DNA-BSA interaction, cytotoxicity and molecular docking of mononuclear zinc complexes with reductively cleaved N2S2 Schiff base ligands

Accepted Manuscript Research paper DNA-BSA interaction, cytotoxicity and molecular docking of mononuclear zinc complexes with reductively cleaved N2S2 Schiff base ligands Saeedeh Asadizadeh, Mehdi Amirnasr, Farzaneh Fadaei Tirani, Alireza Mansouri, Kurt Schenk PII: DOI: Reference:

S0020-1693(18)31131-9 https://doi.org/10.1016/j.ica.2018.08.037 ICA 18437

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

22 July 2018 23 August 2018 23 August 2018

Please cite this article as: S. Asadizadeh, M. Amirnasr, F.F. Tirani, A. Mansouri, K. Schenk, DNA-BSA interaction, cytotoxicity and molecular docking of mononuclear zinc complexes with reductively cleaved N2S2 Schiff base ligands, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.08.037

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DNA-BSA interaction, cytotoxicity and molecular docking of mononuclear zinc complexes with reductively cleaved N2S2 Schiff base ligands

Saeedeh Asadizadeha, Mehdi Amirnasra*, Farzaneh Fadaei Tiranib*, Alireza Mansouria, Kurt Schenkc

a

Department of Chemistry, Isfahan University of Technology, Isfahan 8415683111, Iran.

b

Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. c

Institute of Physics, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

Corresponding authors: M. Amirnasr, F. Fadaei Tirani *E-mail addresses:

[email protected] [email protected]

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Abstract The synthesis of three potentially tetradentate, N2S2 Schiff-base-ligands, containing a disulfide bond, LThioSSLThio(L1), LBrSSLBr (L2) and LDiMeOSSLDiMeO (L3) are reported. These ligands undergo reductive disulfide bond scission upon reaction with PPh3 in the presence of Zn2+ ion. [LDiOMeS]‾, [LThioS]‾ and [LBrS]‾ are the resulting bidentate thiolate-imine anions respectively, which upon reaction with Zn2+ produce three new zinc(II) complexes: [Zn(LThioSN)2] (1), [Zn(LBrSN)2] (2) and [Zn(LDiOMeSN)2] (3). The structures of (L1) and 1–3 complexes were determined by X-ray diffraction. The interaction of 1–3 with CT-DNA have been investigated by absorption, emission, and CD spectroscopic methods and thermal denaturation measurements. The resulting data reveal that 1–3 show effective binding to CT-DNA (Kb = 2.2 × 104 to 1 × 105 L mol–1). The binding mode of DNA with 1–3 has also been investigated by molecular docking. The protein binding ability of 1–3 has been tested by monitoring the tryptophan emission intensity using BSA as a model protein. The quenching mechanism of BSA by the zinc complexes is static (kq = 1.66 to 3.4 × 1013 L mol–1 s–1). It is remarkable that 1–3 exhibit effective cytotoxicity against two human tumour cell lines (HeLa and MCF-7). The potent cytotoxic effects of 2 and 3, with IC50 values of 19.93 and 20.11 respectively, are higher relative to clinically used cisplatin (IC50 = 23.50) against the MCF-7 cell line, indicating that 2 and 3 may have the potential to act as effective metal-based anticancer drugs. Keywords: Reductive cleavage of S-S bond; Zinc thiolate Schiff base complexes; Crystal structure; DNA-BSA interaction; Cytotoxicity; Molecular Docking

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1. Introduction A recognized important component in biological systems is the disulfide bond that contributes to the thiol/disulfide-exchange reactions in cells. Metal coordination centers such as known metalloenzymes and metalloproteins with complete or partial sulfur coordination play vital roles in the chemistry of biological processes [1-3]. Schiff-bases (SB) containing disulfide bonds are potentially very versatile ligands, as they are able to coordinate to a range of metal centers as either neutral ligands, or monoanions after reductive cleavage of the S–S bond and conversion to thiolates [4-6]. Thiolates are extremely useful as model ligands in biological systems, and interest in their metal complexes has expanded dramatically due to their remarkable structural diversity [7], their potential application in the construction of advanced materials [8-10], and as bioinorganic model compounds and biomimetic complexes for the active sites of metalloenzymes [11-13]. In recent years, there have been extensive studies on the therapeutic application of metal thiolate complexes [14-17]. SB-ligands containing the thiolate functional group, have also shown interesting biological activities and have been used as chelators with mixed sulfur and nitrogen donors for the synthesis of metal complexes with potential medical applications [18-20]. Zinc, the second most abundant trace metal in human body after iron, is a border line Lewis acid and capable of binding to nitrogen, oxygen and sulfur donor atoms of the ligands. Zn2+ with its stable d10 electronic configuration is redox silent [21], however the interactions of zinc with thiols are central to the proposed role of zinc in redox-regulated cell signaling [22]. The biochemistry of zinc thiolate complexes relevant to the zinc metalloproteins have been reviewed in detail in numerous papers and elegant review articles [21-26]. These complexes are synthesized either by direct reaction of Zn2+ with the ligands bearing thiol functional groups [27-

