基于两性羧酸配体的Cu(Ⅱ)、Zn(Ⅱ)、Co(Ⅱ)、Mn(Ⅱ)配合物的合成、晶体结构及其与DNA相互作用
English
Synthesis, Crystal Structures and DNA Interaction of Cu(Ⅱ), Zn(Ⅱ), Co(Ⅱ), and Mn(Ⅱ) Complexes Derived from Zwitterionic Carboxylate Ligand
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Key words:
- zwitterionic carboxylate ligand
- / transition metal complexes
- / crystal structures
- / DNA
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0. Introduction
In the past decades, the metal complexes of carboxylates have been attracting considerable interest in biochemistry due to that they contain a bioactive group of carboxylate that has strong coordination ability and diverse coordination modes. Therefore, to date, a large number of metal complexes of carboxylates have been synthesized and some of them have exhibited promising biological activities, such as anti-tumor[1], antibacterial[2-3], hydrolytic or oxidative cleavage of DNA[4-5], DNA detection[6]. It is worth noting that more and more researchers used zwitterionic carboxylate ligands to construct complexes that have unique structures and potential applications in biological probes[7], ion exchange[8], and so on[9-10]. On the other hand, metal complexes that contain conjugated aromatic rings or with positively charged functional groups, such as quaternary ammonium, may have strong DNA binding abilities[11-12]. However, to date, the metal complexes based on the zwitterionic carboxylate ligands interacting with DNA have been reported rarely[13-15]. Therefore, further systematic investigation of these kinds of zwitterionic carboxylate complexes is necessary.
In an earlier study, we reported a water - soluble copper complex based on a zwitterionic carboxylate ligand, N-(4-carboxybenzyl)pyridinium bromide ((HCbp)Br)[16]. We found that this zwitterionic carboxylate complex showed moderate DNA-binding and cleaving activity. In addition, it is known that polypyridyl ligands, such as 2, 2' - bipyridine (bipy) and 1, 10 phenanthroline (phen), are chelating ligands for transition metal ions[17]. Because of the planar, rigid, and hydrophobic features of the polypyridyl ligands, their complexes have been used as intercalating or groove binding agents for DNA, which may contribute to DNA cleaving and anti-tumor activities[18]. Therefore, we decided to increase the size of the fused ring of the ligand which is based on quaternized isoquinoline instead of quaternized pyridinium to obtain complexes with stronger DNA interactions and better DNAcleaving activities. With this thought in mind, herein we describe the synthesis of a new ligand of N - (4 carboxybenzyl)isoquinolinium bromide ((HCbiq)Br, Scheme 1) and its four metal complexes, [Cu2(Cbiq)4 (H2O)2]Br4·2H2O (1), [Zn(Cbiq)2(H2O)2]Br2·Cbiq·H2O (2), and [M3(Cbiq)8(μ-OH)2(H2O)2](ClO4)4·7H2O (M=Co (3), Mn (4)). The interactions of complexes 1 - 4 with DNA were studied.
Scheme 1
1. Experimental
1.1 Generals
1H and 13C NMR spectra were recorded in DMSOd6 using a Varian Mercury 400 spectrometer and TMS as an internal reference. ESI - MS spectra were measured on Waters UPLC/Quattro Premier XE. IR spectra were recorded on a Nicolet Magna-IR 550. Elemental analyses for C, H, and N were performed on an EA1110 CHNS elemental analyzer. Agarose gel electrophoresis (GE) was conducted on a DYY-8C electrophoresis apparatus and DYCP - 31DN electrophoresis chamber and detected on Alpha Hp 3400 fluorescence and visible light digital image analyzer. UV - Vis and fluorescence spectra were measured on a TU - 1901 spectrophotometer and a HITACHI F-2500 spectrofluorimeter, respectively.
Calf - thymus (CT) DNA and plasmid pBR322 DNA were obtained from Sigma - Aldrich and Takara Chemical Co., respectively. Their solutions were prepared in 5 mmol·L-1 Tris - HCl buffer (5 mmol·L-1 NaCl, pH 7.0). The concentration of CT DNA was determined spectrophotometrically using the molar extinction coefficient of 13 200 L·mol-1·cm-1 per base pair (bp) at 260 nm[19]. All the other chemicals and reagents were obtained from commercial sources and used without further purification. Buffer solutions were prepared in triply distilled deionized water.
