2
The dicopper(ii) complex [Cu2(R′SSR)2(SO4)2] (1), where R′SSR is a Schiff base, has been prepared from the reaction of CuSO4·5H2O with the Schiff base N,N′-1,1′-dithiobis(ethylenesalicylaldimine) (H2RSSR) and structurally characterized by X-ray crystallography.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res1
3
The crystal structure of 1 shows two {Cu(R′SSR)}2+ units linked by two sulfate ligands each showing a η3,μ2-binding mode.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res2
4
The Cu⋯Cu distance is 4.562(2) Å with each copper having a square pyramidal (4 + 1) CuNO4 coordination geometry.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res2
5
The monoanionic Schiff base R′SSR has a pendant cationic amine –SCH2CH2NH3+ group which is presumably formed from the hydrolysis of one imine bond of H2RSSR.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con1
6
Complex 1 models the N- and C-terminus domains of bleomycins.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac1
7
The metal centers in 1 are essentially magnetically non-interacting giving a −2J value of 3 cm−1 with the singlet as the ground state.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res3
8
Using complex 1 as a precursor, ternary copper(ii) complexes [Cu(R′SSR)B(SO4)] (2–4) are prepared, characterized and their DNA binding and cleavage properties studied (B: kanamycin A, 2; 2,2′-bipyridine, 3; 1,10-phenanthroline, 4).
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj2
9
IR spectral data suggest a square pyramidal (4 + 1) geometry for the one-electron paramagnetic ternary complexes with the sulfate bound to copper.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res4
10
The complexes are non-conducting in DMF but show conductivity in aqueous medium due to dissociation of the sulfate ligand.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res5
11
They bind to calf thymus DNA in the minor groove giving the relative order: 4 > 2 > 1 ∼ 3 (Kapp = 5.4 × 105 M−1 for 4).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs1
12
The precursor complex 1 does not show any apparent chemical nuclease activity when treated with supercoiled (SC) DNA in the presence of 3-mercaptopropionic acid (MPA).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs2
13
The kanamycin A and phen adducts as such or generated under in situ reaction conditions using 1 and the ligand display efficient chemical nuclease activity in the presence of MPA, while the bpy species shows poor cleavage activity.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs3
14
The ternary kanamycin A complex presents the first synthetic model for three functional domains of bleomycins.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con2
Introduction
15
Bleomycins (BLMs) are the glycopeptide antitumor antibiotics that cleave DNA in an oxidative manner and are clinically used for the treatment of squamous cell and malignant lymphomas.1–3
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
16
Bleomycins, in the presence of ferrous ion and molecular oxygen, cause sequence-selective DNA strand scission.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
17
The structure of bleomycins consists of three major domains playing different functional roles (Scheme 1).
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
18
The N-terminus domain has binding sites for the metal, viz. iron or copper.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
19
Binding of molecular oxygen to the metal occurs in this domain.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
20
The C-terminus domain with a bithiazole unit and a cationic terminal amine moiety show DNA binding affinity and sequence selectivity.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
21
The carbohydrate domain facilitates cell permeability of BLMs and oxygen binding.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
22
Since activation of bleomycins requires a metal ion cofactor such as Fe2+ (or Cu2+ in the presence of dithiothreitol as reductant), the coordination chemistry of low molecular weight transition metal complexes as synthetic models assume paramount importance for gaining better insights into different structural and functional aspects of BLMs.
Type: Motivation |
Advantage: None |
Novelty: None |
ConceptID: Mot1
23
BLM–DNA interactions have been studied using various analogues/conjugates to probe specific roles played by each domain.1–6
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
24
In comparison, synthetic model iron or copper complexes are few and they do not model all the domains of the elephantine structure of BLMs.7–11
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
25
The structural and chemical complexities associated with BLMs have been addressed by Mascharak and co-workers in a series of papers on synthetic model iron and copper complexes.7,8
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
26
The complexes generally model the metal binding N-terminus domain.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
27
Hertzberg and Dervan have reported a model iron complex with a proposed structure that mimics the N- and C-terminus domains.9
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
28
They suggested that methidiumpropyl-EDTA in presence of Fe2+ forms an iron–EDTA complex with a pendant cationic DNA binding site and the complex cleaves DNA in an oxidative manner.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
29
Using a similar synthetic analogue approach, we are successful in forming ternary copper(ii) complexes containing carbohydrate or heterocyclic base (B) using a precursor copper(ii) complex [Cu2(R′SSR)2(SO4)2] (1) that has a pendant arm with a cationic amine moiety.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res6
30
Complex 1 has been structurally characterized by X-ray crystallography.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res7
31
Our proposed kanamycin A bound ternary copper(ii) complex12 is unique in modeling three functional domains of BLMs (Scheme 1).