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29], or by interaction of Zn2+ with disulfide containing ligands under reducing conditions [30]. Thiol groups are prone to oxidative attack, however their reactivity is restricted when coordinated to metal ions like Zn2+, as in some zinc fingers [31-33], or becoming part of a chelate [30, 34]. The cytotoxic activity and DNA binding of zinc thiolate complexes, as potential anticancer agents, have recently become the focus of attention by bioinorganic chemists [34-36]. In continuation of research on the synthesis of metal thiolate complexes [30], herein we report the synthesis and X-ray structures of three monomeric zinc thiolate complexes from disulfide containing SB-ligands using PPh3 as the reducing agent. The affinity of these complexes for BSA and CT-DNA has been investigated, and the anti-oxidative activity of the complexes has been evaluated by determining the IC50 values against the two human tumour cell lines (HeLa and MCF-7). 2. Experimental Section 2.1. Materials and General Methods Starting materials and solvents were supplied by Sigma Aldrich or Alfa Aesar Chemical Companies and used with no further purification. Calf Thymus DNA (CT-DNA) and Bovine Serum Albumin (BSA) were purchased from Sigma Aldrich Chemical Company and used as received. CT-DNA and BSA stock solutions were prepared by dissolving in Tris-buffer (NaCl 50 mM, Tris-HCl 5 mM, pH adjusted to 7.4 with NaOH 0.5 M). CT-DNA solution (5 mg/mL) was standardized spectrophotometrically using its known molar absorption coefficient at 260 nm (6600 mol-1 L cm-1). The ratio of the UV absorbance at 260 and 280 nm, A260/A280, was about 1.9, indicating that DNA was appropriately protein free [37]. The stock solutions were stored at 5 °C and used only for 4 days. A Varian BioCary-100 UV-Vis spectrophotometer was used to conduct Tm measurement using a 1 cm path length cell. An FT-IR JASCO 680 4

spectrophotometer was used to record IR spectra using KBr pellets. The 1H NMR spectra of the ligands and complexes were obtained on BRUKER AVANCE III 300 and 400 MHz spectrometers. Proton chemical shifts are reported in parts per million (ppm) relative to an internal standard of Me4Si. A JASCO V-570 spectrophotometer was used to measure UV-Vis spectra using a 1 cm path length cell. The emission spectra were recorded on a RF-5301PC fluorescence spectrophotometer. Elemental analyses were performed by using a Perkin–Elmer 2400II CHNS–O elemental analyser. The ligands L1, L2 and L3, were synthesized by the reaction of the benzaldehyde derivative with 2,2'-diaminodiphenyl disulfide, Phds, in a 2:1 molar ratio. For the synthesis and characterization details of the ligands and zinc complexes see Supplementary Data 1-7. The molecular structures of L1, 1, 2, and 3, were determined by single crystal X-ray diffraction. For the details of data collections and determinations and refinements of the structures see Supplementary Data Section 8. 2.2. Absorption and emission spectral study Absorption spectra were recorded on a JASCO V-570 spectrophotometer. UV–Vis spectra were recorded using Tris buffer, containing 0.2% v/v MeOH. Spectroscopic titrations were performed at room temperature to determine the binding affinity between CT-DNA and the zinc complexes using two-beam spectrophotometer and reference and sample cuvettes (1 cm path length). The titration was carried out by successive injections of 20 μL CT-DNA (2 × 10-4 M) solution to both reference and sample cells containing 2 mL Tris buffer and zinc complex (2 × 10-5 M) respectively, reaching the final concentration of 0–20 μM for CT-DNA. The emission spectra were recorded from 350 to 600 nm on a RF-5301PC fluorescence spectrophotometer with excitation wavelengths as indicated in Table 2 and excitation and emission slits set at 5 nm and 3 nm, respectively. 5

2.3. Human cell culture The human cervix carcinoma (HeLa), and human breast adenocarcinoma (MCF-7) cell lines was purchased from the National Cell Bank of Pasteur Institute of Iran, and were cultured in a humidified atmosphere containing 5% CO2 at 37 °C in DMEM medium supplemented with 100 U/mL penicillin, 100 μg mL-1 streptomycin as antibiotics and 10% fetal bovine serum in 96-well culture plates in a CO2 incubator. 2.4. MTT assay The in vitro cytotoxicity of the zinc complexes was determined by a MTT (3-(-4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma Aldrich M2128). The complexes were dissolved in DMSO, and tested at 20, 40, and 80 μM concentrations. The solutions were added to the wells 24 h after seeding 1.5 × 104 cells per well in fresh culture medium. DMSO was used as the vehicle control, and was found to be non-toxic to the cells up to 1% concentration. After 24 and 48 h, each well was loaded with 0.1 mg of MTT (in 20 μL of PBS pH = 7.4) for 4 h at 37 °C. The formed formazan crystals were dissolved by addition 100 μL of DMSO to each well and the absorption values were determined on microplate reader under 490/630 nm double wavelength using a 96 well plate reader (680Microplate Reader). Data were collected for three replicates each and were used to calculate the mean. Experiments were carried out in triplicate, and the percentage of cell viability was calculated according to the following equation: Cell viability (%) = [A490 (Sample) / A490 (Control)] × 100 where A490 (sample) refers to the reading from the wells treated with zinc complex and A490 (control) refers to that from the wells treated with medium containing 10% FBS only. The IC50

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value was determined by plotting the percentage viability versus concentration on a logarithmic graph. The average of three duplicate experimental results was taken as the final IC50 value.