1.2 Synthesis of (HCbiq)Br
To a solution of 4-(bromomethyl)benzoic acid (2.15 g, 10 mmol) in acetone (120 mL) was added drop slowly a solution of isoquinoline (1.3 g, 10 mmol) in acetone (30 mL) at room temperature. After stirring for 24 h, the white precipitates formed were collected through filtration and washed with ethyl acetate (5 mL) and ether (5 mL) to afford (HCbiq)Br (3.11 g, 75%). 1H NMR (400 MHz, DMSO-d6): δ 10.38 (s, H-9, 1H), 8.88 (dd, J=6.8, 1.2 Hz, H-1, 1H), 8.65 (d, J=6.8 Hz, H-2, 1H), 8.57 (d, J=8 Hz, H-7, 1H), 8.39 (d, J=8 Hz, H-4, 1H), 8.28-8.32 (m, H-5, 1H), 8.10-8.13 (m, H-6, 1H), 8.00 (d, J=8 Hz, H-13 and H-15, 2H), 7.71 (d, J=8 Hz, H-12 and H-16, 2H), 6.13 (s, H-10, 2H) (Fig.S1, Supporting information). 13C NMR (100 MHz, DMSO-d6): δ 166.6 (C-17), 150.4 (C-9), 138.7 (C-11), 137.2 (C-3), 137.1 (C - 5), 134.8 (C - 1), 131.3 (C - 14) 131.3 (C - 6), 130.6 (C-7), 129.9 (C-13 and C-15), 128.9 (C-12 and C - 16), 127.3 (C- 3 and C- 4), 126.3 (C- 2), 62.6 (C- 10) (Fig.S2). ESI-MS m/z: 264.5 ([M-Br]+). Main IR bands (KBr disc, cm-1): 3 442(sb), 3 048(m), 3 008(m), 1 700 (s), 1 641(w), 1 608(w), 1 444(w), 1 398(w), 1 370(w), 1 222(m), 1 115(s), 1 066(s), 952(m), 822(m), 754(m), 538(m).
1.3 Synthesis of complexes 1-4
(HCbiq) Br (137.7 mg, 0.4 mmol) was dissolved in MeOH (4 mL), and the pH value of the solution was adjusted to 7.0 with 0.1 mol·L-1 NaOH solution. Then a solution of CuSO4·5H2O (60 mg, 0.2 mmol) in MeOH (2 mL) was added. The resulting mixture was stirred for 1 h. The formed precipitates were collected by filtration, re-dissolved in H2O (15 mL), and allowed to stand at ambient temperature for about one week to produce the blue crystals of complex 1 (120 mg, 76%). Elemental Anal. Calcd. for C68H60Br4N4O12Cu2(%): C 51.96, H 3.85, N 3.56. Found(%): C 52.09, H 3.99, N 3.72. Main IR bands (KBr disc, cm-1): 3 443 (sb), 3 130 (sb), 1 610 (m), 1 445(m), 1 399 (s), 1 123 (s), 1 065 (s), 952 (m), 865 (m), 539 (m).
Complex 2 (98 mg, 69%) was prepared from (HCbiq)Br (137.7 mg, 0.4 mmol) and ZnCl2 (27 mg, 0.2 mmol), using similar procedures as described for complex 1. Elemental Anal. Calcd. for C51H45Br2N3O9Zn (%): C 57.29, H 4.24, N 3.93. Found(%): C 57.45, H 4.41, N 3.85. IR (KBr disc, cm-1): 3 443 (sb), 3 065 (m), 1 616 (s), 1 565 (w), 1 396 (m), 1 369 (s), 1 151 (s), 1 070 (s), 954 (m), 860 (m), 811 (w), 544 (m).
(HCbiq) Br (137.7 mg, 0.4 mmol) was dissolved in MeOH (4 mL), and the pH value of the solution was adjusted to 7.0 with 0.1 mol·L-1 NaOH solution. Then a solution of Co(ClO4)2·6H2O (73.2 mg, 0.2 mmol) in MeOH (2 mL) was added. The resulting mixture was stirred at room temperature for 3 h and filtered to give a clear solution. Diethyl ether was allowed to diffuse into the filtrate. After standing at ambient temperature for two days, the formed pick crystals were collected by filtration, washed with Et2O, and dried in vacuo to get complex 3 (81 mg, 56%). Elemental Anal. Calcd. for C136H124Cl4N8O43Co3(%): C 56.77, H 4.34, N 3.89. Found (%): C 56.56, H 4.26, N 3.71. IR (KBr disc, cm-1): 3 444 (sb), 3 127 (w), 1 639 (w), 1 609 (m), 1 563(m), 1 443 (m), 1 403 (m), 1 264 (w), 1 120 (s), 1 066 (s), 995 (m), 951 (m), 865 (m), 819 (w), 563 (m), 540 (m), 515 (m).
Complex 4 (86 mg, 60%) was prepared from (HCbiq)Br (137.7 mg, 0.4 mmol) and Mn(ClO4)2·6H2O (73.2 mg, 0.2 mmol), using similar procedures as described for complex 3. Elemental Anal. Calcd. for C136H124Cl4N8O43Mn3(%): C 57.01, H 4.36, N 3.91. Found(%): C 57.14, H 4.43, N 3.84. IR (KBr disc, cm-1): 3 385 (sb), 1 638 (m), 1 611 (m), 1 566 (w), 1 403 (s), 1 151 (s), 1 072(s), 953 (w), 860 (m), 547 (m).
1.4 Structures determination for complexes 1-4
All the measurements were made on a Rigaku Mercury CCD X - ray diffractometer by using graphite monochromated Mo Kα (λ=0.071 073 nm). Crystals of complexes 1-4 were mounted with grease at the top of glass fiber. Cell parameters were refined by using the program CrystalClear. The collected data were reduced by using the program CrystalStructure while an absorption correction (multiscan) was applied. The structure was solved and refined using the SHELXTL program suite[20], equipped with XS[21] and XL programs[22]. Leastsquares refinement routines were performed in refinement. Hydrogen atoms on carbon atoms were placed in calculated positions, and their coordinates and displacement parameters were constrained to ride on the carrier atom (C—H 0.097 nm, Uiso(H) =1.2Ueq(C) for alkyl H atoms; C—H 0.093 nm, Uiso(H) =1.2Ueq(C)) for aromatic H atoms. Hydrogen atoms on oxygen atoms were located from difference density maps. A summary of the key crystallographic information for complexes 1-4 is listed in Table 1.
