Nickel complexes of aroylhydrazone ligand: synthesis, crystal structure and DNA binding properties

Journal Pre-proof Nickel complexes of aroylhydrazone ligand: synthesis, crystal structure and DNA binding properties

Ping Yang, Li-Lei Zhang, Zi-Zhou Wang, Dan-Dan Zhang, YaMin Liu, Qing-Shan Shi, Xiao-Bao Xie PII:

S0162-0134(19)30549-5

DOI:

https://doi.org/10.1016/j.jinorgbio.2019.110919

Reference:

JIB 110919

To appear in:

Journal of Inorganic Biochemistry

Received date:

17 August 2019

Revised date:

3 November 2019

Accepted date:

11 November 2019

Please cite this article as: P. Yang, L.-L. Zhang, Z.-Z. Wang, et al., Nickel complexes of aroylhydrazone ligand: synthesis, crystal structure and DNA binding properties, Journal of Inorganic Biochemistry (2019), https://doi.org/10.1016/j.jinorgbio.2019.110919

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© 2019 Published by Elsevier.

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Nickel complexes of Aroylhydrazone ligand: synthesis, crystal structure and DNA binding properties

Ping Yang a, 1, Li-Lei Zhang b, 1, Zi-Zhou Wang c, Dan-Dan Zhang a, Ya-Min Liu a,

a

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Qing-Shan Shi a, *, Xiao-Bao Xie a, *

Guangdong Institute of Microbiology, Guangdong Academy of Sciences,

ro

Guangzhou 510070, China.

-p

State Key Laboratory of Applied Microbiology Southern China.

re

Guangdong Provincial Key Laboratory of Microbial Culture

College of Chemistry and Chemical Engineering, Luoyang Normal University,

Luoyang 471000, China.

School of Chemistry and Chemical Engineering, Guangzhou University, 230 Wai

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c

na

b

lP

Collection and Application. Guangdong Open Laboratory of Applied Microbiology.

Huan Xi Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, China.

*

Corresponding author at: Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China. E-mail: [email protected] (Q-S. Shi); [email protected] (X.-B. Xie). 1 Both the authors contributed equally to this work. 1

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Highlights • Three novel nickel complexes of aroylhydrazone ligand (NiL1-3) were synthesized. • The ligands were coordinated by enolate form. • Complex NiL2 and NiL3 exhibit moderate binding affinity toward calf Thymus DNA.

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• Complex NiL2 and NiL3 bind DNA through minor groove binding and

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na

lP

re

-p

ro

intercalation.

2

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Abstract In

this

work,

three

aroylhydrazone

ligands

((E)-2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide, (E)-3-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide, (E)-4-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide,

HL1; HL2; HL3)

and

and their

of

complexes with nickel (Ni(L1)2, NiL1; Ni(L2)2∙2DMF, NiL2; Ni(L3)2∙2DMF, NiL3) were prepared. The single crystal X-ray structures analysis of three compounds

ro

showed that they were neutral. The ligand adopts tridentate chelating mode. The

-p

nickel ion is six-coordinate with two O atoms and four N atoms from two ligands, and

re

forms an octahedral arrangement. The investigation of DNA binding ability by

lP

ultraviolet and fluorescence titrations showed that NiL2 and NiL3 exhibit moderate

na

binding affinity toward calf Thymus DNA. Spectroscopy, molecular docking, and molecular dynamics simulation indicated that NiL2 and NiL3 bind at the minor

Jo ur

groove of DNA through intercalation.

Graphical abstract

DNA binding of nickel aroylhydrazone complexes with different hydroxyl substitution positions was studied by ultraviolet spectra, fluorescence spectra, molecular docking, and molecular dynamics simulation.