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac4
32
The significant result of this study is the observation of efficient DNA cleavage activity of the kanamycin A complex in the presence of mercaptopropionic acid (MPA).
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res8
33
Ternary copper(ii) complexes [Cu(R′SSR)B(SO4)] (2–4) are prepared for the DNA binding and cleavage studies from the reaction of 1 with kanamycin A or heterocyclic bases such as 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) (B: kanamycin A, 2; bpy, 3; phen, 4).
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj3
34
Our choice of 1 as a precursor is based on its novel structural features showing the presence of a cationic DNA-binder arm making it a potential synthetic model for the N- and C-terminus domains of BLMs.
Type: Object |
Advantage: Yes |
Novelty: New |
ConceptID: Obj3
35
Herein we report the synthesis, structure and DNA cleavage properties of 1–4.
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj4
Experimental
Materials
36
All reagents and chemicals were procured from commercial sources and used without further purifications.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
37
Solvents used were purified by standard procedures.13
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
38
Calf thymus (CT) DNA and supercoiled (SC) pUC19 DNA (caesium chloride purified) were purchased from Bangalore Genie (India).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
39
Agarose (molecular biology grade) and ethidium bromide (EB) were from Sigma (USA).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
40
The disulfide Schiff base ligand (H2RSSR) was prepared by a literature procedure using cysteamine hydrochloride (Lancaster, UK) and salicylaldehyde (Aldrich, USA)14.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
Physical measurements
41
The elemental analyses were done using a Thermo Finnigan Flash EA 1112 CHNSO analyser.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp2
42
The IR, electronic, and fluorescence spectral data were obtained from Bruker Equinox 55, Hitachi U-3400, and Perkin Elmer LS-50B spectrometers, respectively.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp3
43
Conductivity measurements were made using Control Dynamics Conductivity Meter.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp4
44
Electrochemical measurements were done at 25 °C on an EG & G PAR model 253 Versa Stat potentiostat/galvanostat with electrochemical analysis software 270 for cyclic voltammetric work using a three-electrode setup consisting of a glassy carbon working, platinum wire auxiliary and saturated calomel reference electrode.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp5
45
Variable temperature magnetic susceptibility data in the temperature range 18–300 K were obtained for polycrystalline samples using a George Associates Inc. Lewis-coil-force magnetometer system (Berkeley, CA) equipped with a closed-cycle cryostat (Air Products) and a Cahn balance.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp6
46
Hg[Co(NCS)4] was used as a standard.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp6
47
Experimental susceptibility data were corrected for diamagnetic contributions and temperature independent paramagnetism.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp6
48
The corrected molar magnetic susceptibilities for the dicopper(ii) complex were theoretically fitted by the modified Bleaney–Bowers expression based on the isotropic form of the Heisenberg–Dirac–van Vleck (HDvV) model giving H = −2JS1S2, where S1 = S2 = ½.15,16
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod1
49
The susceptibility equation used for fitting was: χCu = [Ng2β2/kT][3 + exp(−2J/kT)]−1(1 − ρ) + (Ng12β2/4kT)ρ + Nα, where ρ is the fraction of monomeric impurity.