Table 1
Parameter 1 2 3 4 Molecular formula C68H60Br4N4O12Cu2 C51H45Br2N3O9Zn C136H124Cl4N8O43Co3 C136H124Cl4N8O43Mn3 Formula weight 1 571.92 1 069.09 2 877.02 2 865.05 Crystal system Monoclinic Monoclinic Rhombohedral Rhombohedral Space group C2/c P21 R3 R3 a/nm 3.028 31(11) 1.012 41(12) 3.616 12(11) 3.661 23(12) b/nm 1.710 53(13) 2.720 92(13) 3.616 12(11) 3.661 23(12) c/nm 1.379 12(12) 2.053 81(12) 3.079 59(14) 3.125 32(18) β/(°) 102.121(3) 91.081(3) V/nm3 6.984 6(8) 5.656 6(8) 34.875(2) 36.281(3) Z 4 4 9 9 T/K 291(2) 291(2) 291(2) 291(2) Dc/(g·cm-3) 1.495 1.255 1.233 1.180 μ/mm-1 2.958 1.897 0.463 0.371 Total reflection 31 641 42 020 84 481 92 574 2θmax/(°) 52.00 52.00 52.00 52.00 Unique reflection 6 851 (Rint=0.069 1) 20 940 (Rint=0.018 6) 15 113 (Rint=0.073 3) 15 797 (Rint=0.066 8) Observed reflection [I > 2σ(I)] 4 423 16 594 10 376 11 483 Number of parameters 424 1 271 912 912 Ra 0.061 2 0.050 5 0.063 9 0.058 6 wRb 0.133 2 0.123 3 0.138 9 0.110 0 GOFc 1.072 1.024 1.071 1.075 (Δρ)max, (Δρ)min/(e·nm-3) 780, -424 441, -307 295, -317 295, -251 a R=∑||Fo|-|Fc||/|Fo|; b wR=[∑w(Fo2-Fc2)2/∑w(Fo2)2]1/2; c GOF=[∑w(Fo2-Fc2)2/(n-p)]1/2, where n is the number of reflections and p is the total numbers of parameters refined. CCDC: 2165985, 1; 2165991, 2; 2166081, 3; 2166082, 4.
1.5 DNA cleavage
The cleavage experiments were conducted by using methods similar to those described previously[23-25]. Specifically, a mixture of pBR322 DNA (0.5 g·L-1, 0.7 µL) and each of complexes 1-4 was diluted with 5 mmol· L-1 Tris- HCl buffer (5 mmol·L-1 NaCl, pH 7.0) to 16 µL and incubated at 37 ℃ for 5 h. The reaction was quenched by adding loading buffer containing 0.035% bromophenol blue, 36% glycerol, 30 mmol·L-1 EDTA, and 0.05% xylene cyanol FF. The solution was then loaded on 1% agarose gel containing EB (ethidium bromide, 1.0 mg·L-1) and analyzed with electrophoresis in Tris-acetate-EDTA (TAE) buffer (pH 8.0). Bands were visualized by UV light and photographed.
The kinetics for the DNA cleavage was investigated at 37 ℃ for different intervals of time, by varying the concentrations of complex 1 in 5 mmol·L-1 Tris - HCl buffer (5 mmol·L-1 NaCl, pH 7.0) [23-25]. The percentage of the supercoiled DNA form was determined and plotted against time for each concentration of complex 1. The data were fitted with a single - exponential curve (pseudo-first-order kinetics) to give the kobs values. The kobs values were then plotted versus the concentrations (c1) of complex 1 (Eq.1), allowing the determination of the corresponding maximal first-order rate constant kmax and Michaelis constant KM.
$k_{\mathrm{obs}}=k_{\max } c_1 /\left(K_{\mathrm{M}}+c_1\right) $ (1) For mechanistic investigations, inhibition reactions were carried out in the presence of DMSO (1.0 mol·L-1), MeOH (1.0 mol·L-1), NaN3 (0.1 mol·L-1), KI (0.1 mol·L-1), and EDTA (0.1 mol·L-1), followed by the addition of complex 1.
1.6 DNA binding experiment
EB displacement experiments of (HCbiq)Br and complexes 1-4 were performed by keeping the concentrations of CT DNA and EB constant, while gradually increasing the concentrations of (HCbiq)Br or each of the metal complexes. Thus, to a solution of CT DNA (2.10 µmol·L-1) and EB (2.63 µmol·L-1) in 5 mmol· L-1 Tris - HCl (5 mmol·L-1 NaCl, pH 7.0) were added aliquots of a solution of each compound containing CT DNA (2.10 µmol·L-1) and EB (2.63 µmol·L-1) in the same buffer. The corresponding fluorescence spectra were measured (λex=510 nm) until saturation was observed. The apparent binding constant (Ka) was obtained by analyzing the relative fluorescence intensity (I/I0) as a function of the concentration of each complex[26].