3

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Keywords

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Pyrazine; Aroylhydrazone; Nickel complexes; Single-crystal structure; DNA binding 1 Introduction

ro

Metal complexes play an essential role in cancer treatment[1]. Various

-p

complexes from cisplatin (from the earliest discovery) to ruthenium complexes have

re

entered into their clinical trial stage[2][3][4]. These complexes have potential

lP

anticancer activity due to their strong DNA binding ability[5][6]. These complexes

na

can be tethered to DNA by covalent interaction or bound to DNA by non-covalent interaction, such as π-π stacking, electrostatic binding, H-bonding [7][8]. The

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interaction of metal complexes with DNA is much dependent on the molecular structures of ligands. In earlier studies, the ligands o-phenanthroline and bipyridine were commonly used due to their planar structure[9][10], which allowed them to insert into DNA easily. Besides, thiosemicarbazone complexes have been shown to have significant anti-proliferative effects[11][12]. Aroylhydrazone is an excellent class of metal-chelating ligand [13]. The acyl hydrazone units of the aroylhydrazone have many interesting properties, such as the degree of rigidity, the conjugated π-system, and the protonated-deprotonated site on NH group [14][15]. Some aroylhydrazones have been used as iron chelators to treat 4

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iron overload diseases or thalassemia[16][17]. Aroylhydrazone metal complexes showed various of biological activities, such as DNA/protein interaction[14][18], antioxidant[15][19], antibacterial[20][21] and anticancer[22][23]. Altering the structure and properties of aroylhydrazone compounds (such as electronegativity of heteroatom, ring size, and planarity) can affect its interaction with DNA[14][24].

of

Based on these considerations, three aroylhydrazone ligands were selected to study, which contain the methyl pyrazinylketone moiety and with different

ro

substitution positions of the hydroxyl groups on the benzene ring. The three ligands

-p

are close to planar and are potentially tridentate (N, N, O) chelators. The potential

re

H-bond donor (hydroxyl group) and the potential H-bond acceptor (uncoordinated

lP

pyrazyl N-atom) are favorably disposed to bind DNA. Moreover, the octanol-water

na

partition coefficients (log P) for ligands are about 1.9 [16], which indicates a good water solubility and lipid permeability. If they can be used as candidate drugs for the

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treatment of cancer, they will be well absorbed by the human body. As such, we synthesized their nickel complexes and determined the crystal structure for the complexes. The binding ability and mode of the complexes with DNA were investigated using ultraviolet spectra, fluorescence spectra, molecular docking, and molecular dynamics simulation.

2 Materials and methods 2.1 Reagents and instrumentation Solvents and reagents were purchased from commercial companies and used 5

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without further purification. Nuclear magnetic resonance (NMR) spectra were measured in dimethyl sulfoxide (DMSO) using a Bruker AVANCE III HD 600 (600 M). Mass spectra (MS) were measured using a Bruker maXis impact, equipped with an electrospray ion source (ESI), operated in a negative ion mode. Infrared (IR) spectra were recorded on a Bruker Tensor II using KBr pellets. Ultraviolet (UV)

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spectra were measured using a Thermo Scientific NanoDrop One. Fluorescence spectra were measured using a PerkinElmer LS 45 fluorescence spectrometer.

ro

2.2 Synthesis

-p

(E)-2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL1): According to a

re

method based on literature[25], a solution of 2-acetylpyrazine (1.22 g, 10 mmol) in

lP

ethanol (20 mL) was slowly added to a solution of 2-hydroxybenzohydrazide (1.52 g, 10 mmol) in methanol (20 mL). After the mixture solution was refluxed at 65 °C for 4

na

h, it was cooled down, suction filtered, and then washed with cold ethanol. The final product of 2.1 g with a yield of 82% was obtained by air-drying. MS, NMR, IR, and

Jo ur

UV data are shown in the Supporting information (Section S1).

(E)-3-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL2): A solution of 2-acetylpyrazine (1.22 g, 10 mmol) in ethanol (20 mL) was slowly added to a solution of 3-hydroxybenzohydrazide (1.52 g, 10 mmol) in ethanol (20 mL). The mixture solution was then refluxed at 65 °C for 4 h; after that, it was cooled down, suction filtered, and then washed with cold ethanol. The final product (1.65 g, 64% yield) was obtained by air-drying. MS, NMR, IR, and UV data are shown in the Supporting information (Section S1).