3. Results and discussion 3.1. Synthesis and characterization The zinc thiolate complexes were synthesized by the reaction of the disulfide containing N2S2 Schiff bases, LXS–SLX, with Zn2+ in the presence of PPh3 as the reducing agent. The structures of these complexes clearly show that disulfide containing ligands undergo a reductive disulfide bond scission in the presence of PPh3 and Zn2+ ion to give [LXS]¯ ligands (Scheme 1). The resulting two fragments act as bidentate thiolate-imine monoanionic ligands and coordinate to the Zn2+ ions leading to monomeric pseudotetrahedral complexes with good yields (75-80%). These complexes are air-stable solids with well-defined crystalline structures suitable for X-ray crystallography (vide infra).

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N

S S

S

N N S

S

S [LThioS]

LThioSSLThio (L1)

S S

N

S

N N Br

Br

Br [LBrS]

LBrSSLBr (L2)

N

S S

S N

N

MeO MeO

-

OMe OMe LDiMeOSSLDiMeO (L3)

MeO MeO -

[LDiOMeS]

Scheme 1. The chemical formula of the Schiff base ligands, LXSSLX and [LXS]¯. 3.2. Spectral characterization All compounds are air-stable solids and have good elemental analyses (for the FT-IR, UV-Vis and 1H NMR spectra of the ligands and their zinc complexes see Supplementary Data, Figs. S1 to S6). The FT-IR spectra of the free ligands LThioSSLThio (L1), LBrSSLBr (L2), and LDiMeOSSLDiMeO (L3) exhibit the characteristic imine C=N bands at 1624, 1620 and 1605 cm–1 respectively. In the FT-IR spectra of the zinc thiolate complexes (1-3) however the C=N stretching vibration of the coordinated thiolate Schiff base, [LXS]‾, appears at 1589, 1584 and 1586 cm–1 respectively. Coordination of SB-ligands to metal ion leads to a decrease in the C=N bond order and the vibration frequency shifts to a lower frequency [38]. The electronic spectra of

8

the ligands show the intraligand transitions and the same spectral features are also observed in the spectra of the complexes. The main feature observed in the 1H NMR spectra of the ligands and their zinc complexes is the singlet due to the Himin appearing at 8.22 to 8.60 ppm for the ligands and 8.43 to 8.58 ppm for the zinc complexes. The aromatic protons appear in the appropriate region of 6.57 to 7.99 ppm [30]. Two additional singlets are expected for the methyl protons of the dimethoxy ligand, LDiMeOSSLDiMeO, L3, and its zinc complex [Zn(LDiOMeSN)2], 3. These signals appear at 3.72 and 3.88 ppm for the ligand and 3.99 and 4.02 ppm for the complex. As expected the two singlets are slightly shifted to the lower fields for the coordinated ligand. 3.3 The Crystal Structures of L1, and zinc complexes 1, 2 and 3 LThioSSLThio (L1): After having determined the structure of L1, we realized that it had already been reported [39]. Now we have a structure at T = 125(2) K (Fig. S9) and ours at T = 100(1) K (Fig. S10). Both are good structures and if one compares them with the overlay-option in the Mercury program, one can hardly see a difference. Even more reassuring: both structures suffer from the same shortcomings: elongated displacement ellipsoids in one of the thiophene groups. In order to also make a contribution, we shall try to understand why this is so, and propose a better model. Although L1 has twofold topological symmetry, nature could not find a symmetrical way to pack the molecules. Thus S1 and S4 (Fig. 1) present rather different interactions and S1 has slightly (1Å3) more space available. Fig. S11 shows the two comparably strong interactions for S1 on opposing sides of its cavity (Table S3). So, during growth, the S1 thiophene alternatively chooses one of these two interactions and that leads to the two positions (Fig. S12). Indeed, compared to S1 the S4 thiophene has slightly weaker interactions; in

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exchange it is built in a compacter sort of way into the structure and is held in place by dispersive forces.

Fig. 1. The ligand LThioSSLThio, L1, and its numbering scheme (Ujk scale = 50%). -[Zn(LThioSN)2] (1): (Fig. 2 and Fig. S13) presents an approximate C2 symmetry about a [311] axis through the Zn1 atom. All the phenyl, thiophene and even the chelate rings span planes better than

RMS=0.138Å.

If one rather defines N-C-C-S planes in the chelate rings, one finds a

distance of +0.25Å for the Zn1 atom. The dihedral angles between the phenyl and thiophenes are 42.3 and 46.5°. [Zn(LThioSN)2], 1, is a perfectly ordered structure. Further geometrical items are compiled in Table S2. The structure of 1 is held together by 2D network of weakest

interactions (Table S3), namely S2...C18 and S3...C5 along the c base vector and S3...S4 along the a base vector. There are no significant interactions along the b base vector.

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Fig. 2. The complex [Zn(LThioSN)2], 1, and its numbering scheme (Ujk scale = 50%). [Zn(LBrSN)2] (2): Neither the [Zn(LBrSN)2], 2, complex nor the ZnN2S2 display any point symmetry beyond C1 (Fig. 3 and Fig. S15). The two LBrSN ligands are disposed in an antiparallel manner around the central Zn atom with Br-phe/phe angles of 58 and 70°. The complex resembles a V-shaped angle iron of which the edge is roughly parallel to the a-axis. Two of these angle irons, related by an inversion, build up endless [100] chains, held together by weak C-H... Br hydrogen bonds (it is true that these are very weak (~0.5kcal mol-1, but their character has been confirmed by Brammer et alii’s exhaustive survey [40] in other compounds). Neighbouring (antiparallel) chains are linked by weak C-H...S hydrogen bonds according to Domagała et alii’s careful analysis [41] along the b axis and C-H... interactions along the c axis. All this is shown in Fig. 3, S17, and S18, and some geometrical details are given in Table S2.