1.7 Molecular docking
The molecule structure of complex 1 was constructed and optimized using Molecular Operating Environment (MOE) package[27], while the 3D structure of DNA (PDB ID: 453D) was constructed and optimized by the Chimera package[28]. The initial structures of DNA with compound 1 were manually built by molecular docking in MOE to give the binding energies[29]. The visual analysis of binding modes was obtained in the force field by Python molecule (PyMOL)[30].
2. Results and discussion
2.1 Synthesis of (HCbiq)Br and complexes 1-4
The synthetic route of (HCbiq)Br and complexes 1-4 is shown in Scheme 2. Thus, the reaction of isoquinoline with 4-(bromomethyl)benzoic acid in acetone afforded (HCbiq)Br in 75% yield. Treatment of (HCbiq)Br with 1/2 Equiv. of the corresponding metal salts gave complexes 1-4 in 56%-76% yields.
Scheme 2
(HCbiq) Br was characterized by 1H NMR, 13C NMR, ESI-MS, elemental analyses, and IR, while the structures of complexes 1-4 were confirmed by singlecrystal X-ray crystallography, elemental analyses, and IR. The elemental analyses of (HCbiq)Br and complexes 1 - 4 were consistent with their chemical formula, and the 1H NMR and 13C NMR spectra of (HCbiq)Br were also in full agreement with the given structures. Complexes 1-4 were further characterized by single-crystal X-ray crystallography.
2.2 Crystal structures of complexes 1-4
2.2.1 Structure of complex 1
Complex 1 crystallizes in the monoclinic space group C2/c. In complex 1, each asymmetric unit consists of half a [Cu2(Cbiq)4(H2O)2]2+ dication, two Br- anions, and three H2O molecules (two of them having 0.3 occupancies, one of them having 0.4 occupancies). As depicted in Fig. 1, the centrosymmetric binuclear coppers are bridged by four Cbiq molecules, and each copper ion is further coordinated to one water molecule, thereby forming a paddle - wheel structure with four carboxylate bridges. There is a center symmetry point at the center of the bond Cu1…Cu1i with a bond length of 0.261 4(14) nm. The selected bond distances and angles for complex 1 are shown in Table 2.
Figure 1
Table 2
Cu1—O1i 0.194 2(4) Cu1—O4 0.198 3(4) Cu1—O2 0.198 6(4) Cu1—O3i 0.199 8(4) Cu1—O1W 0.215 4(4) Cu1—Cu1i 0.261 4(14) O1i—Cu1—O4 88.85(17) O1i—Cu1—O2 168.99(16) O4i—Cu1—O2i 91.64(16) O1i—Cu1—O3i 89.65(16) O4—Cu1—O3i 169.83(17) O2—Cu1—O3i 87.94(16) O1i—Cu1—O1W 91.69(16) O4—Cu1—O1W 95.30(15) O2—Cu1—O1W 99.21(16) O3i—Cu1—O1W 94.80(16) O1i—Cu1—Cu1i 83.30(12) O4—Cu1—Cu1i 80.89(12) O2—Cu1—Cu1i 85.91(12) O3i—Cu1—Cu1i 88.94(17) O1W—Cu1—Cu1i 173.74(11) Symmetry code: i 2-x, -y, 2-z. 2.2.2 Structure of complex 2
Complex 2 crystallizes in the monoclinic space group P21 and the asymmetric unit consists of two [Zn(Cbiq)2(H2O)2]2+ dications, two Cbiq molecules, ten Br- anions (three of them having 0.3 occupancies, four of them having 0.4 occupancies and three of them having 0.5 occupancies), and five H2O molecules (each of them having 0.4 occupancies). As depicted in Fig. 2, the center Zn ion is coordinated by two H2O molecules and two O atoms of carboxylate from two unidentate Cbiq molecules, thereby forming a distorted tetrahedron coordination geometry. The selected bond distances and angles for complex 2 are shown in Table 3.
Figure 2
Table 3
Zn1—O1 0.206 1(3) Zn1—O3 0.192 4(3) Zn2—O6 0.202 9(3) Zn2—O8 0.220 2(3) Zn1—O1W 0.199 5(3) Zn1—O2W 0.197 8(3) Zn2—O3W 0.198 8(3) Zn2—O4W 0.193 4(3) O3—Zn1—O2W 98.71(14) O3—Zn1—O1W 112.49(14) O2W—Zn1—O1W 89.26(13) O3—Zn1—O1 133.07(13) O2W—Zn1—O1 111.42(13) O1W—Zn1—O1 103.30(13) O4W—Zn2—O3W 95.95(13) O4W—Zn2—O6 106.37(13) O3W—Zn2—O6 101.11(13) O4W—Zn2—O8 96.79(12) O3W—Zn2—O8 104.12(12) O6—Zn2—O8 143.53(12) 2.2.3 Structure of complexes 3 and 4
Complexes 3 and 4 all crystallize in the trigonal space group R3 and the asymmetric unit consists of half a [M3(Cbiq)8(μ-OH)2(H2O)2]2+ dication (M=Co (3), Mn (4)), two ClO4- anions, nine H2O molecules (two of them having 0.5 occupancies, five of them having 0.4 occupancies, one of them having 0.33 occupancies and one of them having 0.17 occupancies). Because complexes 3 and 4 have similar [M3(Cbiq)8(μ-OH)2(H2O)2]2+ dication structures in which every two M (Ⅱ) ions are bridged by two Cbiq molecules and one hydroxo - O atom and the two peripheric M (Ⅱ) ions further coordinate to two unidentate Cbiq molecules and one H2O molecule, only the perspective view of the molecular structure of complex 3 is depicted in Fig. 3. The selected bond distances and angles for complexes 3 and 4 are listed in Table 4. The similar structure of complex 4 is shown in Fig.S3.