6

Journal Pre-proof (E)-4-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL3): According to a method based on literature[16], we made some modifications. 2-acetylpyrazine (1.22 g, 10 mmol) and 4-hydroxybenzohydrazide (1.52 g, 10 mmol) were mixed with 10 mL of DMSO. The solution mixture was refluxed at 75 °C for 4 h, and was after that cooled down, filtered, and washed with cold ethanol. Two grams of product (78% yield) were obtained by air-drying. MS, NMR, IR, and UV data are shown in the

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Supporting information (Section S1).

ro

NiL1: A solution of Ni(NO3)2∙6H2O (2.91 mg, 0.01 mmol) in methanol (1 mL) was

-p

slowly added to a solution of HL1 (5.12 mg, 0.02 mmol) in N,N-dimethylformamide

re

(DMF) (1 mL). After three weeks of static volatilization at room temperature, single crystals of Ni(L1)2 were obtained with a yield (calculated based on metal ions) of

na

(Section S1).

lP

86%. Elemental analysis and IR data are shown in the Supporting information

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NiL2: A solution of Ni(NO3)2∙6H2O (2.91 mg, 0.01 mmol) in methanol (1 mL) was slowly added to a solution of HL2 (5.12 mg, 0.02 mmol) in DMF (1 mL). After three weeks of static volatilization at room temperature, single crystals of Ni(L2)2∙2DMF were obtained with a yield (calculated based on metal ions) of 80%. Elemental analysis, IR, and UV data are shown in the Supporting information (Section S1).

NiL3: A solution of NiCl2∙6H2O (2.38 mg, 0.01 mmol) in methanol (1 mL) was slowly added into a solution of HL3 (5.12 mg, 0.02 mmol) in DMF (1 mL). After three weeks of static volatilization at room temperature, single crystals of Ni(L3)2∙2DMF were obtained with a yield (calculated based on metal ions) of 88%. 7

Journal Pre-proof Elemental analysis, IR, and UV data are shown in the Supporting information (Section S1).

2.3 Single-crystal X-ray diffraction Data for the complex of NiL1 was recorded with a CCD area detector and Oxford Gemini S Ultra to collect the diffraction data (Mo-Kα, λ = 0.071073 nm) at room temperature. Due to free solvent molecules in the lattice, data for the complexes

of

of NiL2 and NiL3 were recorded with a CCD area detector and Oxford Gemini S

ro

Ultra to collect the diffraction data (Cu-Kα, λ = 0.154178 nm) at room temperature.

-p

The SADABS program was used for absorption correction[26]. The crystal structure

re

was solved through the direct methods [27], and then, the SHELXTL program package [28][29] was used to conduct a full-matrix least-square structure refinement

lP

based on F2. The non-hydrogen atoms were all anisotropically refined. All H atoms

na

were placed in geometrically idealized positions, with C-H = 0.93 Å (sp2), C-H = 0.96 Å (sp3) and O-H = 0.82 Å[30]. Further details of the X-ray structural analyses

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for complexes NiL1, NiL2, and NiL3 are given in Table 1. Selected bond lengths and angles for NiL1, NiL2, and NiL3 are listed in the Supporting information (Table S1).

2.4 UV titration Calf Thymus DNA (ctDNA) was resuspended in 10 mM Tris-HCl buffers, pH 7.0 containing 50 mM NaCl. The ratio of UV absorbance at 260 and 280 nm (A260/A280) of ctDNA stock solution was 1.9, which indicates that it is free of protein contamination. The DNA concentration was expressed as monomer units (nucleotides) and determined by UV absorption at 260 nm using a molar absorptivity of 6600 8