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Fig. 3. The complex [Zn(LBrSN)2], 2, and its numbering scheme (Ujk scale = 50%). [Zn(LDiOMeSN)2] (3): displays an approximate C2 symmetry about a [100] axis through the zinc atom (Fig. 4 and S19). The LDiMeOSN ligands are attached to the zinc atom in a polar, crosswise manner; the dimethoxyphenyl (dmp) moieties are quite parallel. The phenyl and the dmp groups subtend angles of 45° and 59°. Further geometrical items can be found in Table S2. It is noteworthy that one of the dmp in the complex is perfectly ordered (even the methoxies lie naturally (= without restraints) in the plane. The other dmp moiety, however, is disordered between two positions [0.528(2): 0.472(2)] with respect to a binary rotation through the C21A and C24A atoms. In view of the temperature (100 K) and the size of the group this would have to be static disorder created at growth. 3 is a layer structure with chunks built up by the n glide operation (even allowing for a possible (010) cleavage plane) and the layers generated by the 21 screw operation and the inversion. Perhaps, this could, to some extent, explain the disorder in the dmp group. This structure owes its cohesion to the weakest forces available: some C-H...O hydrogen bonds (Table S3) and a plethora of C-H..., H-H and C...S interactions and, of course, van der Waals forces.

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Fig. 4. The complex [Zn(LDiOMeSN)2], 3, and its numbering scheme (Ujk scale = 50%). 3.4. DNA-binding 3.4.1. Absorption spectroscopic measurements The binding ability of the zinc complexes 1–3 to CT-DNA was examined by electronic absorption spectroscopy; absorption spectra of 1–3 in the absence and presence of CT-DNA in Tris-HCl buffer (pH = 7.4) are shown in Fig. 5. As expected for Zn2+ with a d10 electronic configuration, no d-d transition is observed. The absorption band in the 200–225 nm region is attributed to the π–π* transition in the aromatic rings. The two absorptions between 250 and 350 nm are due to the imine π–π* and thiol to phenyl n–π* transitions. The absorption band at 357 nm in the spectrum of 3 is due to the n–π* transition from the methoxy group to the aromatic rings [42]. The intensity of this band is significantly reduced during the titration of 3 with CTDNA providing extra evidence for its partial intercalation as confirmed by competitive fluorescent and molecular docking studies (vide infra). Upon the addition of CT-DNA, the absorption band maxima of 1-3 at 222, 222 and 211 nm, respectively, exhibited hypochromisms of about (23, 17, 29%). Additionally, the band at 211 nm presents a red shift (bathochromism) of

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5 nm (up to 216 nm). These changes have been linked to the effective interaction of the zinc complexes with duplex DNA [43,44]. The interaction between a DNA reacting site (base pair, P) and the Zn(II) complexes C to give the complex (PC) can be described by a simplified 1:1 reaction model (Reaction (1)).

The binding constant for reaction (1), K, can be evaluated by analyzing the spectrophotometric data using Eq. (2)

where [Com] is the total analytical complexes concentration, ΔA = A - A0, A0 is the absorbance in the absence of DNA, [P] is the concentration of DNA free sites ([P] = C P - [PC]) and Δε = εPC – εCom is the change in the molar absorptivity upon binding. According to Eq. (2) a plot of [Com]/ΔA vs. 1/[P] should yield a straight line with Δε = 1/intercept and K = intercept/slope. Note that the calculus requires an iterative procedure as reported in the literature [45]. Fig. 5. (aʹ, bʹ, cʹ) shows an example of data analysis and the analysis results are presented in Table 1. Table 1 Kb, ΔG, and absorption spectral data of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, bound to CT-DNA. Complex λmax (nm) Change in absorbance H% Kb (M-1) ΔG(kJ/mol) 4 Hypochromism 222 23 4.8×10 -26.70 1 4 Hypochromism 222 17 2.2×10 -24.77 2 5 Hypochromism 211 29 1×10 -28.52 3

The calculated Kb values and Gibbs free energies of binding are comparable to those reported for related zinc complexes, and indicate that the interaction of all three complexes with CT-DNA is thermodynamically favorable, with a reasonable to strong binding to CT-DNA. To further

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examine the type of interaction that occurs between CT-DNA and zinc complexes, a fluorescence titration of CT-DNA with zinc complexes was also performed [43, 44, 46].

Fig. 5. Absorbance spectra for zinc complexes upon addition of increasing amounts of DNA, (a) [Zn(LThioSN)2], 1, (b) [Zn(LBrSN)2], 2, and (c) [Zn(LDiOMeSN)2] 3. Fig. 5. (aʹ, bʹ, cʹ) analysis according to Eq. (2) of the binding isotherms from absorbance titration data for zinc complexes/DNA systems. [Com] = 2 × 10-5 M, buffer solution (5 mM TrisHCl/50 mM NaCl at pH 7.4), T = 25 °C.

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3.4.2. Fluorescence studies To further investigate the binding mode of our zinc complexes to CT-DNA, fluorescence titration of CT-DNA with 1–3 was used. Fluorescence spectra were recorded from 350 to 600 nm keeping the concentration of the zinc complex (2 × 10-6 M) constant while varying the DNA concentration from 0 – 15 μM. For [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, the excitation wavelength was set on 331, 314 and 331 nm, respectively (Table 2). The excitation and emission slits were set at 5 nm and 3 nm, respectively.