Figure 3
Table 4
3 Co1—O7i 0.206 3(2) Co1—O7 0.206 3(2) Co1—O6i 0.207 0(2) Co1—O6 0.207 0(2) Co1—O9i 0.211 0(2) Co1—O9 0.211 0(2) Co2—O8 0.201 9(2) Co2—O3 0.209 3(2) Co2—O5 0.209 8(2) Co2—O9 0.212 0(2) Co2—O1 0.214 3(2) Co2—O1W 0.216 8(2) O7i—Co1—O7 180.00(8) O7i—Co1—O6i 90.76(8) O7—Co1—O6i 89.24(8) O7i—Co1—O6 89.24(8) O7—Co1—O6 90.76(8) O6i—Co1—O6 180.00(11) O7i—Co1—O9i 92.49(8) O7—Co1—O9i 87.51(8) O6i—Co1—O9i 92.01(8) O6—Co1—O9i 87.99(8) O7i—Co1—O9 87.51(8) O7—Co1—O9 92.49(8) O6i—Co1—O9 87.99(8) O6—Co1—O9 92.01(8) O9i—Co1—O9 180.00(13) O8—Co2—O3 173.80(8) O8—Co2—O5 89.54(8) O3—Co2—O5 88.81(8) O8—Co2—O9 98.06(8) O3—Co2—O9 88.03(8) O5—Co2—O9 94.64(8) O8—Co2—O1 87.95(8) O3—Co2—O1 93.38(8) O5—Co2—O1 176.09(8) O9—Co2—O1 88.68(8) O8—Co2—O1W 86.70(9) O3—Co2—O1W 87.31(8) O5—Co2—O1W 89.58(9) O9—Co2—O1W 173.65(9) O1—Co2—O1W 87.29(9) 4 Mn1—O7 0.209 4(16) Mn1—O7i 0.209 4(16) Mn1—O6i 0.209 5(16) Mn1—O6 0.209 5(16) Mn1—O9 0.214 5(15) Mn1—O9i 0.214 5(15) Mn2—O8 0.204 7(16) Mn2—O5 0.212 3(17) Mn2—O3 0.212 7(17) Mn2—O9 0.215 4(15) Mn2—O1 0.217 6(16) Mn2—O1W 0.219 2(16) O7—Mn1—O7i 180.00(10) O7—Mn1—O6i 89.33(6) O7i—Mn1—O6i 90.67(6) O7—Mn1—O6 90.67(6) O7i—Mn1—O6 89.33(6) O6i—Mn1—O6 180.0 O7—Mn1—O9 92.29(6) O7i—Mn1—O9 87.71(6) O6i—Mn1—O9 87.84(6) O6—Mn1—O9 92.16(6) O7—Mn1—O9i 87.71(6) O7i—Mn1—O9i 92.29(6) O6i—Mn1—O9i 92.16(6) O6—Mn1—O9i 87.84(6) O9—Mn1—O9i 180.0 O8—Mn2—O5 89.57(7) O8—Mn2—O3 173.99(6) O5—Mn2—O3 88.73(7) O8—Mn2—O9 97.99(6) O5—Mn2—O9 94.55(6) O3—Mn2—O9 87.89(6) O8—Mn2—O1 87.90(6) O5—Mn2—O1 176.18(7) O3—Mn2—O1 93.50(7) O9—Mn2—O1 88.63(6) O8—Mn2—O1W 86.87(6) O5—Mn2—O1W 89.69(7) O3—Mn2—O1W 87.35(7) O9—Mn2—O1W 173.55(7) O1—Mn2—O1W 87.32(7) Symmetry codes: i 1-x, 2-y, -z for 3; i 1-x, 2-y, -z for 4. 2.3 Cleavage of pBR322 DNA
It is known that many metal complexes are capable of catalyzing the cleavage of DNA[31]. We investigated the cleaving activities of complexes 1 - 4 toward pBR322 DNA. Fig. 4 shows the GE patterns for the cleavage of pBR322 DNA by complexes 1-4 at pH 7.0 and 37 ℃. It can be seen that the conversion of the supercoiled DNA (CCC) form (Form Ⅰ) into the open circular (OC) form (Form Ⅱ) was apparent. The DNAcleaving efficiency decreased in the order of complex 1 (Lane 3)
2 (Lane 4) ≈ 3 (Lane 5) ≈ 4 (Lane 6). Of the four complexes, complex 1 was the most active and therefore its DNA-cleaving activity was further explored.$ \gg $ Figure 4
Firstly, we carried out the concentration - dependent DNA cleavage by complex 1 (Fig. 5). It can be seen that complex 1 was capable of efficiently converting pBR322 DNA into OC form and that the cleaving activity increased with the concentration of complex 1 (Lanes 2-7). When the concentration of complex 1 was about 0.5 mmol·L-1, almost all of Form Ⅰ was converted to Form Ⅱ. It should be noted that neither (HCbiq) Br (Lane 8) nor CuSO4 (Lane 9) showed any obvious cleaving activity. Such concentration dependence experiments lent strong support that complex 1 catalyzed the cleavage.