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod1
Synthesis
Preparation of [Cu2(R′SSR)2(SO4)2] (1)
50
The precursor complex 1 was prepared as a green crystalline solid in ∼55% yield (220 mg) from the reaction of CuSO4·5H2O (0.25 g, 1.0 mmol) with the Schiff base N,N′-1,1′-dithiobis(ethylenesalicylaldimine) (H2RSSR, 0.36 g, 1.0 mmol) in CH2Cl2–MeOH (12 cm3, 9 ∶ 1 v/v) at 25 °C for 1 h magnetic stirring in dark under nitrogen atmosphere.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
51
The product, obtained on slow concentration of the solution, was washed with methanol and finally dried in vacuum over P4O10 (Found: C, 31.8; H, 3.7; N, 6.9, C22H32Cu2N4O10S6 (1) requires C, 31.7; H, 3.9; N, 6.7%).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
52
Conductivity (ΛM/Ω−1 cm2 mol−1) in H2O = .39017
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
53
UV-vis in H2O [λmax/nm (ε/dm3 mol−1 cm−1)]: 631 (90), 321 (3850), 269 (8250), 237 (12300), 218 (14200).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
54
IR (KBr phase, cm−1): 2930w, 1632s, 1542m, 1475m, 1452m, 1402w, 1349w, 1315w, 1200s, 1144vs, 1130vs, 1115vs, 1052w, 1000m, 936m, 912w, 760m, 656w, 620m, 586w, 486w, 456w (vs, very strong; s, strong; m, medium; w, weak).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
55
μeff per Cu = 2.0 at 300, 1.9 μB at 18 K [theoretical fit: −2J = 3 cm−1, g = 2.18, g1 = 2.2, ρ = 0.004].
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
Synthesis of [Cu(R′SSR)B(SO4)] (B: kanamycin A, 2)
56
The ternary copper(ii) kanamycin A adduct was prepared from reaction of 1 (1.0 mmol, 0.83 g) with kanamycin A (2.0 mmol, 1.16 g) in water (20 cm3) at 25 °C on magnetic stirring for 12 h.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
57
The solution was filtered and the filtrate on slow concentration gave brownish-green solid which was isolated, washed with aqueous methanol (1 ∶ 1 v/v) and dried in vacuum over P4O10 giving ∼62% (0.55 g) yield (Found: C, 39.1; H, 3.9; N, 9.5, C29H37 CuN6O16S3 (2) requires C, 39.3; H, 4.2; N, 9.5%).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
58
Conductivity (ΛM/Ω−1 cm2 mol−1) in DMF–H2O (1 ∶ 4 v/v) = 145.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
59
UV-vis in DMF [λmax/nm (ε/dm3 mol−1 cm−1)]: 630 (175), 369 (1090), 310 (3970).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
60
IR (KBr phase, cm−1): 3337br, 3054w, 1622s, 1538m, 1465m, 1450m, 1400m, 1345w, 1315m, 1282w, 1205m, 1145vs, 1109vs, 1000m, 939m, 906w, 756m, 613s, 483w, 453w (br, broad).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
Synthesis of [Cu(R′SSR)B(SO4)] (B: bpy, 3; phen, 4)
61
Complexes 3 and 4 were prepared by following a general procedure in which the heterocyclic base (2.0 mmol: 0.31 g bpy or 0.4 g phen) was reacted with an aqueous solution (20 cm3) of 1 (1.0 mmol, 0.83 g).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
62
The solution was magnetically stirred for 5 h at 25 °C.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
63
The solution was filtered.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
64
The product as a green solid was isolated on slow concentration of the filtrate on a rotary evaporator.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
65
The solid was washed with aqueous ethanol (1 ∶ 1 v/v) and dried in vacuum over P4O10 (Yield: 74%, 0.42 g for 3; 68%, 0.4 g for 4).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
66
Characterization data for 3: Found: C, 44.3; H, 4.0; N, 9.6, C21H24CuN4O5S3 requires C, 44.1; H, 4.2; N, 9.8%.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
67
Conductivity (ΛM/Ω−1 cm2 mol−1) in DMF–H2O (1 ∶ 4 v/v) = 140.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
68
UV-vis in DMF [λmax/nm (ε/dm3 mol−1 cm−1)]: 611 (150), 371 (830), 274 (2300).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