Fig. 6. Changes in the fluorescence spectra of zinc complexes (2 × 10-6 M), with increasing concentrations of DNA (2 × 10-5 M) in buffer solution (5 mM TrisHCl/50 mM NaCl at pH 7.4) (a) [Zn(LThioSN)2], 1, (b) [Zn(LBrSN)2], 2, and (c) [Zn(LDiOMeSN)2], 3.

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These zinc complexes are luminescent in the absence of DNA in Tris-HCl buffer at room temperature. Complexes 1 and 2 do show a noticeable increase in their emission intensity upon addition of DNA (Fig. 6 a, b). The enhancement in the fluorescence intensity shows that the two zinc complexes are protected from solvent water molecules (quenchers) by binding to DNA and being surrounded by a hydrophobic environment. This is a potential clue to the fact that the interaction between the zinc complexes and DNA is possibly through hydrophobic pocket along the major and minor grooves [47,48]. Similar to the quenching process, the enhancement constant can be obtained through the equation (3) [49, 50]: F0/F = 1- KE[E]

(3)

In the case that a dynamic process contributes to the enhancing mechanism, equation (3) can be written as follows: F0/F=1 - KD[E] =1 - kBτ0[E]

(4)

where F0 and F represent the fluorescence intensities in the absence and presence of an enhancer (E), respectively, and [E] is the concentration of E. KD is the dynamic enhancement constant (like a dynamic quenching constant), kB the bimolecular enhancement constant (like a bimolecular quenching constant) and τ0 the fluorescence life-time. KD for the zinc complexes 1 and 2 were calculated (Table 2) by means of (4). Based on a typical τ0 of 10 ns [48,49], the bimolecular enhancement constants of 1 and 2 were calculated from KD = kBτ0 (Table 2). Since our values are greater than the largest possible value for the bimolecular enhancement constant (1.0 × 1010 L mol–1 s–1) in aqueous medium [48,49], it is reasonable to assume that the fluorescence enhancement is controlled by a static process. Contrary to 1 and 2, complex 3, which is also luminescent in the absence of DNA in Tris-HCl buffer at room temperature, shows an appreciable decrease in emission upon addition of DNA (Fig. 6 c); this decrease can be

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attributed to a partial quenching of the emissive state by DNA. The corresponding KSV and kq values were obtained by means of the Stern–Volmer fluorescence quenching equation (5) (Table 2) [37]. F0/F= 1 + Ksv[Q] = 1 + kqτ0[Q]

(5)

where F and F0 are the fluorescence intensities of complex 3 in the presence and absence of DNA, respectively. kq, KSV, τ0 and [Q] are the biomolecule quenching rate constant, the dynamic quenching constant, the fluorescence time in the absence of quencher, and the quencher concentration, respectively. KSV can be obtained from the plot of F0/F vs. [DNA]. Values of kq, computed with τ0 =10 ns and kq = KSV/ τ0 are given in Table 2. Table 2 Fluorescence spectral properties of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, bound to CT-DNA. λex (nm)

λem (nm)

KD (L mol–1)

kB (L mol–1 s–1)

1 2

331 314

389 375

3

331

389

3.7×104 1.3×105 Ksv (L mol–1) 1.39×105

3.7×1012 1.3×1013 kq (L mol–1 s–1) 1.39×1013

Complex

Our kq for complex 3 is greater than the largest possible value of the bimolecular collisionquenching constant (2.0 × 1010 L mol–1 s–1) for biomolecules [46], which indicates a static quenching process. The observed decrease in the emission intensity of 3 by DNA is attributed to the partial intercalation of complex 3 via π–π stacking. Upon intercalation of 3 into DNA base pairs, the π* orbital of the coordinated ligand interacts with the π orbitals of the base pairs, lowering the π–π* transition probability in the zinc complex and reducing the emission intensity of this emissive state. The gradual disappearance of the n–π* absorption band at 357 nm during the titration of complex 3 by DNA, as shown in Fig. 5c (vide supra), also gives additional hint to

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the competition between DNA base pairs and methoxy lone pairs for electron transfer to the π* orbital of the coordinated ligand. Further support for the partial intercalation of the complex 3 comes from the competitive fluorescent and molecular docking studies (vide infra).

Fig .7. The emission spectra of the MB–DNA system, in the presence of (a) [Zn(LThioSN)2], 1, (b) [Zn(LBrSN)2], 2, and (c) [Zn(LDiOMeSN)2], 3. [DNA] = 5 × 10-5 M, [complex] = 0 – 1 × 10-5 M, [MB] = 5 × 10-6 M. The black arrows indicate the changes in the emission intensity upon increasing the concentration of the complexes. 3.4.3. Competitive fluorescent studies In order to interpret the spectral results and determine the exact mode of interaction of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, with DNA, a complementary study using methylene blue (MB) was carried out. MB is a planar cationic dye molecule and a familiar 19