Figure 5
Secondly, to gain insight into the cleaving activity of complex 1, the kinetics of pBR322 DNA degradation was studied. Fig. 6a shows that the extent of supercoiled DNA cleavage into Form Ⅱ varied exponentially with the reaction time (0.5 mmol·L-1), giving pseudofirst - order kinetics with a rate constant of (0.494± 0.032) h-1. The saturation kinetics profile (Fig. 6b) gave the kmax and KM obtained from the saturation ((2.80± 0.97) h-1 and (3.22±0.76) mmol·L-1) for complex 1. Thus, the catalytic efficiency (kmax/KM) was 0.88 L· mmol-1·h-1 for complex 1. In particular, complex 1 could catalyze the cleavage at a rate acceleration of about 108 fold over uncatalyzed cleavage of supercoiled DNA cleavage (k=3.6×10-8 h-1 for cleavage of a phosphodiester bond in double-stranded DNA under physiological condition)[32-34].
Figure 6
Thirdly, to gain further insight into the cleaving activity of complex 1, its mechanism of action toward the cleavage of pBR322 DNA was investigated. It is known that nucleic acid can be cleaved through either an oxidative or hydrolytic pathway. In general, oxidative cleavage of plasmid DNA may lead to the formation of reactive singlet oxygen (1O2), hydrogen peroxide (H2O2), and/or hydroxyl radical (·OH) species. These species contain a photo or redox active center, which causes damage to the sugar and/or base[34-35]. Therefore, to distinguish the probable mechanism of action of complex 1, we conducted the cleavage reactions in the presence of hydroxyl radical scavengers DMSO and MeOH, singlet oxygen scavenger NaN3, hydrogen peroxide scavenger KI and metal ion - chelating agent EDTA[36] (Fig. 7). As a result, EDTA (Lane 6) efficiently inhibited DNA cleavage, indicating that complex 1 was obligatory in DNA cleavage reaction. When DMSO (Lane 3) and MeOH (Lane 4) were added to the reaction mixture, no significant influence on the DNA cleavage was observed, strongly suggesting that hydroxyl radical was not involved in the DNA cleavage. In the presence of NaN3 (Lane 7), the DNA cleavage was significantly inhibited, suggesting that singlet oxygen was likely to be the reactive species responsible for the nuclease activity[37]. Similarly, in the presence of KI (Lane 5), the cleavage was repressed, suggesting that H2O2 might be the reactive species in the cleavage process. Taken together, these results strongly suggest that DNA cleavage by complex 1 proceeds via an oxidative mechanism[38]. Thus, the proposed mechanism may be that the copper centers strongly bind O2 to form reactive oxygen species (ROS), such as singlet oxygen and superoxide, which can further activate the cleavage of supercoiled DNA to form nicked DNA[39].
Figure 7
2.4 DNA binding
It is widely recognized that DNA binding is a critical step for DNA cleavage in most cases. Thus, we estimated the binding affinities of complexes 1-4 by means of EB displacement experiments. EB emits intense fluorescent light in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs. Competitive binding of other drugs to DNA leads to the displacement of bound EB and a decrease in fluorescence intensity[40]. Under the measuring conditions, (HCbiq)Br and complexes 1-4 induced decreases in the fluorescence intensity (FI) of EB (Fig.S10-S19), indicating that they were capable of substituting EB bound to CT DNA. Their binding constants were obtained by analyzing the relationship between the relative fluorescence intensity and the concentrations of each complex (Table 5). As a result, complex 1 had the highest affinity with the binding constant being (8.63±3.82) × 105 L·mol-1, 10-100 fold higher than those of complexes 2-4 and (HCbiq)Br, which was consistent with their DNA cleaving activities.
Table 5
Compound Ka/(L·mol-1) (HCbiq)Br (4.59±0.88)×103 1 (8.63±3.82)×105 2 (3.16±0.51)×103 3 (6.86±1.38)×104 4 (2.02±1.02)×104 2.5 Molecular docking
Molecule simulation is an efficient way to predict the binding mode and the interaction region[41]. Molecular docking simulates the bonding form between the molecule and target DNA. The negative values of the binding energy of docked compounds suggest that the compounds bind to DNA strands. The binding free energy of complex 1 was -49.87 kJ·mol-1. Fig. 8 shows that complex 1 binds DNA through hydrogen bonds and π-π stacking between aromatic rings from bases of CT DNA and benzene rings from complex 1. Complex 1 binds to the large groove of CT DNA and intercalates with it, forming eight aromatic centers (0.35-0.56 nm) and two hydrogen bonds (0.29-0.34 nm). The hydrophobic interaction between CT DNA and complex 1 was also formed, which was not shown in the figure. Thus, computer- aided molecular docking studies show there are many interactions between CT DNA and complex 1. It has been reported that most synthesized metal complexes could bind to DNA with intercalation mode[42-43], which would affect the stability of DNA, thus exhibiting DNA cleavage capacity.
