentiometric data in the same pH range the CuLH can coexist
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with a dimeric Cu2L2H2 complex which is a minor species but it can be recognizable by EPR and ESI MS techniques (see the paragraphs 3.3.2, 3.3.3). In the high pH region, the CuL and CuLH-1 complexes can be found. The first complex can exist in the keto form with the 7-OH
ED
and 3’-OH groups deprotonated or in the enolate form with the 7-OH deprotonated and the 3’OH group still protonated. On the other hand, in the second one the only donor atom set could
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be the (O, N, O-enol) and the 7-OH and 3’-OH fully deprotonated. Complex formation can be seen in UV-Vis spectra (Fig. 7).
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Fig. 7.
According to the speciation diagram at pcH above 3 initially CuLH2 is formed. The [C(5)-O- C(4)-N] coordination mode, i.e. one oxygen and one nitrogen as donor atoms, is supported by the d-d electron transition energy corresponding to λmax = 640 nm which is similar to many other Cu(II) systems [44,45]. This species coexists in low pH with aqua copper(II) ions therefore λmax registered at 3.50 pH is slightly shifted to lower energy (see Fig. 7). With the increasing pcH the same donor atom set is also clearly seen in the other complexes. The hypsochromic shift of the band can be caused by both a specific coordination environment and the coexistence of different species. 3.3.2. EPR studies The Cu(II)-HHSB system with the ratio 1:1 was investigated in DMSO/water 40:60 v/v (Fig. S05). The anisotropic EPR spectra recorded in the system confirm the formation of the species [CuLHx] with x = 2, 1, 0, -1. The resonances of two species are observable with gz = 2.277, Az = 181.6104 cm1 and gz = 2.243, Az = 188.8104 cm1. The difference between these compounds is due to the deprotonation of the keto complex with the donor set (O, N, 15
ACCEPTED MANUSCRIPT Oket) or the enolate coordination (O, N, Oenol) (see Scheme 2a or b, respectively) which results in a decrease of gz and an increase of Az. The superhyperfine coupling with
14
N
nucleus is observable in the enol complex but not in the keto complex. To clarify the number
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of nitrogen atoms coordinated to copper, EPR spectra with 63CuSO4·5H2O were recorded; the
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comparison of the first parallel resonance in the spectra of the enol complex with natural and enriched (63Cu 99.7%) copper is reported in Fig. 8. It can be noticed that with
63
Cu isotope
only three resonances are detected with the expected intensity ratio of 1:1:1 due to the
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coupling with one 14N nucleus (I = 1). Therefore, the multiplet observed with natural copper is due to the overlap of the two triplets given by the coupling with
63
Cu and
65
Cu, which
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accounts for the experimental ratio of 1:3:3:2 between the signals (Fig. 9a). Fig. 9.
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The values of 14.5 and 15.3104 cm1 for A||N and AN are in good agreement with those reported in the literature [46-48]. The complete spectrum of the enol complex is shown in Fig. 9b. The asterisks indicate the resonances of the parallel and perpendicular region where
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the coupling with the nitrogen nucleus is observed. The decrease of the spectral intensity and the transition in the ΔM = 2 region above neutral pH support the formation of the dinuclear
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species with the stoichiometry Cu2L2H2 (Fig. S06). The theoretical calculations suggest the enolic coordination mode (O, N, Oenol).
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3.3.3. ESI MS spectra
Mass spectrometry showed that the ligand itself was pure, proved the formation of copper complexes and clearly confirmed the stoichiometry 1:1 and 2:2 of the species formed in HHSB-Cu(II) system (Fig. 10A). This finding is in the agreement with potentiometric and EPR results. The presence of CuL and Cu2L2 complexes is supported by the mono-charged m/z signals at 482.1 and 965.1, respectively. The signal attribution was confirmed not only by the comparison of the experimental mass of the clusters with their simulations (Fig. 10B, where the simulated elemental compositions are CuC23H19N2O6 for the for CuL and Cu2C46H36N4O6 for Cu2L2), but also in a method of the analysis of their isotopic pattern which is quite specific for copper complexes, due to the fact that this metal has two major naturally occurring isotopes, 63Cu and 65Cu. The spectra were measured at pH range from 3 to 9, and the differences between them are negligible; mass spectrometry is unable to detect different protonation states, due to the ionisation process itself, which involves the evaporation of solvent.