intercalator, which is often used as a sensitive fluorescence probe to detect the mode of binding of small molecules to DNA. The emission intensity of MB in the presence of DNA is considerably reduced owing to the strong intercalative interaction [51]. In the competitive intercalation of a metal complex to DNA, the emission intensity of the MB-DNA system is enhanced, apparently due to the release of MB. In this context, the fluorescence spectrum of DNA bound to MB, in the presence of varying concentrations of 1-3, was monitored (Fig. 7). As evident from Fig. 7a and 7b 1 and 2 have very little effect on the emission intensity of MB-DNA indicating that MB and 1 and 2 independently bind to DNA, with no binding competition [52]. Complex 3 on the other hand shows a tendency for intercalation, leading to the gradual release of methylene blue and an enhancement of the fluorescence (Fig. 7c) [53,54]. Since Kb(3-DNA) = 1 × 105 M is more than 5 times that of and Kb(MB-DNA) = 1.89 × 104 M, it is reasonable to assume that the observed emission enhancement in Fig. 7c is presumably due to replacement of MB by complex 3 confirming partial intercalation of this complex, with exchange efficacy of about 25%. 3.4.4. Thermal denaturation of DNA The thermal behaviour of DNA with zinc complexes provides information regarding the strength of DNA-complex-interaction. Indeed, a foreign substance can modify the base-pairing and hydrogen bonding of DNA, thus altering the helix-coil transition. A melting temperature, Tm, is defined as the temperature at which 50 per cent of the double stranded DNA has unwound. We determine Tm and the width of the transition, Tm, by following the absorbance of the electronic transition at 260 nm in CT-DNA and then extracting the values by means of several tangents applied to the sigmoid curve. Tm and Tm can be used to narrow down the interaction mechanism between molecules and DNA. The intercalation of molecules to DNA results in

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noticeable increases in Tm [55] and Tm. Fig. 8 shows the effect of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, on Tm of CT-DNA in TrisHCl buffer; Tm of CT-DNA alone was 82.3(2) °C. After 1-3 were added, Tm increased by 2.2, 3.1 and 3.8 °C, respectively. These Tm-values are smaller than those for common DNA-metallo-intercalators, for which ∆Tm >10 °C is reported [55-57]. Our ∆Tm values then suggest the existence of a moderate interaction between the zinc complexes and DNA. Complex 3 manifests stronger binding to DNA with respect to 1 and 2. This conclusion is in line with other experimental results confirming its partial intercalation [56,57].

Fig. 8. Plots of the absorbance at 260 nm of CT-DNA (7.5 × 10-6 M) upon heating in the absence and the presence of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3. (37.5 × 10-6 M) in 5 mM TrisHCl with 50 mM NaCl. 3.4.5. Studies of the Circular Dichroism (CD) CD is a sensitive technique for investigating the binding of a metal complex to DNA and the ensuing conformational changes. The observed CD spectrum of CT-DNA consists of a positive band at 275 nm, due to π-π interaction of bases, and a negative band at 245 nm which characterizes the helicity of B-type DNA [55,58]. These bands depend crucially on the DNA interactions with metal complexes and on the interaction type. To examine the effect of 1-3 on 21

the conformation of the secondary structure of DNA, the CD spectra were monitored in a buffer solution of 50 mM of Tris in the absence and after addition of the zinc complexes (Fig. 9). The decrease in the intensity of the negative band, upon addition of 1 and 2, indicates partial unwinding of the DNA helix, while the simultaneous increase in the positive band intensity specifies binding of DNA to the complexes [55,58,59]. These results can be attributed to a conformational conversion from a more B-like to a more A-like structure within the DNA molecules [58,59], and suggest the existence of a non-intercalative mode (groove binding) or a mixture of groove and partial intercalation between the zinc complexes 1 and 2 and the nucleic acids of DNA. In the presence of complex 3, however, the positive peak intensity of the CD spectrum of DNA increased with a slight displacement which can be associated with an interaction between the aromatic rings of the complex 3 and some base-pairs of DNA leading to its partial intercalation as confirmed by other experimental results [60].

Fig. 9. CD of CT-DNA (1×10-4 M) in the absence and the presence of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, (5×10-5 M) in 5 mM TrisHCl with 50 mM NaCl (pH = 7.4). 3.5. Protein Binding Studies 3.5.1. Tryptophan quenching experiment 22

The protein albumin in the circulatory system is a substantial element in metallo-drug transportation, since albumin undergoes strong interactions with these drugs. Bovine serum albumin (BSA) and human serum albumin (HSA) are structurally very similar. BSA is therefore an interesting protein for studying metallo-drug transportation and should furnish information on the mechanism of drug-action, their therapeutic responses and their pharmacokinetics [44,46,61,62]. Since the three aromatic amino acids phenylalanine, tyrosine and tryptophan are part of proteins, the latter inherently show fluorescence and this characteristic of many proteins is usually caused by tryptophan alone. BSA has a strong fluorescence emission with a broad peak related to the tryptophan residues, around 345 nm upon excitation at 280 nm. Fluorescence spectral analysis has been used for evaluating the interaction between metal complexes and BSA [44,46,61,62].