Figure 8
3. Conclusions
In summary, the new ligand of (HCbiq)Br and its four metal complexes have been synthesized and fully characterized. The efficiency of (HCbiq)Br and its metal complexes in promoting the cleavage of DNA was monitored by the use of agarose GE. Kinetic assays indicated that complex 1 was capable of efficiently cleaving plasmid pBR322 DNA, most probably through an oxidative mechanistic pathway. Molecular docking exposed the binding mode of complex 1 with DNA.
The results presented in this study highlight the fact that zwitterionic carboxylate metal complexes exhibit biological activities. Efforts aimed at developing diverse zwitterionic carboxylate metal complexes are currently in progress with a view toward the design of new synthetic nucleases with promising antitumor activity.
Supporting information is available at http://www.wjhxxb.cn
Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (Grants No. 21874064, 21871203), and the Medical Scientific Research Foundation of Guangdong Province of China (Grant No. A2021267). -
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[1]
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Figure 5 Agarose GE patterns for the cleavage of pBR322 DNA by complex 1 of increasing concentrations in 5 mmol·L-1 Tris-HCl (5 mmol·L-1 NaCl, pH 7.0) at 37 ℃ (5 h)
Lane 1, DNA alone; Lanes 2-7, DNA+complex 1 at the concentrations of 0.05, 0.1, 0.2, 0.5, 0.75, and 1.0 mmol·L-1, respectively; Lane 8, DNA+(HCbiq)Br (4 mmol·L-1); Lane 9, DNA+CuSO4 (2 mmol·L-1)
Figure 6 (a) Time course of pBR322 DNA cleavage promoted by complex 1 (0.5 mmol·L-1) in 5 mmol·L-1 Tris-HCl (5 mmol·L-1 NaCl, pH 7.0) at 37 ℃; (b) Saturation kinetics plot of kobs versus the concentration of complex 1
xForm Ⅰ is the percentage of supercoiled DNA (Form Ⅰ); Inset: agarose GE patterns of the time-variable reaction products; Lane 1, DNA alone; Lanes 2-8, reaction time was 0.5, 1, 1.5, 2, 2.5, 3.5, and 4.5 h, respectively
Table 1. Crystallographic data for complexes 1-4
Parameter 1 2 3 4 Molecular formula C68H60Br4N4O12Cu2 C51H45Br2N3O9Zn C136H124Cl4N8O43Co3 C136H124Cl4N8O43Mn3 Formula weight 1 571.92 1 069.09 2 877.02 2 865.05 Crystal system Monoclinic Monoclinic Rhombohedral Rhombohedral Space group C2/c P21 R3 R3 a/nm 3.028 31(11) 1.012 41(12) 3.616 12(11) 3.661 23(12) b/nm 1.710 53(13) 2.720 92(13) 3.616 12(11) 3.661 23(12) c/nm 1.379 12(12) 2.053 81(12) 3.079 59(14) 3.125 32(18) β/(°) 102.121(3) 91.081(3) V/nm3 6.984 6(8) 5.656 6(8) 34.875(2) 36.281(3) Z 4 4 9 9 T/K 291(2) 291(2) 291(2) 291(2) Dc/(g·cm-3) 1.495 1.255 1.233 1.180 μ/mm-1 2.958 1.897 0.463 0.371 Total reflection 31 641 42 020 84 481 92 574 2θmax/(°) 52.00 52.00 52.00 52.00 Unique reflection 6 851 (Rint=0.069 1) 20 940 (Rint=0.018 6) 15 113 (Rint=0.073 3) 15 797 (Rint=0.066 8) Observed reflection [I > 2σ(I)] 4 423 16 594 10 376 11 483 Number of parameters 424 1 271 912 912 Ra 0.061 2 0.050 5 0.063 9 0.058 6 wRb 0.133 2 0.123 3 0.138 9 0.110 0 GOFc 1.072 1.024 1.071 1.075 (Δρ)max, (Δρ)min/(e·nm-3) 780, -424 441, -307 295, -317 295, -251 a R=∑||Fo|-|Fc||/|Fo|; b wR=[∑w(Fo2-Fc2)2/∑w(Fo2)2]1/2; c GOF=[∑w(Fo2-Fc2)2/(n-p)]1/2, where n is the number of reflections and p is the total numbers of parameters refined. Table 2. Selected bond lengths (nm) and angles (°) for complex 1