16
ACCEPTED MANUSCRIPT 3.3.4. Theoretical calculations Coordination sphere of copper ions was based on the model structures for x-ray salicylaldehyde acetylhydrazone bonded to copper ion [49]. Initial structure was fully
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geometry optimized using PM6 semi-empirical method and using DFT method (B3LYP/6-
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31G*). Energy was minimized at B3LYP/6-31G* level of theory structure of Cu2L2H2 complex is shown in Fig. 11. Unlike in the salicylaldehyde acetylhydrazone [49] hydrazone hesperetin Schiff base does not form planar Cu2L2H2 complex. Highly distortion form
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planarity of coordination sphere of cooper ions is caused probably by steric repulsion between hydrogen atoms from phenyl ring of one ligand and O5 oxygen atom from the another ligand
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(see green arrows Fig. 11). Such a steric clash does not appear in the structures of salicylaldehyde acetylhydrazone complexed with copper ion [49] when dinuclear complex
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were shown to dominate in the broad range of pH. However in another studies when (E)-N’(2-oxy-3-methoxybenzylidene)benzohydrazide was used as a ligand, more complicated tetranuclear complexes (cuban-like) were observed [39]. Our results as well as Monfared et al.
ED
[39] clearly suggest that the presence of benzoic group attached to hydrazide moiety destabilizes the formation of planar complexes that includes two or more ligands because of
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steric repulsion.
The destabilization of Cu2L2H2 is clearly supported by results presented in Fig. 6
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where the concentration of dinuclear complex is very low as to compare to another species. DFT (B3LYP/6-31G*) and semi-empirical calculations (PM6) clearly show that in Cu2L2H2 complex the ligand exists in an enolic form (see Fig. 11). All attempts of geometry optimization models of Cu2L2H2 complex when ligand was in the keto form lead to the complex dissociation and the formation of two separate CuLH molecules (data not shown). Free ligand exists in the keto form (at least in acidic pH) as it was shown in X-ray studies presented in this paper (see Fig. 1). In comparison to the complex of salicylaldehyde acetylhydrazone with copper ions [49] the ligand exists in keto form. However, in the case of the complex of (E)-N’-(2-oxy-3-methoxybenzylidene) benzohydrazide with the copper(II) only enolic form of ligand is observed [39]. Keto form of the ligand is destabilized by the repulsion interaction between hydrogen atom from benzoic group and hydrazide hydrogen (place of such a repulsion interaction is marked in Fig. 11 by violet arrows). In the case of the free ligand (see Fig. 1) such a repulsion does not exist, however probably when the complex is formed the bond lengths as well as the valence angles change in a such way that coplanarity of benzoic group with hydrazide moiety is no longer possible for the keto-type ligand. 3.3.5. Circular dichroism study 17
ACCEPTED MANUSCRIPT The results of electronic absorption and luminescence studies supported that hesperetin Schiff base and its metal complexes can bind to DNA via intercalation mode [10]. But for understanding the mechanism of compounds binding to DNA and helical
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conformation of nucleic acids circular dichroism is an efficient spectroscopic technique to test
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effects of synthetic agents induced on DNA three-dimensional structure [50]. Analysis of the CD spectra is very useful in the investigation of morphological changes in double-stranded DNA caused by the interaction of small molecules. The negative
SC
band near 244 nm with the positive band near 275 nm can be used as a spectral discriminator for the B forms, right handed helicity duplexes. [51]. The positive band near 265 nm and the
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negative one near 236 nm have been ascribed to the exciton splitting of the π-π* transitions resulting from base stacking [52]. Any change in the base stacking pattern or the helicity of
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the strands is manifested by either a change in the band position, the intensity, or both. A simple electrostatic interaction or binding to the grooves has almost no or very slight effect on the band at 275 nm, while the same band undergoes significant variations in intensity due
ED
to intercalation by small molecules [53]. In Fig. 12 the spectra registered at increasing concentration of CuHHSB complex and the constant CDNA are presented. The CD spectrum of
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DNA undergoes some changes. The band at 275 nm is blue shifted to 269 nm, changed in the intensity and at the ratio of DNA/CuHHSB 4 a new band above 300 nm appears.
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Fig. 12.
The spectrum at the ratio of DNA/CuHHSB 9.4 is slightly different from that of DNA alone but after 20 hrs an impact of the complex on DNA is more significant. The comparison of the DNA-CuHHSB spectra with those of CuHHSB clearly indicates some interactions between DNA and the complex. The CuHHSB may be bound externally to the phosphate backbone or in the minor/major grooves or intercalated between DNA bases. On the base of the results it is difficult to indicate unequivocally an intercalative or covalent cross-linker binding mode of CuHHSB complex to DNA. It is supposed that combinations of all possibilities are feasible. 3.3.6. Cleavage of pEGFP-C1 DNA The biological activity of metal complexes is often related to their ability to cleave DNA. They are able to bind to DNA either specifically or sequence independent and cleave one or both strands by either a radical or a hydrolytic pathway, the latter similar to the one of natural nucleases [54-57]. The cleavage of plasmid DNA (4731 bp) by HHSB and CuHHSB was monitored by agarose gel electrophoresis. Strand cleavage of the naturally occurring supercoiled DNA (Form I SC) led either to an open circular relaxed form (Form II OC) upon single strand cleavage or to a linear form (Form III) upon double strand cleavage. The 18
ACCEPTED MANUSCRIPT different forms could be distinguished by agarose gel electrophoresis, as form I migrated faster than the other forms, while form II was the slowest of the three due to its relaxed structure (Fig. 13a) [58].