Fig. 10 (a, b, c). Fluorescence emission spectra of BSA in the absence (dashed line) and presence (solid lines) of (a) [Zn(LThioSN)2], 1, (b) [Zn(LBrSN)2], 2, and (c) [Zn(LDiOMeSN)2], 3. 23

[BSA]

=

2 × 10-6 M, [complex] = 0- 1.82 ×10-6 M. (d) The plot of F0/F versus the concentration of 1-3 for the titration of BSA by the complex. Fig. 10 shows the effect of increasing the concentrations of added 1-3 on the fluorescence spectrum of BSA. The gradually decreasing emission intensity of BSA indicates a significant interaction between the complex and protein, and in the case of 3, an isoemissive point is observed at 392 nm Fig. 10c [63]. This binding of the complex to BSA can change the secondary structure of the protein and lead to the quenching of its intrinsic fluorescence [44,63,64]. The quenching process can be described by the Stern Volmer equation (5). KSV can be obtained from the plot of F0/F vs. [Q]. Since the fluorescence-, τ0, of a biopolymer is 10-8 s [44,46], the values of the quenching rate constant, kq, were calculated according to kq = KSV/τ0. The calculated values of KSV and kq for the interaction of 1-3 with BSA are listed in Table 3. The quenching rate constant (scatter collision quenching constant), kq, depends on the probability of a collision between a quencher and a fluorophore and expresses the response-rate of tryptophan residues to the metallo-drug. The kq values (Table 3) testify that the zinc complexes possess good quenching ability of the albumin fluorescence. The highest kq of various quenchers with the biopolymer is 2×1010 L mol-1 s-1 [44,46]. Our calculated kq values (Table 3) are greater than typical kq for biopolymers. Thus, the results indicate that quenching can be induced by static mechanisms. Table 3 The quenching constant, binding constant and number of binding sites for the interactions of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, with BSA. Ksv kq Ka Complex λmax (nm) n -1 5 -1 -1 13 (L mol )×10 (L mol s )×10 (L mol-1)×105 343 1.98 1.98 1.67 1.23 1 24

345 344

2 3

3.4 1.66

3.4 1.67

3.27 2.03

0.99 0.75

The number of binding sites (n) and the binding constant (Ka) for the static quenching interaction between zinc complexes and BSA have been calculated using the Scatchard equation (6) [43,44]. –

(6)

The plots of log [(F0-F)/F] vs log [Q] give straight lines (Fig. 11), and n and Ka can be calculated from the slope and the intercept of the doubly logarithmic regression, respectively (Table 3). The distribution of a drug in the blood plasma can be checked by calculating the drug-BSA binding constant (Ka); its value should be high enough such that the compound is bound to the BSA for transport, but less than 1015 L mol-1, such that the compound can be released from the BSA upon arrival at the target cells [64]. The Ka values Table 3 for the BSA interaction with the zinc complexes lie within a reasonable range for an effective binding. In addition, n values for zinc complexes (1-3) average out to be 1, which hints at the existence of only one available binding site in each albumin.

Fig. 11. Plot of log [(F0-F)/F] versus log [Q] for [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3. 25

3.5.2. Absorption spectral studies A strong interaction between a protein and a metal complex leading to structural changes and a static quenching process, can also be detected by changes in the UV−Vis spectrum of the protein in the presence of the metal complex. For a dynamic quenching mechanism, however, no noticeable changes should be observed in the UV−Vis spectrum of the protein. The electronic spectrum of BSA shows two absorption bands at 220 and 280 nm. These bands are associated with BSA polypeptide structure and the aromatic amino acids (Trp, Tyr, and Phe), respectively. The absorption maximum of BSA at 280 nm is highly sensitive to the surrounding microenvironment and any fine change in this absorption band represents a perturbation of an αhelix. The perturbation of the secondary structure of the protein is associated with changes in the absorption band in the 220-240 nm range [65]. Spectral changes of BSA were monitored after adding different concentrations of 1-3 in the range of UV–Vis absorption (Fig. S19). According to the (Eq. 7) [45]:

Where [BSA] is the total analytical BSA concentration, ΔA = A – A0, A0 is the absorbance in the absence of zinc complex, [Com] is the concentration of unreacted zinc complex, and Δε = ε(BSAComp)

– εBSA. According to Eq. (7) a plot of [BSA]/ΔA vs. 1/[Com] should yield a straight line

with Δε = 1/intercept and K = intercept/slope (Fig. S19). The values of Kapp for 1-3 are estimated to be 5 × 104, 3.33 × 104 and 3 × 104 L mol-1, respectively. These results indicate a reasonably effective interaction between the complexes and BSA and show that BSA can be considered a good carrier for transfer of zinc complexes in vivo. 3.6. Molecular Docking

26

Molecular docking was used as a preliminary computational tool for exploring suitable sites for the interaction of 1-3 with DNA. In particular, we attempt to investigate to what extent 1-3 can attach themselves to the dodecamer ACCGACGTCGGT of BPV E2-R3 (PDB ID: 423D) [66], thus altering or blocking one or more active sites of this potentially carcinogenic virus. The geometry of the complexes was considered flexible in the docking calculations but the structure of the DNA was constrained to be rigid. The structural parameters of the complexes were those described in Table S2 and Section SD-8. The Lamarckian Genetic Algorithm (LGA) was used for the docking calculations. A grid box with XYZ dimensions of 126 × 88 × 82 Å3 and a grid point spacing of 0.375 Å was generated, then affinity maps of the present atoms as well as electrostatic maps were computed with the help of AutoGrid [67]. Based on these results free energies of binding were computed by means of the AutoDock 4.2 software [67], namely -7.73 for 1, -8.30 for 2 and -8.52 kcal mol-1 for 3. Fig. 12 shows the molecular docking and the binding site of the complexes interacting with DNA.

Fig. 12. The zinc complexes [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, and [Zn(LDiOMeSN)2], 3, are docked to the active sites of DNA.