Journal Pre-proof L∙mol−1∙cm−1. The complex was dissolved in DMSO and then diluted with water. Due to lower water solubility of the complex, the concentration of DMSO in the final solution was kept lower than 5% (v/v) after the complexes were mixed with DNA. All measurements were performed at room temperature. For titration, 500 μL of 0.47 mM ctDNA solutions were mixed with 500 μL of H2O and different concentrations of the complex solutions (0.176 mM, 0.352 mM, 0.528 mM, 0.704 mM, 0.880 mM, 1.056

of

mM, 1.232 mM, 1.408 mM, 1.584 mM, and 1.760 mM). The absorbance (λmax = 260

ro

nm) of ctDNA was then measured. Comparatively, 500 μL of 0.088 mM NiL2 and

-p

0.176 mM NiL3 solutions were mixed with 500 μL of H2O and different

re

concentrations of ctDNA solution (0.047 mM, 0.141 mM, 0.235 mM, 0.329 mM, and

lP

0.423 mM). It is to study the effect of ctDNA in the absorption of the complexes. The

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λmax = 389 and 309 nm.

na

absorption of NiL2 was measured at λmax = 383 nm, and that of NiL3 was measured at

2.5 Fluorescence titration

To further clarify the interaction of the complexes with DNA, steady-state competitive binding experiment was carried out. The GelStain GS101-02 dye was purchased from TransGen Biotech, Beijing, China. Dye (10×) was mixed with 4.7 mM ctDNA for 1 h and then diluted 1:10 to obtain working solution of ctDNA-dye complex (concentration = 0.47 mM). Then, 200 μL of the working solution of ctDNA-dye complex was mixed with 200 μL of H2O and various concentrations of the complex solutions (0.176 mM, 0.352 mM, 0.528 mM, 0.704 mM, 0.880 mM, and 9

Journal Pre-proof 1.056 mM). After that, the change of fluorescence emission spectra at λem = 600 nm (λex = 254) of the mixture solutions was recorded to study the effect of the complexes on the binding of ctDNA to dye. To further confirm the binding mode, the fluorescence titration of DAPI-DNA (DAPI is 4',6-diamidino-2-phenylindole) and methyl green-DNA by complexes NiL2 and NiL3 were carried out. DAPI and methyl green are minor and major groove’s binders

of

of DNA, respectively[31][32][33]. 200 μL ctDNA (0.47 mM) reacted with 8 μL DAPI

ro

staining solution (C1005) and 2 μL methyl green staining solution (C0115, Beyotime

-p

Biotech, Shanghai, China) for 5 minutes, respectively. Then the solution of

re

DAPI-DNA and methyl green-DNA were mixed with 200 μL H2O and complex

lP

solutions (0.088mM, 0.176 mM, 0.352 mM, 0.528 mM, 0.704 mM, and 0.880 mM)

na

respectively. The fluorescence emission spectra of λem = 454 nm (λex = 364) and λem =

Jo ur

677 nm (λex = 633) were recorded.

2.6 In silico studies

The initial conformations of NiL2 and NiL3 were obtained from the single crystal X-ray structure data. The crystal structure of human B-DNA (PDB ID: 1BNA) (The sequence is CGCGAATTCGCG) was downloaded from the RCSB Protein Data Bank[34][35]. The heteroatoms and water molecules were removed from the B-DNA before docking simulation. Molecular docking studies on ligands and B-DNA were carried out using the AutoDock Vina program[36]. During the process of docking simulation, the whole B-DNA crystal structure was considered active sites. Finally, 10

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NiL2 and NiL3 were docked into the B-DNA to find and select the lowest energy among 20 docking modes given by cluster analysis, respectively. Molecular dynamics (MD) simulation of DNA-NiL2 / DNA-NiL3 complex was carried out using GROMACS 2019 software package[37]. The lowest binding energy conformation for each complex was employed as the initial conformation. The

of

AMBER99SB-ILDN force field was applied to describe the DNA receptor, whereas the universal force field was performed to describe ligands. Each system was

ro

immersed in a periodic water box of a cube shape. To hold the system at an

-p

electrically neutral state, we added appropriate numbers of sodium ions. The

re

simulation was started by energy minimization for 500 steps with the steepest descent

lP

method, followed by 100 ps restricted dynamics relaxation. Then, each system was

na

gradually heated from 0 K to 310 K within 200 ps. A 10 ns MD simulation with a time step of 2 fs was performed. During the simulation, the PME (Particle Mesh Ewald)

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algorithm was employed to deal with the long-range electrostatic interactions. The velocity-rescaling method was used to control the temperatures of the DNA-NiL2 / DNA-NiL3 complex and the remaining atoms in this system.