Cu1—O1i 0.194 2(4) Cu1—O4 0.198 3(4) Cu1—O2 0.198 6(4) Cu1—O3i 0.199 8(4) Cu1—O1W 0.215 4(4) Cu1—Cu1i 0.261 4(14) O1i—Cu1—O4 88.85(17) O1i—Cu1—O2 168.99(16) O4i—Cu1—O2i 91.64(16) O1i—Cu1—O3i 89.65(16) O4—Cu1—O3i 169.83(17) O2—Cu1—O3i 87.94(16) O1i—Cu1—O1W 91.69(16) O4—Cu1—O1W 95.30(15) O2—Cu1—O1W 99.21(16) O3i—Cu1—O1W 94.80(16) O1i—Cu1—Cu1i 83.30(12) O4—Cu1—Cu1i 80.89(12) O2—Cu1—Cu1i 85.91(12) O3i—Cu1—Cu1i 88.94(17) O1W—Cu1—Cu1i 173.74(11) Symmetry code: i 2-x, -y, 2-z. Table 3. Selected bond lengths (nm) and angles (°) for complex 2
Zn1—O1 0.206 1(3) Zn1—O3 0.192 4(3) Zn2—O6 0.202 9(3) Zn2—O8 0.220 2(3) Zn1—O1W 0.199 5(3) Zn1—O2W 0.197 8(3) Zn2—O3W 0.198 8(3) Zn2—O4W 0.193 4(3) O3—Zn1—O2W 98.71(14) O3—Zn1—O1W 112.49(14) O2W—Zn1—O1W 89.26(13) O3—Zn1—O1 133.07(13) O2W—Zn1—O1 111.42(13) O1W—Zn1—O1 103.30(13) O4W—Zn2—O3W 95.95(13) O4W—Zn2—O6 106.37(13) O3W—Zn2—O6 101.11(13) O4W—Zn2—O8 96.79(12) O3W—Zn2—O8 104.12(12) O6—Zn2—O8 143.53(12) Table 4. Selected bond lengths (nm) and angles (°) for complexes 3 and 4
3 Co1—O7i 0.206 3(2) Co1—O7 0.206 3(2) Co1—O6i 0.207 0(2) Co1—O6 0.207 0(2) Co1—O9i 0.211 0(2) Co1—O9 0.211 0(2) Co2—O8 0.201 9(2) Co2—O3 0.209 3(2) Co2—O5 0.209 8(2) Co2—O9 0.212 0(2) Co2—O1 0.214 3(2) Co2—O1W 0.216 8(2) O7i—Co1—O7 180.00(8) O7i—Co1—O6i 90.76(8) O7—Co1—O6i 89.24(8) O7i—Co1—O6 89.24(8) O7—Co1—O6 90.76(8) O6i—Co1—O6 180.00(11) O7i—Co1—O9i 92.49(8) O7—Co1—O9i 87.51(8) O6i—Co1—O9i 92.01(8) O6—Co1—O9i 87.99(8) O7i—Co1—O9 87.51(8) O7—Co1—O9 92.49(8) O6i—Co1—O9 87.99(8) O6—Co1—O9 92.01(8) O9i—Co1—O9 180.00(13) O8—Co2—O3 173.80(8) O8—Co2—O5 89.54(8) O3—Co2—O5 88.81(8) O8—Co2—O9 98.06(8) O3—Co2—O9 88.03(8) O5—Co2—O9 94.64(8) O8—Co2—O1 87.95(8) O3—Co2—O1 93.38(8) O5—Co2—O1 176.09(8) O9—Co2—O1 88.68(8) O8—Co2—O1W 86.70(9) O3—Co2—O1W 87.31(8) O5—Co2—O1W 89.58(9) O9—Co2—O1W 173.65(9) O1—Co2—O1W 87.29(9) 4 Mn1—O7 0.209 4(16) Mn1—O7i 0.209 4(16) Mn1—O6i 0.209 5(16) Mn1—O6 0.209 5(16) Mn1—O9 0.214 5(15) Mn1—O9i 0.214 5(15) Mn2—O8 0.204 7(16) Mn2—O5 0.212 3(17) Mn2—O3 0.212 7(17) Mn2—O9 0.215 4(15) Mn2—O1 0.217 6(16) Mn2—O1W 0.219 2(16) O7—Mn1—O7i 180.00(10) O7—Mn1—O6i 89.33(6) O7i—Mn1—O6i 90.67(6) O7—Mn1—O6 90.67(6) O7i—Mn1—O6 89.33(6) O6i—Mn1—O6 180.0 O7—Mn1—O9 92.29(6) O7i—Mn1—O9 87.71(6) O6i—Mn1—O9 87.84(6) O6—Mn1—O9 92.16(6) O7—Mn1—O9i 87.71(6) O7i—Mn1—O9i 92.29(6) O6i—Mn1—O9i 92.16(6) O6—Mn1—O9i 87.84(6) O9—Mn1—O9i 180.0 O8—Mn2—O5 89.57(7) O8—Mn2—O3 173.99(6) O5—Mn2—O3 88.73(7) O8—Mn2—O9 97.99(6) O5—Mn2—O9 94.55(6) O3—Mn2—O9 87.89(6) O8—Mn2—O1 87.90(6) O5—Mn2—O1 176.18(7) O3—Mn2—O1 93.50(7) O9—Mn2—O1 88.63(6) O8—Mn2—O1W 86.87(6) O5—Mn2—O1W 89.69(7) O3—Mn2—O1W 87.35(7) O9—Mn2—O1W 173.55(7) O1—Mn2—O1W 87.32(7) Symmetry codes: i 1-x, 2-y, -z for 3; i 1-x, 2-y, -z for 4. Table 5. Binding constants (Ka) with CT DNA of (HCbiq)Br and complexes 1-4*
Compound Ka/(L·mol-1) (HCbiq)Br (4.59±0.88)×103 1 (8.63±3.82)×105 2 (3.16±0.51)×103 3 (6.86±1.38)×104 4 (2.02±1.02)×104
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