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Fig. 13
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To prevent DNA aggregation, the final concentration of CuHHSB in each experiment was never higher than 100 μM. The results presented in Fig. 13a show that the complex was able to cut DNA effectively under applied conditions in contrast to the ligand HHSB (Fig. 13b).
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The complex behaves as a chemical nuclease by nicking the DNA Form I into Form III. It is suggested that the CuHHSB compound may cut the DNA strand at two positions. Although
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the cleavage reaction promoted by the complex does not require additional external agents, we carefully investigated the effects of scavengers on the DNA cleavage ability of the
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complex to gain information if any oxidative cleavage occurs. Hydroxyl radical scavengers (DMSO, glycerol), hydrogen peroxide scavenger (KI) and a singlet oxygen scavenger (NaN3) were added into the samples under the same conditions. The electrophoresis gel results are
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shown in Lines 3-6 (Fig. 13c). The experiments reveal that hydroxyl radical and hydrogen peroxide inhibitors can’t prevent the DNA degradation, while the presence of NaN3 efficiently
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inhibits the DNA scission (Lane 6, Fig. 13c.). Hence, the singlet oxygen species might be involved in an oxidative cleavage pathway. It may indicate that the localized generation of
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singlet oxygen or singlet oxygen-like entity, possibly a copper-peroxide complex, rather than free hydroxyl radical probably plays a role in the CuHHSB -induced DNA strand breaks as it was observed with other copper complexes [59]. However, according to some authors this result should not be interpreted as an indication that 1O2 is also participating in the process; it is probably due to the affinity of sodium azide for transition metals [60]. Therefore, hydrolytic pathway can’t be ruled out as well. DNA cleavage experiments in the presence and absence of DAPI and methyl green, these recognized minor and major groove’s binders of DNA, indicated that CuHHSB is probably not groove binder (Fig. S07). Therefore, the obtained data may point out to its action as intercalator or covalent cross-linker. 3.3.7. Anticancer Activity Anticancer drugs may act specifically on particular types of cancer. Therefore, the anticancer activity of the HHSB ligand and the CuHHSB complex was tested against two different tumor cell lines: HeLa (cervix carcinoma) and K562 (leukemia). The cells were challenged with each of the compound, over a range of concentrations from 10nM to 1mM for 72h and cell viability was measured by MTT assay. Cells incubated with different concentration of doxorubicin served as a reference sample. 19
ACCEPTED MANUSCRIPT Both HHSB and CuHHSB complex decreased cell viability (Fig. S08), with IC50 values in the micromolar range (Table 3). They have lower cytotoxic activity than doxorubicin. The comparison of IC50 values indicates that CuHHSB complex exhibits greater
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inhibitory effect on investigated human cancer cell lines than HHSB. Therefore one can
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consider that the observed cytotoxicity results from the activity of the complex CuHHSB rather than the HHSB ligand only. Interestingly, published data point out that copper(II) ions have very low cytotoxicity against cancer cells (HeLa) [61]. The compound HHSB was the
SC
least toxic toward HUVEC cells and it was impossible to determine IC50 in our experimental settings. This result is very promising and it could be of interest for further investigation.
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3.3.8. Antimicrobial activity
For in vitro antimicrobial activity, the investigated compounds were tested against the
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bacteria: Salmonella Enteritidis ATCC 13076, Salmonella Typhimurium, ATCC 14028, Escherichia coli ATCC 8739 and two Gram-positive strains Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19111. In Table 4, bacteria growth inhibition by
ED
CuHHSB complex at a concentration of 250 μg mL-1was observed for all tested bacteria. The values of the zones of inhibition of bacterial growth were comparable to the control, which
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was a solution of CuCl2 (250 μg mL-1).