27

It is the Zn-complex with the strongest hydrogen bond acceptors and the highest flexibility (3) which furnishes the largest binding energy. It is noteworthy that 3 presents the least number of phenyl-moieties in a position capable of .... stacking interactions (SI), but this one seems to be of the intercalation type. 1 and 2 have two SI, but stretch much of their atoms away from the doublecoils. The sulfur atoms in 2 seem to participate less in the bonding than the bromine atoms. One is tempted to say that none of the complexes seems very comfortable inside the active site and rather appears to flee instead of snuggling down into the double-strand. Despite the similarity of the coordination sphere of Zn in 1-3 and Pt in cisplatine, the attachment types are quite different. It is surprising that the rather unwieldy and non-covalent 1-3, compared to the very compact and spherical cis-diammine-dichlorideo(II), Cl2(NH3)2Pt, still present comparably good cytotoxicity. 3.7. Cytotoxicity By using an MTT assay, the anticancer activities of 1–3 were investigated against HeLa and MCF-7 cancer cells in aqueous buffer solution and the famously feared drug cisplatin was used as a positive control under identical conditions. The IC50 values of the test compounds and cisplatin were calculated from the data obtained from MTT assays and are collected in Table S4. As shown in Fig. 13, all three complexes exhibit significant cytotoxic activity on MCF-7 cell lines, and, to a lesser extent, on the Hela cell line. The potent cytotoxic effects of 2 and 3 with IC50 values of 19.93 and 20.11 respectively (Table S4) are higher relative to cisplatin (IC50 = 23.50) against the MCF-7 cell line, indicating that 2 and 3 have the potential to act as effective metal-based anticancer drugs.

28

Fig. 13. The histogram of IC50 values (μM) for the anticancer effect of [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, [Zn(LDiOMeSN)2], 3, and cisplatin on the MCF-7 and the Hela cell lines.

4. Conclusion Three mononuclear zinc(II) complexes of bidentate thiolate Schiff-base-ligands produced from the reductive S–S bond cleavage of three N2S2 disulfide SB have been synthesized. The crystal structures of the ligand L1 and the three zinc thiolate complexes [Zn(LThioSN)2], 1, [Zn(LBrSN)2], 2, [Zn(LDiOMeSN)2], 3, were determined by X-ray diffraction revealing a distorted tetrahedral geometry around Zn(II). The relatively high binding affinity of the zinc complexes toward CT-DNA and BSA has been confirmed by using various physico-chemical techniques. The interaction of the complexes 1 and 2 with CT-DNA, as revealed by various experiments, is non-covalent via the groove binding mode. Competitive binding studies with MB have revealed that only complex 3 has the ability to displace the typical intercalator MB from the MB–DNA system suggesting partial intercalation as a possible mode of its interaction with DNA. This was also confirmed by molecular docking results. The interaction of the complexes with BSA suggests the opportunity of being transferred to the relevant potential biological targets and the possibility of their release. The biological activity of these complexes was evaluated by assessing

29

the in vitro cytotoxicity against two human tumour cell lines (HeLa and MCF-7). The anticancer activities of 1-3 were comparable or higher relative to the clinically-used cisplatin; 1-3 may therefore be regarded as potential anticancer drugs. Acknowledgments Partial support of this work by the Isfahan University of Technology Research Council (grant number 500/95/24305) is gratefully acknowledged. Appendix A. Supplementary data CCDC numbers 1844120-23 for compounds L1, 1, 2 and 3 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. Supplementary Data (SD) associated with this article: synthesis, characterization and spectroscopic data of the ligands and their zinc complexes, X-ray crystal structure determination and related tables, and cytotoxicity data can be found in the online version, at http://dx.doi.org/10.1016/j.ica. References [1] B. Krebs, G. Henkel, Angew. Chem. Inr. Ed. Engl. 30 (1991) 769-788. [2] S. Yao, R.M. Flight, E.C. Rouchka, H.N.B. Moseley, Proteins 58 (2017) 885-907. [3] K. D. Mjos, C. Orvig, Chem. Rev. 114 (2014) 4540–4563. [4] T. Nguyen, A. Panda, M.M. Olmstead, A.F. Richards, M. Stender, M. Brynda, P.P. Power, J. Am. Chem. Soc. 127 (2005) 8545–8552. [5] E.C.M. Ording-Wenker, M. van der Plas, M.A. Siegler, S. Bonnet, F.M. Bickelhaupt, C. Fonseca Guerra, E. Bouwman, Inorg. Chem. 53 (2014) 8494–8504. [6] M. Gennari, B. Gerey, N. Hall, J. Pécaut, M.-N. Collomb, M. Rouzières, R. Clérac, M. Orio, C. Duboc, Angew. Chem. Int. Ed. 53 (2014) 5318 –5321. [7] J.A. Denny, M.Y. Darensbourg, Chem. Rev. 115 (2015) 5248–5273. [8] O. Crespo, M.C. Gimeno, A. Laguna, F.J. Lahoz, C. Larraz, Inorg. Chem. 50 (2011) 9533– 9544. 30

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Research highlights  Synthesis of [Zn(LXSN)2] from disulfide Schiff bases L1-L3 in the presence of PPh3.  L1-L3 undergo reductive S–S bond cleavage and form zinc(II) thiolate complexes 1-3.  X-ray crystal structures of the L1 and pseudotetrahedral 1-3 have been determined.  The binding of 1-3 toward DNA and BSA has been confirmed by different analyses.

 1–3 exhibit effective cytotoxicity against HeLa and MCF-7 cell lines, comparable to cisplatin.

35