69
IR (KBr phase, cm−1): 3700br, 3037w, 1618s, 1595m, 1565w, 1532m, 1470m, 1442s, 1320m, 1242w, 1116vs, 1022m, 980w, 906w, 769s, 730m, 660w, 620s, 470w, 456w.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
70
Characterization data for 4: Found: C, 46.2; H, 3.7; N, 9.5, C23H24N4CuO5S3 requires C, 46.3; H, 4.0; N, 9.4%.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
71
Conductivity (ΛM/Ω−1 cm2 mol−1) in DMF–H2O (1 ∶ 4 v/v) = 135.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
72
UV-vis in DMF [λmax/nm (ε/dm3 mol−1 cm−1)]: 666 (105), 372 (1820), 318 (1350), 268 (3260).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
73
IR (KBr phase, cm−1): 3740br, 3050w, 1621s, 1541m, 1515m, 1473m, 1450m, 1424m, 1348m, 1330w, 1147vs, 1103vs, 1040w, 987w, 912w, 855s, 760m, 722s, 616s.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
Crystal structure determination of 1
Crystal data
74
C22H32Cu2N4O10S6, Mr = 831.96, green rectangular (0.50 × 0.32 × 0.28 mm), monoclinic, space group P21/c, a = 12.771(7), b = 15.664(9), c = 8.544(5) Å, β = 105.971(9)°, U = 1643.2(16) Å3, Z = 2, Dc = 1.682 g cm−3, μ = 1.732 mm−1, min./max.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp10
75
transmission = 0.48/0.64, 2θmax = 50°, λ(Mo-Kα) = 0.71073 Å, T = 293(2) K.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp10
Data collection and processing
76
Crystals of 1 were obtained from the mother-liquor on slow concentration.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp11
77
Intensity data were measured in frames with increasing ω (width of 0.3° frame−1, scan speed of 12 s frame−1) on a Bruker SMART APEX CCD diffractometer.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp11
78
The data were corrected for absorption18.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp11
Structure solution and refinement
79
The structure was solved and refined with SHELX system of programs.19
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
80
The non-hydrogen atoms were refined anisotropically.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
81
The hydrogen atoms were located from the difference Fourier maps and were refined isotropically.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
82
The final full-matrix least-squares refinement converged to R1 = 0.0506, wR2 = 0.1162 for 2895 reflections with I > 2σ(I) and 263 parameters [R1 (all data) = 0.0652; wR2 = 0.1229 (all data)], weighting scheme: w = 1/[σ2(Fo2) + (0.063P)2 + 0.7566P], where P = [Fo2 + 2Fc2]/3.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
83
The goodness-of-fit and the largest difference peak are 1.142 and 0.625, respectively.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
84
The perspective view of the complex was obtained using ORTEP.20
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
85
CCDC reference number 236447.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
86
See http://www.rsc.org/suppdata/dt/b4/b414639e/ for crystallographic data in CIF or other electronic format.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
DNA-binding and cleavage experiments
87
The concentration of the calf thymus DNA (125 µM) was obtained from its absorption intensity at 260 nm with a known ε value of 6600 dm3 mol−1 cm−1.21
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res9
88
The binding of the complexes 1–4 to calf thymus (CT) DNA has been studied by fluorescence spectral method using the emission intensity of ethidium bromide (EB).
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj5
89
The apparent binding constant (Kapp) value for the phen complex was estimated from the equation: KEB[EB] = Kapp[complex] using the Kapp value of EB as 107 M−1.22
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res10
90
The DNA cleavage activity of the complexes was studied by agarose gel electrophoresis.