3 Results and discussion 3.1 Spectroscopic characterization Compared with the raw materials’ spectra, the IR spectrum of HL1-HL3 showed new peaks at 1608, 1613, and 1609 cm-1 due to the stretching vibration of imine in hydrazone. For HL1, a peak at 752 cm-1 belongs to the out-of-plane bending vibration 11

Journal Pre-proof of the ortho-disubstituted benzene ring. For HL2, two peaks at 745 and 681 cm-1 belong to the out-of-plane bending vibration of the meta-disubstituted benzene ring. For HL3, a peak at 847 cm-1 belongs to the out-of-plane bending vibration of the para-disubstituted benzene ring. The carbonyl vibration at 1651, 1658, and 1656 cm-1 of HL1-HL3 disappeared in their complexes. The IR spectra of NiL1-NiL3 appeared new bands at 1596, 1580, and 1596 cm-1, which indicates that -NH-C=O was

of

adjusted to an enolate form -N=C-OH. The result is consistent with the single crystal

ro

X-ray analysis (see below). The IR spectra of these complexes are typical of other

-p

aroylhydrazone analogs[16].

re

The UV absorption peaks of HL1-HL3 in DMSO at room temperature appeared

lP

at 316 nm (ε = 2.34 × 104 L∙mol−1∙cm−1), 306 nm (ε = 4.67 × 104 L∙mol−1∙cm−1) and

na

309 nm (ε = 4.75 × 104 L∙mol−1∙cm−1), corresponding to π-π* transition. Similar peaks at 307 nm (ε = 3.25 × 104 L∙mol−1∙cm−1) and 315 nm (ε = 3.68 × 104 L∙mol−1∙cm−1)

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were also observed in the spectra of NiL2 and NiL3, respectively (Due to poor solubility of NiL1, it was not tested). New peaks at 423 nm (ε = 1.33 × 104 L∙mol−1∙cm−1) and 423 nm (ε = 1.88 × 104 L∙mol−1∙cm−1) in the spectra of NiL2 and NiL3, respectively, were attributed to metal-to-ligand charge transfer (MLCT) transition[19]. Consequently, the complex solutions show a yellow-green color, which is different from the colorless ligand solutions. It also shows that the complexes are stable in solution.

3.2 Crystal structure 12

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As shown in Table 1, the complexes NiL1-NiL3 belong to the orthorhombic system. The nickel center is six-coordinate with two O atoms and four N atoms from two aroylhydrazone ligands and forms an octahedron structure (Fig. 1d). In free aroylhydrazone ligand, the methyl group (C6) is in a syn position to the pyrazine ring nitrogen atom (N1)[16][25]. Here, the C6 and N1 of the complex is an anti-

of

conformation. Because the ligand is disposed to coordinate a metal ion through the pyrazinyl N-atom (N1), imine N-atom (N3), and carbonyl O-atom (O1) after rotation

ro

of the pyrazine group by 180° about C4–C5. The torsion angle of N1–C4–C5–C6 is in

-p

the ranges −175.0(5) to −179.4(5)°. In each case, the ligands bind Ni by a tri-dentate

re

chelating, and the NH group next to the carbonyl deprotonated to generate an enolate

lP

form of the ligand (Fig. 1). The structure is like that found for the related Co, Ni, and

na

Zn complex[38][39][40]. The oxygen atom (O2) on the phenolic group did not deprotonate and stuck to the outside of the coordination sphere to form intermolecular