Table 4
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A surprising result was obtained for S. Enteritidis, S. aureus and L. monocytogenes, whose growth was limited by the ligand. But the ligand did not inhibit the growth of the bacteria E. coli and S. Typhimurium. This phenomenon can be explained by individual lipophilic nature of the complexes. Such increased activity of the metal chelates can be explained on the basis of Overtone’s concept and Tweedy’s chelation theory [62]. According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only lipid soluble materials due to which liposolubility is considered to be an important factor that controls the antimicrobial activity. On chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of metal ion with donor groups [63,64]. Further, it increases the delocalization of the π electrons over the whole chelate ring and enhances the lipophilicity of the complex. This increased lipophilicity enhances the penetration of the complexes into cell lipid membrane [65]. They can disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism [66]. The variation in the activity of different complexes against different organisms depend either on the impermeability of the cells of the microbes or difference in ribosomes of microbial cells. The results have also 20
ACCEPTED MANUSCRIPT indicated that if Cu(II) ions have strong effect on bacteria growth inhibition (Escherichia coli, Salmonella Typhimurium) then the impact of the CuHHSB complex is also higher (no effect of the ligand is observed). This may mean that the HHSB can act as a transporter of Cu(II)
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ions (copper-ionophore) increasing in this way intracellular metal concentration and leading
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to a reduction of the bacterial growth. On the other hand when Cu(II) ions have lower effect on the activity of bacteria (Salmonella Enteritidis Staphylococcus aureus) then the impact of the HHSB and CuHHSB can be observed.
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Table 5 shows the results of the reducing growth of three selected test bacterial cultures: Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 8739 and
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Staphylococcus aureus ATCC 25923 after 24 hours. Growth strains of S. Typhimurium and E. coli was effectively inhibited by CuCl2. In the presence of the copper complex of the growth
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of S. Typhimurium was at the same level as innokulum (4.6 - 5.5 Log CFU mL-1). In the case of the bacterial count of E. coli after 24 hours of exposure to the complex remained at the same level (4.7-4.6 Log CFU mL-1). This shows the bacteriostatic effect of the copper
ED
complex on the bacteria E. coli and S. Typhimurium. Bacteria from genus S.aureus were able to grow in the presence of either CuCl2 or the complex.
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During the test of the permeability of cell membranes by fluorescent method (LIVE/DEAD BacLight bacterial viability kit) the ability of bacteria to form cell aggregates in
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the presence of both ligand and copper complex was observed (Fig. S09). Aggregation test was done for the three selected bacteria strains. The results are shown in Table 6. The aggregation of SalmonellaTyphimurium in the presence of CuCl2 was low (4.0 %). On the other hand, the aggregation ability of SalmonellaTyphimurium was the highest in the presence of the ligand (73%). The copper complex showed the aggregation similar to the control probe. In the case of Escherichia coli, the highest bacteria aggregation was observed in the presence of the ligand and the copper complex, respectively 73.7% and 75.2%. The aggregation of Staphylococcus aureus was different, the highest values of aggregation were observed in case of CuCl2 (53.7%) and CuHHSB (65.3%). The aggregation of Staphylococcus aureus in the presence of the ligand was low and comparable to that of the control probe. The results have indicated that the interactions of the compounds and microorganisms are individual. They depend on the pair: compound and organism. At this stage of our studies we can suppose that the compounds HHSB or CuHHSB may influence both surface proteins and enzymes regulated their presence. It is related to the differences in cell-wall structure of the Gram-positive (Staphylococcus aureus) and Gramnegative (Salmonella Typhimurium and Escherichia coli) bacteria. The differences in the 21
ACCEPTED MANUSCRIPT percentage of aggregation within a given bacteria group (Gram-negative for example, see Table 6) can be driven from subtle chemical differences to their wall fabric. One can suppose that fimbrial filament of the bacterial cell surface may play critical role in interactions with
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HHSB or CuHHSB and influences the aggregation of bacteria. Each fimbrial filament on the
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bacterial surface is composed of approximately 500 to 3,000 copies of a major fimbrial subunit protein [67]. The HHSB and CuHHSB may affect chemically these proteins, (e.g. by changing secondary structure) and this can reduce the virulence of bacteria.
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4. Conclusions
In summary, the crystallographic characteristic of hydrazone hesperetin Schift base -
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HHSB and solution speciation of HHSB and Cu(II)-HHSB chelate have been made. Our results are the first report evaluating the coordinating ability of hydrazone hesperetin Schiff
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base towards copper(II) ion in solution. The crystal structure of the ligand shows spatial arrangement of the potentially chelating atoms. The protonation and overall stability constants of hydrazone hesperetin Schiff base and its copper complexes have been determined,
ED
respectively. The higher values of ionization constants found by the extrapolating Yasuda– Shedlovsky method for aqueous solution can be related to the increased solvent stabilization
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of the acidic species in DMSO-rich mixtures. This interaction is favored by the large dipole moment of the DMSO molecule. The copper(II) ions form complexes with different
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stoichiometry depending on the pH range. The chelation reaction in acid medium promotes the deprotonation of the ligand at the C(5)-OH group and the simultaneous copper binding to the nearest nitrogen situated in the C(4) position. The species with variously ionized ligand can acquire two possible coordination modes: (O, N, Oket) or (O, N, O-enol). Theoretical calculations have shown that the most stable dimer structure is that one containing the enol form of the ligand.