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj6
91
Supercoiled pUC19 DNA (0.8 µl, ∼500 ng) in Tris-HCl buffer (50 mM, pH 7.2) containing NaCl (50 mM) was treated with the complex taken in DMF in the presence or absence of additives.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
92
The oxidative DNA cleavage by the complex was studied in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
93
The sample was incubated for 1 h at 37 °C, added loading buffer (25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3 µl)) and loaded on 0.8% agarose gel containing 1.0 µg ml−1 EB.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
94
Electrophoresis was carried out at 40 V for 2.0 h in Tris-acetate EDTA (TAE) buffer.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
95
Bands were visualized by UV light and photographed.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
96
The cleavage efficiency was measured by determining the ability of the complex in relaxing the SC DNA to its nicked circular (NC) form.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
97
The proportion of DNA in the SC and NC form after electrophoresis was estimated quantitatively from the intensities of the bands using UVITEC Gel Documentation System with due correction of the low level of NC present in the original sample and the low affinity of EB binding to SC compared to NC and linear forms of DNA.23
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
98
Control experiments were carried out in the dark to detect any hydrolytic cleavage of DNA.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp14
99
Religation experiments were carried out to exclude the possibility of hydrolytic cleavage.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp15
100
In these experiments, the NC DNA, obtained from the hydrolytic cleavage reaction, was recovered from the agarose gel using a gel extraction kit and this was followed by addition of 5X ligation buffer and T4 DNA ligase (1 µl, 4 units).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp15
101
The solution was incubated for 10 h at 16 °C prior to gel electrophoresis.24
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp15
102
In the inhibition reactions, the additive such as distamycin or DMSO was added initially to the SC DNA and incubation was done for 15 min at 37 °C prior to the addition of the complex and MPA.25
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp16
103
Considering the good solubility of 1 in water, the DNA binding and cleavage reactions of the adducts 2–4 were studied under in situ conditions in which complex 1 was treated with the ligand (B) in a Tris buffer medium.
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj7
Results and discussion
Synthesis and general aspects
104
Ternary copper(ii) complexes [Cu(R′SSR)B(SO4)] (2–4) are prepared by reacting the dimeric precursor [Cu2(R′SSR)2(SO4)2] (1) with kanamycin A or a heterocyclic base such as 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) (B: kanamycin A, 2; bpy, 3; phen, 4) (Scheme 2).
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met1
105
Our choice of 1 as a precursor is based on its novel structural features showing the presence of a cationic amine pendant arm which is expected to show good DNA binding ability.
Type: Method |
Advantage: Yes |
Novelty: New |
ConceptID: Met1
106
Complex 1 is prepared from a reaction of CuSO4·5H2O with the Schiff base N,N′-1,1′-dithiobis(ethylenesalicylaldimine) (H2RSSR) (Scheme 2).
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met2
107
The complexes 1–4 are characterized by analytical and spectral methods.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met3
108
Complex 1 is structurally characterized by X-ray crystallography.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met4
109
The crystal structure of 1 shows the dimeric nature of the complex in which two {Cu(R′SSR)}2+ units are bridged by two sulfates (Fig. 1).
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res11
110
The Cu⋯Cu distance is 4.562(2) Å and each copper atom has a square pyramidal (4 + 1) CuNO4 coordination geometry with the sulfate atom O(2) as the axial ligand.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res11
111
The pendant –SCH2CH2NH3+ group is presumably formed from the hydrolysis of one imine bond of H2RSSR.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con3
112
The disulfide moiety does not show any apparent interaction with the metal atoms.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res12
113
The phenolato oxygen atom O(1), the terminal amine group and the uncoordinated oxygen atom O(5) of the sulfate are involved in hydrogen bonding interactions in the solid state.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res13
114
Selected bond parameters for 1 are given in Table 1.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res14
115
We have probed the formation of the ternary complexes in solution by electronic spectral and cyclic voltammetric studies.
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj8
116
The conductivity value of 1 in H2O suggests the formation of two monomeric units in solution.17
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res15
117
The two near UV bands at 360 and 320 nm for a solution of 1 in water disappear on addition of the ligand and a new band is observed at 380 nm for kanamycin A and 389 nm for phen (Fig. 2).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs4
118
Cyclic voltammetric experiments show a cathodic peak for 1 at −0.62 V with an anodic counterpart at −0.04 V vs. SCE at 50 mV s−1 in H2O–0.1 M KCl.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs5
119
Addition of kanamycin A to 1 causes a shift of the cathodic peak to −1.04 V with a reduced current and no corresponding anodic response.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs6
120
The voltammetric responses of the ternary species are significantly different from those of CuSO4 in the presence or absence of kanamycin A. The ternary structure of complexes are found to be stable in water even on addition of ten-fold excess quantity of kanamycin A or heterocyclic base (bpy, phen).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs7
121
Although the formation of bis(phen)copper(ii) species is a possibility from the reaction of 1 with excess phen, the cyclic voltammetric measurements rule out any such complex formation as the voltammetric response is not characteristic of this binary complex.26
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res16
122
The one-electron paramagnetic (μeff ∼ 1.8 μB) complexes 2–4 are non-electrolytic in DMF but show 1 ∶ 1 electrolytic behavior in aqueous DMF (4 ∶ 1 v/v).