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hydrogen bonds, which is conducive to the growth and stability of crystals. Hydrogen bonds are shown in Table S2, Table S3, and Table S4. Since NiL1 contains a strong intramolecular hydrogen bond O2-H2A∙∙∙N4 (2.553(7) Å, 147.7°), its solubility is weak. The Ni-O and Ni-N bond lengths are in the ranges 2.080(2)-2.097(4) and 1.973(2)-2.144(5) Å (Table S1). Bond length is close to the statistic value of (N, N, O) tridentate chelated nickel complexes (N–Ni 2.093±0.06, N(middle)–Ni 1.973±0.041, O–Ni 2.107±0.083 Å) in CCDC (Sample is 311). The angle between coordination bonds was like that of nickel analogs [38] (Table S1). They deviate from 180° and 90°, indicating that the octahedron is a little distorted. 13

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Table 1. Crystal data and structure refinement for NiL1-NiL3. NiL2

NiL3

Empirical formula

C26H22N8NiO4

C32H36N10NiO6

C32H36N10NiO6

Formula weight

569.22

715.42

715.42

Temperature (K)

298(2)

298(2)

298(2)

Wavelength (Å)

0.71073

1.54178

1.54178

Crystal system

Orthorhombic

Orthorhombic

Orthorhombic

Space group

Aba2

Pbcn

Pbcn

a (Å)

12.5534(7)

11.6963(2)

11.6656(3)

b (Å)

18.8700(13)

9.40620(10)

9.6490(3)

c (Å)

10.0629(8)

31.3167(5)

30.3106(8)

Volume (Å3) Z

2383.7(3)

3445.39(9)

3411.80(16)

4

4

4

Density (calculated) (Mg/m3) Absorption coefficient (mm-1)

1.586

1.379

1.393

1.302

1.315

F(000)

1176

1496

1496

Reflections collected

2400

Independent reflections, Rint

1542, 0.0269

Data / restraints / parameters Goodness-of-fit on F2 R1 [I > 2σ(I)]

ro -p

0.867

6435

2773, 0.0236

2697, 0.0197

1542 / 1 / 179

2773 / 0 / 226

2697 / 0 / 226

1.039

1.066

1.039

0.0359

0.0439

0.0377

0.0959

0.1189

0.1036

0.268 and -0.220

0.446 and -0.349

0.274 and -0.253

re

19218

lP

wR2 (all data)

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na

Largest diff. peak and hole (e.Å-3)

of

NiL1

14

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Figure 1 ORTEP drawing of complex NiL1 (a), NiL2 (b), NiL3 (c) and polyhedral of central nickel (d) with thermal ellipsoids 30% probability. For clarity, hydrogen atoms and solvent have been neglected. Symmetry code: A: -x, 1-y, z (in NiL1); -x, y, 3/2-z (in NiL2); -x, y, 1/2-z (in NiL3).

of

3.3 UV titration To investigate the binding affinity with the complex and DNA, UV titration was

ro

carried out. At a constant ctDNA concentration, the characteristic absorption of

-p

double-stranded DNA at 260 nm increased from the increasing concentration of the

re

complexes (Fig. 2a and Fig. 2b). At a constant complex concentration, the

lP

characteristic absorption of the complexes NiL2 and NiL3 at 383nm/389nm increased

na

from the increase in ctDNA concentration (Fig. 2c and Fig. 2d). Hyperchromicity has also been reported in the literature[41][42][43]. It appears that the complexes undergo

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a distortion of the coordination sphere after binding to DNA resulting in an enhancing of the MLCT bands[44]. The characteristic absorption of the complex NiL3 at 309 nm decreased with the increase of ctDNA concentration (Fig. 2d). The hypochromism may be due to an increasing of electron cloud density around the complexes after binding to DNA, leading to a decreasing in the transition probability of π-π* intraligand[45][46][47]. Although the Ni(II) complex showed hypochromism of about 9.8% without any shift at 309 nm, the complexes interact with DNA most likely through an intercalation mode[19]. The DNA binding constants of NiL2 and NiL3 complexes, calculated by the equation: [DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb (εb − 15