CD spectrum analysis indicates that the copper complex with HHSB alters the DNA structure as it induces the changes of the position and the intensity of the DNA band. The CuHHSB complex may partially intercalate into DNA base-pairs with an unclassical intercalative mode and exhibits efficient cleavage of supercoiled DNA (pEGFP-C1 DNA) in the absence of any external agents via oxidative/hydrolytic (or both) mechanism. In vitro studies revealed that hydrazone hesperetin Schiff base and the copper complex exhibit cytotoxic and antimicrobial activities. The CuHHSB complex has a greater cytotoxic activity against HeLa and K562 cells than the HHSB ligand. On the other hand, the ligand does not exhibit cytotoxicity toward HUVEC cells in the experimental conditions. Beside the mechanistic speculations, which need to be supported by further investigations, the main 22
ACCEPTED MANUSCRIPT feature of the present system is the high reactivity in the absence of cofactors which makes the CuHHSB complex a very promising candidate for biological applications in vivo, especially in the therapeutic area, and for all those applications which are not compatible with
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the use of cofactor activated DNA-cleaving agents.
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The results of antimicrobial activity lead to the conclusion that HHSB and CuHHSB display antimicrobial activities against tested strains of bacteria, especially S. aureus is more sensitive to CuHHSB in comparison to other bacteria however the synthesized compounds
SC
exhibit lower activity compared to that one of the control drugs. The HHSB and CuHHSB trigger the bacteria aggregation which can limit the virulence of the bacteria. The mechanism
NU
for the antimicrobial activities of these compounds needs further study and the experimental results above would provide basic data for pharmacological research of HHSB and its
MA
copper(II) complex.
ED
Acknowledgement
AC CE
PT
Financial support of this work by Statute Funds No. I28/DzS/9184.
23
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SC
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AC CE
PT
ED
MA
NU
[66]
27
ACCEPTED MANUSCRIPT
Table 1. Ionization constants of hydrazone hesperetin Schiff base (HHSB) in (DMSO/H2O
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T
40%/60% v/v as pKa and in aqueous solution as pK aw ) and hesperetin for comparison
Ligand
Species
LogβHnL
pKa1
SC
(standard deviations are in parentheses). (I = 0.1M (KCl), T = 298K).
pKa2
pKa3
w ( pK a2 )
w ( pK a3 )
7-OH
3’-OH
5-OH
7.65
9.39
10.58
(7.24)
(9.18)
(10.21)
7.63
8.33
11.29
This work
6.80
10.40
>11.50
[36]
6.67
8.76
11.54
[37]
LH2-
19.97(±0.02)
LH3
27.62(±0.02)
LH2-
ED
10.58(±0.01)
11.29(±0.02)
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Hesperetin
LH2-
PT
HHSB
MA
NU
w ( pK a1 )
LH2-
19.62(±0.03)
LH3
27.25(±0.03)
28
Ref.
This work
ACCEPTED MANUSCRIPT
Table 2. Stability constants (logβpqr) of copper(II) complexes with HHSB (standard deviations are in parentheses) and spectral parameters
logβpqr
UV-Vis
Coordination mode
US
Species
CR
IP
T
(I = 0.1 M (KCl), T = 298 K).
-1
MA N
λ/nm -1
(ε/M cm ) 640(22)
CuLH
19.85(±0.02)
625(23)
Cu2L2H2
43.55(±0.08)
CuL
9.68(±0.02)
CuLH-1
-1.71(±0.04)
O, N, Oket
Az / 104 cm1
H / Gaussa
2.277
181.6
37.2
2.243
188.8
38.2
O, N, Oket/O, N, Oenol O, N, Oenol
O, N, Oket/O, N, Oenol
AC
625(25)
gz
TE D
26.81(±0.02)
CE P
CuLH2
EPR
a
625(25)
O, N, Oenol
Linewidth of the first parallel hyperfine resonance measured on the complexes formed with 63Cu.
29
ACCEPTED MANUSCRIPT
Table 3. The IC50 values for HHSB and CuHHSB complex against all used cell lines calculated from the dose-response curves.
IP
T
Doxorubicin was used as a reference compound.