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res17
123
Complex 1 shows three IR bands for the sulfate which displays a η3,μ2-binding mode to the metal centers.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res18
124
We have also observed two well resolved IR bands for the sulfate ligand in 2 and 4 indicating its bonding to the metal atom.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res19
125
We propose that the sulfate binding could be at the elongated axial site considering its labile nature in aqueous medium.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con4
126
The complexes display a visible band in the spectral range 611–666 nm.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs8
127
This band is assignable to the d–d transition.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res20
128
While complex 1 shows good solubility both in water and DMF, complexes 2–4 are soluble in DMF.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs9
129
Variable temperature magnetic susceptibility data in the range 18–300 K show the essentially paramagnetic nature of the metal atoms in 1.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res21
130
The theoretical fit of the data shows a −2J value of 3 cm−1 indicating the non-interacting nature of the spins in the absence of any superexchange pathway(s) involving the sulfate ligands.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con5
DNA binding and cleavage studies
131
The propensity of binding of 1–4 to calf thymus (CT) DNA has been studied by fluorescence spectral method using the emission intensity of ethidium bromide (EB).
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj9
132
The DNA binding plot gives the relative order: 4 > 2 ≥ 1 ≈ 3 (Kapp: 5.4 × 105 M−1 for 4) (Fig. 3).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs10
133
Complex 1 alone is cleavage inactive when reacted with supercoiled (SC) pUC19 DNA and MPA as a reductant, but it cleaves DNA efficiently on addition of kanamycin A or phen under in situ reaction conditions (Fig. 4).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs11
134
Similar cleavage efficiency is observed using adducts 2 and 4 in the presence of MPA.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs12
135
A 5 µM solution of 1 cleaves SC DNA (∼500 ng) to the extent of ∼90% on addition of 30 µM phen or 50 µM kanamycin A and 5 mM MPA.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs13
136
We have probed different aspects of the DNA binding and cleavage reactions from control experiments using various additives (Fig. 5).
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj10
137
The reaction of copper sulfate with kanamycin A in the presence of MPA shows no apparent cleavage of SC DNA.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs14
138
Similarly, no cleavage of DNA occurs by Cu2+ (aq) or kanamycin A alone in presence of MPA.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs15
139
Distamycin addition is found to inhibit the cleavage activity of the ternary complexes suggesting minor groove directing nature of 1–4 similarly to BLMs.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con6
140
A similar inhibition of DNA cleavage is observed in the presence of hydroxyl radical scavengers such as DMSO indicating an oxidative cleavage pathway involving hydroxyl radicals.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con7
141
Although the true identity of the reactive oxygen species is yet to be conclusively established, it could be either free hydroxyl radical or a metal bound oxo or hydroxo moiety effecting hydrogen abstraction from the deoxyribose sugar moiety.1,27
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con8
142
The oxidative nature of the DNA cleavage is evidenced from the T4 ligase experiments which show no religation of the nicked circular DNA to its original SC form thus excluding the possibility of any hydrolytic cleavage.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res22
143
The disulfide bond of the Schiff base R′SSR is found to be stable under reducing conditions in the presence of excess MPA as the control experiments show enhancement of DNA cleavage efficiency of the ternary complex on increasing the concentration of MPA.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res23
Conclusions
144
In conclusion, the ternary kanamycin A complex, obtained from a dicopper(ii) precursor containing a pendant cationic amine moiety, exemplifies the first potential model for three functional domains of BLMs.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con9
145
While the precursor complex is cleavage inactive, the ternary complexes containing kanamycin A and phen show efficient oxidative DNA cleavage in the presence of MPA.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res24
146
Binding of kanamycin A to copper significantly enhances the DNA cleavage activity of 2 in comparison to the precursor or its bpy adduct thus mimicking the facilitating role of the sugar unit in the DNA cleavage activity of BLMs.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con10
147
The higher cleavage efficiency of the phen complex in comparison to 1–3 could be related to the efficient DNA groove binding ability of this planar heterocyclic base.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con11