Journal Pre-proof εf)[48][49], were 2 ×104 and 1×104 M−1. The binding constants have the same order of magnitude as that of the six-coordinated nickel analogs (~104) [50] but are lower than that of the four-coordinated nickel analogs (~105) [13]. Gibbs Free Energy of the binding of the complexes NiL2 and NiL3 with ctDNA were calculated to be −24.5 and −22.8 kJ/mol (−5.8 and −5.4 kcal/mol), using the equation: ΔG = −RTlnKb, where

of

R = 8.314 J·K−1·mol−1, and T = 298 K. The results indicate spontaneous binding

3

2

1

0 240

260

280

300

320

340

360

Absorbance

2.0

1.5

1.0

0.5

420

-p 3

2

1

0 240

440

260

280

300

320

340

360

380

400

420

440

Wavelength (nm)

0.423mM ctDNA 0.329mM ctDNA 0.235mM ctDNA 0.141mM ctDNA 0.047mM ctDNA 0mM ctDNA

(d) 3.5

0.423mM ctDNA 0.329mM ctDNA 0.235mM ctDNA 0.141mM ctDNA 0.047mM ctDNA 0mM ctDNA

3.0

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(c) 2.5

400

na

Wavelength (nm)

380

4

2.5

Absorbance

Absorbance

4

5

Absorbance

5

re

(a)

1.760mM NiL3 1.584mM NiL3 1.408mM NiL3 1.232mM NiL3 1.056mM NiL3 0.880mM NiL3 0.704mM NiL3 0.528mM NiL3 0.352mM NiL3 0.176mM NiL3 0mM NiL3

(b)

lP

1.760mM NiL2 1.584mM NiL2 1.408mM NiL2 1.232mM NiL2 1.056mM NiL2 0.880mM NiL2 0.704mM NiL2 0.528mM NiL2 0.352mM NiL2 0.176mM NiL2 0mM NiL2

ro

between these compounds and ctDNA.

2.0 1.5 1.0 0.5 0.0 240 260 280 300 320 340 360 380 400 420 440 460 480 500

0.0 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Wavelength (nm)

Wavelength (nm)

Figure 2 UV spectra: (a) titration of NiL2 against ctDNA; (b) titration of NiL3 against ctDNA; (c) titration of ctDNA against NiL2; and (d) titration of ctDNA against NiL3.

3.4 Fluorescence titration Further support concerning the binding of complexes to DNA was obtained using 16

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a fluorescence titration to have a better insight into the binding effects. The ctDNA bound to dye emitted a robust orange light of 600 nm. However, the fluorescence intensity was weakened and quenched after the complex was added. This phenomenon suggests that the complexes can replace the dye on the DNA-dye (Fig. 3a and Fig. 3b). The quenching process conforms to the linear Stern-Volmer

of

equation[51][52]: I0/I = 1 + Ksqr, where I0 and I represent the fluorescence intensity without and with the addition of the complex, Ksq is the static quenching constant, and

ro

r is the concentration ratio of the complex and DNA. The Ksq of the complexes NiL2

analog

(E)-N'-(1-(3-methylpyrazin-2-yl)ethylidene)benzohydrazide

re

the

-p

and NiL3 were 1.17 and 0.68 (Fig. 3c and Fig. 3d). The Ksq value is close to that of nickel

lP

complexes[52]. The Ksq of HL2 and HL3 was 0.41 and 0.38 (Fig. S1). The results

na

suggested that the binding interaction of the Ni(II) complex with ctDNA was stronger than that of the free ligand. The formation of nickel complexes could enhance the

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biological activity of free aroylhydrazone[23].

160 140

Intensity

120

180 160 140 120

Intensity

180

0mM NiL3 0.176mM NiL3 0.325mM NiL3 0.528mM NiL3 0.704mM NiL3 0.880mM NiL3 1.056mM NiL3

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(d) 2.8 (c) 4.0

y = 0.6868x + 0.9893 R2 = 0.987

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y = 1.1769x + 0.9988 R2 = 0.989

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Figure 3 (a) and (b) are fluorescence titration of NiL2 and NiL3 against ctDNA-dye.