K562
HUVEC
IC50
IC50
10 µM ± 1.27
ND*
6 µM ± 0.76
8 µM
0,06 µM ± 0.011
0.03 µM
CR
HeLa
Compound
70 µM ± 6.65
CuHHSB
30 µM ± 2.7
Doxorubicin
1 µM ± 0.15
TE D
MA N
HHSB
US
IC50
*
AC
CE P
ND-not determined
30
ACCEPTED MANUSCRIPT
Table 4. Antibacterial activity of test compounds.
IP
T
Inhibition zone diameters (mm, mean value, n=3)
Concentration Salmonella
Escherichia
Staphylococcus
Listeria
Enteritidis
Typhimurium
coli
aureus
monocytogenes
7.30.91
10.60.47
11.00.0
5.30.94
0.00.0
0.00.0
7.32.35
9.02.00
MA N
US
Salmonella
250
7.71.24
HHSB
250
8.73.77
CuCl2
250
8.31.88
10.61.41
14.00.0
8.00.50
9.00.90
Doxycycline
30
20.01.80
19.51.41
26.50.47
29.00.90
36.02.30
Kanamycin
30
16.00.47
14.00.47
17.51.41
12.00.82
18.00.47
DMSO
20000
0.00.0
0.00.0
0.00.0
0.00.0
0.0
AC
TE D
CuHHSB
CE P
(μg mL-1)
CR
Sample
31
ACCEPTED MANUSCRIPT Table 5. Growth of selected bacteria in the presence of the test compounds after 24 hours incubation.
Salmonella
Staphylococcus
CuCl2A
2.90.04b
HHSB A
8.40.31c
CuHHSB A
5.30.11a
Control
9.20.18c
4.70.20a
6.40.03b
2.80.85b
7.90.05c
9.50.20c
6.60.59b
4.60.08a
9.60.07d
9.50.44c
coli
4.11.48a
PT
ED
concentration 250 g mL-1, SD – standard deviation, a,b,c significantly different, p≤0.05.
AC CE
A
NU
4.60.13a
MA
Inoculum
aureus
SC
Typhimurium
Escherichia
RI P
Sample
T
Growth [Log10 CFU mL-1] SD
32
ACCEPTED MANUSCRIPT
Table 6. Aggregation of selected bacteria in the presence of the test compounds. Aggregation [%] SD Sample
Staphylococcus
RI P
T
Salmonella
4.03.46a
HHSB A
73.03.89b
CuHHSBA
34.613.06c 21.31.3c
coli
53.73.19a
37.316.05a
27.610.17b
73.72.8b
65.39.45a
75.23.25a
16.32.04b
30.47.71b
PT
ED
concentration 250 g mL-1, SD – standard deviation, a,b,c significantly different, p≤0.05.
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A
MA
Control
NU
CuCl2A
aureus
SC
Typhimurium
Escherichia
33
ACCEPTED MANUSCRIPT Scheme/Figure Legends Scheme 1. Keto-enol tautomerism of N-[(E)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-
T
phenyl)chroman-4-ylidene]amino]benzamide (HHSB).
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Scheme 2. Proposed structure for the keto (a) and enolate (b) coordination. The charges are omitted, X symbolises a solvent molecule, R ring B with C(3’)-OH protonated/deprotonated.
SC
Fig.1. The molecule HHSB, DMF and water in the asymmetric unit. The hydrogen bonds are shown by dash lines.
NU
Fig.2. The unit cell packing of the compound HHSB.
MA
Fig.3. Yasuda–Shedlovsky extrapolations of HHSB in DMSO–water mixtures (40, 50, 60, 70% v/v) at 25°C and an ionic strength of 0.1 M KCl. The values of the dielectric constants have been taken from Ref. [19].
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Fig. 4. Species distribution diagram for the HHSB system as a function of pcH. CL =1×10-3 M.
PT
Fig. 5. Electronic absorption (UV-Vis) spectra recorded in DMSO/water mixture (40%/60% v/v fraction) at different pcH corresponding for dominant species of HHSB;
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CL=1×10-5 M. The values of pcH are indicated above the arrows. Fig. 6. Species distribution curves for the system Cu(II)-HHSB. Ligand/metal = 1/1, CL= 110-3 M; CCu(II) = 1×10-3 M Fig. 7. Electronic absorption (UV-Vis) spectra recorded at different pcH; Cu(II)/L = 1/1; CCu(II) = 1×10-3 M; CL = 1×10-3 M (DMSO/Water: 40%/60% v/v fraction). Fig. 8. First parallel hyperfine resonance of the enol complex (Cu concentration 2102 M): a) system with natural copper (69.2% of 63Cu and 30.8% of 65Cu) and b) system with 63Cu (99.7% of
63
Cu). The triplet due to the superhyperfine coupling between the nuclei of
63
Cu and 65Cu with 14N nucleus are indicated.