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λex = 254 nm. (c) and (d) are the variation of fluorescence intensity ratio with the

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concentration ratio of NiL2 and NiL3 to DNA at 600 nm.

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The fluorescence titration of NiL2 and NiL3 to DAPI-DNA showed that the

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fluorescence intensity decreases with the increase of complex concentration (Fig. 4a

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and Fig. 4b). But the fluorescence intensity of methyl green-DNA unchanged with the

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increase of complex concentration (Fig. 4c and Fig. 4d). These results indicate that

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NiL2 and NiL3 bind to the minor groove of DNA.

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Figure 4 Fluorescence spectra of DAPI-DNA with NiL2 (a), DAPI-DNA with NiL3

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(b), methyl green-DNA with NiL2 (c), and methyl green-DNA with NiL3 (d).

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3.5 Molecular docking and molecular dynamics (MD) simulation

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To better understand the binding mode and force of the complex and DNA,

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molecular docking and MD simulation were carried out using the structures of B-DNA (PDB ID: 1BNA) and aroylhydrazone complexes NiL2 and NiL3. The

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docking data indicated that the least relative binding energies of DNA-NiL2 and

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DNA-NiL3 were −8.6 and −8.8 kcal/mol. The calculated binding energy difference between DNA-NiL2 and DNA-NiL3 is small (0.2 kcal/mol), which is an agreement with the experimental difference (0.4 kcal/mol) obtained from the UV spectroscopic data. It indicated that the binding affinity of the two complexes to DNA is similar. After 10 ns MD simulation, the systems of DNA-NiL2 and DNA-NiL3 were equilibrated with no visible RMSD fluctuations (Fig. 5). Video of molecular movements can be seen in the supporting information. The results support that parallel-displaced π-π stacking interaction is the dominant force in stabilizing the combination of DNA and complexes (Fig. 6). It is consistent with the experimental data of spectra titration. NiL2 and NiL3 bind to the minor groove of DNA through 19

Journal Pre-proof inserting phenolic group into DC3:DG22 – DG4:DC21 and DG2:DC23 – DC3:DG22 base pairs [53][54][55]. The position of the hydroxyl group has little effect on the binding mode of nickel complexes with DNA. (a)

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Figure 5 MD simulation of NiL2 (a) and NiL3 (b) with B-DNA (PDB ID: 1BNA)

Figure 6 The lowest binding energy conformation of NiL2 (A) and NiL3 (B) bound to B-DNA (PDB ID: 1BNA). DA, light gray; DT, pink; DG, green; DC, gray.

4 Conclusion Three nickel aroylhydrazone complexes were synthesized, and their crystal structures were determined. The metal to ligand ratio of complexes is 1:2. The nickel center is a six-coordinate octahedral configuration. The acyl hydrazone unit of the ligand was converted into an enol structure. The ligands are tridentate chelate metal 20

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ions. The spectroscopy, molecular docking, and molecular dynamics simulation results showed that the complex NiL2 and NiL3 bind to the minor groove of DNA through intercalation.

Acknowledgement

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This work was supported by the National Natural Science Foundation of China (Grant 41701349), GDAS' Project of Science and Technology Development

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(2018GDASCX-0912) and Nanyue Talent Fund (GDIMYET20180205).

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Supplementary material

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Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge

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CB21EZ, UK. Copies of the data can be obtained free of charge on quoting the

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depository numbers CCDC-1906506, CCDC-1906519, and CCDC-1906530 for NiL1, NiL2 and NiL3 (Fax: +44-1223-336-033; E-Mail: [email protected], http://www.ccdc.cam.ac.uk).

MS, NMR, IR, UV, and selected bond lengths and angles of compounds were shown in the Supporting information. The video is in another uploaded file.

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Journal Pre-proof Conflict of Interest

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There are no conflicts to declare.

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