Fig. 9. a) Origin of the multiplet in the first parallel hyperfine resonance measured with natural copper on the enol complex. b) Anisotropic X-band EPR spectrum recorded
34
ACCEPTED MANUSCRIPT at 100 K on 1:1 solid complex in the enol form dissolved in DMSO/water (40%/60% v/v). With an asterisk are indicated the resonances of the parallel and
T
perpendicular region where the coupling with the 14N nucleus is observed.
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Fig. 10. A) ESI-MS spectra of the system containing copper and HHSB at the ratio 1/1. Cligand= 110-4 M, m/z ratio of all shown species = 1. B) The simulated isotopic
SC
patterns.
Fig. 11. Structure of Cu2L2H2 complex obtained from DFT (B3LYP/6-31G*) calculations.
NU
The copper(II) ions are shown in cyan, oxygen, carbon, nitrogen hydrogen atoms are
MA
shown in red, black, blue and white, respectively. Green arrows show the possible repulsive interactions that may destabilize the complex (see the text for details).
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Violet arrows show the places in molecule where the repulsive interactions could lead to a destabilization of the keto form of the ligand (see the text for details).
PT
Fig. 12. CD spectra recorded in DNA-Tris, DNA-CuHHSB-Tris and CuHHSB-Tris systems after: a) 15 min of incubation, b) 15 min of incubation in the larger scales of molar
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ellipticity and wavelength and c) 20 hrs of incubation. The concentrations of DNA 9.4×10-5M, CuHHSB 1×10-5M or 2.35×10-5M. Fig. 13 (a) Quantification of gel electrophoresis bands originating from SC, OC and linear DNA in cleavage experiments for different concentrations of CuHHSB. Line 1DNA (pEGFP-C1 0.25 µg/µL; 10 mM Tris-buffer, pH 7.0), line 2- DNA+ 25μM CuHHSB, line 3- DNA+50 μM CuHHSB, line 4- DNA+100 μM CuHHSB. (b) Quantification of gel electrophoresis bands originating from SC and OC DNA in cleavage experiments for different concentrations of HHSB. Line 1- DNA, line 2DNA+ 25μM, line 3- DNA+50 μM, line 4- DNA+100 μM, line 5- DNA+150 μM, line 6- DNA+200 μM. (c) Gel electrophoresis diagrams showing the cleavage of supercoiled pEGFP-C1 DNA (0.25 µg/µL; 10 mM Tris-buffer, pH 7.0) by CuHHSB
35
ACCEPTED MANUSCRIPT (100 µM): with/without scavengers including DMSO (0.4 M), glycerol (0.4 M), KI (10 mM) and NaN3 (10 mM) in Tris–HCl buffer at pH 7.0 and 37 °C with incubation
T
time of 20 h. Line 1 DNA control; line 2 DNA+CuHHSB; line 3 DNA + CuHHSB +
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DMSO; line 4 DNA+CuHHSB+glycerol; line 5 DNA + CuHHSB+ KI; line 6
AC CE
PT
ED
MA
NU
SC
DNA+CuHHSB+NaN3.
36
ACCEPTED MANUSCRIPT
T
Scheme 1.
3'
O
4'
8
O
C
A
6
4
2 3
N NH 2" 3"
O
keto
OH
N
A
6
HHSB forms in solution
6"
4"
AC CE
PT
ED
5"
37
OH 3' 4'
2'
B
O
C
7
6'
5
OH
8
HO
5'
NU
7
B
MA
HO
CH3
SC
2'
RI P
OH
4
5'
2
6'
3
5
N 2"
HO
enol
3" 6"
4" 5"
O
CH3
ACCEPTED MANUSCRIPT
Scheme 2.
O-
T
O
X
Cu
RI P
X
O(H) O
Cu
O
N
O
O
R
AC CE
PT
ED
MA
(a)
38
O
N
R
Ph
NU
H
Ph
SC
N
N
(b)
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 1.
39
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 2.
40
ACCEPTED MANUSCRIPT
Fig. 3.
T
pKa1 pKa2
14
RI P
pKa3
SC NU
12
11
10
0.0132
0.0134
0.0136
0.0138
1/
AC CE
PT
ED
9 0.0130
MA
pKa+log[H2O]
13
41
0.0140
0.0142
0.0144
ACCEPTED MANUSCRIPT
RI P
T
Fig. 4.
0.0010
LH3
0.0009
0.0007
LH
L
2-
NU
0.0006 0.0005
MA
0.0004 0.0003 0.0002
ED
0.0001 0.0000
7
PT
6
8
9
pc H
AC CE
concentration of species
0.0008
SC
-
LH2
42
10
11
3-
ACCEPTED MANUSCRIPT Fig. 5.
0.35 2-
LH3
-
LH2
0.25
L
8.35
3-
12.05
0.20
A
SC
0.15 0.10
0.00 300
350
400
NU
6.05 0.05
T
10.05
0.30
RI P
LH
450
AC CE
PT
ED
MA
[nm]
43
500
550
600
ACCEPTED MANUSCRIPT
CR
IP
T
Fig. 6.
0.0010
CuLH2
MA N
0.0008 0.0007 0.0006
US
LH3
Cu
2+
TE D
0.0005 0.0004
0.0002 0.0001 0.0000 3
4
5
CuLH-1
CuL
CuLH
Cu2L2H2
CE P
0.0003
AC
concentration of species
0.0009
LH
-
LH2 6
7
pc H
44
8
9
10
2-
free L 11
3-
ACCEPTED MANUSCRIPT Fig. 7.
0,3
8.50
RI P
0,2
T
5.50
SC
3.50
NU
0,1
500
600
MA
0,0
700
PT
ED
nm
AC CE
Absorbance
11.05
45
800
900
1000
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 8.
46
ACCEPTED MANUSCRIPT
T
Fig. 9.
NU
SC
RI P
a
63
Cu
0.31
0.69
0.31
0.31
1.00
1.00
b
AC CE
PT
Tot.
0.31
ED
65
0.69
MA
Cu
47
0.69
0.69
ACCEPTED MANUSCRIPT
Fig. 10
Cu: hespertin (L) = 2:1, pH 7
Intens. x10 9
Cu2L2 965.1
A)
965.1
CuL
T
482.1 482.1
RI P
1.5
SC
1.0
NU
0.5
421.1 321.8
902.2 836.3
0.0 200
400
600
800
1000
1200
Intens. x10 9
17_01_Marzena_K3_CID50_z ekstra Cu_000001.d: +MS
experimental
482.1
1.5
ED
1.0
484.1
0.5 483.1
485.1
0.0
PT
17_01_Marzena_K3_CID50_z ekstra Cu_000001.d: C23H19N2O6Cu, M ,482.05
482.1
2000
1500
1000
AC CE
B)
m/z
MA
CuL
1400
simulation
484.1
483.1
500
0
480
482
485.1
484
486
488
m/z
Cu2L2
Intens. x10 9
17_01_Marzena_K3_CID50_000001.d: +MS
965.1
963.1
experimental
1.5
1.0 964.1 966.1
967.1
0.5
968.1 969.1 0.0
17_01_Marzena_K3_CID50_000001.d: (C23H18N2O6)2HCu2, M ,963.10
2500
simulation 963.1
2000
965.1
1500
964.1
1000
966.1
500
967.1 968.1 969.1
0 960
962
964
966
968
970
972
974
48
m/z
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 11
49
ACCEPTED MANUSCRIPT Fig. 12
a)
4
T
3
0 -1
SC
DNA DNA/CuHHSB = 9.4 DNA/CuHHSB = 4 -5 CCuHHSB=2.3510 M
-2 -3
-5
CCuHHSB=110 M
NU
-4 -5
240
250
22 20 16 14 10 8 6
0
AC CE
-2
280
290
300
ED
12
2
270
PT
-1
molar ellipticity [M cm
-1
18
4
260
MA
b)
RI P
1
-1
molar ellipticity [M cm
-1
2
-4
250
275
300
325
350
375
400
wavelength [nm]
c)
-1 -1 molar ellipticity [M mcm ]
2
DNA/CuHHSB = 9.4 1
0
DNA -1 DNA_20h DNA DNA:CuHHSB=9.4:1_20h
-2
-3 250
275
300
325
350
375
Wavelength [nm]
50
400
425
450
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
Fig. 13.
51
ACCEPTED MANUSCRIPT
AC CE
PT
ED
MA
NU
SC
RI P
T
GRAPHICAL ABSTRACT
52
ACCEPTED MANUSCRIPT Highlights: Cu(II)-hesperetin Schiff base complex shows nuclease activity
T
The complex distinctly suppresses cancer cell viability.
AC CE
PT
ED
MA
NU
SC
RI P
HHSB and CuHHSB reveal some antimicrobial activities.
53
ACCEPTED MANUSCRIPT Graphical Abstract (synopsis). Please provide a synopsis of 50 words maximum that illustrates your work submitted.
X-ray characteristic of novel hesperetin Schiff base is reported. Different
T
species in copper(II)-hesperetin Schiff base-water-DMSO system are
RI P
characterized. Hesperetin Schiff base and Cu(II) complex have revealed
AC CE
PT
ED
MA
NU
SC
nucleolytic, antimicrobial and cytotoxic activities.
54
